CN1852921A - Soluble fragments of the SARS-cov spike glycoprotein - Google Patents

Abstract

The invention relates to the spike protein from the virus (SARS-CoV) that is etiologically linked to severe acute respiratory syndrome (SARS); polypeptides and peptide fragments of the spike protein; nucleic acid segments and constructs that encode the spike protein, polypeptides and peptide fragments of the spike protein, and coupled proteins that include the spike protein or a portion thereof; peptidomimetics; vaccines; methods for vaccination and treatment of severe acute respiratory syndrome; antibodies; aptamers; and kits containing immunological compositions, or antibodies (or aptamers) that bind to the spike protein.

Description

The soluble fragments of sars coronavirus spike glycoprotein

The claimed right of priority to No. the 60/524th, 642, No. the 60/489th, 166, the U. S. application of submitting on July 21st, 2003 and the U. S. application of submitting on November 25th, 2003 of the application, they are attached to herein by reference in full.

Governmental fund

The research and development of invention as described herein are subjected to subsidy healthy and human service department.United States Government has certain right to the present invention.

Invention field

The coded spike glycoprotein of the coronavirus that present invention relates in general to be associated (being SARS-CoV here) with severe acute respiratory syndrome (SARS) etiology.The invention further relates to polypeptide, nucleic acid and their conservative property varient with aminoacid sequence consistent with the spike glycoprotein fragment of SARS-CoV.The present invention also relates to produce the antibody of identification SARS-CoV spike glycoprotein, and produce the vaccine of anti-SARS-CoV with it with these nucleic acid, polypeptide, varient and fragment.But another aspect of invention relates to the spike protein fragment that causes SARS-CoV and zooblast fusion.

Background of invention

Severe acute respiratory syndrome (SARS) is a kind of infectious severe acute respiratory syndrome of confirming in the patient of 32 countries and regions recently.Anetiological severe acute respiratory syndrome is found as far back as the Guangdong Province of China.Subsequently, this serious heat generation respiratory tract disease of propagating has been reported in Hong Kong, Vietnam, Xin Jiapo, Canada and Beijing in succession in kinsfolk and medical personnel.This disease is afterwards by The World Health Organization's called after " severe acute respiratory syndrome (SARS) ".To on May 19th, 2003,29 countries altogether to WHO report 7864 routine SARS cases.Total death report is that (case fatality rate is 643 examples: 8.2%).

Investigators have all over the world carried out sequencing to the genome from the SARS pathogenic virus of global different areas, and this virus is classified into coronavirus.Coronavirus is divided into 3 classes according to the cross reaction and the gene data of its antibody.The human virus of two kinds of previously knowns and SARS-CoV are in different groups.The coronavirus that causes SARS is different from known any group.But self forms a new group.To the phylogenetics analysis prompting of the viral protein of prediction, this virus is not similar closely to any known three class coronavirus.Most of coronavirus or cause respiratory tract disease or cause the intestinal tract disease that fecal oral route is propagated.

Be 2 to 7 days the latent period of SARS.Infection characteristic is heating, dry cough, breathes hard and the slight unusual and rabat realification of auscultation.Lymphopenia, oligoleukocythemia, thrombopenia, liver enzyme increase with the creatinine kinases and increase also existence in most of cases.The symptom relevant with gi tract also can be seen in patient SARS.

The pathological research of dying from patient SARS to Guangdong, Hong Kong, Beijing, Singapore shows, it is obvious characteristic that the wetting property alveolar of lung damages.Due to illness in the serious and dead individuality of feelings, the II type i alveolar cell that is dispersed in demonstrates tangible cytology to be changed, and comprises multinucleation, giant cellsization, macronucleusization, nuclear chromatin clarification (clearing) and the kernel of giving prominence at those.Though these variations are very serious, but still in the scope that the epithelial cell that other wetting property alveolars damage changes.The morphological change of differentiating comprises: bronchial epithelial cell degrades, cilium is lost and squamous metaplasia.Other discoveries comprise focus intra-alveolar hemorrhage, hemocytophagia, stingy road gangrenosum acne inflammatory fragment, organized pneumonia or Secondary cases bacterial pneumonia.

This sick pathomechanism is still waiting to determine, yet the mechanism of acute lung injury may relate to virus attack endothelium or epithelial cell and direct infringement that alveolus wall is produced.Optionally, virus can infect inflammatory cell by cytokine, interleukin or tumor necrosis factor alpha mediation causing damage.The tissue injury that yet might SARS causes does not have direct relation with virus to the infection of tissue, but the adjacent tissue inductive cytokine of virus beyond lung tissue and the secondary effect of other factors.

Pathological evaluation to death shows hepatic cell fattydegeneration, liver cloudy swelling, apoptosis and spotty necrosis, with Kupffer hyperplasia and portal vein lymphocytic infiltration.Gastrointestinal tract wall patient has local hemorrhage, the congestion of blood vessel and lymphocytic infiltration.

Because SARS-CoV can propagate through the air approach,, therefore, press for the method appearance of immunity and treatment SARS-CoV the infected in immunity before infecting, diagnose infections, the course of infection so it has caused special threat to the numerous crowds of All Around The World.

Summary of the invention

Obtained solution in the invention that these need and other need to describe here.The invention provides polypeptide, peptide fragment, viral fusion inhibitor, coupling protein, immune peptide, immune composition, imitative polypeptide drug, nucleic acid fragment, expression cassette, nucleic acid construct, recombinant virus, viral vaccine, peptide vaccine, microorganism vaccine, dna vaccination, antibody, fit, medicinal compositions, the method for immune animal, the method for treatment severe acute respiratory syndrome (SARS), method and the test kit of diagnosis SARS.

The invention provides the polypeptide of the aminoacid sequence that the has polypeptide unanimity relevant with the SARS etiology.Preferred polypeptide can suppress the fusion of SARS and zooblast and/or cause immune response in animal body from the spike protein of SARS-CoV.In some embodiments, polypeptide is the soluble polypeptide from the spike protein of SARS-CoV.In other embodiments, polypeptide comprises the 17-757 amino acids from the spike protein of SARS-CoV.In some embodiments, polypeptide comprises the 762-1189 amino acids from the spike protein of SARS-CoV.In other embodiments, polypeptide comprises the 17-757 amino acids from the spike protein of SARS-CoV.In some embodiments, polypeptide comprises the 17-276 amino acids from the spike protein of SARS-CoV.In other embodiments, polypeptide comprises the 303-537 amino acids from the spike protein of SARS-CoV.In some embodiments, polypeptide comprises the 317-517 amino acids from the spike protein of SARS-CoV.In other embodiments, polypeptide comprises the 272-537 amino acids from the spike protein of SARS-CoV.In some embodiments, polypeptide comprises the 17-537 amino acids from the spike protein of SARS-CoV.In other embodiments, polypeptide comprises the 17-1189 amino acids (relevant with SEQ ID NO:1) from the spike protein of SARS-CoV.Polypeptide of the present invention can suppress the fusion of SARS-CoV and zooblast.Nucleic acid of the present invention and peptide vaccination animal capable cause immune response.In some embodiments, trigger cell immune response behind nucleic acid of the present invention and the peptide vaccination animal.In other embodiments, cause humoral immune reaction behind nucleic acid of the present invention and the peptide vaccination animal.Animal can be a Reptilia.In certain embodiments, animal can be a bird.In certain embodiments, animal is a Mammals, is the people sometimes.

The invention provides the peptide fragment from the SARS-CoV spike protein, the preferred peptide fragment is solvable in the aqueous solution.Peptide fragment of the present invention can be the aminoacid sequence of the complete spike protein of SARS-CoV but an amino-acid residue disappearance is arranged.In some embodiments, peptide fragment length is at least 3 amino acid.In other embodiments, peptide fragment length is at least 10 amino acid.In some embodiments, peptide fragment length is at least 20 amino acid.In other embodiments, peptide fragment length is at least 30 amino acid.In some embodiments, peptide fragment length is at least 40 amino acid.In other embodiments, peptide fragment length is at least 50 amino acid.In some embodiments, peptide fragment length is at least 60 amino acid.Peptide fragment also can be that single amino acids unit adds in the fragment of a certain-length, and for example, peptide fragment length can be 3,4,10,11,21,22,31 or 32 amino acid.Peptide fragment of the present invention can cause immune response after can suppressing the fusion of SARS CoV and zooblast or being used to inoculate animal body.Can cause immunoreactive example behind the inoculation animal and comprise that for example sequence is that D24 peptide and the sequence of DVQAPNYTQHTSSMRGC (SEQ ID NO:58) are the D540 peptide of PSSKRFQPQQFGRDC (SEQ ID NO:59).In certain embodiments, peptide fragment of the present invention is used to inoculate trigger cell immune response behind the animal, and in other embodiments, peptide fragment of the present invention causes humoral immune reaction after being used to inoculate animal.Animal can be a Reptilia.In certain embodiments, animal can be an avian species.In other embodiments, animal can be a Mammals.In further embodiment, animal can be the people.

The invention provides coupling protein, this coupling protein comprises a carrier proteins that is connected on second polypeptide.Preferably, this carrier proteins is a solubility.In some embodiments, after being used to inoculate animal, this carrier proteins strengthens the immune response to proteic second polypeptide of coupling.In other embodiments, after being used to inoculate animal, this carrier proteins is induced the cell immune response to proteic second polypeptide of coupling.In some embodiments, after being used to inoculate animal, this carrier proteins is induced the humoral immune reaction to proteic second polypeptide of coupling.This second polypeptide can be polypeptide of the present invention or peptide fragment, or its conservative property varient.Animal can be a Reptilia.In certain embodiments, animal can be an avian species.In other embodiments, animal can be a Mammals.In further embodiment, animal can be the people.

The invention provides and comprise polypeptide of the present invention or peptide fragment or its conservative property varient and be connected to immune peptide on ethanoyl, picryl, arsanilic acid, the Sulphanilic Acid.In some embodiments, immune peptide is connected on ethanoyl or the picryl.In other embodiments, immune peptide is connected on arsanilic acid or the Sulphanilic Acid.Preferably, immune peptide is a solubility.Preferably, immune peptide causes immune response after being used to inoculate patient.In other embodiments, after being used to inoculate animal, the immune response of immune Toplink primosome liquid.In some embodiments, after being used to inoculate animal, the immune response of epidemic disease Toplink trigger cell.Animal can be a Reptilia.In certain embodiments, animal can be an avian species.In other embodiments, animal can be a Mammals.In further embodiment, animal can be the people.

The invention provides the peptide mimics of polypeptide and peptide fragment and its conservative variant among the present invention, its peptide bond is replaced by non-peptide bond.In some embodiments, peptide mimics can suppress SARSCo-V and zooblast fusion.In other embodiments, after being used to inoculate animal, peptide mimics can cause immune response, and for example, after being used to inoculate animal, peptide mimics can the trigger cell immune response.Washability ground, after being used to inoculate animal, peptide mimics can cause humoral immune reaction.Animal can be a Reptilia.In certain embodiments, animal can be an avian species.In other embodiments, animal can be a Mammals.In further embodiment, animal can be the people.

The invention provides the composition that contains adjuvant and nucleic acid of the present invention, polypeptide, peptide fragment or peptide mimics, in some embodiments, composition can suppress the fusion of SARS-CoV and zooblast.In other embodiments, after being used to inoculate animal, composition causes immune response.In some embodiments, after being used to inoculate animal, the immune response of composition trigger cell.In other embodiments, after being used to inoculate animal, composition causes humoral immune reaction.Animal can be a Reptilia.In certain embodiments, animal can be an avian species.In other embodiments, animal can be a Mammals.In further embodiment, animal can be the people.

The invention provides the nucleic acid fragment of coding polypeptide of the present invention and peptide fragment and their conservative variant.

Be connected to the expression cassette of nucleic acid fragment of the present invention with the invention provides being operated property of promotor.In some embodiments, promotor is a composition.In other embodiments, promotor is derivable.

The invention provides the nucleic acid construct that contains carrier and nucleic acid fragment of the present invention, nucleic acid construct can comprise expression cassette of the present invention.In some embodiments, carrier can be a virus.In other embodiments, carrier can be a plasmid.In further embodiment, carrier can be an expression vector.

The invention provides the recombinant virus that contains virus vector and nucleic acid fragment of the present invention.In some embodiments, virus vector is a simplexvirus.In other embodiments, virus vector is canary pox virus (canary pox virus).In other embodiments, virus vector is an adenovirus.In further embodiment, virus vector is a vaccinia virus.

The invention provides the viral anti-SARS vaccin that contains virus vector, nucleic acid fragment of the present invention and pharmaceutical carrier.In some embodiments, virus vector is a simplexvirus.In other embodiments, virus vector is a canary pox virus.In some embodiments, virus vector is an adenovirus.In further embodiment, virus vector is a vaccinia virus.Preferably, pharmaceutical carrier is used for injection.Preferably, after being used to inoculate animal, this viral vaccine causes immune response.In some embodiments, after being used to inoculate animal, this virus vaccines trigger cell immune response.In other embodiments, after being used to inoculate animal, this virus vaccines causes humoral immune reaction.Animal can be a Reptilia.In certain embodiments, animal can be an avian species.In other embodiments, animal can be a Mammals.In further embodiment, animal can be the people.

The invention provides the anti-SARS peptide vaccine that contains peptide mimics of the present invention, polypeptide and peptide fragment or its conservative property varient and pharmaceutical carrier.Preferably, pharmaceutical carrier is formulated into injection.Preferably, peptide vaccine is made into unit dosage form.Preferably, after being used to inoculate animal, peptide vaccine causes immune response.In some embodiments, after being used to inoculate animal, the immune response of peptide vaccine trigger cell.In other embodiments, after being used to inoculate animal, peptide vaccine causes humoral immune reaction.Animal can be a Reptilia.In certain embodiments, animal can be an avian species.In other embodiments, animal can be a Mammals.In further embodiment, animal can be the people.

The invention provides to contain and express polypeptide of the present invention or the microorganism of peptide fragment or their conservative variant and the anti-SARS microorganism vaccine of pharmaceutical carrier.Preferably, this microorganism is an attenuation.In some embodiments, microorganism is salmonella (Salmonella).In other embodiments, microorganism is that listeria spp belongs to (Listeria).In some embodiments, microorganism is Listeria monocytogenes (Listeria monocytogenes).In some embodiments, pharmaceutical carrier is formulated into injection.In other embodiments, pharmaceutical carrier is formulated into oral dosage form.Preferably, microorganism vaccine is made into unit dosage form.Preferably, after being used to inoculate animal, microorganism vaccine causes immune response.In some embodiments, after being used to inoculate animal, the immune response of microorganism vaccine trigger cell.In other embodiments, after being used to inoculate animal, microorganism vaccine causes humoral immune reaction.Animal can be a Reptilia.In certain embodiments, animal can be an avian species.In other embodiments, animal can be a Mammals.In further embodiment, animal can be the people.

The invention provides and contain the carrier that is inserted with nucleic acid fragment of the present invention and the anti-SARS dna vaccination of pharmaceutical carrier.This dna vaccination can contain adjuvant.Dna vaccination can contain the muscular death agent.For example, the muscular death agent can be a bupivacaine.In other embodiments, the muscular death agent can be a cardiotoxin.Carrier can be, and is for example viral.In some embodiments, carrier is that bacterium is had a liking for thalline.In further embodiment, carrier is a plasmid.Containing the segmental carrier of insertion can prepare in eukaryotic cell.In some embodiments, containing the segmental carrier of insertion can prepare in prokaryotic cell prokaryocyte.For example, containing the segmental carrier of insertion can prepare in bacterium.In some embodiments, pharmaceutical carrier is used to prepare the mucosa delivery agent.In other embodiments, pharmaceutical carrier is used to prepare injection.Preferably, dna vaccination is made into unit dosage form.Preferably, after being used to inoculate animal, this dna vaccination causes immune response.In some embodiments, after being used to inoculate animal, this microorganism vaccine causes humoral immune reaction.In other embodiments, after being used to inoculate animal, this microorganism vaccine trigger cell immune response.Animal can be a Reptilia.In certain embodiments, animal can be an avian species.In other embodiments, animal can be a Mammals.In further embodiment, animal can be the people.

The invention provides and polypeptide of the present invention or peptide fragment, or their varient bonded antibody.In some embodiments, antibody is a kind of and antigen bonded antibody fragment.In other embodiments, antibody is polyclonal antibody.In further embodiment, antibody is single-chain antibody.In other embodiments, antibody is monoclonal antibody.In some preferred embodiments, antibody is humanized antibody.Antibody can combine with a marker that is used to detect, and for example, the certification mark thing is an isotopic label.In some embodiments, the certification mark thing is an affinity tag.In other embodiments, the certification mark thing is an enzyme.In other embodiments, the certification mark thing is a fluorescent marker.Antibody also can link to each other with toxin.

The invention provides and polypeptide of the present invention or peptide fragment, or their conservative property varient bonded is fit.This is fit can to combine with the certification mark thing.For example, the certification mark thing can be an isotopic label.In some embodiments, the certification mark thing is an affinity tag.In other embodiments, the certification mark thing is an enzyme.In further embodiment, the certification mark thing is a fluorescin.In some preferred embodiments, the certification mark thing is a fluorescent marker.Fitly also can combine with toxin.

The invention provides the medicinal compositions or the test kit that contain antibody of the present invention, S polypeptide or fit and pharmaceutical carrier.Preferably, this medicinal compositions is formulated into injection.

The accompanying drawing summary

This patent or application text contain at least one width of cloth coloured picture.This office will be as requested and provides this patent or the patent application text that has coloured picture under the situation of essential cost collecting.

Figure 1A is the agarose electrophoresis figure that contains the segmental DNA construct of insertion of the spike protein of the present invention of encoding.Swimming lane from left to right: road 1 is the dna molecular amount mark of 1kb (being 0.5,1.0,1.6,2.0,3.0,4.0 from top to bottom); Road 2 shows by the postdigestive DNA construct of BamHI/XbaI, the dna fragmentation (band down) of tangible carrier strap (going up band) and coding spike protein occurs; Road 3 shows by the postdigestive DNA construct of HindIII, because the dna fragmentation of carrier and coding spike protein has the restriction enzyme site of HindIII, has produced a less band and a bigger band of expection.

Figure 1B is the monomeric synoptic diagram of SARS-CoV S glycoprotein of total length, has shown removal signal sequence (1-16 position residue, SEQ ID NO:60) various soluble polypeptide fragments afterwards.Soluble fragments is the spike protein fragment, called after " S ", continue after be the spike protein amino acid figure place that reaction constitutes this fragment end.Like this, " S756 " refers to that one originates in the 17th amino acids (immediately following after the signal sequence), ends at the solubility spike protein fragment of the 756th amino acids." TM " refers to that transmembrane segment, arrow have indicated the possible cleavage site between 758-761 (sequence RNTR) amino acids." RBD " refers to the potential receptor binding domain between 272-537 (SEQ IDNO:57) amino acids, is positioned at probably between the upstream residue of downstream residue and the 537th (SEQ ID NO:61) amino acids of the 303rd amino acids.

Fig. 2 has shown the denaturing polyacrylamide electrophorogram that the peptide fragment of SARS-CoV spike protein is expressed in the intestinal bacteria.This peptide fragment is the 17-446 amino acids of SEQ ID NO:1.The nucleic acid fragment of coding 17-446 amino acids is cloned into pRSET carrier, is built into pRSET-S (17-446), expresses in the BL21DE3 cell.Left-hand digit and arrow indication are that the molecular weight of unit indicates with the kilodalton.It is that molecular weight indicates that swimming lane contains following polypeptide: M; Swimming lane 1 and 2 contrasts colibacillary polypeptide for the nucleotide sequence of the 17-446 amino acids residue that do not contain coding SEQ ID NO:1 with without isopropylthiogalactoside (IPTG) inductive; Swimming lane 3 is for the nucleotide sequence of the 17-446 amino acids residue that do not contain coding SEQ ID NO:1 but contrast colibacillary polypeptide through isopropylthiogalactoside (IPTG) inductive; Swimming lane 4 is analyzed for the pRSET carrier of the nucleotide sequence of the 17-446 amino acids residue that contains coding SEQ ID NO:1 and through IPTG inductive intestinal bacteria.The arrow indication on the right and the position of the corresponding peptide fragment of 17-446 amino acids residue of the SEQ ID NO:1 of escherichia coli expression.

Fig. 3 is the slot blot analysis chart that the described polypeptide fragment of the spike protein of SARS-CoV is expressed in mammalian cell.The segmental nucleic acid fragment of encoded peptide is cloned into pSecTag2B carrier, holds the mouse k chain leader sequence, the C end that contain the tool secreting function to contain to be useful on expression N and detects and the c-Myc determinant of affinitive layer purification and the peptide fragment of histidine mark thing.This nucleic acid construct is entered HEK293 and VeroE6 cell by conversion.The segmental expression of specified polypeptide detects by the slot blot analysis with anti-c-Myc antibody.The numeral of the left and right sides is indicated the amino-acid residue of detected peptide fragment.One expression of hurdle representative peptide fragment in the HEK293 cell on the left side, the expression of hurdle representative peptide fragment in the VeroE6 cell on the right.First hurdle representative is obtained from the sample (secretory protein) of cell culture medium, and second hurdle representative is obtained from the sample (intracellular protein) of cell lysate.The positive contrast of PC is provided by the producer of the plasmid that the PSA that contains C end band c-Myc marker is provided.The negative contrast of NC contains from the total length spike protein of SARS-CoV but does not have the c-Myc determinant or the histidine mark thing.

Fig. 4 A is the slot blot analysis that the specified polypeptide fragment of spike protein is expressed in people's 293 cells or monkey VeroE6 cell.In the presence of the T7 polysaccharase that has or do not have recombined vaccinia virus to express transfection the culture supernatant of 293 and VeroE6 cell of plasmid of coding S fragment (S276, S537 and S756) be transferred on the nitrocellulose filter also that the antibody with anti-c-Myc determinant detects.The numeral indication of the left and right sides is detected the amino-acid residue of peptide fragment.PSA PC is the PSA positive control that contains C end band c-Myc marker.PCDNA-S NC contains SARS-CoV total length spike protein but does not have the c-Myc determinant or the negative control of histidine mark thing.Swimming lane thes contents are as follows: (1) does not use people's 293 cells of VTF7.3 vaccinia virus infection, (2) people's 293 cells of usefulness VTF7.3 vaccinia virus infection, (3) do not use the monkey VeroE6 cell of VTF7.3 vaccinia virus infection, (4) are with the monkey VeroE6 cell of VTF7.3 vaccinia virus infection.

Fig. 4 B as described in Fig. 4 A with the supernatant of transfectional cell with Ni-NTA sepharose 4B incubation, clean and use with Fig. 4 A in identical anti--c-Myc epitope antibody carry out the test of Western trace.

Fig. 4 C is for detecting the segmental diagram of S with the anti-respectively rabbit polyclonal antibody that originates in the peptide of the 24th residue (D24, middle groups) and the 540th residue (P540, the right group) of two strains.One group of the left side is the Western trace contrast with anti-c-Myc determinant antibody test S537 and S756.

Fig. 5 is by the diagram of the relevant S protein expression of the film of FCM results show total length at cell surface with rabbit polyclonal antibody P540.The nucleic acid of coding total length S glycoprotein is used to rotaring redyeing 293 cell, and then uses the VTF7.3 transfection.Collecting cell and hatch altogether with P540 polyclonal antibody and the second antibody that has a FITC after, through washing, analyze with flow cytometer again.The same plasmid of the plasmid with expressing S that does not contain the nucleic acid of the S that encodes is used to the cell of transfection as negative control (NC) in control experiment, transfection has the cell of the nucleic acid that contains coding total length S glycoprotein to indicate with S.

Fig. 6 A and 6B show that S glycoprotein does not have abiogenous cutting substantially.The lysate of 293 cells of the last cleer and peaceful expression S glycoprotein of the rotaring redyeing 293 cell of expression S756, Se carries out the Western trace with P540 antibody, and the cutting of observing discovery S and Se is at baseline values.To be sample be used for the diagram of Western engram analysis cutting spectrum with monitoring nonspecific protease active effect at 4 ℃ after depositing 3 days to Fig. 6 A.As a comparison, Fig. 6 B is the engram analysis diagram of carrying out immediately after the specimen preparation.

What Fig. 7 A-C showed is S glycoprotein cell fusion mediated.A no S inserts segmental plasmid based on pCDNA3 and is used as the plasmid contrast, and the fusion of S express cell and ACE2-ecto express cell is used as negative control.This pCDNA3-ACE2-ecto construct is only expressed the solubility ecto functional zone that have the peptide-labeled thing of C9 of ACE2.Fig. 7 A shows that transfection has no syncytium formation between the 293T cell of pSecTag2B-S and pCDNA3-ACE2-Ecto.By contrast, Fig. 7 B shows that transfection has between the 293T cell of pSecTag2B-S and pCDNA3-ACE2 and has syncytium to form.Fig. 7 C is the diagram of the cytogamy measured based on the test of reporter gene with one, and as shown in the figure, the S glycoprotein of pCDNA3 and pSecTag2B vector expression all can measure in testing based on cell-cytogamy of β-gal reporter gene.

Fig. 8 A-C shows that the acceptor-land (RBD) of S glycoprotein is between the 272nd and 537 residue.Fig. 8 A is two different solubility S fragments (S537 and S756) and 293 cells and Vero E6 cell bonded diagram.Fig. 8 B is various S fragments and Vero E6 cell bonded diagram, the OD of background negative control ₄₀₅Value is from the segmental OD of each S ₄₀₅Reduce the OD that obtains in the value ₄₀₅Value is represented with the per-cent of relative S537.Fig. 8 C is the S polypeptide fragment that records with the ELISA method and the interactional diagram of solubility ACE2 behind the purifying.In all experiments, negative control (NC) is not for encoding any albumen its processing mode and the identical sample of other samples except the plasmid that is used for transfection.Here at least three independently experiments of data presented representative.The OD of all samples ₄₀₅Per-cent with relative S537 is represented.

Dimerized diagram verifiedly between the N-terminal fragment of S glycoprotein of SARS-CoV takes place by co-immunoprecipitation and cross connection in Fig. 9 A-D.All N-terminal fragments are except that the fragment (S317-517) of the minimum that contains receptor binding domain, by P540 antibody and S756 co-immunoprecipitation.P540 antibody is the rabbit polyclonal antibody that resists the peptide of the 540-555 position residue that contains S glycoprotein, and it can combine with the non-N-terminal fragment of S756 polypeptide.

Among Fig. 9 A, coding N-terminal segmental plasmid (the numeral name that stops amino-acid residue with the numeral that stops amino-acid residue or initial sum) be used to independent transfection (6 roads, the left side) or with plasmid co-transfection (4 roads, the right) the 293T cell of coding S756, these cells are used the transfection of VTF7.3 vaccinia virus again, after hatching, the collecting cell substratum is with discerning the diagram that all segmental mouse anti c-Myc determinant antibody carry out the Western engram analysis.

Fig. 9 B is all N-terminal S fragments, except that the minimal segment that contains the receptors bind functional zone (S317-517), by the diagram of P540 antibody and S756 co-immunoprecipitation.At first be used to carry out immunoprecipitation with the media samples that used substratum is the same among Fig. 9 A with the P540 polyclonal antibody of only discerning S756, antibody with anti-c-Myc determinant carries out the Western engram analysis to these throw outs then, to determine by the N-terminal fragment of co-immunoprecipitation.

What Fig. 9 C showed is under the situation that has or do not have DTT to exist, the new band that the molecular weight size is consistent with dimeric forms.In order to get rid of the possibility of the non-specific disulfide linkage formation that may cause co-immunoprecipitation, DTT is used in one of them co-immunoprecipitation experiment.DTT does not have effect to immunoprecipitation or the co-immunoprecipitation of excretory S756 (left road) or S756+S276 (right road).Contain excretory S756 (left road) or the segmental media samples of S756+S276 (right road) and under the situation that has or do not have 2mM DTT to exist, carry out immunoprecipitation with P540 antibody.

Fig. 9 D is the synoptic diagram of S polypeptide oligomer size.(IL) X-bracing as described in the embodiment after the SDS-PAGE separation, detects the monomer of S537 and its oligomer with anti-c-Myc antibody by the Western trace for Pierce, Rockford for S537 fragment and BS3.Swimming lane as Fig. 9 D the right is shown, a new band occurred after the X-bracing agent adds.The molecular weight of this new band and dimer are consistent and with more the high-grade oligomer is different.

Figure 10 A is the Dimerized diagram by the detected N-terminal fragment of exclusion chromatography S537.Indicate standard check and correction eluted position of albumen and molecular weight with arrow and numeral on the elution profile of S537 and S317-517.

The diagram of Figure 10 B for the S537 that collects and S317-517 flow point being carried out the Western trace with anti-c-Myc determinant antibody.

Figure 11 A-B shows that the N-terminal functional zone of distal-most end are essential by the cell-cytogamy of S glycoprotein mediation.Figure 11 A is the diagram of deletion mutantion of S glycoprotein and the summary of cell-cytogamy testing data, and wherein RBD represents the approximate location of receptor binding domain.The signal that occurs because merge does not detect the signal that is higher than background level minus sign (-) expression with plus sige (+) expression.Have only the polypeptide that contains the 17-1255 amino acids of wild-type to have fusion-activity.Contain 103-1255 (Del1) and contain the equal amixis activity of the amino acid whose deletion mutant of 311-1255 (Del2).Figure 11 B shows the total length S glycoprotein that records with the Western analytical method and the expression level of its mutant.Each sample is used for detecting with the cell lysate application of sample of same amount, rabbit polyclonal antibody P540.Figure 11 C is the total length S glycoprotein that records with flow cytometer and Del1 and the Del2 deletion mutant diagram at cell surface expression.Though surface expression is lower, only transfection three curves of negative control and other of cell of empty plasmid have obviously different.

Figure 12 A-B show dimer S1 and the unit price fragment that contains receptor binding domains mutually specific energy more effectively combine with acceptor ACE2.Figure 12 A has shown that with transfection the 200 μ l culture supernatant of cell of S276, S319-518 and S537 construct are by the segmental relative expression's level of the detected different S of ELISA method.Anti-Histidine antibody and anti-c-Myc determinant antibody are used to the S albumen that sandwich ELISA detects excretory tape label thing.Figure 12 B shows S fragment and the ACE2 bonded level that records with the ELISA method.The ACE2 that has mark is by bag is fixing onboard by the anti-C9 antibody on plate in advance, transfection the cells and supernatant of the proteic cells of various S mixed after, add elisa plate, under the condition that has (hachure) or the anti-c-Myc antibody of nothing (blank) to exist, hatch.The highest expression or combination water horizontal sliding are decided to be 100%.As shown in the figure, it is more effective with combining of ACE2 than the S319-518 fragment that only contains receptor binding domain to have S537 fragments of Dimerized and two functional zone of receptors bind of N-terminal.

Figure 13 A-B is presented at that solubility S extracellular domain is a tripolymer under the condition of exclusion chromatography.Among Figure 13 A, the Se of purifying passes through with the corrected gel filtration chromatography of the albumen of known molecular amount.The BSA of same amount is used as internal contrast.In Figure 13 B,, analyze by the Western trace from the different flow point that gel-filtration column is collected.In some contain the segmental flow point of Se of known molecular amount, detect the band of two S polypeptide, represent Se fragment self (following band) and its polymer (top band).

Figure 14 A shows that dna vaccination of the present invention can cause the anti-SARS-CoV serum of very high titre in the mouse body.Mouse 1A-5A contains the segmental dna immunization of S319-518 of spike protein receptor binding domain (RBD) with coding; The mouse 1B-5B syzygy immunity of RBD-coding DNA (S319-518 fragment) and the segmental nucleic acid of coding Fc; Mouse 1C-3C inoculates empty plasmid (no S sheet segment DNA).Collect antiserum(antisera), detect to determine the titre of different isolates with the ELISA method.In Figure 14 A, first digit is represented single mouse, and each immune group, the used extent of dilution of last digitized representation represented in letter.Antiserum(antisera) dilutes by 50,250,1250 and 7250 dilution factor, shown in the X-axis of this post figure.These digital prompt are carried out immune induction with the DNA of coding S proteic receptor binding domain and are gone out the very strong immune response at SARS-CoV.

Figure 14 B shows can prevent the S cell fusion mediated from the antiserum(antisera) that obtains with RBD-coding DNA mice immunized.Cell (293T) is hatched with the antiserum(antisera) that is obtained from the mouse that crosses with the segmental dna immunization of coding spike protein receptor binding domain polypeptide (S319-518), then this cell suspension and the proteic cytomixis of expression S.The mensuration that merges is (also see, Xiao etc., BBRC 2003) as described in example 20 above.The active per-cent of each fusion reaction (1=100% here) is plotted in Y-axis, and the per-cent that does not have the fusion of any inhibition is designated as 100%.PC represents the not positive control of increase serum.To #1 is to #2 mouse serum in each group, used serum dilution factor is 10 (being appointed as 0.1), 100 (being appointed as 0.01) and 1000 (being appointed as 0.001).#3 3# mouse serum in #5 mouse serum and the control group during A group and B organized, used serum dilution factor is 20 (being appointed as 0.05) and 100 (being appointed as 0.01).These Notes of Key Datas are carried out immunity with the DNA of coding S proteic receptor binding domain and can be prevented SARS-CoV to infect.

Figure 15 shows that solubility S glycoprotein fragment suppresses the S cell fusion mediated.Various S fragments and the ACE2-express cell of 10 μ g/ml were at first at room temperature hatched 10 minutes.This ACE2-express cell mixes with the S express cell again, and test for fusion carries out with the method for describing among the embodiment.Y-axis is that each sample has deducted the OD behind the background noise ₅₉₅Value.The initial sum of each polypeptide of digitized representation of each construct stops residue.

Detailed Description Of The Invention

SARS is important public health problem. The method that the patient of SARS-CoV has been infected in diagnosis and treatment provides chance for the further propagation that prevents or control SARS-CoV infects. Because SARS-CoV infects human body by the approach of air, so these method particular importances. The invention provides the nucleic acid of the amino acid sequence fragment of encoding SARS-CoV spike protein. The present invention also provides amino acid sequence the polypeptide consistent with the amino acid fragment sequence of SARS-CoV spike protein. The present invention also provides the fragments of peptides of SARS-CoV spike protein and their conservative variant, also has in addition amino acid sequence part coupling protein and the peptide mimics consistent with spike protein.

Spike protein is very important albumen, is because it is positioned at the surface of complete S ARS-CoV. Therefore, it can be used as virus have an opportunity to suppress or remove before the infection cell target of intact virus.

The spike protein of nucleic acid provided by the invention and the relative total length of polypeptide obvious advantage is arranged, because nucleic acid is easy to produce, polypeptide of the present invention also can prepare in a large number with the form of solubility. The relatively natural spike protein of polypeptide of the present invention also has another advantage, and they can be prepared to has the more on the whole form of drag to the degraded in the animal body. Polypeptide of the present invention also can strengthen its antigenicity by preparation makes it become more effective antigen at animal body, causes immune response such as human body.

So, the invention provides to be formulated into and can be used in immunity and treatment SARS-CoV the infected's vaccine and nucleic acid and the polypeptide antigen of immune composition. In addition, the present invention also provides the antibody that can be used for diagnosis, immunity and treatment SARS-CoV the infected of being combined with the spike protein of SARS-CoV.

Definition:

" adjuvant " is defined as adding the immunoreactive material of strong antigen generally speaking non-specificly. Various adjuvants can be used for immune peptide of the present invention and immune fragopeptides. Most of adjuvants contain protection antigen in design not by the material of tachymetabolism; such as aluminium hydroxide or mineral oil; with the immune response stimulant, as lipid A, pertussis thin Salmonella (Bortadella pertussis) or come source protein in conjunction with mycobacterium (Mycobacterium tuberculosis). On sale on the suitable adjuvant market, for example, Freund ' s Freunds incomplete adjuvant and Freund's complete adjuvant (Difco Laboratories, Detroit, Mich.); Merck adjuvant 65 (Merck and Company, Inc., Rahway, N.J.); Aluminium salt such as gel aluminum hydroxide (alum) or aluminum phosphate; Calcium salt, molysite and zinc salt; Insoluble acidylate tyrosine suspension; Acidylate sugar; The polysaccharide of cation or anionic derivative; Polyphosphazene; Biodegradable microspheres; Monophosphoryl lipid A and quil A; Cell factor also can be used as adjuvant such as GM-CSF or proleulzin ,-7 or-12.

" animal " refers to and can transfer immunoreactive organism for the attack of antigen. For example, reptile, avian species and mammal can produce antibody response to the attack of antigen. The antibody that produces in inhuman organism is considered to be conducive to reduce and get rid of cross reaction for diagnostic test.

" fit " is peptide, polypeptide or the nucleic acid (RNA or DNA) of being combined with polypeptide of the present invention or fragments of peptides.

" carrier protein " refers to be connected to the polypeptide that forms coupling protein on polypeptide of the present invention or the fragments of peptides. Carrier protein can be for the solubility that strengthens polypeptide or fragments of peptides and immunogenicity and is connected on polypeptide or the fragments of peptides. For example, biotin can be used as carrier protein be connected on polypeptide or the fragments of peptides form one can be by separating with the interaction of Avidin, or by using the fluorescently-labeled affine coupling protein that usually detects. In another example, the carrier protein that is connected with antibody can be connected to and form a coupling protein on polypeptide or the fragments of peptides, and antibody links to each other with the carrier protein that is connected albumen this coupling protein with antibody is continuous.

The present invention comprises nucleic acid, peptide, polypeptide or the albumen of purifying separation or basic. In content of the present invention, the polypeptide of the nucleic acid of " separation ", DNA or RNA molecule or " separation " refers to be present in nucleic acid, DNA, RNA molecule or the polypeptide in the non-natural environment, is not natural products therefore. The nucleic acid, DNA, RNA molecule or the polypeptide that separate can exist with the form of purifying or be present in the non-natural environment, for example, and in the genetically modified host cell. Other cellular materials or culture medium should be substantially do not contained when the nucleic acid molecules of " separation ", peptide, polypeptide or albumen or its fragment are produced with recombinant technique, precursor or other chemical substances should be substantially do not contained when producing with chemical synthesis. In one embodiment, the nucleic acid of " separation " is not for containing the natural flanking sequence of this nucleic acid on it originates organic genomic DNA (namely be positioned at this nucleic acid 5 ' and the sequence of 3 ' end). In various embodiments, the nucleic acid molecules of separation can contain the flanking sequence less than this nucleotide sequence on its derived cell genomic DNA of about 5kb, 4kb, 3kb, 2kb, 1kb, 0.5kb or 0.1kb. Substantially albumen, peptide or the polypeptide that does not contain cellular material comprises albumen, peptide or the polypeptide formulations that contains less than the contaminating protein of about 30%, 20%, 10% or 5% (dry weight). When albumen of the present invention or their biologically-active moiety are when producing by the method for restructuring, preferred culture medium contains less than the precursor of 30%, 20%, 10% or 5% (dry weight) or non-destination protein chemical substance.

Term polypeptide, peptide and albumen are here used with mutually.

Peptide or polypeptide " fragment " are used in reference to the little fragment of peptide, polypeptide or albumen than total length here. For example, peptide or polypeptide fragment can contain at least 3, at least 4, at least 5, at least 10, at least 20, at least 30, at least 40 amino acid on length, or their single unit length. For example, fragment can be 6,7,8,9,10,11,12,13,14,15,16,17 or amino acids more on length, and fragments of peptides is no maximum in size. But in some embodiments, fragments of peptides can be less than 500 amino acid, less than 400 amino acid, less than 300 amino acid, less than 250 amino acid on length. The preferred peptide fragment is used for can inducing immune response behind the inoculation animal. Fragments of peptides be used for to cause immune response can be by joining fragments of peptides and adjuvant and rear inoculation animal, the fragments of peptides of namely being combined with adjuvant, or and the fragments of peptides of arsanilic acid, sulfanilic acid, acetyl group or a trinitrophenyl coupling. Can contain non-amido link in the fragments of peptides, and can be peptide mimics.

Term " solvable " is used for referring to that polypeptide has the ability of dissolving at the aqueous solution here that for example, soluble polypeptide can mix with aqueous medium, makes the peptide that has at least part to measure in the aqueous medium. The detection of peptide can realize by routine techniques, such as optical absorption method, fluorescence method, with the binding ability of dyestuff, reduce ability and other similar approach of silver ion.

Term " specific binding " refer to be combined with single antigenic determinat and not with the antibody of being combined more than one antigenic determinant, therefore, with the antibody that a kind of polypeptid specificity is combined an antigenic determinant on this polypeptide is combined, and the antigenic determinant on another polypeptide is not combined.

I. polypeptide of the present invention, fragments of peptides, coupling protein, immune peptide and peptide mimics

The invention provides and contain the polypeptide of amino acid sequence of consensus amino acid sequence of levying the spike protein of the relevant virus (SARS-CoV) of (SARS) aetology with serious acute respiratory system synthesis, its representative amino acid sequence is SEQ ID NO:1, and this sequence is enumerated in lower for reference:

   1  MFIFLLFLTL TSGSDLDRCT TFDDVQAPNY TQHTSSMRGV

  41  YYPDEIFRSD TLYLTQDLFL PFYSNVTGFH TINHTFGNPV

81 IPFKDGIYFA ATEKSNVVRG WVFGSTMNNK SQSVIIINNS

121 TNVVIRACNF ELCDNPFFAV SKPMGTQTHT MIFDNAFNCT

161 FEYISDAFSL DVSEKSGNFK HLREFVFKNK DGFLYVYKGY

201 QPIDVVRDLP SGFNTLKPIF KLPLGINITN FRAILTAFSP

241 AQDIWGTSAA AYFVGYLKPT TFMLKYDENG TITDAVDCSQ

281 NPLAELKCSV KSFEIDKGIY QTSNFRVVPS GDVVRFPNIT

321 NLCPFGEVFN ATKFPSVYAW ERKKISNCVA DYSVLYNSTF

361 FSTFKCYGVS ATKLNDLCFS NVYADSFVVK GDDVRQIAPG

401 QTGVIADYNY KLPDDFMGCV LAWNTRNIDA TSTGNYNYKY

441 RYLRHGKLRP FERDISNVPF SPDGKPCTPP ALNCYWPLND

481 YGFYTTTGIG YQPYRVVVLS FELLNAPATV CGPKLSTDLI

521 KNQCVNFNFN GLTGTGVLTP SSKRFQPFQQ FGRDVSDFTD

561 SVRDPKTSEI LDISPCAFGG VSVITPGTNA SSEVAVLYQD

601 VNCTDVSTAI HADQLTPAWR IYSTGNNVFQ TQAGCLIGAE

641 HVDTSYECDI PIGAGICASY HTVSLLRSTS QKSIVAYTMS

681 LGADSSIAYS NNTIAIPTNF SISITTEVMP VSMAKTSVDC

721 NMYICGDSTE CANLLLQYGS FCTQLNRALS GIAAEQDRNT

761 REVFAQVKQM YKTPTLKYFG GFNFSQILPD PLKPTKRSFI

801 EDLLFNKVTL ADAGFMKQYG ECLGDINARD LICAQKFNGL

841 TVLPPLLTDD MIAAYTAALV SGTATAGWTF GAGAALQIPF

881 AMQMAYRFNG IGVTQNVLYE NQKQIANQFN KAISQIQESL

921 TTTSTALGKL QDVVNQNAQA LNTLVKQLSS NFGAISSVLN

961 DILSRLDKVE AEVQIDRLIT GRLQSLQTYV TQQLIRAAEI

1001 RASANLAATK MSECVLGQSK RVDFCGKGYH LMSFPQAAPH

1041 GVVFLHVTYV PSQERNFTTA PAICHEGKAY FPREGVFVFN

1081 GTSWFITQRN FFSPQIITTD NTFVSGNCDV VIGIINNTVY

1121 DPLQPELDSF KEELDKYFKN HTSPDVDLGD ISGINASVVN

1161 IQKEIDRLNE VAKNLNESLI DLQELGKYEQ YIKWPWYVWL

1201 GFIAGLIAIV MVTILLCCMT SCCSCLKGAC SCGSCCKFDE

1241 DDSEPVLKGV KLHYT

The present invention also provides and contains the peptide fragment of aminoacid sequence of consensus amino acid sequence of levying the spike protein of the relevant virus (SARS-CoV) of (SARS) etiology with serious acute respiratory system synthesis, and these aminoacid sequences comprise the sequence of SEQ ID NO:13,14,15,20-59 and 61-63 representative.The peptide fragment of SEQ ID NO:1 on the length also can be amino acid more than three or three, and can produce immune response when being used to inoculate animal.These peptide fragment are three amino acid with those length, or more longer single amino acids unit, as length be 4,5,6,7,8,9,10 amino acid and with the sequence of the consensus amino acid sequence of SEQ ID NO:1 on lack an amino acid whose aminoacid sequence and be the typical case.

The present invention also provides and contains carrier proteins and be connected to coupling protein on polypeptide of the present invention and the peptide fragment.Carrier proteins can be used to strengthen the solubility of coupling protein.Carrier proteins also can be used for strengthening the immunogenicity of coupling protein with the output of increase with polypeptide of the present invention or peptide fragment bonded antibody.Carrier proteins also can be used for separating and detects coupling protein, and therefore, coupling protein is can be by the interaction of other compositions of partly combining with the carrier proteins of coupling protein detected or separate.For example, coupling protein contains the carrier proteins of avidin, can detect with vitamin H and separates by known method.Many carrier proteinss can be used to prepare coupling protein of the present invention.The example of these carrier proteinss comprises keyhole  hemocyanin, bovine serum albumin, Protalbinic acid, mice serum albumin, albumin rabbit serum and its analogue.Carrier proteins can pass through form and of the present invention polypeptide or the peptide fragment coupling of the method for reorganization with fusion rotein.Carrier proteins also can pass through method and the polypeptide of the present invention or the peptide fragment coupling of chemistry, or by using the chemical linkers coupling.Harlow etc. is seen in the description known in the art and existing of these coupling methods, Antibodies:ALaboratory Manual, page 319 (Cold Spring Harbor Pub.1988); Taylor, Protein Immobilization, Marcel Dekker, Inc., New York, (1991).

The invention provides polypeptide of the present invention or peptide fragment and be connected to arsanilic acid, Sulphanilic Acid, acetyl group, or the immune peptide of picryl, the method that is connected with these groups is known and existing report, sees Weigle, J.Exp.Med., 116:913-928 (1962); Weigle, J.Exp.Med., 122:1049-1062 (1965); Weigle, J.Exp.Med., 121:289-308 (1965).

Polypeptide of the present invention and peptide fragment can be glycosylation and non-glycosylated form.Polypeptide of the present invention and peptide fragment can be soluble or insoluble in the aqueous solution.Polypeptide of the present invention and peptide fragment can be the conservative property varients, the conservative property varient refers to derive from full-length polypeptide, as polypeptide and the peptide fragment of exemplary sequence SEQ ID NO:1, by deletion (what is called is blocked), add or deduct one or more amino acid at the N-terminal and/or the C-terminal of full-length polypeptide; Or deletion, add or at the N-terminal of full-length polypeptide and/or C-terminal one place or many places deduct one or more amino acid.These varients can by, for example, gene pleiomorphism cause or artificially handle due to, such method of operating is this area institute likewise known.For example, the variant amino acid sequence body of SEQ ID NO:1 can prepare by the DNA of sudden change coded polypeptide.The method of sudden change and change nucleotide sequence is this area institute likewise known.See, for example, Kunkel, Proc.Natl.Acad.Sci.USA, 82,488 (1985); Kunkel etc., Methods in Enzymol., 154:367 (1987); U.S. Patent number 4,873,192; Walker and Gaastra, eds., Techniques in Molecular Biology.MacMillan Publishing Company, NewYork (1983) and the reference of quoting here.The guide of suitable aminoacid replacement can be at the Atlas of Protein Sequence and Structure of Dayhoff etc., Natl.Biomed.Res.Found., and Washington finds in C.D. (1978) model, and is incorporated herein by reference here.Conservative property replaces, and is preferred as another amino acid of aminoacid replacement with a similar performance.For example, replace another, or replace another with a hydrophilic amino acid with a hydrophobic amino acid.Polypeptide or peptide fragment from SEQ ID NO:1 after conventional shaker test can be used to determine to replace are used for whether producing immune response behind the animal.The example of this shaker test is known in the art, comprise enzyme linked immunosorbent assay, radioimmunoassay, chromium-release test and similar test, these tests are at Harlow et al., Antibodies:ALaboratory Manual, page 319 (Cold Spring Harbor Pub.1988) go up existing the description.

The invention provides the peptide mimics of polypeptide of the present invention or peptide fragment, peptide mimics is a peptide analogs, as those pharmacy industries commonly used, property class is similar to the non-peptide medicament (Fauchere of its template peptide, J., Adv.Drug Res., 15:29 (1986) and Evans etc., J.Med.Chem., 30:1229 (1987)).Peptide mimics is structurally similar with polypeptide or peptide fragment, contains peptide bond but one or more peptides connection keys is arranged by methods known in the art quilt--CH ₂NH--,--CH ₂S--,--CH ₂--CH ₂--,--CH=CH--(genial trans),--COCH ₂--,--CH (OH) CH ₂--and--CH ₂The such connection key option of SO--replaces.The advantage of the natural relatively polypeptide of peptide mimics comprise more economically produce, bigger stability, altered specificity and reinforced pharmacy characteristic, as transformation period, absorptivity, intensity and effect.

Polypeptide of the present invention, peptide fragment, coupling protein and peptide mimics can be modified to be reduced in intravital degraded and are used in the body by add encapsulant at its amino and/or carboxyl terminal.This way its end before polypeptide enters cell tends to by meeting under the situation of proteasome degradation useful.Such encapsulant includes but not limited to be connected to the relevant or uncorrelated peptide sequence that adds of polypeptide, peptide fragment, coupling protein and the peptide mimics that will use, this can pass through the method that those of ordinary skill is familiar with, finish in the building-up process of polypeptide, peptide fragment or coupling protein with chemical process, or finish with recombinant DNA technology.Washability, encapsulant such as Pyrrolidonecarboxylic acid and other molecules known in the art can be connected on aminoterminal and/or the carboxyl terminal residue, or the amino group of aminoterminal and/or carboxyl terminal residue can be replaced by different molecules, therefore, the invention provides polypeptide or the peptide fragment that aminoterminal and carboxyl terminal residue are closed.

Polypeptide of the present invention or peptide fragment cause immunoreactive ability can be tested by the method for many this areas approval, and for example, they induce the ability of antibody generation and the ability of irritation cell toxic T lymphocyte reaction.

Polypeptide of the present invention and peptide fragment can be used to shaker test with discriminating or separate and the spike protein bonded antibody of polypeptide of the present invention or peptide fragment or SARS-CoV.For example, polypeptide or peptide fragment can be used for phage display technique and test and separate and polypeptide or peptide fragment bonded antibody.In another example, polypeptide of the present invention and peptide fragment are incorporated on the solid support that an antibody can touch, like this, can just be fixed on the solid support with polypeptide and peptide fragment bonded antibody, these antibody can elute from solid support later.Polypeptide of the present invention and peptide fragment can be used for separation antibody by other many methods known in the art.

Can be used for the small-scale of coupling protein of the present invention, polypeptide or peptide fragment or the expression system of scale operation includes but not limited to: cell or microorganism that the recombinant nucleic acid construct that contained nucleic acid fragment of the present invention has transformed.The example of recombinant nucleic acid construct comprises bacteriophage DNA, plasmid DNA, glutinous grain DNA or virus expression carrier.Can be comprised bacterium (for example, intestinal bacteria or subtilis) by the example of cell transformed and microorganism; Yeast (for example, yeast belong and pichia spp); Insect cell system (for example, baculovirus); The vegetable cell system; Mammal cell line system (for example, COS, CHO, BHK, 293, VERO, HeLa, MDCK, W138 and NIH 3T3 cell).Can be used as host cell also have transfection have plasmid vector or infection have virus vector directly from former generation in Mammals source or inferior for cell.The example of suitable expression vector comprising but be not limited to plasmid and virus vector, as simplexvirus, retrovirus, vaccinia virus, attenuation vaccinia virus, canary pox virus, adenovirus, adeno-associated virus, slow virus and simplexvirus.The synthetic method also can be used to produce polypeptide of the present invention and peptide fragment, and known these methods and existing report are seen Merrifield, Science, 85:2149 (1963).

II. nucleic acid fragment of the present invention, expression cassette and nucleic acid construct

The invention provides the isolating nucleic acid fragment of coding polypeptide of the present invention, peptide fragment and coupling protein.Nucleic acid fragment of the present invention also comprises owing to the degeneracy of the gene codon same amino acid whose fragment of encoding, and for example, the amino acid Threonine is by ACU, ACC, and therefore ACA and ACG coding are degeneracies.The present invention has a mind to comprise all varients of same amino acid whose polynucleotide passage of encoding.Like this sport (Watson etc. known in the art, Molecular Biology of the Gene, Benjamin Cummings 1987), sudden change also comprises the nucleic acid fragment after the change then the conservative amino acid of encoding, for example, Isoleucine is replaced and similar replacement by leucine, and such sporting is known in the art.So, gene of the present invention and nucleotide sequence comprise abiogenous sequence and mutant nucleotide sequence the two.

Nucleic acid fragment of the present invention can be contained on the carrier, and carrier can include but not limited to: any plasmid, phasmid, the F-factor, virus, glutinous grain or two strands or strand wire and cyclic can or cannot oneself's infiltration or mobile phages.Carrier also can transform protokaryon or eucaryon host by the form that is integrated into cellular genome or exist with karyomit(e) outward (autonomously replicating plasmid that promptly contains the duplicate field).

Preferably, the nucleic acid fragment on the carrier under the control of suitable promotor or other regulatory elements or being operated property be connected to promotor or other regulatory elements with in external or host cell such as eukaryotic cell or microorganism, as transcribing in the bacterium.Carrier can be can be in multiple host the shuttle vectors of functionating, carrier can also be typically to contain the cloning vector that one or several restriction enzyme site, foreign DNA can insert in the mode of determining, such insertion can not cause the forfeiture of the basic biological function of cloning vector.Cloning vector also can contain and is applicable to the marker gene that this carrier cell transformed is differentiated and screened, and the example of marker gene has tsiklomitsin and ampicillin resistance gene.Many cloning vectors can buy from the market (Stratagene, New England Biolabs, Clonetech).

Nucleic acid fragment of the present invention also can be inserted into expression vector.Typical expression vector contains the prokaryotic cell prokaryocyte DNA element of coding bacterium replication region and is used to increase and screens the antibiotics resistance gene of the expression vector in the host bacterium; Contain control and start the regulatory element such as the promotor of transcribing; The DNA element such as intron or the Transcription Termination/polyadenylic acid sequence that contain the processing of controlling transcripton.

There are the method (Sambrook etc. of ready-made carrier that nucleic acid fragment is packed into this area, Molecular Cloning:A Laboratory Manual, 3rd edition, Cold SpringHarbor Press, Cold Spring Harbor, N.Y. (2001)), say simply, the carrier that is inserted into nucleic acid fragment is handled with one or more Restriction Enzymes (restriction enzyme), produce contain flat terminal, with 5 ' or the linear carrier of 3 ' outstanding surplus " glue " ends or their any combination.Carrier is used modifying enzyme after can handling with Restriction Enzyme again, handles as polysaccharase, restriction endonuclease, dephosphorylation enzyme or kinases, has the linear carrier that helps nucleic acid fragment is connected into the feature of carrier to obtain one.The nucleic acid fragment that is inserted into carrier is handled with one or more Restriction Enzymes (restriction enzyme), produce contain flat terminal, with 5 ' or 3 ' outstanding surplus " glue " end or the linear fragment of their any combination.After nucleic acid fragment also can be handled with Restriction Enzyme, then handle with other dna modification enzymes, these dna modification enzymes have the nucleic acid fragment that helps nucleic acid fragment is connected into the feature of carrier including, but not limited to polysaccharase, restriction endonuclease, dephosphorylation enzyme or kinases to obtain one.

Then, with this area available method (Sambrook etc., Molecular Cloning:ALaboratory Manual, 3rd edition, Cold Spring Harbor Press, Cold SpringHarbor, N.Y. (2001)), treated carrier is connected together the construct that formation contains nucleic acid fragment with nucleic acid fragment.Say that simply processed nucleic acid fragment and processed carrier are mixed together in the condition that has suitable damping fluid and ligase enzyme to exist, under suitable condition, hatch then, nucleic acid fragment is connected on the carrier to allow ligase enzyme.

The present invention also provides the expression cassette that contains the nucleotide sequence that can instruct special nucleus acid fragment expression of the present invention in external or host cell, as SEQ ID NO:2.Simultaneously, nucleic acid fragment of the present invention also can be inserted into expression cassette to produce an antisense information.Expression cassette is an independently unit, can and can be used for in-vitro transcription and the translation test for wire.(Madison, Wisconsin) company buys from Promega Corp. to carry out the material of these tests and method.For example, in-vitro transcription can place nucleotide sequence under the control of T7 promotor, begins in-vitro transcription with the T7 polysaccharase then.These transcriptons can be translated by adding rabbit reticulocyte lysate.Optionally, expression cassette can be loaded into carrier and duplicate in host cell and draw together and increase, or at in-vitro transcription and translation nucleic acid fragment.

Such expression cassette can contain one or more restriction sites, allows nucleic acid fragment to be placed under the adjusting of regulating sequence.Expression cassette also can contain termination signal and the required adjusting sequence of the normal translation of nucleic acid fragment that functionally is connected to nucleic acid fragment.The contained nucleic acid fragment of expression cassette can be a mosaic type, and promptly it component is ectogenic with respect to its other component at least.Expression cassette can be natural but obtain to make things convenient for expression of exogenous gene with the form of recombinating.The expression of nucleic acid fragment in expression cassette can be subjected to the control of constitutive promoter or inducible promoter, and inducible promoter only just starts when host cell is exposed to special exogenous irritant transcribes.

Expression cassette can comprise transcribing of 5 '-3 ' direction, transcribes and translate the promoter region for one, the transcribing and the translation termination district an of nucleic acid fragment and a performance function in external and/or body.This terminator can be transcription initiation region natural all, can be nucleic acid fragment natural all, or from other the source gained.

Regulating sequence can be including, but not limited to enhanser, promotor, repressor binding site, translation leader sequence, intron and polyadenylic acid signal sequence.They can comprise natural and associating sequence synthetic sequence and natural and composition sequence.Regulate sequence and be not limited to promotor, some are regulated sequence and comprise constitutive promoter, inducible promoter, adjustment type promotor, organizing specific type promotor, viral promotors and synthetic promoter.

Promotor is one section nucleotide sequence, transcribes the expression of necessary recognition site control encoding sequence by RNA polymerase and other are provided.Promotor comprises a minimal promoter, contain all the most basic initial necessary elements of transcribing, as TATA box and/or initial son, initial son is one section short dna sequence, contain other sequences of TATA box and sign transcription initiation site, regulatory element combines with it with control and expresses.Promotor can derive from a natural gene fully, or by forming from the different elements of different natural promoters, even form by the synthetic dna fragmentation.Promotor can contain and protein factor bonded sequence, and such protein factor can be reacted to physics or developmental condition and be controlled the validity of transcription initiation.

The invention provides the construct that contains carrier and expression cassette.Carrier can be selected from but be not limited to the carrier that any front had been described.Method (the Sambrook etc. that can describe by method known in the art and front, Molecular Cloning:A Laboratory Manual, 3rd edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (2001)) expression cassette is inserted carrier.In one embodiment, the adjusting sequence of expression cassette can derive from beyond the carrier of this expression cassette insertion.In another embodiment, the formation that contains the construct of carrier and expression cassette is nucleic acid fragment of the present invention to be inserted self have the carrier of regulating sequence, so expression cassette is formed when nucleic acid fragment inserts carrier.Having the carrier of regulating sequence can buy on market, and its using method is known in the art.

III. immune composition of the present invention and vaccine

The invention provides the immunoreactive immune composition and the vaccine that are used for to produce behind the animal body virus relevant on the anti-etiology with severe acute respiratory syndrome.Immune response can be humoral immune reaction or cell immune response.

Immune composition of the present invention can comprise adjuvant and nucleic acid, polypeptide, peptide fragment, peptide mimics, coupling protein and immune peptide or their any associating.Immune composition can contain the adjuvant that is connected with method non-chemically with polypeptide of the present invention, peptide fragment, peptide mimics, coupling protein and immune peptide.Immune composition can contain the adjuvant that chemically is connected with polypeptide of the present invention, peptide fragment, peptide mimics, coupling protein and immune peptide.Immune composition of the present invention also can contain pharmaceutically acceptable diluent or carrier.

Immune composition can be by conventional method preparation, and particularly, the nucleic acid in the composition, polypeptide, peptide fragment, peptide mimics, coupling protein and immune peptide or their any associating can combine with pharmaceutically acceptable diluent or carrier.The example of pharmaceutically acceptable diluent or carrier comprises water or salts solution, as phosphate buffered saline buffer (PBS).Usually, select pharmaceutically acceptable diluent according to the mode of administration and the pharmacy practice of approach and standard.The thing that pharmaceutically acceptable diluent and carrier and all that pharmaceutical compositions must be used has description in the canonical reference book Remington ' of this area s PharmaceuticalSciences.

Immune composition can contain adjuvant described herein and known in the art.Aluminum compound can be used as adjuvant, and these aluminum compounds comprise aluminium hydroxide, aluminum phosphate, hydroxide phosphoric acid aluminium and analogue thereof.Nucleic acid, polypeptide, peptide fragment, peptide mimics, coupling protein and immune peptide or their any associating can be by standard method absorption or be deposited in aluminum compound.Other adjuvants comprise polyphosphonitrile (WO 95/2415); DC-chol (3-β-[N-(N '; N '-dimethylamino methylmethane) formamyl] cholesterol) (U.S. Patent number 5; 283; 185 and WO96/14831); QS-21 (WO 88/9336) and from ImmunoChem (Hamilton, RIBI Montana).The immunostimulatory oligonucleotide that contains non-methylated CpG dinucleotides (" CpG ") the adjuvant that is used as system or mucosa delivery known in the art (WO 96/02555, and EP 468520, etc., J.Immunol., 160:870 (1998); McCluskie and Davis, J.Immunol., 161:4463 (1998).When CpG is prepared into immune composition or vaccine, usually in free solution with free antigen administration.Immunol., 161:4463 (1998)) or with antigen covalently bound (PCT Publication No.WO 98/16247), or with carrier such as aluminium hydroxide preparation back administration.(Brazolot-Millan et al.，Proc.Natl.Acad.Sci.，95：15553(1998)).

The present invention also provides the vaccine that comprises nucleic acid of the present invention, polypeptide, peptide fragment, peptide mimics, coupling protein and immune peptide or their any associating.These vaccines can be prepared with the method known to method as described herein or the vaccine field.For example, can construct the virus vaccines of expressing polypeptide of the present invention, peptide fragment or coupling protein according to method known in the art, the example that is used as virus vector can comprise adenovirus, simplexvirus, vaccinia virus, canary bird simplexvirus and similar virus.Vaccine also can be made into liposome, and its compound method is known to those skilled in the art.See Liposomes:A Practical Approach.RRCNew Ed, IRL press (1990).

The present invention also provides the vaccine based on nucleic acid of expressing polypeptide of the present invention, peptide fragment or coupling protein.For example nucleic acid vaccine can be expressed the fragment of SEQ ID NO:1,13,14,15,20-59,61-63 polypeptide or SEQ ID NO:1.Nucleic acid construct inoculation animal with coding polypeptide of the present invention, peptide fragment or coupling protein can induce antigenic body fluid or the cell-mediated immune response of being encoded, it is considered to the professional antigen presenting cell of derived from bone marrow by the nucleic acid construct transfection, and the antigen that is encoded is through transcribing and translate due to the immunogenicity polypeptide that generates the inducing specific reaction.A feature of nucleic acid vaccine is that it can induce very strong cytotoxic T lymphocyte (CTL) reaction.This reaction is because polypeptide synthetic cause in transfected cell of nucleic acid encoding.In addition, the nucleic acid construct of producing in bacterium is rich in the non-methylated CpG Nucleotide that can be taken as foreign matter by scavenger cell, and therefore, scavenger cell brings out the natural immunity that can strengthen the secondary immunity, so, even nucleic acid vaccine is also very effective under the situation of no adjuvant.

Expression cassette is injected directly into host cell alive, expresses nucleic acid and the expressing gene product that is imported into thereby many cells are transformed.Transfected cell can be cell surface and I class and the main histocompatibility antigen of II class with the antigen fragment biography of expressing and be combined into mixture.

Before nucleic acid construct being injected animal, comprising the people, can be by inducing the muscle degraded more effectively with nucleic acid construct transfered cell (Vitadello etc., Hum.Gene.Ther., 5:11 (1994); Danko and Wolff, Vaccine, 12:1499 (1994); Davis etc., Hum.Gene.Ther., 4:733 (1993)).For example, such processing is considered to improve transfection efficiency to 40 times.Two kinds of the most frequently used muscular death agent (myonecrotic) are local anesthetic bupivacaine and cardiotoxin (Danko and Wolff, Vaccine, 12:1499 (1994); Davis etc., Hum.Gene.Ther, 4:733 (1993)).Other many technology also are used to nucleic acid construct is transduceed in the muscle, and these technology comprise retroviral vector, adenovirus carrier and liposome.But in intracellular expression, the direct injection naked nucleic acid is that to be that these import in mechanism the most effective for the transduction of exogenous nucleic acid and nucleic acid.

Nucleic acid construct can be with pharmaceutically acceptable carrier medication.Pharmaceutically acceptable carrier is suitable for being used for people or other mammiferous media for biologically compatible, as physiological saline.The treatment significant quantity be nucleic acid construct can in by the animal of administration, cause immunoreactive amount (as, strengthening t cell responses or antibody produces), know as medical field, dosage to any one patient depends on many factors, comprises patient's height, body surface area, age, the special compound that will use, sex, administration time and approach, general health situation and other medicines that uses simultaneously.Dosage can change, but the preferred dose of nucleic acid construct is about 10 ⁶To 10 ¹²The nucleic acid construct of copy.If desired, this dosage can repeat administration.

The administration of nucleic acid construct can have many approach, its example comprises that intramuscular, intravenously, intestines are outer, in the intracutaneous, nose and subcutaneous injection, these approach cause in the chicken body once that all the immune response of resisiting influenza virus hemagglutinin (HA) (sees the summary of Pardoll and Beckerkleg, Immunity 3 (1995), 165-169).Vaccine based on nucleic acid also can pass through polymer, Biodegradable microparticles or micro-capsule importing carrier, and its optimised is arrived phagocytic cell, big or small as the best of macrophage phagocytic.For example, can use PLGA (poly-lacto-co-glycolide) particulate of diameter as 1-10 μ m.Nucleic acid construct is wrapped in these particulates, by macrophage phagocytic and in cell biological degradation takes place gradually, so discharge nucleic acid construct.In case discharge, nucleic acid construct cognition is expressed in cell.Another approach that makes nucleic acid construct enter cell is that liposome can prepare by standard method by the use liposome.Nucleic acid construct can be separately, also can be incorporated into these with tissue specificity antibody imports carrier.Washability ground also can prepare nucleic acid construct and be attached to the coupling molecule of poly-L Methionin by static or covalent force, and poly-l-lysine can combine (Cristano etc. (1995), J.Mol.Med.73,479) with the part of acceptor on the target cell.Washability ground, Lymphoid tissue specific target tropism can realize by using the special transcription regulatory element (TRE) of Lymphoid tissue, as bone-marrow-derived lymphocyte, T lymphocyte or specific for dendritic cells TRE, lymphocyte specific TRE knownly (sees Thompson etc., Mol.Cell.Biol., 12:1043 (1992); J.Exp.Med. such as Toll, 177:1663 (1993); Penix etc., J.Exp.Med., 178:1483 (1993)).

The present invention also provides the vaccine based on microorganism.In general, these vaccines relate to the microorganism that the nucleic acid construct of being expressed polypeptide of the present invention, peptide fragment or coupling protein has transformed.For example, the monocyte hyperplasia listeria spp is used as the immunity of carrier inducing T cell, because it can infect antigen presenting cell, and because it infects for mucosal infections, sees Lieberman and Frankel, Vaccine, 20:2007-10 (2002).So, can use the nucleic acid construct transfection listeria bacteria of polypeptide, peptide fragment or coupling protein, remove to induce humoral immune reaction at causing the coronavirus that serious acute respiratory system synthesis is levied.Can prepare highly attenuated listeria bacteria according to this area reported method, see Lieberman and Frankel, Vaccine, 20:2007-10 (2002).Salmonellas also can be used as carrier and induce at the cytotoxic T lymphocyte reaction that causes the coronavirus that serious acute respiratory system synthesis is levied.See Pasetti etc., Infect Immun., 70:4009 (2002).

Immune composition or vaccine can be by the conventional route administrations in vaccine field.For example, immune composition or vaccine can be by oral or intravenous drips, or subcutaneous, intramuscular, enteron aisle are outer, in the anus, in the intravaginal, nose, in the gi tract, in the tracheae or administration in cardiovascular.The selection of route of administration is depended on all multifactor, as the character of active principle; The composition of polypeptide, peptide fragment, peptide mimics, coupling protein, immune peptide, dna vaccination; Or with above-mentioned molecule bonded adjuvant.The administration of immune composition can be single dose or dosage within a certain period of time once or administration several times.Proper dosage changes according to the difference of factor; these factors comprise processed individuality (adult or child), immune composition or antigen itself, administration mode and frequency, have the adjuvant of denying, if any; the type of adjuvant and the effect of expection (as protection or treatment) will be decided by those skilled in the art.

IV. antibody of the present invention and fit

The invention provides fragment or its conservative property varient bonded antibody with aminoacid sequence shown in SEQ ID NO:1,13,14,15, the 20-59,60,61,62,63 or SEQ ID NO:1.These antibody can be used for diagnosis, immunoprophylaxis and treatment severe acute respiratory syndrome (SARS).In certain embodiments, antibody combines with the peptide of tool SEQ ID NO:58 or 59 sequences.Effective with peptide P540 (SEQ ID NO:59) bonded antibody height, even behind dilution for many times, still can detect the furcella polypeptide.For example, still can detect the furcella polypeptide behind 1: 10000 times of the P540 antibody dilution.

Antibody can be by preparing as immunizing antigen with complete desired polypeptides or peptide fragment.The polypeptide or the peptide fragment that are used for immunity can be through the cDNA translations or through synthetic.If necessary, polypeptide or peptide fragment can be incorporated on the carrier proteins, and these normally used carrier proteinss that are connected on the peptide with chemical mode comprise keyhole  hemocyanin (KLH), thyroglobulin, bovine serum albumin and tetanus toxin.Coupling protein can be used for immune animal (as mouse, rat or rabbit).

If necessary, polyclone or monoclonal antibody can further be purified, and for example, are used to produce the polypeptide of antibody or the matrix phase of peptide fragment combines with being combined with, and elute from this matrix.Those skilled in the art understands that the purifying that immune field is commonly used and/or the technology of concentrated polyclonal antibody and monoclonal antibody (see Coligan etc., Unit9, Current protocolin Immunology, Wiley Interscience, 1991, combination by reference).

The monoclonal antibody that produces an analogue antigen determinant by the antiidiotype technology also is possible.For example, the antiidiotype of anti-first monoclonal antibody will contain the combined function district of hypervariable region, and these functional zone are " mirror shadows " of first monoclonal antibody institute bonded determinant.

Be fit to polypeptide or peptide fragment bonded antibody be special to certain regional part of polypeptide at least.For example, those skilled in the art can remove to produce antibody suitable among the present invention by enough peptide fragment.Antibody of the present invention comprises the fragment of polyclonal antibody, monoclonal antibody and polyclone and monoclonal antibody.

The preparation polyclonal antibody by those skilled in the art known (see Green etc., Production of Polyclonal Antisera, in Immunochemical Protocols (Manson, ed.), pages 1-5 (Humana Press 1992); Coligan etc., Productionof Polyclonal Antisera in Rabbits, Rats, Mice and Hamsters, in CurrentProtocols in Immunology, section 2.4.1 (1992), incorporated herein by reference).For example, polypeptide or peptide fragment are injected into host animal, preferably, according to the embodiment that is predetermined, carry out the one or many booster immunization again, and stage is taken a blood sample to animal.The specific antibody of anti-polypeptide or peptide fragment can carry out affinity chromatography with the suitable solid support that is combined with polypeptide or peptide fragment, thereby antibody purifying from these antiserum(antisera)s is come out.

MONOCLONAL ANTIBODIES SPECIFIC FOR be equally routinely (see Kohler ﹠amp; Milstein, Nature, 256:495 (1975); Coligan etc., sections 2.5.1-2.6.7; With Harlow etc., Antibodies:A Laboratory Manual, page 726 (Cold Spring Harbor Pub.1988)), by reference in conjunction with therewith.Say simply, monoclonal antibody can be by injecting mouse with containing antigenic composition, after confirming that from the serum specimen of gathering antibody exists, get its spleen and obtain bone-marrow-derived lymphocyte, bone-marrow-derived lymphocyte and myeloma cell are merged the formation hybridoma, the clone hybridization oncocyte, screening produces the positive colony at antigenic antibody, separation antibody from the substratum of hybridoma.Can be by multiple known method separation and purification antibody from the substratum of hybridoma.These isolation technique comprise with the albumin A agarose carries out affinity chromatography, molecular exclusion chromatography and ion exchange chromatography.(see Coligan etc., sections 2.7.1-2.7.12 and sections 2.9.1-2.9.3; Barnes etc., Purification ofImmunoglobulin G (IgG), in Methods in Molecular Biology, Vol.10, pages 79-104 (Humana Press 1992)) monoclonal antibody method that increases is in vitro and in vivo known by those skilled in the art, amplification in vitro can be in suitable medium, as Dulbecco ' s Modified Eagle Medium and PRMI 1640 substratum, an amount of mammalian blood serum such as foetal calf serum and the trace element of adding, and add and keep the supplementation material of growth such as the peritoneal exudate cells of normal mouse, splenocyte and bone marrow macrophage.Produced in vitro provides pure relatively antibody preparation, and can add extensive antibody with the mass production needs.Extensive hybridoma is cultivated and can be realized by cultivating at airlift reactor, continuous-stirring reactor or immobilization or entrapped cell.Amplification can for example, in the osyngeneic mouse body, be grown the tumour that produces antibody by cell clone being injected in the mammalian body compatible with parent cell tissue in the body.Earlier use hydrocarbon polymer, particularly oils such as tetramethyl-pentadecane to activate animal before the optional injection, monoclonal antibody is reclaimed in one to three all backs from the body fluid of animal.

Antibody also can prepare by phage display technique, in an example, use antigen, as polypeptide of the present invention or organism of peptide fragment immunity, from by isolated lymphocytes in the spleen of organism of immunity, from lymphocyte, extract total RNA, be contained in mRNA among total RNA and be reversed and record into complementary deoxyribonucleotide (cDNA).The light chain of coding immunoglobulin (Ig) and the cDNA of variable region of heavy chain draw together with polymerase chain reaction (PCR) and increase, produce the antibody variable region fragment (scFV) of strand, the amplified production of light chain and heavy chain can connect in the suitable carriers by shearing the complete sequence of overlapping extension PCR generation then, carrier transformed into escherichia coli with coding scFV, and then it is carried out transfection with a helper phage, produce the phage that has antibody on the surface.Washability ground, in order to produce a complete Fab (Fab), the heavy chain amplified production can merge with the nucleotide sequence of coding phage outer membrane protein, light chain is drawn together the volume increase thing and can be cloned in the suitable carriers, the carrier of drawing together the volume increase thing with the expression light chain transforms the intestinal bacteria of expressing the heavy chain that merges with the phage outer membrane protein, and light chain and heavy interchain disulfide linkage are connected in the colibacillary periplasm and form.The result of this method produces one greatly to 10 ⁹Clone's antibody library, the size of antibody library can also increase to 10 drawing together from the immune response of the additional immunity of identical or different organism by increasing ¹⁸Individual phage.The antigenic antibody of identification specificity can screen by the method for elutriation, briefly, with complete antibody library be exposed to expectation can with the solidified antigen of antibodies, do not produce with the phage of antigen bonded antibody will be by flush away, expression will be attached on the solidified antigen with the phage of antigen bonded antibody, and these phages will be eluted and draw together in intestinal bacteria to be increased.Can repeat the phage-infest of this process with enrichment expression and antigen-specific bonded antibody.Behind the phage of isolating expression and antigen-specific bonded antibody, the carrier that contains the sequence of encoding antibody can extract from phage particle, is advanced a suitable carrier to produce soluble antibody by the clone once more.In another example, people's phage library can be used to screen antibody, as with the spike protein bonded monoclonal antibody of SARS-CoV.Say that simply from infecting or do not infect the human body separating Morr. cell of SARS-CoV, with above description and method founder's phage library known in the art, these methods can be used for obtaining the spike protein bonded human monoclonal antibodies with SARS-CoV.Phage display technique is used to separate the method description known in the art and existing of antigen and antibody and (sees Gram etc., Proc.Natl.Acad.Sci., 89:3576 (1992); Kay etc., Phage display of peptides and proteins:Alaboratory manual.San Diego:Academic Press (1996); Kermani etc., Hybrid, 14:323 (1995); Schmitz etc., Placenta, 21Suppl.A:S106 (2000); Sanna etc., Proc.Natl.Acad.Sci., 92:6439 (1995)).

Antibody of the present invention can come from the monoclonal antibody of " humanization ", and Humanized monoclonal antibodies is by transferring to the complementary determining region of the heavy chain of rat immune globulin and light chain people's variable region, and the residue of personnel selection replaces the residue of mouse framework region and gets then.From the use of the antibody component of Humanized monoclonal antibodies, the potential problems of having avoided the immunogenicity of mouse constant region to cause.The general technology of relevant clone's mouse immune globulin variable zone is existing to be described (see Orlandi etc., Proc.Nat ' l Acad.Sci.USA, 86:3833 (1989) integral body by reference is incorporated into this).Jones etc. is seen in the description that produces the technology of Humanized monoclonal antibodies., Nature, 321:522 (1986); Riechmann etc., Nature, 332:323 (1988); Verhoeyen etc., Science, 239:1534 (1988); Carter etc., Proc.Nat ' l Acad.Sci.USA, 89:4285 (1992); Sandhu, Crit.Rev.Biotech., 12:437 (1992); With Singer etc., J.Immunol., 150:2844 (1993), they are incorporated herein by reference.

In addition, antibody of the present invention can be from human monoclonal antibodies, and these antibody are from being obtained at the transgenic mouse that antigenic stimulation produce specific people's antibody by genetic engineering technique is transformed.In this technology, the locus that people's heavy chain and light chain gene seat are imported into its endogenous heavy chain and light chain is directed in the mouse in ruinate embryonic stem cell source, this transgenic mouse can synthesize the special people's antibody of human antigen, and this mouse can be used to produce the hybridoma of secretion people antibody.The method of obtaining people's antibody from transgenic mouse is described in Green etc., Nature Genet., 7:13 (1994); Lonberg etc., Nature, 368:856 (1994); With Taylor etc., Int.ImmunoL.6:579 (1994), they are incorporated herein by reference.

Antibody fragment of the present invention can prepare by protease hydrolysis antibody or the expression of DNA in intestinal bacteria by encode fragment.Can digest complete antibody with stomach en-or papain by the method for routine and obtain antibody fragment.For example, by can obtain F (ab) 2 fragments of a 5S with stomach en-cutting antibody, this fragment is further cut with the sulfo-reduction factor, and washability ground with the blocking groups sealing because disulfide linkage is cut the mercapto groups of generation, can produce the Fab unit price fragment of 3.5S.Washability ground is by also can directly producing two unit price Fab and a Fc fragment with the stomach en-cutting.The existing description of these methods (seen U.S. Patent number 4,036,945; 4,331,647; With 6,342,221 and be contained in this reference; Porter, Biochem.J., 73:119 (1959); Edelman etc., Methods in Enzymology, Vol.1, at sections 2.8.1-2.8.10and 2.10.1-2.10.4 such as page 422 (Academic Press 1967) and Coligan).

The method of other cutting antibody, as separate heavy chain and form the method for unit price light chain segments, the method further method of cutting fragment, or other zymetologys, chemistry or gene engineering also can be used, as long as the fragment that forms can combine with the antigen that complete antibody is discerned.

For example, the Fv fragment contains the V that links together _HAnd V _LChain, this connection can the covalently bound (Inbar etc. of right and wrong, Proc.Nat ' l Acad.Sci.USA, 69:2659 (1972)), variable chains can washability ground connect by intramolecular disulfide bond or by chemical substance such as glutaraldehyde cross connection (Sandhu, Crit.Rev.Biotech., 12:437 (1992)).The preferred segmental V of Fv _HAnd V _LChain connects by peptide bond.These single chain antigen coupling proteins (sFv) can contain coding V by making up one _HAnd V _LChain, the middle structure gene that links to each other with oligonucleotide, this structure gene is inserted expression vector, again this carrier is imported host cell, as intestinal bacteria, the promptly synthetic single polypeptide chain that contains middle with two continuous V functional zone of connection peptides of the host cell of this reorganization.The method of relevant generation sFvs see description (Whitlow etc., Methods:ACompanion to Methods in Enzymology, Vol.2, page 97 (1991); Bird etc., Science, 242:423 (1988), Ladner etc., U.S.patent No.4,946,778; Pack etc., Bio/Technologv, 11:1271 (1993) and Sandhu, Crit.Rev.Biotech., 12:437 (1992)). the another kind of form of antibody fragment is the peptide that forms single complementary determining region (CDR), and CDR peptide (" atom ") can obtain by the gene that makes up the coding target antibody.Preparing such gene can pass through, for example, and with polymerase chain reaction synthetic variable region (Vol.2, page 106 (1991) for Larrick etc., Methods:ACompanion to Methods in Enzymology) from the RNA of antibody produced cell.

Antibody of the present invention can combine with certain toxin, and such antibody can be used for the treatment of and infected the animal of levying relevant virus on etiology with serious acute respiratory system synthesis, comprises the people.For example, with be connected tetanus toxin with spike protein bonded antibody that serious acute respiratory system synthesis is levied the coronavirus that is associated on the etiology, be used to infect the animal that above-mentioned virus is arranged, the antibody that is combined with toxin combines with the spike protein of infected cell surface, then infected cell is killed.

Antibody of the present invention can combine with certain certification mark thing, and such antibody can be used for diagnostic test to determine animal, as the people, whether has infected SARS-CoV.The example of certification mark thing has fluorescin (being green fluorescent protein, red fluorescent protein, yellow fluorescence protein).Fluorescent mark (being fluorescein isothiocyanic acid, rhodamine, texas Red), radio-labeled are (promptly ³H, ³²P, ¹²⁵I), enzyme (beta-galactosidase enzymes, horseradish peroxidase, β-glucuronic acid Glycosylase alkaline phosphatase), or affinity labelling (being avidin, vitamin H, Streptavidin).Antibody is known in the art with the method that the certification mark thing is connected.See Harlow etc., Antibodies:A Laboratory Manual, page 319 (Cold Spring HarborPub.1988).

The present invention also provides the fit of polypeptide of the present invention or peptide fragment.Of the present invention fit can be the fit or aptamer of peptide.Peptide is fit to be and polypeptide of the present invention or peptide fragment bonded peptide, and its bonding force is normal suitable with monoclonal antibody-antigenic compound.Similar, aptamer is the nucleic acid that very strong bonding force is arranged with polypeptide of the present invention or peptide fragment, and its bonding force is normal suitable with monoclonal antibody-antigenic compound.

In an example, aptamer can obtain by using random oligonucleotide sequence library to separate.Screen this library to determine that some oligonucleotide sequences combine with S polypeptide of the present invention and peptide fragment.The bonded oligonucleotide elutes from solidified polypeptide or peptide fragment, increases with PCR then.This process can repeat repeatedly to filter out polypeptide of the present invention and peptide fragment are had the fit of high-bond.Determine this fit nucleotide sequence of coding then, and its clone is entered in the suitable carriers to help this fit production and preservation.

Peptide is fit can show that screening contain promotor, initiator codon and encode at random that the library of the nucleotide sequence of peptide obtains by mRNA.In certain embodiments, the nucleic acid fragment of encoding histidine marker is also contained in this DNA library.Transcribe as t7 rna polymerase with suitable polysaccharase in this library.The poly A joint of transcribing a purine-containing mycin after finishing is added to the 3 ' end of new synthetic mRNA.When these mRNA after external translation, the fusion molecule of the covalently bound formation of newly-generated peptide and tetracycline joint mRNA-peptide.With Ni-NTA agarose and oligo-dT Mierocrystalline cellulose the fusion molecule of mRNA-peptide is carried out purifying.MRNA to fusion molecule partly carries out reverse transcription again.Then, hatch altogether with the DNA/RNA-peptide fusion molecule of two strands and peptide of the present invention or peptide fragment, unconjugated fusion molecule will be by flush away, the bonded fusion molecule elutes from solidified peptide or peptide fragment, increase with PCR, this process can repeat to filter out with peptide of the present invention or peptide fragment the fit of height bonding force arranged again.Can determine this fit nucleotide sequence of encoding then, and it is cloned into suitable carriers.Prepare existing description of the fit method of peptide and (see (Wilson etc., Proc.Natl.Acad.Sci., 98:3750 (2001)).So, the invention provides and can discern the fit of peptide of the present invention and peptide fragment.

V. medicinal compositions of the present invention

The invention provides the fragment or their the conservative variant bonded antibody and the medicinal compositions of pharmaceutically acceptable carrier that contain with aminoacid sequence shown in SEQ ID NO:1,13,14,15, the 20-59,60,61,62,63 or SEQ ID NO:1.In certain embodiments, antibody combines with the peptide with SEQ ID NO:58 or 59 sequences.Effective with peptide P540 (SEQ ID NO:59) bonded antibody height, even under the condition of dilution for many times, also can detect the furcella polypeptide.For example, P540 antibody still can detect the furcella polypeptide under the condition of 1: 10000 times of dilution.

Medicinal compositions of the present invention can be prepared to various ways, comprises tablet, hard or soft gelatine capsule, aqueous pharmaceutical, suspension, liposome and other sustained release preparations, if any the form height molecular glue.Also can make a kind of oral dosage dosage form, antibody is discharged enter enteron aisle in stomach after.Such preparation is at U.S. Patent number 6,306, on 434 and be contained in the reference of this paper description is arranged.

Oral liquid pharmaceutical composition can be these forms, for example, and water-based or butyrous suspension, solution, emulsion, syrup or elixir, or the drying products that uses after using preceding water or other suitable media to be in harmonious proportion.These liquid pharmaceutical compositions can contain conventional additives, as suspension agent, emulsifying agent, non-aqueous media (they can comprise edible oil) or sanitas.

Antibody can be made into the preparation (for example, by injection, as disposable injection or continuous drip) of intestines external administration, is loaded on the form of unitary dose in the container of the instillation container of syringe, low capacity of ampoule, pre-can or multiple doses, and adds preservatives.This medicinal compositions can be suspension, solution or oiliness or aqueous emulsion, and can contain the such forming agent of suspension agent, stablizer and/or dispersion agent.The preparation that is suitable for the anus innerlich anwenden can be prepared to the suppository of unitary dose.Suitable carriers comprises salt solution and other materials that this area is commonly used.

For inhalation, antibody can import at an easy rate with the mode that gas atomization was packed or imported easily to insufflator, spraying gun, pressure.The pressure packing can have suitable propelling agent such as Refrigerant 12, Trichloromonofluoromethane, dichloro tetrafluoro ethane, carbonic acid gas and other suitable gas.With under the situation of pressurised aerosol, can determine dose unit by providing air valve to import metered amount.

Washability ground, for sucking or being blown into administration, antibody can be the composition of dry powder form, for example, conditioning agent and suitable dry powder matrix such as the pulverulent mixture of lactose or starch.Pulverulent mixture can be loaded in capsule and the cartridge case with the form of unitary dose, or for example in gelatin or the Blister Package, medicinal powder can the administration from these containers by sucker or insufflator.For intranasal administration, antibody can pass through liquid spray, for example by a plastics atomizer.

Medicinal compositions of the present invention also can contain other compositions, as seasonings, tinting material, biocide or sanitas.The amount of the antibody that is used for the treatment of not only will be different because selected carrier is different, and can be different because of route of administration, the state of an illness of being treated, patient's age and patient's situation, and this is understandable.Finally may determine suitable dose by medical supplier.In addition, medicinal compositions can be made into single unit dosage form.

VI. the method for immunity, treatment and the diagnosis of the animal of levying at serious acute respiratory system synthesis

The invention provides the method that immune animal is levied with opposing serious acute respiratory system synthesis.This method relates to and gives the animal dose therapeutically effective and fragment aminoacid sequence shown in SEQ ID NO:1,13,14,15, the 20-59,60,61,62,63 or SEQ ID NO:1 or their varient bonded antibody; Give the immune composition of animal dose therapeutically effective; Give the virus vaccines of animal dose therapeutically effective; Give the nucleic acid vaccine of animal dose therapeutically effective.Animal can be a Mammals, as the people.The method that gives vaccine and immune composition has description and known in the art here.

According to the present invention, also can infect the animal of SARS-CoV by the method treatment of passive immunotherapy.For example, the animal that will infect SARS-CoV with fragment or their the conservative property varient bonded antibody of aminoacid sequence shown in SEQ ID NO:1,13,14,15, the 20-59,60,61,62,63 or SEQ ID NO:1 is as the people.Such administrated method may be suitable for patient's immunity and be suppressed, and can not transfer effective anti-SARS-CoV immunity, or to vaccine or the nonreactive situation of immune composition.

The invention provides the diagnosis animal and whether suffer from the method that serious acute respiratory system synthesis is levied, this method relates to use with the fragment of aminoacid sequence shown in SEQ ID NO:1,13,14,15, the 20-59,60,61,62,63 or SEQ ID NO:1 or their conservative property varient bonded antibody and contacts with biological sample from this animal, as tissue sample, blood, mucus or saliva, determine whether this antibody combines with biological sample.It is known in the art to utilize antibody to come whether have antigen to exist in the detection of biological sample.Say simply, antibody of the present invention can be fixed on a surface, biological sample contacts with solidified antibody so that antigen in the sample and antibodies form immune complex then, washing sample in washability ground is removed unconjugated material, second antibody of the present invention is combined with a certification mark thing, as enzyme or radioactively labelled substance, can combine with immune complex and make enzyme or radioactively labelled substance be fixed to the surface.Can detect this certification mark thing then and decide whether contain antigen in the biological sample.In another example, biological sample can be fixed in a surface, and then, the antibody of the present invention that is connected with the certification mark thing contacts with the solidified biological sample, and flush away binding substance not, the existence by detecting the certification mark thing is to determine whether whether contain antigen in the biological sample.The example of these tests is known in the art, comprises enzyme linked immunosorbent assay, radioimmunoassay test and similar test.

Method based on nucleic acid also can be used for diagnosing serious acute respiratory system synthesis to levy.In an example, polymerase chain reaction (PCR) can be used to diagnose SARS-CoV to infect.Say simply, in animal body, obtain biological sample such as tissue sample, blood, mucus or saliva, use ordinary method, as the nucleic acid in organic extraction method extraction sample, it is that the typical buffers that template is extended the composition of forward and reverse primer is mixed with SARS nucleic acid that the nucleic acid that extracts and the forward and reverse primer, polysaccharase, the Nucleotide that are connected to the proteic nucleotide sequence of encoding SARS and containing of can annealing allow polysaccharase, and whether the existence that detects the forward that amplifies and the DNA between the reverse primer then is to contain the nucleic acid that SARS originates in definite sample.Nucleic acid hybridization technique also can be used for the existence of SARS nucleic acid in the detection of biological sample as Northern and Southern trace.

VII. test kit

The invention provides contain wrapping material and with the fragment of aminoacid sequence shown in the SEQ ID NO:1,13,14,15,45,46 or 47,58,59,61,62,63 or SEQ ID NO:1 or the test kit of their conservative property varient bonded antibody.Test kit can contain syringe so that the antibody in the test kit is injected to animal, as the people.In another embodiment, the invention provides contain wrapping material and with the fragment of aminoacid sequence shown in SEQ ID NO:1,13,14,15, the 20-59,60,61,62,63 or SEQ ID NO:1 or the test kit of their conservative property varient bonded antibody, and be made into and be used for animal, as people's preparation.In certain embodiments, antibody combines with the aminoacid sequence of SEQ ID NO:59.In other embodiments, antibody combines with the aminoacid sequence of SEQ ID NO:58.Such test kit can contain syringe to be injected to animal, as the people with the convenient antibody that test kit is contained.

The invention provides and contain packing material and contain aminoacid sequence shown in the coding SEQ ID NO:1,13,14,15,45,46 or 47,58,59,61,62,63 or fragment or the dna molecular of their conservative property varient or the dna vaccination of expression vector of SEQID NO:1.This test kit also can contain the device (as syringe and particle gun) that uses dna vaccination and be used for animal with the convenient vaccine that test kit is contained, as the people.

The present invention also provides and contains packing material and comprise fragment with the aminoacid sequence shown in the SEQ ID NO:1,13,14,15,45,46 or 47,58,59,61,62,63 or SEQID NO:1 or the vaccine composition of the polypeptide of their conservative property varient.This test kit also can contain vaccine using appts (as syringe) and be used for animal with the convenient vaccine that test kit is contained, as the people.

The invention provides one contains packing material and has the fragment of aminoacid sequence shown in the SEQ ID NO:1,13,14,15,45,46 or 47,58,59,61,62,63 or SEQID NO:1 or the polypeptide of their conservative property varient, be used to detect the test kit that SARS-CoV infects.Polypeptide can be fixed on the solid support.Such test kit can be used to detect the antibody of direct anti-SARS-CoV in infection animal or people's the serum.This test kit also includes these antibody and the S polypeptide bonded method of detecting.The aminoacid sequence of the furcella of the total length of VIII.Tor2SARS-CoV virus isolated strain (S) albumen (amino acid/11-1255)

MFIFLLFLTLTSGSDLDRCTTFDDVQAPNYTQHTSSMRGVYYPDEIFRSD

TLYLTQDLFLPFYSNVTGFHTINHTFGNPVIPFKDGIYFAATEKSNVVRG

WVFGSTMNNKSQSVIIINNSTNVVIRACNFELCDNPFFAVSKPMGTQTHT

MIFDNAFNCTFEYISDAFSLDVSEKSGNFKHLREFVFKNKDGFLYVYKG

YQPIDVVRDLPSGFNTLKPIFKLPLGINITFRAILTAFSPAQDIWGTSAAA

YFVGYLKPTTFMLKYDENGTTTDAVDCSQNPLAELKCSVKSFEIDKGIYQ

TSNFRVVPSGDVVRFPNITNLCPFGEVFNATKFPSVYAWERKKISNCVAD

YSVLYNSTFFSTFKCYGVSATKLNDLCFSNVYADSFVVKGDDVRQIAPG

QTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGK

LRPFERDISNVPFSPDGKPCTPPALNCYWPLNDYGFYTTTGIGYQPYRVV

VLSFELLNAPATVCGPKLSTDLIKNQCVNFNFNGLTGTGVLTPSSKRFQP

FQQFGRDVSDFTDSVRDPKTSEILDISPCAFGGVSVITPGTNASSEVAVLY

QDVNCTDVSTAIHADQLTPAWRIYSTGNNVFQTQAGCLIGAEHVDTSYE

CDIPIGAGICASYHTVSLLRSTSQKSIVAYTMSLGADSSIAYSNNTIAIPTN

FSISITTEVMPVSMAKTSVDCNMYICGDSTECANLLLQYGSFCTQLNRAL

SGIAAEQDRNTREVFAQVKQMYKTPTLKYFGGFNFSQILPDPLKPTKRSF

IEDLLFNKVTLADAGFMKQYGECLGDINARDLICAQKFNGLTVLPPLLT

DDMIAAYTAALVSGTATAGWTFGAGAALQIPFAMQMAYRFNGIGVTQ

NVLYENQKQIANQFNKAISQIQESLTTTSTALGKLQDVVNQNAQALNTL

VKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLI

RAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQAAPHGVV

FLHVTYVPSQERNFTTAPAICHEGKAYFPREGVFVFNGTSWFITQRNFFS

PQIITTDNTFVSGNCDVVIGIINNTVYDPLQPELDSFKEELDKYFKNHTSP

DVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKW

PWYVWLGFIAGLIAIVMVTILLCCMTSCCSCLKGACSCGSCCKFDEDDSE

PVLKGVKLHYT(SEQ ID NO：1)

IX. the nucleotide sequence of the furcella of total length (S) albumen (amino acid/11-3768)

ATGTTTATTTTCTTATTATTTCTTACTCTCACTAGTGGTAGTGACCTTG

ACCGGTGCACCACTTTTGATGATGTTCAAGCTCCTAATTACACTCAAC

ATACTTCATCTATGAGGGGGGTTTACTATCCTGATGAAATTTTTAGAT

CAGACACTCTTTATTTAACTCAGGATTTATTTCTTCCATTTTATTCTAA

TGTTACAGGGTTTCATACTATTAATCATACGTTTGGCAACCCTGTCAT

ACCTTTTAAGGATGGTATTTATTTTGCTGCCACAGAGAAATCAAATGT

TGTCCGTGGTTGGGTTTTTGGTTCTACCATGAACAACAAGTCACAGTC

GGTGATTATTATTAACAATTCTACTAATGTTGTTATACGAGCATGTAA

CTTTGAATTGTGTGACAACCCTTTCTTTGCTGTTTCTAAACCCATGGG

TACACAGACACATACTATGATATTCGATAATGCATTTAATTGCACTTT

CGAGTACATATCTGATGCCTTTTCGCTTGATGTTTCAGAAAAGTCAGG

TAATTTTAAACACTTACGAGAGTTTGTGTTTAAAAATAAAGATGGGTT

TCTCTATGTTTATAAGGGCTATCAACCTATAGATGTAGTTCGTGATCT

ACCTTCTGGTTTTAACACTTTGAAACCTATTTTTAAGTTGCCTCTTGGT

ATTAACATTACAAATTTTAGAGCCATTCTTACAGCCTTTTCACCTGCT

CAAGACATTTGGGGCACGTCAGCTGCAGCCTATTTTGTTGGCTATTTA

AAGCCAACTACATTTATGCTCAAGTATGATGAAAATGGTACAATCAC

AGATGCTGTTGATTGTTCTCAAAATCCACTTGCTGAACTCAAATGCTC

TGTTAAGAGCTTTGAGATTGACAAAGGAATTTACCAGACCTCTAATTT

CAGGGTTGTTCCCTCAGGAGATGTTGTGAGATTCCCTAATATTACAAA

CTTGTGTCCTTTTGGAGAGGTTTTTAATGCTACTAAATTCCCTTCTGTC

TATGCATGGGAGAGAAAAAAAATTTCTAATTGTGTTGCTGATTACTCT

GTGCTCTACAACTCAACATTTTTTTCAACCTTTAAGTGCTATGGCGTT

TCTGCCACTAAGTTGAATGATCTTTGCTTCTCCAATGTCTATGCAGAT

TCTTTTGTAGTCAAGGGAGATGATGTAAGACAAATAGCGCCAGGACA

AACTGGTGTTATTGCTGATTATAATTATAAATTGCCAGATGATTTCAT

GGGTTGTGTCCTTGCTTGGAATACTAGGAACATTGATGCTACTTCAAC

TGGTAATTATAATTATAAATATAGGTATCTTAGACATGGCAAGCTTA

GGCCCTTTGAGAGAGACATATCTAATGTGCCTTTCTCCCCTGATGGCA

AACCTTGCACCCCACCTGCTCTTAATTGTTATTGGCCATTAAATGATT

ATGGTTTTTACACCACTACTGGCATTGGCTACCAACCTTACAGAGTTG

TAGTACTTTCTTTTGAACTTTTAAATGCACCGGCCACGGTTTGTGGAC

CAAAATTATCCACTGACCTTATTAAGAACCAGTGTGTCAATTTTAATT

TTAATGGACTCACTGGTACTGGTGTGTTAACTCCTTCTTCAAAGAGAT

TTCAACCATTTCAACAATTTGGCCGTGATGTTTCTGATTTCACTGATT

CCGTTCGAGATCCTAAAACATCTGAAATATTAGACATTTCACCTTGCG

CTTTTGGGGGTGTAAGTGTAATTACACCTGGAACAAATGCTTCATCTG

AAGTTGCTGTTCTATATCAAGATGTTAACTGCACTGATGTTTCTACAG

CAATTCATGCAGATCAACTCACACCAGCTTGGCGCATATATTCTACTG

GAAACAATGTATTCCAGACTCAAGCAGGCTGTCTTATAGGAGCTGAG

CATGTCGACACTTCTTATGAGTGCGACATTCCTATTGGAGCTGGCATT

TGTGCTAGTTACCATACAGTTTCTTTATTACGTAGTACTAGCCAAAAA

TCTATTGTGGCTTATACTATGTCTTTAGGTGCTGATAGTTCAATTGCTT

ACTCTAATAACACCATTGCTATACCTACTAACTTTTCAATTAGCATTA

CTACAGAAGTAATGCCTGTTTCTATGGCTAAAACCTCCGTAGATTGTA

ATATGTACATCTGCGGAGATTCTACTGAATGTGCTAATTTGCTTCTCC

AATATGGTAGCTTTTGCACACAACTAAATCGTGCACTCTCAGGTATTG

CTGCTGAACAGGATCGCAACACACGTGAAGTGTTCGCTCAAGTCAAA

CAAATGTACAAAACCCCAACTTTGAAATATTTTGGTGGTTTTAATTTT

TCACAAATATTACCTGACCCTCTAAAGCCAACTAAGAGGTCTTTTATT

GAGGACTTGCTCTTTAATAAGGTGACACTCGCTGATGCTGGCTTCATG

AAGCAATATGGCGAATGCCTAGGTGATATTAATGCTAGAGATCTCAT

TTGTGCGCAGAAGTTCAATGGACTTACAGTGTTGCCACCTCTGCTCAC

TGATGATATGATTGCTGCCTACACTGCTGCTCTAGTTAGTGGTACTGC

CACTGCTGGATGGACATTTGGTGCTGGCGCTGCTCTTCAAATACCTTT

TGCTATGCAAATGGCATATAGGTTCAATGGCATTGGAGTTACCCAAA

ATGTTCTCTATGAGAACCAAAAACAAATCGCCAACCAATTTAACAAG

GCGATTAGTCAAATTCAAGAATCACTTACAACAACATCAACTGCATT

GGGCAAGCTGCAAGACGTTGTTAACCAGAATGCTCAAGCATTAAACA

CACTTGTTAAACAACTTAGCTCTAATTTTGGTGCAATTTCAAGTGTGC

TAAATGATATCCTTTCGCGACTTGATAAAGTCGAGGCGGAGGTACAA

ATTGACAGGTTAATTACAGGCAGACTTCAAAGCCTTCAAACCTATGT

AACACAACAACTAATCAGGGCTGCTGAAATCAGGGCTTCTGCTAATC

TTGCTGCTACTAAAATGTCTGAGTGTGTTCTTGGACAATCAAAAAGA

GTTGACTTTTGTGGAAAGGGCTACCACCTTATGTCCTTCCCACAAGCA

GCCCCGCATGGTGTTGTCTTCCTACATGTCACGTATGTGCCATCCCAG

GAGAGGAACTTCACCACAGCGCCAGCAATTTGTCATGAAGGCAAAGC

ATACTTCCCTCGTGAAGGTGTTTTTGTGTTTAATGGCACTTCTTGGTTT

ATTACACAGAGGAACTTCTTTTCTCCACAAATAATTACTACAGACAAT

ACATTTGTCTCAGGAAATTGTGATGTCGTTATTGGCATCATTAACAAC

ACAGTTTATGATCCTCTGCAACCTGAGCTCGACTCATTCAAAGAAGA

GCTGGACAAGTACTTCAAAAATCATACATCACCAGATGTTGATCTTG

GCGACATTTCAGGCATTAACGCTTCTGTCGTCAACATTCAAAAAGAA

ATTGACCGCCTCAATGAGGTCGCTAAAAATTTAAATGAATCACTCAT

TGACCTTCAAGAATTGGGAAAATATGAGCAATATATTAAATGGCCTT

GGTATGTTTGGCTCGGCTTCATTGCTGGACTAATTGCCATCGTCATGG

TTACAATCTTGCTTTGTTGCATGACTAGTTGTTGCAGTTGCCTCAAGG

GTGCATGCTCTTGTGGTTCTTGCTGCAAGTTTGATGAGGATGACTCTG

AGCCAGTTCTCAAGGGTGTCAAATTACATTACACATAA(SEQ ID NO：2)

Embodiment 1

The clone of spike protein

The nucleotide sequence of coding total length spike protein obtains with the method for overlapping polymerase chain reaction (PCR).The overlapping clone that contains spike protein obtains from British Columbia Cancer Agency (Vancouver, British Columbia).Following primer is used to the nucleotide sequence of amplification coding total length SARS-CoV spike protein in the PCR reaction: clone 1: forward primer: 5 '-AGTC GGA TCCGGT AGG CTT ATC ATT AGA G-3 ' (SEQ ID NO:3); Reverse primer: 5 '-CCA TCA GGG GAG AAA GGC AC-3 (SEQ ID NO:4).Clone 2: forward primer: 5 '-GTG CCT TTC TCC CCT GAT GG-3 ' (SEQ ID NO:5); Reverse primer: 5 '-GAA GAG CAG CGC CAG CAC C-3 ' (SEQ ID NO:6).Clone 3: forward primer: 5 '-GGT GCT GGC GCT GCT CTT C-3 ' (SEQ IDNO:7); Reverse primer: 5 '-A CTG TCT AGAGTT CGT TTA TGT GTAATG-3 (SEQ ID NO:8).

By the above primer of overlapping pcr amplification between the nucleic acid fragment that nucleic acid fragment produced contain 1 to 1255 amino-acid residue levying the spike protein of the relevant virus of etiology (SARS-CoV) with serious acute respiratory system synthesis.The primer sequence representative that has underscore is used for amplified fragments is cloned into the Restriction Enzyme BamH1 of pCDN3 (+) plasmid and the restriction enzyme site of XbaI.

The clone of this total length spike protein gene is as shown in Fig. 1, and Fig. 1 shows is glue figure (road 2:BamH1 and XbaI enzyme cutting after being limited property of pCDN3.1 (+) the carrier enzyme enzyme that inserted the nucleic acid fragment of coding spike protein total length is cut; Road 3:HindIII enzyme is cut).

Embodiment 2

N terminal fragment of total length spike protein (S1) and the segmental generation of carboxyl terminal (S2)

Determine the aminoterminal (S1) of spike protein and the potential function cut-point of carboxyl terminal (S2) with Computer Analysis, the cut-point between S1 and the S2 SEQ ID NO:1 758 and 761 ( ⁷⁵⁸RNTR ⁷⁶¹) between.The method of PCR is used to prepare the encode n terminal fragment (S1) and carboxyl terminal (S2) fragment of spike protein.

Following primer is right, the S1 forward primer: 5 '-AGTC GGA TCCGAC CGG TGCACC ACT TTT G-3 ' (SEQ ID NO:9) and S1 reverse primer: 5 '-AGTC GGG CCCCTG TTC AGC AGC AAT ACC-3 ' (SEQ ID NO:10) is used to prepare the nucleic acid fragment of the 17th to 757 amino acids residue of spike protein of encoding.Two restricted point of contacts, BamHI and ApaI (parts of two primer sequence band underscores) are used to clone the nucleic acid fragment (S1) of the n terminal fragment of this coding spike protein gene and express in the pSecTaG2B plasmid.

Following primer is right, the S2 forward primer: 5 '-ACTG GGATCC GAA GTG TTCGCT CAA GTC-3 ' (SEQ ID NO:11) and S2 reverse primer: 5 '-ACTGTCTAGA TTG CTC ATA TTT TCC C-3 ' (SEQ ID NO:12) is used to the PCR reaction nucleic acid fragment with preparation coding spike protein the 762nd to 1189 amino acids residue.Two restricted point of contacts, BamHI and XbaI (parts of two primer sequence band underscores) are used to clone the nucleic acid fragment (S2) of the carboxyl-terminal fragment of this coding spike protein gene and express in pCDN3.1 (+) plasmid.

In order to obtain to contain the fragment of the 272nd to 537 residue, following primer is to being used to pcr amplification: primer: 5 ' GATCGGATCCGGTACAATCACAG 3 ' (SEQ IDNO:64) and primer 5 ' GATCGGGCCCGACACACTGGTTC 3 ' (SEQ IDNO:65).This amplified fragments is with BamHI and ApaI digestion, and is connected with pSecTag2B with same enzymic digestion.The diagram among Figure 1B is seen in the segmental position of many solubility spike proteins of total length spike protein.

In some cases, natural leader sequence (the 1st to 16 amino acids of spike protein of the nucleic acid of the fragment of coding S and the S polypeptide of total length, MFIFLLFLTLTSGSDL (SEQID NO:60) is replaced (METDTLLLWVLLLWVPGSTGD) (SEQ ID NO:16) to facilitate secretion, just as described below by mouse k chain leader sequence.

Embodiment 3

Lack kytoplasm tail and the acquisition of striding the complete solubility spike protein (sS) in film district

Following primer is to being used to obtain the nucleic acid fragment that coding lacks the 17th to 1189 amino acids that contains SEQ ID NO:1 of kytoplasm tail: the S1 forward primer: 5 '-AGTC GGATCCGAC CGG TGC ACC ACT TTT G-3 ' (SEQ ID NO:9), and reverse primer: 5 ' ACTG TCTAGATTG CTC ATA TTT TCC C-3 ' (SEQ ID NO:12).

Embodiment 4

The aminoterminal of spike protein and the expression of carboxyl-terminal fragment

By with containing pSecTag2 or pCDN3.1 (+) plasmid and coding aminoterminal (S1), carboxyl terminal (S2) fragment, or the segmental expression construct transfection 293 of SARS-CoV spike protein or the E6Vero cell that lack the kytoplasm tail and stride the film district are expressed.The removal of striding the film district is considered to cause polypeptide and peptide fragment solvable in the aqueous solution.Segmental expression efficiency then is encoded test.In case obtain positive signal with electrophoretic analysis, will set up a stable transfectional cell series.The spike protein of total length and its fragment will be carried out purifying according to the proteic method that is used for the purifying high glycosylation of routine, be used for mass production as lens lectin post.The protein that obtains: the S2 (sS2) and the complete S (sS) of the S1 of solubility (sS1), solubility will contain following aminoacid sequence.The signal peptide that the boldface type representative can be cut, oozy albumen will not contain it.

The aminoacid sequence of the solubility n terminal fragment of spike protein (17-757 amino acids)

DRCTTFDDVQAPNYTQHTSSMRGVYYPDEIFRSDTLYLTQDLFLPFYSN

VTGFHTINHTFGNPVIPFKDGIYFAATEKSNVVRGWVFGSTMNNKSQSVI

IINNSTNVVIRACNFELCDNPFFAVSKPMGTQTHTMIFDNAFNCTFEYISD

AFSLDVSEKSGNFKHLREFVFKNKDGFLYVYKGYQPIDVVRDLPSGFNT

LKPIFKLPLGINITNFRAILTAFSPAQDIWGTSAAAYFVGYLKPTTFMLKY

DENGTITDAVDCSQNPLAELKCSVKSFEIDKGIYQTSNFRVVPSGDVVRF

PNITNLCPFGEVFNATKFPSVYAWERKKISNCVADYSVLYNSTFFSTFKC

YGVSATKLNDLCFSNVYADSFVVKGDDVRQIAPGQTGVIADYNYKLPD

DFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDISNVPFSPD

GKPCTPPALNCYWPLNDYGFYTTTGIGYQPYRVVVLSFELLNAPATVCG

PKLSTDLIKNQCVNFNFNGLTGTGVLTPSSKRFQPFQQFGRDVSDFTDSV

RDPKTSEILDISPCAFGGVSVITPGTNASSEVAVLYQDVNCTDVSTAIHAD

QLTPAWRIYSTGNNVFQTQAGCLIGAEHVDTSYECDIPIGAGICASYHTV

SLLRSTSQKSIVAYTMSLGADSSIAYSNNTIAIPTNFSISITTEVMPVSMAK

TSVDCNMYICGDSTECANLLLQYGSFCTQLNRALSGIAAEQ(SEQ IDNO：13)

The aminoacid sequence of the solubility carboxyl-terminal fragment of spike protein (762-1189 amino acids)

EVFAQVKQMYKTPTLKYFGGFNFSQILPDPLKPTKRSFIEDLLFNKVTLA

DAGFMKQYGECLGDINARDLICAQKFNGLTVLPPLLTDDMIAAYTAALV

SGTATAGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKQIANQ

FNKAISQIQESLTTTSTALGKLQDVVNQNAQALNTLVKQLSSNFGAISSV

LNDILSRDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAAT

KMSECVLGQSKRVDFCGKGYHLMSFPQAAPHGVVFLHVTYVPSQERNF

TTAPAICHEGKAYFPREGVFVFNGTSWFITQRNFFSPQIITTDNTFVSGNC

DVVIGIINNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVV

NIQKEIDRLNEVAKNLNESLIDLQELGKYEQ(SEQ ID NO：14)

Contain the 17-757 amino acids of SEQ ID NO:1 and the aminoacid sequence of 762-1189 amino acids solubility spike protein (lacking signal peptide and potential cleavage site)

DRCTTFDDVQAPNYTQHTSSMRGVYYPDEIFRSDTLYLTQDLFLPFYSN

VTGFHTINHTFGNPVIPFKDGIYFAATEKSNVVRGWVFGSTMNNKSQSVI

IINNSTNVVIRACNFELCDNPFFAVSKPMGTQTHTMIFDNAFNCTFEYISD

AFSLDVSEKSGNFKHLREFVFKNKDGFLYVYKGYQPIDVVRDLPSGFNT

LKPIFKLPLGINITNFRAILTAFSPAQDIWGTSAAAYFVGYLKPTTFMLKY

DENGTITDAVDCSQNPLAELKCSVKSFEIDKGIYQTSNFRVVPSGDVVRF

PNITNLCPFGEVFNATKFPSVYAWERKKISNCVADYSVLYNSTFFSTFKC

YGVSATKLNDLCFSNVYADSFVVKGDDVRQIAPGQTGVIADYNYKLPD

DFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDISNVPFSPD

GKPCTPPALNCYWPLNDYGFYTTTGIGYQPYRVVVLSFELLNAPATVCG

PKLSTDLIKNQCVNFNFNGLTGTGVLTPSSKRFQPFQQFGRDVSDFTDSV

RDPKTSEILDISPCAFGGVSVITPGTNASSEVAVLYQDVNCTDVSTAIHAD

QLTPAWRIYSTGNNVFQTQAGCLIGAEHVDTSYECDIPIGAGICASYHTV

SLLRSTSQKSIVAYTMSLGADSSIAYSNNTIAIPTNFSISITTEVMPVSMAK

TSVDCNMYICGDSTECANLLLQYGSFCTQLNRALSGIAAEQDEVFAQVK

QMYKTPTLKYFGGFNFSQILPDPLKPTKRSFIEDLLFNKVTLADAGFMKQ

YGECLGDINARDLICAQKFNGLTVLPPLLTDDMIAAYTAALVSGTATAG

WTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKQIANQFNKAISQI

QESLTTTSTALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRL

DKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVL

GQSKRVDFCGKGYHLMSFPQAAPHGVVFLHVTYVPSQERNFTTAPAICH

EGKAYFPREGVFVFNGTSWFTTQRNFFSPQIITTDNTFVSGNCDVVIGIINN

TVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRL

NEVAKNLNESLIDLQELGKYEQ(SEQ ID NO：15)

Embodiment 5

The generation of the additional soluble fragments of spike protein

Obtain to contain the nucleotide sequence of polypeptide of the 17-757 amino acids of coding SEQ ID NO:1 by polymerase chain reaction (PCR).Following primer is used at this nucleotide sequence of PCR amplification: forward primer: 5 ' AGCT GGA TCCGAC CGG TGC ACC ACTTTT G 3 ' (SEQ TD NO:9); And reverse primer: 5 ' AGCT GGG CCCCTGTTC AGC AGC AAT ACC 3 ' (SEQ ID NO:10).This PCR product is cut with BamHI and ApaI enzyme, the polypeptide of coding and SEQ ID NO:43 consensus amino acid sequence.Be connected with pSecTag2B with same enzymic digestion by postdigestive PCR product (Invitrogen, Carlsbad, California).The pSecTag2B construct coded polypeptide SEQ ID NO:46 that contains the PCR product of insertion, the aminoterminal of this polypeptide contains mouse k chain leader sequence (METDTLLLWVLLLWVPGSTGD) (SEQ ID NO:16), and carboxyl terminal contains the myc determinant (EQKLISEEDL) (SEQ ID NO:17) that is useful on affinitive layer purification and adds histidine mark thing (HHHHHH) (SEQ ID NO:18).

Obtain to contain the nucleotide sequence of polypeptide of the 17-276 amino acids of coding SEQ ID NO:1 by polymerase chain reaction (PCR).Following primer is used at this nucleotide sequence of PCR amplification: forward primer: 5 ' AGCT GGA TCCGAC CGG TGC ACC ACTTTT G3 ' (SEQ ID NO:9); Reverse primer: 5 ' CTAG CTC GAGCAA CAGCAT CTG TG 3 ' (SEQ ID NO:19).This PCR product is cut with BamHI and XhoI enzyme, and coding contains the polypeptide of SEQ ID NO:44 aminoacid sequence.Be connected with pSecTag2B with same enzymic digestion by postdigestive PCR product (Invitrogen, Carlsbad, California).The pSecTag2B construct coded polypeptide SEQID NO:47 that contains the PCR product of insertion, the aminoterminal of this polypeptide contains mouse k chain leader sequence (METDTLLLWVLLLWVPGSTGD) (SEQ ID NO:16), and carboxyl terminal contains the myc determinant (EQKLISEEDL) (SEQ ID NO:17) that is useful on affinitive layer purification and adds histidine mark thing (HHHHHH) (SEQ ID NO:18).

The nucleotide sequence that coding contains the polypeptide of SEQ ID NO:1 17-537 amino acids obtains by the nucleotide sequence that digests coding SEQ ID NO:43 (as described above), and this nucleic acid fragment coding contains the polypeptide of SEQ ID NO:45.This nucleic acid fragment is connected with pSecTag2B with BamHI and EcoRV digestion.The pSecTag2B construct coded polypeptide SEQ ID NO:48 that contains the PCR product of insertion, the aminoterminal of this polypeptide contains mouse k chain leader sequence (METDTLLLWVLLLWVPGSTGD) (SEQ ID NO:16), and carboxyl terminal contains the myc determinant (EQKLISEEDL) (SEQ ID NO:17) that is useful on affinitive layer purification and adds histidine mark thing (HHHHHH) (SEQ ID NO:18).

Fig. 3 has shown the expression of these polypeptide fragments in mammalian cell, and these peptide fragment of data presentation can be secreted in the substratum of the cell growth of expressing these peptide fragment.Fig. 3 has shown that also peptide fragment is solvable in the aqueous solution.

Table 1

The embodiment of additional peptide fragment of the present invention

SEQ ID No	Amino acid position with respect to SEQ ID NO:1	The amino-acid residue that the aminoacid sequence black matrix is represented (amino-acid residue 1-16) representation signal sequence
			20	1-100	MFIFLLFLTLTSGSDLDRCTTFDDVQAP NYTQHTSSMRGVYYPDEIFRSDTLYLTQ DLFLPFYSNVTGFHTINHTFGNPVIPFKD GIYFAATEKSNVVRG
21	101-200	WVFGSTMNNKSQSVIIINNSTNVVIRACN FELCDNPFFAVSKPMGTQTHTMIFDNAF NCTFEYISDAFSLDVSEKSGNFKHLREFV FKNKDGFLYVYKGY
			22	201-300	QPIDVVRDLPSGFNTLKPIFKLPLGINITN FRAILTAFSPAQDIWGTSAAAYFVGYLK PTTFMLKYDENGTITDAVDCSQNPLAEL KCSVKSFEIDKGIY
23	301-400	QTSNFRVVPSGDVVRFPNITNLCPFGEVF NATKFPSVYAWERKKISNCVADYSVLY NSTFFSTFKCYGVSATKLNDLCFSNVYA DSFVVKGDDVRQIAPG
			24	401-500	QTGVIADYNYKLPDDFMGCVLAWNTRN IDATSTGNYNYKYRYLRHGKLRPFERDI SNVPFSPDGKPCTPPALNCYWPLNDYGF YTTTGIGYQPYRVVVLS
25	501-600	FELLNAPATVCGPKLSTDLIKNQCVNFN FNGLTGTGVLTPSSKRFQPFQQFGRDVS DFTDSVRDPKTSEILDISPCAFGGVSVITP GTNASSEVAVLYQD
			26	601-700	VNCTDVSTAIHADQLTPAWRIYSTGNNV FQTQAGCLIGAEHVDTSYECDIPIGAGIC ASYHTVSLLRSTSQKSIVAYTMSLGADS SIAYSNNTIAIPTNF
27	701-800	SISITTEVMPVSMAKTSVDCNMYICGDST ECANLLLQYGSFCTQLNRALSGIAAEQD RNTREVFAQVKQMYKTPTLKYFGGFNF SQILPDPLKPTKRSFI
			28	801-900	EDLLFNKVTLADAGFMKQYGECLGDIN ARDLICAQKFNGLTVLPPLLTDDMIAAY TAALVSGTATAGWTFGAGAALQIPFAM QMAYRFNGIGVTQNVLYE

SEQ ID No	Amino acid position with respect to SEQ ID NO:1	The amino-acid residue that the aminoacid sequence black matrix is represented (amino-acid residue 1-16) representation signal sequence
			29	901-1000	NQKQIANQFNKAISQIQESLTTTSTALGK LQDVVNQNAQALNTLVKQLSSNFGAISS VLNDILSRLDKVEAEVQIDRLITGRLQSL QTYVTQQLIRAAEI
30	1001-1100	RASANLAATKMSECVLGQSKRVDFCGK GYHLMSFPQAAPHGVVFLHVTYVPSQE RNFTTAPAICHEGKAYFPREGVFVFNGT SWFITQRNFFSPQIITTD
			31	1101-1189	NTFVSGNCDVVIGIINNTVYDPLQPELDS FKEELDKYFKNHTSPDVDLGDISGINASV VNIQKEIDRLNEVAKNLNESLIDLQELGK YEQ
32	1-200	MFIFLLFLTLTSGSDLDRCTTFDDVQAP NYTQHTSSMRGVYYPDEIFRSDTLYLTQ DLFLPFYSNVTGFHTINHTFGNPVIPFKD GIYFAATEKSNVVRGWVFGSTMNNKSQ SVIIINNSTNVVIRACNFELCDNPFFAVSK PMGTQTHTMIFDNAFNCTFEYISDAFSLD VSEKSGNFKHLREFVFKNKDGFLYVYK GY
			33	201-400	QPIDVVRDLPSGFNTLKPIFKLPLGINITN FRAILTAFSPAQDIWGTSAAAYFVGYLK PTTFMLKYDENGTITDAVDCSQNPLAEL KCSVKSFEIDKGIYQTSNFRVVPSGDVVR FPNITNLCPFGEVFNATKFPSVYAWERK KISNCVADYSVLYNSTFFSTFKCYGVSA TKLNDLCFSNVYADSFVVKGDDVRQIAP G
34	401-600	QTGVIADYNYKLPDDFMGCVLAWNTRN IDATSTGNYNYKYRYLRHGKLRPFERDI SNVPFSPDGKPCTPPALNCYWPLNDYGF YTTTGIGYQPYRVVVLSFELLNAPATVC GPKLSTDLIKNQCVNFNGLTGTGVLT PSSKRFQPFQQFGRDVSDFTDSVRDPKTS EILDISPCAFGGVSVITPGTNASSEVAVLY QD

SEQ ID No	Amino acid position with respect to SEQ ID NO:1	The amino-acid residue that the aminoacid sequence black matrix is represented (amino-acid residue 1-16) representation signal sequence
			35	601-800	VNCTDVSTAIHADQLTPAWRIYSTGNNV FQTQAGCLIGAEHVDTSYECDIPIGAGIC ASYHTVSLLRSTSQKSIVAYTMSLGADS SIAYSNNTIAIPTNFSISITTEVMPVSMAK TSVDCNMYICGDSTECANLLLQYGSFCT QLNRALSGIAAEQDRNTREVFAQVKQM YKTPTLKYFGGFNFSQILPDPLKPTKRSFI
36	801-1000	EDLLFNKVTLADAGFMKQYGECLGDIN ARDLICAQKFNGLTVLPPLLTDDMIAAY TAALVSGTATAGWTFGAGAALQIPFAM QMAYRFNGIGVTQNVLYENQKQIANQF NKAISQIQESLTTTSTALGKLQDVVNQN AQALNTLVKQLSSNFGAISSVLNDILSRL DKVEAEVQIDRLITGRLQSLQTYVTQQLI RAAEI
			37	1001-1189	RASANLAATKMSECVLGQSKRVDFCGK GYHLMSFPQAAPHGVVFLHVTYVPSQE RNFTTAPAICHEGKAYFPREGVFVFNGT SWFITQRNFFSPQIITTDNTFVSGNCDVVI GIINNTVYDPLQPELDSFKEELDKYFKNH TSPDVDLGDISGINASVVNIQKEIDRLNE VAKNLNESLIDLQELGKYEQ
38	1-400	MFIFLLFLTLTSGSDLDRCTTFDDVQAP NYTQHTSSMRGVYYPDEIFRSDTLYLTQ DLFLPFYSNVTGFHTINHTFGNPVIPFKD GIYFAATEKSNVVRGWVFGSTMNNKSQ SVIIINNSTNVVIRACNFELCDNPFFAVSK PMGTQTHTMIFDNAFNCTFEYISDAFSLD VSEKSGNFKHLREFVFKNKDGFLYVYK GYQPIDVVRDLPSGFNTLKPIFKLPLGINI TNFRAILTAFSPAQDIWGTSAAAYFVGY LKPTTFMLKYDENGTITDAVDCSQNPLA ELKCSVKSFEIDKGIYQTSNFRVVPSGDV VRFPNITNLCPFGEVFNATKFPSVYAWE RKKISNCVADYSVLYNSTFFSTFKCYGV SATKLNDLCFSNVYADSFVVKGDDVRQI APG

SEQ ID No	Amino acid position with respect to SEQ ID NO:1	The amino-acid residue that the aminoacid sequence black matrix is represented (amino-acid residue 1-16) representation signal sequence
			39	1-600	MFIFLLFLTLTSGSDLDRCTTFDDVQAP NYTQHTSSMRGVYYPDEIFRSDTLYLTQ DLFLPFYSNVTGFHTINHTFGNPVIPFKD GIYFAATEKSNVVRGWVFGSTMNNKSQ SVIIINNSTNVVIRACNFELCDNPFFAVSK PMGTQTHTMIFDNAFNCTFEYISDAFSLD VSEKSGNFKHLREFVFKNKDGFLYVYK GYQPIDVVRDLPSGFNTLKPIFKLPLGINI TNFRAILTAFSPAQDIWGTSAAAYFVGY LKPTTFMLKYDENGTITDAVDCSQNPLA ELKCSVKSFEIDKGIYQTSNFRVVPSGDV VRFPNITNLCPFGEVFNATKFPSVYAWE RKKISNCVADYSVLYNSTFFSTFKCYGV SATKLNDLCFSNVYADSFVVKGDDVRQI APGQTGVIADYNYKLPDDFMGCVLAWN TRNIDATSTGNYNYKYRYLRHGKLRPFE RDISNVPFSPDGKPCTPPALNCYWPLND YGFYTTTGIGYQPYRVVVLSFELLNAPA TVCGPKLSTDLIKNQCVNFNFNGLTGTG VLTPSSKRFQPFQQFGRDVSDFTDSVRDP KTSEILDISPCAFGGVSVITPGTNASSEVA VLYQD

SEQ ID No	Amino acid position with respect to SEQ ID NO:1	The amino-acid residue that the aminoacid sequence black matrix is represented (amino-acid residue 1-16) representation signal sequence
			40	1-800	MFIFLLFLTLTSGSDLDRCTTFDDVQAP NYTQHTSSMRGVYYPDEIFRSDTLYLTQ DLFLPFYSNVTGFHTINHTFGNPVIPFKD GIYFAATEKSNVVRGWVFGSTMNNKSQ SVIIINNSTNVVIRACNFELCDNPFFAVSK PMGTQTHTMIFDNAFNCTFEYISDAFSLD VSEKSGNFKHLREFVFKNKDGFLYVYK GYQPIDVVRDLPSGFNTLKPIFKLPLGINI TNFRAILTAFSPAQDIWGTSAAAYFVGY LKPTTFMLKYDENGTITDAVDCSQNPLA ELKCSVKSFEIDKGIYQTSNFRVVPSGDV VRFPNITNLCPFGEVFNATKFPSVYAWE RKKISNCVADYSVLYNSTFFSTFKCYGV SATKLNDLCFSNVYADSFVVKGDDVRQI APGQTGVIADYNYKLPDDFMGCVLAWN TRNIDATSTGNYNYKYRYLRHGKLRPFE RDISNVPFSPDGKPCTPPALNCYWPLND YGFYTTTGIGYQPYRVVVLSFELLNAPA TVCGPKLSTDLIKNQCVNFNFNGLTGTG VLTPSSKRFQPFQQFGRDVSDFTDSVRDP KTSEILDISPCAFGGVSVITPGTNASSEVA VLYQDVNCTDVSTAIHADQLTPAWRIYS TGNNVFQTQAGCLIGAEHVDTSYECDIPI GAGICASYHTVSLLRSTSQKSIVAYTMSL GADSSIAYSNNTIAIPTNFSISITTEVMPVS MAKTSVDCNMYICGDSTECANLLLQYG SFCTQLNRALSGIAAEQDRNTREVFAQV KQMYKTPTLKYFGGFNFSQILPDPLKPT KRSFI

SEQ ID No	Amino acid position with respect to SEQ ID NO:1	The amino-acid residue that the aminoacid sequence black matrix is represented (amino-acid residue 1-16) representation signal sequence
			41	1-1000	MFIFLLFLTLTSGSDLDRCTTFDDVQAP NYTQHTSSMRGVYYPDEIFRSDTLYLTQ DLFLPFYSNVTGFHTINHTFGNPVIPFKD GIYFAATEKSNVVRGWVFGSTMNNKSQ SVIIINNSTNVVIRACNFELCDNPFFAVSK PMGTQTHTMIFDNAFNCTFEYISDAFSLD VSEKSGNFKHLREFVFKNKDGFLYVYK GYQPIDVVRDLPSGFNTLKPIFKLPLGINI TNFRAILTAFSPAQDIWGTSAAAYFVGY LKPTTFMLKYDENGTITDAVDCSQNPLA ELKCSVKSFEIDKGIYQTSNFRVVPSGDV VRFPNITNLCPFGEVFNATKFPSVYAWE RKKISNCVADYSVLYNSTFFSTFKCYGV SATKLNDLCFSNVYADSFVVKGDDVRQI APGQTGVIADYNYKLPDDFMGCVLAWN TRNIDATSTGNYNYKYRYLRHGKLRPFE RDISNVPFSPDGKPCTPPALNCYWPLND YGFYTTTGIGYQPYRVVVLSFELLNAPA TVCGPKLSTDLIKNQCVNFNFNGLTGTG VLTPSSKRFQPFQQFGRDVSDFTDSVRDP KTSEILDISPCAFGGVSVITPGTNASSEVA VLYQDVNCTDVSTAIHADQLTPAWRIYS TGNNVFQTQAGCLIGAEHVDTSYECDIPI GAGICASYHTVSLLRSTSQKSIVAYTMSL GADSSIAYSNNTLAIPTNFSISITTEVMPVS MAKTSVDCNMYICGDSTECANLLLQYG SFCTQLNRALSGIAAEQDRNTREVFAQV KQMYKTPTLKYFGGFNFSQILPDPLKPT KRSFIEDLLFNKVTLADAGFMKQYGECL GDINARDLICAQKFNGLTVLPPLLTDDMI AAYTAALVSGTATAGWTFGAGAALQIP FAMQMAYRFNGIGVTQNVLYENQKQIA NQFNKAISQIQESLTTTSTALGKLQDVVN QNAQALNTLVKQLSSNFGAISSVLNDILS RLDKVEAEVQIDRLITGRLQSLQTYVTQ QLIRAAEI

SEQ ID No	Amino acid position with respect to SEQ ID NQ:1	The amino-acid residue that the aminoacid sequence black matrix is represented (amino-acid residue 1-16) representation signal sequence
			42	1-1189	MFIFLLFLTLTSGSDLDRCTTFDDVQAP NYTQHTSSMRGVYYPDEIFRSDTLYLTQ DLFLPFYSNVTGFHTINHTFGNPVIPFKD GIYFAATEKSNVVRGWVFGSTMNNKSQ SVIIINNSTNVVIRACNFELCDNPFFAVSK PMGTQTHTMIFDNAFNCTFEYISDAFSLD VSEKSGNFKHLREFVFKNKDGFLYVYK GYQPIDVVRDLPSGFNTLKPIFKLPLGINI TNFRAILTAFSPAQDIWGTSAAAYFVGY LKPTTFMLKYDENGTITDAVDCSQNPLA ELKCSVKSFEIDKGIYQTSNFRVVPSGDV VRFPNITNLCPFGEVFNATKFPSVYAWE RKKISNCVADYSVLYNSTFFSTFKCYGV SATKLNDLCFSNVYADSFVVKGDDVRQI APGQTGVIADYNYKLPDDFMGCVLAWN TRNIDATSTGNYNYKYRYLRHGKLRPFE RDISNVPFSPDGKPCTPPALNCYWPLND YGFYTTTGIGYQPYRVVVLSFELLNAPA TVCGPKLSTDLIKNQCVNFNFNGLTGTG VLTPSSKRFQPFQQFGRDVSDFTDSVRDP KTSEILDISPCAFGGVSVITPGTNASSEVA VLYQDVNCTDVSTAIHADQLTPAWRIYS TGNNVFQTQAGCLIGAEHVDTSYECDIPI GAGICASYHTVSLLRSTSQKSIVAYTMSL GADSSIAYSNNTIAIPTNFSISITTEVMPVS MAKTSVDCNMYICGDSTECANLLLQYG SFCTQLNRALSGIAAEQDRNTREVFAQV KQMYKTPTLKYFGGFNFSQILPDPLKPT KRSFIEDLLFNKVTLADAGFMKQYGECL GDINARDLICAQKFNGLTVLPPLLTDDMI AAYTAALVSGTATAGWTFGAGAALQIP FAMQMAYRFNGIGVTQNVLYENQKQIA NQFNKAISQIQESLTTTSTALGKLQDVVN QNAQALNTLVKQLSSNFGAISSVLNDILS RLDKVEAEVQIDRLITGRLQSLQTYVTQ QLIRAAEIRASANLAATKMSECVLGQSK RVDFCGKGYHLMSFPQAAPHGVVFLHV TYVPSQERNFTTAPAICHEGKAYFPREG VFVFNGTSWFITQRNFFSPQIITTDNTFVS GNCDVVIGIINNTVYDPLQPELDSFKEEL DKYFKNHTSPDVDLGDISGINASVVNIQ KEIDRLNEVAKNLNESLIDLQELGKYEQ
43	17-100	DRCTTFDDVQAPNYTQHTSSMRGVYYP DEIFRSDTLYLTQDLFLPFYSNVTGFHTI NHTFGNPVIPFKDGIYFAATEKSNVVRG

SEQ ID No	Amino acid position with respect to SEQ ID NO:1	The amino-acid residue that the aminoacid sequence black matrix is represented (amino-acid residue 1-16) representation signal sequence
			44	17-200	DRCTTFDDVQAPNYTQHTSSMRGVYYP DEIFRSDTLYLTQDLFLPFYSNVTGFHTI NHTFGNPVIPFKDGIYFAATEKSNVVRG WVFGSTMNNKSQSVIIINNSTNVVIRACN FELCDNPFFAVSKPMGTQTHTMIFDNAF NCTFEYISDAFSLDVSEKSGNFKHLREFV FKNKDGFLYVYKGY
45	17-400	DRCTTFDDVQAPNYTQHTSSMRGVYYP DEIFRSDTLYLTQDLFLPFYSNVTGFHTI NHTFGNPVIPFKDGIYFAATEKSNVVRG WVFGSTMNNKSQSVIIINNSTNVVIRACN FELCDNPFFAVSKPMGTQTHTMIFDNAF NCTFEYISDAFSLDVSEKSGNFKHLREFV FKNKDGFLYVYKGYQPIDVVRDLPSGFN TLKPIFKLPLGINITNFRAILTAFSPAQDIW GTSAAAYFVGYLKPTTFMLKYDENGTIT DAVDCSQNPLAELKCSVKSFEIDKGIYQ TSNFRVVPSGDVVRFPNITNLCPFGEVFN ATKFPSVYAWERKKISNCVADYSVLYNS TFFSTFKCYGVSATKLNDLCFSNVYADS FVVKGDDVRQIAPG
			46	17-600	DRCTTFDDVQAPNYTQHTSSMRGVYYP DEIFRSDTLYLTQDLFLPFYSNVTGFHTI NHTFGNPVIPFKDGIYFAATEKSNVVRG WVFGSTMNNKSQSVIIINNSTNVVIRACN FELCDNPFFAVSKPMGTQTHTMIFDNAF NCTFEYISDAFSLDVSEKSGNFKHLREFV FKNKDGFLYVYKGYQPIDVVRDLPSGFN TLKPIFKLPLGINITNFRAILTAFSPAQDIW GTSAAAYFVGYLKPTTFMLKYDENGTIT DAVDCSQNPLAELKCSVKSFEIDKGIYQ TSNFRVVPSGDVVRFPNITNLCPFGEVFN ATKFPSVYAWERKKISNCVADYSVLYNS TFFSTFKCYGVSATKLNDLCFSNVYADS FVVKGDDVRQIAPGQTGVIADYNYKLPD DFMGCVLAWNTRNIDATSTGNYNYKYR YLRHGKLRPFERDISNVPFSPDGKPCTPP ALNCYWPLNDYGFYTTTGIGYQPYRVV VLSFELLNAPATVCGPKLSTDLIKNQCV NFNFNGLTGTGVLTPSSKRFQPFQQFGR DVSDFTDSVRDPKTSEILDISPCAFGGVS VITPGTNASSEVAVLYQD

SEQ ID No	Amino acid position with respect to SEQ ID NQ:1	The amino-acid residue that the aminoacid sequence black matrix is represented (amino-acid residue 1-16) representation signal sequence
			47	17-800	DRCTTFDDVQAPNYTQHTSSMRGVYYP DEIFRSDTLYLTQDLFLPFYSNVTGFHTI NHTFGNPVIPFKDGIYFAATEKSNVVRG WVFGSTMNNKSQSVIIINNSTNVVIRACN FELCDNPFFAVSKPMGTQTHTMIFDNAF NCTFEYISDAFSLDVSEKSGNFKHLREFV FKNKDGFLYVYKGYQPIDVVRDLPSGFN TLKPIFKLPLGINTTNFRAILTAFSPAQDIW GTSAAAYFVGYLKPTTFMLKYDENGTIT DAVDCSQNPLAELKCSVKSFEIDKGIYQ TSNFRVVPSGDVVRFPNITNLCPFGEVFN ATKFPSVYAWERKKISNCVADYSVLYNS TFFSTFKCYGVSATKLNDLCFSNVYADS FVVKGDDVRQIAPGQTGVIADYNYKLPD DFMGCVLAWNTRNIDATSTGNYNYKYR YLRHGKLRPFERDISNVPFSPDGKPCTPP ALNCYWPLNDYGFYTTTGIGYQPYRVV VLSFELLNAPATVCGPKLSTDLIKNQCV NFNFNGLTGTGVLTPSSKRFQPFQQFGR DVSDFTDSVRDPKTSEILDISPCAFGGVS VITPGTNASSEVAVLYQDVNCTDVSTAI HADQLTPAWRIYSTGNNVFQTQAGCLIG AEHVDTSYECDIPIGAGICASYHTVSLLR STSQKSIVAYTMSLGADSSIAYSNNTIAIP TNFSISITTEVMPVSMAKTSVDCNMYICG DSTECANLLLQYGSFCTQLNRALSGIAA EQDRNTREVEAQVKQMYKTPTLKYFGG FNFSQILPDPLKPTKRSFI

SEQ ID No	Amino acid position with respect to SEQ ID NO:1	The amino-acid residue that the aminoacid sequence black matrix is represented (amino-acid residue 1-16) representation signal sequence
			48	17-1000	DRCTTFDDVQAPNYTQHTSSMRGVYYP DEIFRSDTLYLTQDLFLPFYSNVTGFHTI NHTFGNPVIPFKDGIYFAATEKSNVVRG WVFGSTMNNKSQSVIIINNSTNVVIRACN FELCDNPFFAVSKPMGTQTHTMIFDNAF NCTFEYISDAFSLDVSEKSGNFKHLREFV FKNKDGFLYVYKGYQPIDVVRDLPSGFN TLKPIFKLPLGINITNFRAILTAFSPAQDIW GTSAAAYFVGYLKPTTFMLKYDENGTIT DAVDCSQNPLAELKCSVKSFEIDKGIYQ TSNFRVVPSGDVVRFPNITNLCPFGEVFN ATKFPSVYAWERKKISNCVADYSVLYNS TFFSTFKCYGVSATKLNDLCFSNVYADS FVVKGDDVRQIAPGQTGVIADYNYKLPD DFMGCVLAWNTRNIDATSTGNYNYKYR YLRHGKLRPFERDISNVPFSPDGKPCTPP ALNCYWPLNDYGFYTTTGIGYQPYRVV VLSFELLNAPATVCGPKLSTDLIKNQCV NFNFNGLTGTGVLTPSSKRFQPFQQFGR DVSDFTDSVRDPKTSEILDISPCAFGGVS VITPGTNASSEVAVLYQDVNCTDVSTAI HADQLTPAWRIYSTGNNVFQTQAGCLIG AEHVDTSYECDIPIGAGICASYHTVSLLR STSQKSIVAYTMSLGADSSIAYSNNTIAIP TNFSISITTEVMPVSMAKTSVDCNMYICG DSTECANLLLQYGSFCTQLNRALSGIAA EQDRNTREVFAQVKQMYKTPTLKYFGG FNFSQILPDPLKPTKRSFIEDLLFNKVTLA DAGFMKQYGECLGDINARDLICAQKFN GLTVLPPLLTDDMIAAYTAALVSGTATA GWTFGAGAALQIPFAMQMAYRFNGIGV TQNVLYENQKQIANQFNKAISQIQESLTT TSTALGKLQDVVNQNAQALNTLVKQLS SNFGAISSVLNDILSRLDKVEAEVQIDRLI TGRLQSLQTYVTQQLIRAAEI

SEQ ID No	Amino acid position with respect to SEQ ID NO:1	The amino-acid residue that the aminoacid sequence black matrix is represented (amino-acid residue 1-16) representation signal sequence
			49	17-1189	DRCTTFDDVQAPNYTQHTSSMRGVYYP DEIFRSDTLYLTQDLFLPFYSNVTGFHTI NHTFGNPVIPFKDGIYFAATEKSNVVRG WVFGSTMNNKSQSVIIINNSTNVVIRACN FELCDNPFFAVSKPMGTQTHTMIFDNAF NCTFEYISDAFSLDVSEKSGNFKHLREFV FKNKDGFLYVYKGYQPIDVVRDLPSGFN TLKPIFKLPLGINITNFRAILTAFSPAQDIW GTSAAAYFVGYLKPTTFMLKYDENGTIT DAVDCSQNPLAELKCSVKSFEIDKGIYQ TSNFRVVPSGDVVRFPNITNLCPFGEVFN ATKFPSVYAWERKKISNCVADYSVLYNS TFFSTFKCYGVSATKLNDLCFSNVYADS FVVKGDDVRQIAPGQTGVIADYNYKLPD DFMGCVLAWNTRNIDATSTGNYNYKYR YLRHGKLRPFERDISNVPFSPDGKPCTPP ALNCYWPLNDYGFYTTTGIGYQPYRVV VLSFELLNAPATTVCGPKLSTDLKNQCV NFNFNGLTGTGVLTPSSKRFQPFQQFGR DVSDFTDSVRDPKTSEILDISPCAFGGVS VITPGTNASSEVAVLYQDVNCTDVSTAI HADQLTPAWRIYSTGNNVFQTQAGCLIG AEHVDTSYECDIPIGAGICASYHTVSLLR STSQKSIVAYTMSLGADSSIAYSNNTIAIP TNFSISITTEVMPVSMAKTSVDCNMYICG DSTECANLLLQYGSFCTQLNRALSGIAA EQDRNTREVFAQVKQMYKTPTLKYFGG FNFSQILPDPLKPTKRSFIEDLLFNKVTLA DAGFMKQYGECLGDINARDLICAQKFN GLTVLPPLLTDDMIAAYTAALVSGTATA GWTFGAGAALQIPFAMQMAYRFNGIGV TQNVLYENQKQIANQFNKAISQIQESLTT TSTALGKLQDVVNQNAQALNTLVKQLS SNFGAISSVLNDILSRLDKVEAEVQIDRLI TGRLQSLQTYVTQQLIRRAAEIRASANLA ATKMSECVLGQSKRVDFCGKGYHLMSF PQAAPHGVVFLHVTYVPSQERNFTTAPA ICHEGKAYFPREGVFVFNGTSWFITQRNF FSPQIITTDNTFVSGNCDVVIGIINNTVYD PLQPELDSFKEELDKYFKNHTSPDVDLG DISGINASVVNIQKEIDRLNEVAKNLNES LIDLQELGKYEQ

SEQ ID No	Amino acid position with respect to SEQ ID NQ:1	The amino-acid residue that the aminoacid sequence black matrix is represented (amino-acid residue 1-16) representation signal sequence
			50	17-276	DRCTTFDDVQAPNYTQHTSSMRGVYYP DEIFRSDTLYLTQDLFLPFYSNVTGFHTI NHTFGNPVIPFKDGIYFAATEKSNVVRG WVFGSTMNNKSQSVIIINNSTNVVIRACN FELCDNPFFAVSKPMGTQTHTMIFDNAF NCTFEYISDAFSLDVSEKSGNFKHLREFV FKNKDGFLYVYKGYQPIDVVRDLPSGFN TLKPIFKLPLGINITNFRAILTAFSPAQDIW GTSAAAYFVGYLKPTTFMLKYDENGTIT DAV
51	17-446	DRCTTFDDVQAPNYTQHTSSMRGVYYP DEIFRSDTLYLTQDLFLPFYSNVTGFHTI NHTFGNPVIPFKDGIYFAATEKSNVVRG WVFGSTMNNKSQSVIIINNSTNVVIRACN FELCDNPFFAVSKPMGTQTHTMIFDNAF NCTFEYISDAFSLDVSEKSGNFKHLREFV FKNKDGFLYVYKGYQPIDVVRDLPSGFN TLKPIFKLPLGINITNFRAILTAFSPAQDIW GTSAAAYFVGYLKPTTFMLKYDENGTIT DAVDCSQNPLAELKCSVKSFEIDKGIYQ TSNFRVVPSGDVVRFPNITNLCPFGEVFN ATKFPSVYAWERKKISNCVADYSVLYNS TFFSTFKCYGVSATKLNDLCFSNVYADS FVVKGDDVRQIAPGQTGVIADYNYKLPD DFMGCVLAWNTRNIDATSTGNYNYKYR YLRHG

SEQ ID No	Amino acid position with respect to SEQ ID NO:1	The amino-acid residue that the aminoacid sequence black matrix is represented (amino-acid residue 1-16) representation signal sequence
			52	17-537	DRCTTFDDVQAPNYTQHTSSMRGVYYP DEIFRSDTLYLTQDLFLPFYSNVTGFHTI NHTFGNPVIPFKDGIYFAATEKSNVVRG WVFGSTMNNKSQSVIIINNSTNVVIRACN FELCDNPFFAVSKPMGTQTHTMIFDNAF NCTFEYISDAFSLDVSEKSGNFKHLREFV FKNKDGFLYVYKGYQPIDVVRDLPSGFN TLKPIFKLPLGINITNFRAILTAFSPAQDIW GTSAAAYFVGYLKPTTFMLKYDENGTIT DAVDCSQNPLAELKCSVKSFEIDKGIYQ TSNFRVVPSGDVVRFPNITNLCPFGEVFN ATKFPSVYAWERKKISNCVADYSVLYNS TFFSTFKCYGVSATKLNDLCFSNVYADS FVVKGDDVRQIAPGQTGVLADYNYKLPD DFMGCVLAWNTRNIDATSTGNYNYKYR YLRHGKLRPFERDISNVPFSPDGKPCTPP ALNCYWPLNDYGFYTTTGIGYQPYRVV VLSFELLNAPATVCGPKLSTDLJKNQCV NFNFNGLTGTGV

EQ ID No	Amino acid position with respect to SEQ ID NO:1	The amino-acid residue that the aminoacid sequence black matrix is represented (amino-acid residue 1-16) representation signal sequence
			53	17-757 adds N end mouse K chain targeting sequencing and C end myc epitope and polyhistidine tag thing	METDTLLLWVLLLWVPGSTGDDRCTTF DDVQAPNYTQHTSSMRGVYYPDEIFRSD TLYLTQDLFLPFYSNVTGFHTINHTFGNP VIPFKDGIYFAATEKSNVVRGWVFGSTM NNKSQSVIIINNSTNVVIRACNFELCDNPF FAVSKPMGTQTHTMIFDNAFNCTFEYIS DAFSLDVSEKSGNFKHLREFVFKNKDGF LYVYKGYQPIDVVRDLPSGFNTLKPIFKL PLGINITNFRAILTAFSPAQDIWGTSAAA YFVGYLKPTTFMLKYDENGTITDAVDCS QNPLAELKCSVKSFEIDKGIYQTSNFRVV PSGDVVRFPNITNLCPFGEVFNATKFPSV YAWERKKISNCVADYSVLYNSTFFSTFK CYGVSATKLNDLCFSNVYADSFVVKGD DVRQIAPGQTGVIADYNYKLPDDFMGC VLAWNTRNIDATSTGNYNYKYRYLRHG KLRPFERDISNVPFSPDGKPCTPPALNCY WPLNDYGFYTTTGIGYQPYRVVVLSFEL LNAPATVCGPKLSTDLIKNQCVNFNFNG LTGTGVLTPSSKRFQPFQQFGRDVSDFT DSVRDPKTSEILDISPCAFGGVSVITPGTN ASSEVAVLYQDVNCTDVSTAIHADQLTP AWRIYSTGNNVFQTQAGCLIGAEHVDTS YECDIPIGAGICASYHTVSLLRSTSQKSIV AYTMSLGADSSIAYSNNTIAIPTNFSISITT EVMPVSMAKTSVDCNMYICGDSTECAN LLLQYGSFCTQLNRALSGIAAEQEQKLS EEDLHHHHHH
54	17-276 adds N end mouse K chain targeting sequencing and C end myc epitope and polyhistidine tag thing	METDTLLLWVLLLWVPGSTGDDRCTTF DDVQAPNYTQHTSSMRGVYYPDEIFRSD TLYLTQDLFLPFYSNVTGFHTINHTFGNP VIPFKDGIYFAATEKSNVVRGWVFGSTM NNKSQSVIIINNSTNVVIRACNFELCDNPF FAVSKPMGTQTHTMIFDNAFNCTFEYIS DAFSLDVSEKSGNFKHLREFVFKNKDGF LYVYKGYQPIDVVRDLPSGFNTLKPIFKL PLGINITNFRAILTAFSPAQDIWGTSAAA YFVGYLKPTTFMLKYDENGTITDAVEQK LISEEDLHHHHHH

SEQ ID No	Amino acid position with respect to SEQ ID NO:1	The amino-acid residue that the aminoacid sequence black matrix is represented (amino-acid residue 1-16) representation signal sequence
			55	17-537 adds N end mouse K chain targeting sequencing and C end myc epitope and polyhistidine tag thing	METDTLLLWVLLLWVPGSTGDDRCTTF DDVQAPNYTQHTSSMRGVYYPDEIFRSD TLYLTQDLFLPFYSNVTGFHTINHTFGNP VIPFKDGIYFAATEKSNVVRGWVFGSTM NNKSQSVIIINNSTNVVIRACNFELCDNPF FAVSKPMGTQTHTMIFDNAFNCTFEYIS DAFSLDVSEKSGNFKHLREFVFKNKDGF LYVYKGYQPIDVVRDLPSGFNTLKPIFKL PLGINITNFRAILTAFSPAQDIWGTSAAA YFVGYLKPTTFMLKYDENGTITDAVDCS QNPLAELKCSVKSFEIDKGIYQTSNFRVV PSGDVVRFPNITNLCPFGEVFNATKFPSV YAWERKKISNCVADYSVLYNSTFFSTFK CYGVSATKLNDLCFSNVYADSFVVKGD DVRQIAPGQTGVIADYNYKLPDDFMGC VLAWNTRNIDATSTGNYNYKYRYLRHG KLRPFERDISNVPFSPDGKPCTPPALNCY WPLNDYGFYTTTGIGYQPYRVVVLSFEL LNAPATVCGPKLSTDLIKNQCVNFNFNG LTGTGVEQKLISEEDLHHHHHH

EQ ID No	Amino acid position with respect to SEQ ID NO:1	The amino-acid residue that the aminoacid sequence black matrix is represented (amino-acid residue 1-16) representation signal sequence
			56	17-756 N end does not have signal peptide	DRCTTFDDVQAPNYTQHTSSMRGVYYP DEIFRSDTLYLTQDLFLPFYSNVTGFHTI NHTFGNPVIPFKDGIYFAATEKSNVVRG WVFGSTMNNKSQSVIIINNSTNVVIRACN FELCDNPFFAVSKPMGTQTHTMIFDNAF NCTFEYISDAFSLDVSEKSGNFKHLREFV FKNKDGFLYVYKGYQPIDVVRDLPSGFN TLKPIFKLPLGINITNFRAILTAFSPAQDIW GTSAAAYFVGYLKPTTFMLKYDENGTIT DAVDCSQNPLAELKCSVKSFEIDKGIYQ TSNFRVVPSGDVVRFPNITNLCPFGEVFN ATKFPSVYAWERKKISNCVADYSVLYNS TFFSTFKCYGVSATKLNDLCFSNVYADS FVVKGDDVRQIAPGQTGVIADYNYKLPD DFMGCVLAWNTRNIDATSTGNYNYKYR YLRHGKLRPFERDISNVPFSPDGKPCTPP ALNCYWPLNDYGFYTTTGIGYQPYRVV VLSFELLNAPATVCGPKLSTDLIKNQCV NFNFNGLTGTGVLTPSSKRFQPFQQFGR DVSDFTDSVRDPKTSEILDISPCAFGGVS VITPGTNASSEVAVLYQDVNCTDVSTAI HADQLTPAWRIYSTGNNVFQTQAGCLIG AEHVDTSYECDIPIGAGICASYHTVSLLR STSQKSIVAYTMSLGADSSIAYSNNTIAIP TNFSISITTEVMPVSMAKTSVDCNMYICG DSTECANLLLQYGSFCTQLNRALSGIAA E
57	272-537	ITDAVDCSQNPLAELKCSVKSFEIDKGIY QTSNFRVVPSGDVVRFNITNLCPFGEVFN ATKFPSVYAWERKKISNCVADYSVLYNS TFFSTFKCYGVSATKLNDLCFSNVYADS FVVKGDDVRQIAPGQTGVIADYNYKLPD DFMGCVLAWNTRNIDATSTGNYNYKYR YLRHGKLRPFERDISNVPFSPDGKPCTPP ALNCYWPLNDYGFYTTTGIGYQPYRVV VLSFELLNAPATVCGPKLSTDLIKNQCV NFNFNGLTGTGV
			58	24-39 D24 peptide	DVQAPNYTQHTSSMRGC
59	540-555 P540 peptide	PSSKRFQPFQQFGRDC

SEQ ID No	Amino acid position with respect to SEQ ID NO:1	The amino-acid residue that the aminoacid sequence black matrix is represented (amino-acid residue 1-16) representation signal sequence
			60	1-16 furcella signal sequence	MFIFLLFLTLTSGSDL
61	303-537 contains receptor binding domains	SNFRVVPSGDVVRFPNITNLCPFGEVFNA TKFPSVYAWERKKISNCVADYSVLYNST FFSTFKCYGVSATKLNDLCFSNVYADSF VVKGDDVRQIAPGQTGVIADYNYKLPD DFMGCVLAWNTRNIDATSTGNYNYKYR YLRHGKLRPFERDISNVPFSPDGKPCTPP ALNCYWPLNDYGFYTTTGIGYQPYRVV VLSFELLNAPATVCGPKLSTDLIKNQCV NFNFNGLTGTGV
			62	319-517 contains receptor binding domains	ITNLCPFGEVFNATKFPSVYAWERKKISN CVADYSVLYNSTFFSTFKCYGVSATKLN DLCFSNVYADSFVVKGDDVRQIAPGQT GVIADYNYKLPDDFMGCVLAWNTRNID ATSTGNYNYKYRYLRHGKLRPFERDISN VPFSPDGKPCTPPALNCYWPLNDYGFYT TTGIGYQPYRVVVLSFELLNAPATVCGP KLST
63	319-518 contains receptor binding domains	ITNLCPFGEVFNATKFPSVYAWERKKISN CVADYSVLYNSTFFSTFKCYGVSATKLN DLCFSNVYADSFVVKGDDVRQIAPGQT GVIADYNYKLPDDFMGCVLAWNTRNID ATSTGNYNYKYRYLRHGKLRPFERDISN VPFSPDGKPCTPPALNCYWPLNDYGFYT TTGIGYQPYRVVVLSFELLNAPATVCGP KLSTD

Embodiment 6

The structure of spike protein

In order to understand fully proteic characteristic of SARS-CoVS and function, the nucleic acid of coding total length Tor2 strain isolated is cloned expression vector into described above, the The genome sequence of the SARS-associated coronavirus that this Tor2 strain isolated is delivered at Marra etc., Scienee 300:1399-1404 has more descriptions on (2003).The clone who obtains comprises and contains total length S albumen (1255 residues), removed the amino terminal extracellular outskirt Se (residue 17-1189) that only contains the proteic extracellular region of S that strides tail in film district and the kytoplasm of the spike protein of generally acknowledging, contain aminoterminal 276 (SEQ ID NO:50), 537 (SEQ ID NO:52) and 756 (SEQ ID NO:56) amino acids residue (are respectively S276, S537, and S756) and have the fragment of the signal sequence of 16 residues or mouse k chain leader sequence and contain clone's (seeing Figure 1B) of the interior segments (S272-537) of 272-537 position residue (SEQ IDNO:57).

Amino-acid residue 758-761 (RNTR) forms the motif of following precursor conversion enzyme cutting:

K/R-Xn-K/R

Wherein X is any amino-acid residue, n=0,2,4 or 6.

S1 subunit almost is included in the S756 fragment, and size conforms to mouse coronavirus S1 subunit size, and as the JHM strain, its S1 is 769 residues, and human corona virus OC43 is 778 residues, sees Gallagher ﹠amp; .Buchmeier, Coronavirns spike proteins inviral entry andpathogenesis, Virology 279:371-374 (2001); Kunkel ﹠amp; Herrler, Structural and functional analysis of the surface protein of humancoronavirus OC43, Virology 195417:195-202 (1993).But, the S1 of human corona virus 229E is considered to be made up of short fragment that contains 547 residues, corresponding with S537, see Bonavia etc., Identification of a receptor-binding domain of thespike glycoprotein of human coronavirus HCoV-229E, J.Virol.77:2530-2538 (2003).

The molecular weight that its position was all estimated apparently higher than their when all S glycoprotein fragments and total length S glycoprotein were used the SDS-PAGE gel electrophoresis points out these polypeptide to carry out posttranslational modification probably.The apparent molecular weight of S276 polypeptide is about 75kDa, and the apparent molecular weight of S537 is about 100-110kDa, and the apparent molecular weight of S756 is about 130-140kDa, and the apparent molecular weight of Se and S is about 200kDa or higher (Fig. 4 and 6).The band of these polypeptide correspondences also broad (Fig. 6 under the situation of low exposure; Some data are not shown).These Notes of Key Datas have tangible glycosylation to take place, as observed on S glycoprotein and its fragment.As if based on the roughly estimation to molecular weight, S2 subunit is not as the glycosylated degree height of S756 (constituting S1 subunit).It should be noted that the S276 degree of glycosylation is very high if glycosylation is the factor of unique increase molecular wt.

The S glycoprotein of the most of SARS-CoV that obtain from cells and supernatant is not cut, though the weak band that small protein forms can be seen on SDS-PAGE glue.One of them weak band and S756 point out non-effective cutting (Fig. 4 and 6) at same position.The cutting at random of proteolytic enzyme may take place, and requires further study to determine whether the cutting of S glycoprotein is essential by its function.

Embodiment 7

The expression of peptide fragment in the intestinal bacteria

The nucleic acid fragment of the 17-446 amino acids residue that contains SEQ ID NO:1 of coding SEQ ID NO:51 peptide fragment is cloned into pRSET carrier (Invitrogen, San Diego, CA) be built into plasmid pRSET-S (17-446), with pRSET-S (17-446) transfection Escherichia coli BL21DE3, induce with IPTG, Fig. 2 has shown the inductive result.

Embodiment 8

The expression of the peptide fragment of the present invention of being cloned with the T7 promoters driven

Transfection the day before yesterday, people's 293 cells and monkey Vero E6 cell grow into 1.2 * 10 in 5ml DMEM+10%FBS substratum ⁶Cell/T25 culturing bottle (60mm ware), the method that provides according to producer is with Polyfect (Qiagen) transfection reagent box then, the segmental pSecTag2B construct of insertion (each 6 μ g) that will contain the various spike protein peptide fragment of encoding is transfected into cell, and the preparation of these constructs as described above.

After the transfection 4 hours, amount with MOI (infective dose) 20 (is seen Fuerst etc. with a vaccinia virus transfectional cell that has the T7 polysaccharase, Proc.Natl.Acad.Sci., 93:11371 (1986)), this step is purposes to be provided rather than to be weak a lot of CMV promotor (Nussbaum etc. for the T7 promotor on the pSecTag2B carrier, J.Virol., 68:5411 (1994)).Infected back 3 hours, the 1.5ml fresh culture is added into cell, and cell forwarded in 31 ℃ of incubators cultivates.Cell continued to cultivate after 24 hours, collected substratum.

In with any S nucleic acid construct cells transfected, all do not observe (data are not shown) detectable pathological change, that points out total length may not have the significant cytotoxicity effect with solubility S glycoprotein fragment, yet, this effect is possible during high level expression, and plasmodial being formed with described below may cause necrocytosis.

Embodiment 9

The furcella specific antibody

The various polypeptide immune new zealand rabbits that its immunogenicity carried out screening by computer program with 0.1mg.The serum that obtains from new zealand rabbit detects its reactivity with ELISA and Western trace.Serum by the rabbit of two kinds of peptide immunity demonstrates very high and special anti-spike glycoprotein activity, and they are used to further research.The antibody of D24 by name and D540 is induced generation by peptide DVQAPNYTQH TSSMRGC (SEQ ID NO:58) and PSSKRFQPFQQFGRDC (SEQ ID NO:59) respectively.The polyclonal antibody IMG-542 of another anti-SARS-CoVS glycoprotein, the 288-303 amino acid of identification S glycoprotein, (San Diego CA) buys from IMGENEX.

Embodiment 10

The immunoprecipitation of spike protein and purifying

Solubility furcella polypeptide fragment obtains from the substratum of Vero E6 or 293cell, but the spike glycoprotein of total length is only found in cell lysate.

The substratum of cell of the segmental nucleic acid of various solubility S of encoding of having collected transfection, 1000 leave the heart removed the cell relic in 10 minutes, clarifying substratum and Ni-NTA sepharose 4B (Qiagen, Valencia, CA) or immunoprecipitating antibody sugaring Protein G-sepharose 4B (Sigma, St.Louis MO) was hatched 2 hours for 4 ℃.Pearl mixes with the SDS glue sample buffer of measuring together then, boils 3 minutes, and carries out electrophoretic analysis.For the S glycoprotein of total length, cell is at first with being added with the PBS of 1%NP-40 and 0.5mM PMSF 4 ℃ of cracking 1 hour.Then 14000 rev/mins on desk-top Eppendorf whizzer centrifugal 20 minutes.Clarifying lysate at first is used for immunoprecipitation or is directly used in the Western trace.

Embodiment 11

Western trace and slot blot

The cell of expressing S glycoprotein is as mentioned above at first used the NP40 lysis buffer cracking based on PBS, and relic is clarified with centrifugal method.For solubility S fragment, collect substratum and clarify with aforesaid method.To slot blot, (method that CA) provides is used directly in trace on the fine little plain film of nitric acid to clarifying lysate for Bio-Rad, Hercules, and the method as conventional Western trace detects film with antibody then according to producer with the substratum of taking from supernatant.For the Western trace, monoclonal antibody (the Invitrogen of anti-c-Myc epi-position, Carlsbad, C (A) or be obtained from the many antibody that can swell of anti-spike protein rabbit with the rabbit of furcella peptide immunity with the dilution of TBST damping fluids, antibody and film were hatched 2 hours, wash film, film was hatched 1 hour with the second antibody that is combined with HRP more then, washed film 4 times (each 15 minutes), used ECL reagent (Pierce at last, Rockford, IL) colour developing.

Embodiment 12

Cell is in conjunction with test and ELISA

Collection contains the segmental substratum of solubility S, centrifugally makes its clarification.Vero E6 cell or other cells (5 * 10 ⁶) contain the antibody that the segmental clarifying substratum of solubility S and 2 μ g are conjugated with the anti-c-Myc epi-position of HRP with 0.5ml and hatched 2 hours for 4 ℃.Wash cell three times and centrifugal collecting cell with ice-cold PBS then.The cell precipitation thing is with (Indianapolis was hatched 10 minutes under ABTS substrate room temperature IN), and the centrifugal clarification substrate is also measured its optical density value at the 405nm wavelength available from Roche.

For ELISA, the ACE2 (R﹠amp of purifying; D, Minneapolis, MN is adsorbed onto on the Maxisorp elisa plate with the concentration in the 100ng/ hole damping fluid with pH9.6.Containing anti-c-155Myc epitope antibodies that segmental substratum 154 of various solubility S (150 μ l) and 0.6 μ g be combined with HRP adds in each hole 37 ℃ and hatched 2 hours.Hole flushing also adds 60 μ lABTS substrates in each hole, measure its optical density value (OD after 20 minutes ₄₀₅).

Embodiment 13

Distribute again cytogamy test of fluorescence dye

Transfection the Hela or 293 cells of plasmid of coding S glycoprotein, with Calcein AM (molecular probe) load, Calcein AM can be transformed into Calcein in cell green, cell and the substratum that contains 1 μ g/mlCalcein AM are after hatching 1 hour under the condition of 37 ℃ and 5%CO2, through being resuspended in the fresh culture after the washing.The inoculation target cell is in plate, Vero E6 contaminates with CMAC, the substratum of Vero E6 and the CMAC (molecular probe) that contains 1 μ g/ml is after hatching 1 hour under the condition of 37 ℃ and 5%CO2, with twice of substratum washed cell, after in fresh culture, hatching 20 minutes, washed cell adds the 0.5ml substratum in every then hole once more.Load has the S express cell of Calcein to be added in the target cell, at 37 ℃ and 5%CO ₂Condition under hatched 1,2 or 4 hour.Weigh cytogamy with the double staining cell with the ratio between the target cell sum that contacts the S express cell.Use from MethaMorph 4.0 softwares of Universal Imaging and take pictures.

Embodiment 14

Cell-cytogamy test based on β galactase reporter gene

293T cell (1.5 * 10 ⁶) being inoculated in the T25 culturing bottle, next day, (CA) method that provides according to producer changes pCDNA3-S, pSectag2B-S, pCDNA3-ACE2 and pCDNA3-ACE2-Ecto in these cells over to respectively for Qiagen, Valencia with Polyfect transfection reagent box.After the transfection 4 hours, transfection the cell of S construct with the vaccinia virus transfection of expressing the T7 polysaccharase, transfection the cell of ACE-2 with vaccinia virus (VCB21R) transfection of coding β-gal.After the transfection 2 hours, cell hatched and changes over to 31 ℃ of overnight incubation with fresh culture.Second day, S P-glycoprotein expression cell and ACE2 express cell were hatched with 1: 1 mixed and at 37 ℃.After 3 hours, adding NP-40 is 0.5% lysing cell to final concentration, and cell pyrolysis liquid mixes with the CPRG substrate of equivalent, measures its OD after 1 hour ₅₉₅

Embodiment 15

Mammalian cell furcella polypeptide expression

To some test, remove all albumen of total length S glycoprotein and all use c-Myc epitope and histidine mark substance markers.With expressing these albumen behind corresponding plasmid transfection 293 and the Vero E6 cell, express the T7 polysaccharase with vaccinia virus afterwards and infect.

With anti-c-Myc monoclonal antibody (Fig. 4) certification mark albumen.As shown in Figure 4, the T7 promotor is height effectively start of expressing S glycoprotein.In these trials, the expression level that the T7 promotor produces is higher than the CMV promotor, and in most of the cases it is strong promoter (Fig. 4 A).As shown in Figure 4, the S fragment is a soluble fragments, and their concentration in the substratum supernatant and their size are inversely proportional to.

Embodiment 16

Anti-furcella antibody

In order to detect unlabelled albumen, confirm to have prepared rabbit polyclonal antibody by anti--possible antigen site of data and location that c-myc antibody obtains.In these antibody two, D24 and P540 are respectively the anti-antibody that originates in the peptide of the 24th and the 540th residue.D24 and P540 antibody preparation are discerned specific soluble fragments specifically, (Fig. 4 C).As expectation, D24 discerns all fragments; P540 identification S756, Se and S, but the littler fragment (Fig. 4 C, some data is not shown) of nonrecognition.The D24 antibody preparation relatively a little less than, even and the P540 preparation dilute 1: 10000 times also quite responsive, be widely used in the experiment as described herein.

P540 be used to detect S glycoprotein whether in cell, extracellular and cell surface expression.As shown in Figure 5, when measuring with flow cytometer, the S glycoprotein of total length has expression at cell surface, but expression level is lower.

Embodiment 17

The spike protein mediated cell merges

The S glycoprotein of total length mediates the cytogamy with the expressed receptor molecule when neutral pH, carry out cell-cytogamy and test that to prove conclusively total length S glycoprotein be functional, confirms whether the proteic fusion-activity of S needs other viral proteins and low pH.

All can cause the fusion of ACE2 express cell effectively with the total length S glycoprotein of pCDNA3-S and two kinds of vector expressions of pSectag2B-S, the plasmodial formation by all size and be susceptible of proof based on the test (Fig. 7) of β-gal reporter gene.What is interesting is, the leader sequence of S glycoprotein is induced synplasm to form comparatively fast by the pSectag2B-S construct that mouse k chain leader sequence replaces, and, the pCDNA3-S inductive of the S glycoprotein that the synplasm of formation is encoded natural big and more (data are not shown).By test detects based on reporter gene, the degree of the protein induced fusion of S that pSectag2B-S expresses is also than pCDNA3-S inductive height (Fig. 7 B).The natural S glycoprotein of these Notes of Key Datas may not be transferred to cell surface effectively.These study also prompting, and β as described herein-gal test can be used as differentiates that inhibition SARS-CoV enters the quick and quantitative methods of cell inhibiting agent, also can be used as research SARS-CoV simultaneously and enter the instrument of the mechanism of cell.

Note,, when only expressing the acceptor of natural concentration, form the fusion that test all fails to detect Vero E6 cell with β-gal test or synplasm when the transfection of cell ACE2 expression plasmid of no use.For understand fully this result whether since the susceptibility of these two tests too low due to, enabled another test again.This new test is based on the distribution test again of fluorescence dye, can detect individual cells and merge.Promptly use the test based on fluorescence dye, also not found transfection has tangible significant difference between the cell of coding total length S glycoprotein and the various negative control.Some negative controls have comprised transfection coding solubility S fragment (data are not shown) under low pH condition.Significantly cell-cytogamy has had only when cell transfecting the plasmid of coding ACE2 just can detect, and prompting may be crucial for cytogamy by the acceptor high level expression that reorganization ACE2 causes.Generally, the S glycoprotein of these results suggest reorganization can merge by mediated cell, and fusion can take place when neutral pH, and the efficient that merges depends on the concentration of acceptor molecule.

Further, solubility S glycoprotein fragment suppresses the S cell fusion mediated.As shown in Figure 15, add the S fragment S272-537 and the S17-537 that contain receptor binding domain described below, suppress the S cell fusion mediated.In this test, the tool restraint of S272-537 (SEQ IDNO:57) fragment.The S17-276 fragment that does not contain receptor binding domain has only very little or the effect of unrestraint S cell fusion mediated.The S polypeptide fragment that these Notes of Key Datas contain receptor binding domain may suppress the fusion of SARS-CoV and cell, and then suppresses or the prevention cell infection.

Like this, the activity with anti--RBD antibody, S polypeptide, S peptide or fit blocking-up, manipulation or inhibition spike protein receptor binding domain can effectively prevent and treat SARS-CoV and infect.

Embodiment 18

The discriminating of spike protein receptor binding domain

The receptor binding domain that present embodiment has been illustrated spike protein is positioned 272-537 position residue (SEQ ID NO:57) and may be positioned 303-537 position residue (SEQ ID NO:61).The experiment of back shows that the fragment (SEQ ID NO:62) that contains the 319-517 residue also has receptor-binding activity.

Experiment based on the Vero E6 cell that various soluble fragments is attached to expressed receptor is established, with the receptor binding domain (RBD) of location S glycoprotein.This test relates to the antibodies of measuring anti-S polypeptide and arrives the relevant fluorescence of Vero E6 cell, and carries out before the acceptor of differentiating SARS-CoV.The Vero E6 cell of SARS-CoV susceptible and the S polypeptide and the various solubility S fragment of total length are hatched, several to SARS-CoV not the cell of susceptible equally also hatch with the S polypeptide of total length and its soluble fragments.Shown in Fig. 8 A and 8B, all S fragments all combine with Vero E6 cell, except a S fragment (S276) of minimum.When several SARS-CoV is not detected such combining when the S polypeptide of clone and the total length of susceptible and its soluble fragments are not hatched.Be directly proportional with combining of Vero E6 cell, be inversely proportional to segmental size with the level of fragment expression.These find that prompting RBD is between the 272nd and 537 residue.

For further location RBD, the antibody (IMG542) that the peptide that usefulness contains the 288-303 residue produces is used.This antibody does not suppress combining of S537 and Vero E6 cell, though it can combine (Fig. 8 B, some data are not shown) really with the S537 fragment, RBD is between 303 and 537 in prompting.Because antibody is relatively large and the cause of steric hindrance, very possible RBD is positioned at the downstream of 303 residues.Recently, the RBD of HCoV-299E is positioned on the fragment that contains amino-acid residue 407-547, see .A novelcoronavirus associated with severe acute respiratory syndrome such as Ksiazek, N.Engl.J.Med.348:1953-1966 (2003); Characterization of a novelcoronavirus associated with severe acute respiratory syndrome such as Rota, Science300:1394-1399 (2003).By contrast, the RBD of murine hepatitis virus is positioned N and holds 330 amino acid.

Contain RBD SARS-CoV S1 glycoprotein fragment (as, S272-537) and whether exist between the RBD of HCoV-229E or hepatitis virus structural similar, and this similar whether be related to duplicate also with identical host need further research.These two kinds viruses are used different acceptors.Simple direct cell combining method described herein also may be helpful to the virus receptor of differentiating other.

Recently, the researchist reports as the evaluation of SARS-CoV functional receptor ACE2, see Angiotensin-converting enzyme 2is a functionalreceptorfor the SARS coronavirns such as Li, Nature 426:450-54 (2003).The result that ACE2 provides more than further having confirmed as the evaluation of acceptor is correct.Shown in Fig. 8 C, when the ACE2 of purifying be used to ELISA with measure in conjunction with the time, observe with cell in conjunction with the identical binding pattern of test, all tested S fragments are not always the case.

Here the result of Zhan Shiing not only provides new tool that the research SARS virus enters cell, has confirmed that ACE2 is the acceptor of SARS-CoV S1 glycoprotein and has located RBD, and provides convenience for the medicine of developing new vaccine immunogens and prevention and treatment SARS.

Embodiment 19

The N-terminal of S glycoprotein and C-terminal oligomerization

Present embodiment has been set forth the utmost point far-end N-terminal fragment in the RBD upstream of S glycoprotein may bring into play effect in fusion, and the tripolymer that the S extracellular region forms may pass through 6-helical bundle intermediate mediates fusion.

Material and method

Antibody and plasmid: the rabbit that is used for Western and facs analysis is anti--and S blood-serum P 540 prepares by method described above by the inventor, also sees Biochem.Biophys.Res.Comm.312:1159-65 (2003) such as Xiao.(Carlsbad CA) buys anti-Myc epitope antibodies from Invitrogen.Anti-ACE2 goat polyclonal antibody is from R﹠amp; (Minneapolis MN) buys Dsystem, is used for the Western trace and detects.

Site-directed mutagenesis technique be used on the total length SARS-CoV S glycoprotein on the pCDNA3 to make one with HIV-1 after birth glycoprotein (Env) and the consistent total cleavage site of some coronavirus, the method that provides according to producer uses that (La Jolla, the Quick Change test kit of CA) buying carries out from Straagene.For the segmental expression of various N end S, corresponding gene fragment with the pcr amplification rear clone advance the pSecTag2 expression vector (Invitrogen, Carlsbad).The pCDNA3-ACE2-ecto that expression has the ACE2 solubility extracellular fragment of C9 peptide is given by Michael Farzan (Harvard University, Boston MA) friendship.

Protein expression and purifying: the various N end fragments of S glycoprotein are advanced the pSecTag2 expression vector by subclone, be transfected into the 293T cell, that continues infects with VTF7.3, as Xiao etc. described in the Biochem.Biophys.Res.Comm.312:1159-65 (2003).The expression justacrine enters the albumen HiTrap Ni of substratum ⁺⁺-Chelating post is purifying under natural condition.The albumen of purifying is preserved after the PBS dialysis, as further analyzing.

Detect the interaction of the Dimerized of S glycoprotein and itself and ACE2 by co-immunoprecipitation: segmental Dimerized for S, different S glycoprotein constructs, separately or co-transfection go into the 293T cell, as Xiao etc. described in the Biochem.Biophys.Res.Comm.312:1159-65 (2003).Containing the segmental substratum of S is used for carrying out the immunoprecipitation test with the anti-S polyclonal antiserum of rabbit P540.For the test of some co-immunoprecipitations, add DTT and create a reductive condition, intermolecularly interact to get rid of by disulfide linkage.The S fragment of immunoprecipitation detects by Western with anti-Myc epi-position monoclonal antibody.The expression of solubility ACE2-C9 is very similar, expresses the ACE2-C9 enter substratum and is directly used in various S fragments and hatched 2 hours under 4 ℃ of conditions.Continue after, ACE2 carried out immunoprecipitation in 1 hour by hatching under 4 ℃ of conditions with anti-C9 monoclonal antibody and Protein G-sepharose 4B.Sepharose 4B is suspended from the SDS-PAGE sample buffer with PBS washing 4 times, boils to be used for the glue separation after 3 minutes.The ACE2 or the S that exist in the sample detect with Western, as Xiao etc. described in the Biochem.Biophys.Res.Comm.312:1159-65 (2003).

Flow cytometer detects: transfection the S glycoprotein of total length S glycoprotein or each N end disappearance, and the cell and the P540 rabbit anti-S polyclonal antibody that have infected with VTF7.3 and the goat anti-rabbit antibodies that combines FITC in containing the PBS of 1%BSA 4 ℃ hatched 2 hours, use ice-cold PBS washed cell 4 times then, with FacsCalibur (Becton Dickinson, SanJose California) analyzes.

The segmental glue filter analysis of S: after being replaced as PBS by HiTrap Ni-Chelating column purification, damping fluid, with sample on the S fragment to the Superose 1210/300GL post (Pharmacia that has crossed with the PBS balance, Uppsala, Sweden) on, albumen is collected elution fraction with PBS wash-out under the flow velocity that 0.5ml/ divides with the 0.5ml amount.These Superose 12 posts are demarcated with molecular weight of albumen standard 669,440,232,158,67,44 and 25kD.From each component, get 10 μ l and be used for the Western engram analysis.

Crosslinked: the S537 fragment of purifying is diluted to the concentration of 0.2 μ g/ml, BS with PBS ³(Pierce, Rockford, IL) be added in the S537 solution to final concentration be 1mg/ml, and hatch 1 minute on ice.Sample mixes with the 4XSDS-PAGE sample-loading buffer of equivalent then, analyzes with Western.

Cytogamy β-gal reporter gene test: transfection pSecTag2B-S or pCDNA3-ACE2 and the cell trysinization infected with VTF7.3 and VCB21R respectively, with the PBS washing once, cell is suspended from the conventional DMEM substratum of pH7.4 then, mixes.Lysing cell after hatching in 4 hours is measured β-gal activity with CPRG, as Xiao etc. described in the Biochem.Biophys.Res.Comm.312:1159-65 (2003).

ELISA: two kinds of ELISA methods are used.In sandwich ELISA, the elisa plate antibody sandwich of anti--His marker, the S fragment is added in the plate then, and detects with anti-c-Myc epitope antibodies.This method is used to detect the S fragment.In second kind of ELISA method, the ACE2 that will have the C9 mark by anti-C9 antibody is coated on the plate, adds the S fragment then, after washing plate, detects with anti-c-Myc epitope antibodies.Be room temperature 2 hours with hatching of anti-c-Myc epitope antibodies in all experiments.Measure OD value and normalization to maximum value.

The result

The N end fragment of S glycoprotein RBD upstream forms dimer.Solubility S1 (similar SU) fragment that had proved another coronavirus (MHV) in the past forms dimer, 330 amino-acid residues that contain receptor binding domain of N end distal-most end have participated in Dimerized, have only dimer to combine, see Lewicki ﹠amp with CEACAM; Gallagher, J.Biol.Chem.277:19727-34 (2002).Yet present inventor and other people have been positioned at the receptor binding domain of SARS-CoV the downstream of N end utmost point far-end, Biochem.Biophys.Res.Comm.312:1159-65 such as Xiao (2003); J.Biol.Chem.279:3197-3201 such as Wong (2004); J.Virol.78:4552-4560 such as Babcock (2004).

In order to explore the possibility that contains the segmental oligomerization reaction of receptor binding domain and to detect its function in the mediation film merges, segmental oligomerization reaction detects to several S.These S fragments have comprised the fragment (residue 17 to 276 of not holding utmost point far-end with acceptor ACE2 bonded N, S276 by name, SEQ ID NO:50) and contain the 319th to 517 residue and remain with the fragment (by name S319-517, SEQ ID NO:62) of receptor-binding activity.Select these fragments in part because they can independently fold justacrine in cells and supernatant, although their expression efficiency has notable difference (Fig. 9 A, the left side), and their concentration can reduce (Fig. 9 A, the right) when with the S756 coexpression.

In order whether to find these fragments and to contain the receptors bind subunit that is equivalent to envelope glycoprotein and (be generally SU, coronavirus is S1) maximum segment (S756) oligomerization takes place, these polypeptide fragments of coexpression carry out immunoprecipitation with the mixture in the cells and supernatant with P540 then.Described as previous examples, this rabbit polyclonal antibody preparation is the preparation peptide (SEQ ID NO:59) that comes to contain on the anti-S glycoprotein 540 to 555 residues.P450 antibody combines with the S756 polypeptide but does not combine (Fig. 9 B, the left side) with other polypeptide.All contain the N end fragment of receptor binding domain except that minimum fragment (S319-517), all can by P540 co-immunoprecipitation (Fig. 9 B, the right) take place with S756.In order to get rid of the possibility that forms the nonspecific reaction that causes co-immunoprecipitation because of disulfide linkage, DTT has been added into one of them co-immunoprecipitation experiment, DTT does not have influence (Fig. 9 C, left side group) to immunoprecipitation or the co-immunoprecipitation of excretory S756 (left road) or S756+S276 (right road).

In order to understand fully the size of oligomer, one of them fragment (S537) is crosslinked with BS3.Fig. 9 C the right group shows that the molecular weight of emerging band is consistent with dimer, and is not inconsistent with the oligomer of higher category.For the dimer of may and further proving conclusively of getting rid of the crosslinked false appearance that causes forms, the S537 fragment is also analyzed with glue filtering method, observe two filtering elution peaks of glue: a peak is caused by the material that molecular weight is about 230kDa, another material by 110kDa causes, and is consistent with dimer and monomeric size respectively.What form contrast is that the minimal segment that contains receptor binding domain (S319-517) that elutes has only monomer, molecular weight to be about 35kDa (Fig. 2 A, following group).Generally, these results suggest solubilities SU is a dimer, and Dimerized zone is within the upstream and receptor binding domain of N end utmost point far-end 317 residues.

Dimerized N petiolarea is essential by the cell-cytogamy of S mediation.Because the Dimerized zone of inferring is in the upstream of the receptor binding domain of S1, and syncretizing mechanism is in S2, and people can suppose that Dimerized is not that mediates fusion is needed.In order to verify this hypothesis, prepared deletion mutantion of two total length S glycoprotein, 103 residues of N end lack from a fragment, and 311 residues of N end like this, have been removed the Dimerized zone of inferring from another fragment disappearance (Fig. 9 A).Two mutons are compared none and are demonstrated fusion-activity (Fig. 9 A) with the wild type full-length S glycoprotein with fusion-activity.In order to detect the disappearance that whether has caused observable fusion-activity because of the difference of expression level, with flow cytometer and Western trace surface and total expression level are measured, the sub expression level with wild-type of two deletion mutantions of the Notes of Key Data that obtains from two experiments does not have obvious difference (Figure 11 B and C).These results suggest N end utmost point far-end is by relating to or not relate to Dimerized mechanism essential by fusion.

Dimerized S1 is effective with combining more than the monomer that contains receptor binding domain of ACE2.Had only Dimerized S1 to combine Lewicki ﹠amp to the research prompting of another coronavirus (MHV) in the past with its acceptor CEACAM; Gallagher, J.Biol.Chem.277:19727-734 (2002).Experimentize to understand fully how the S1 under the dimer state influences fusion with the SARS-CoV fragment.Specifically, observe unit price or the S1 fragment of two valency forms and combining of ACE2 by using anti-c-Myc epitope antibodies that unit price S1 fragment is changed into two valencys.Segmental one of them (the S319-517 of these S1, SEQ ID NO:62) there be not any combining of measuring with solidifying in the ACE2 on surface, unless it is with after anti-c-Myc epitope antibodies combines, this antibody before itself and acceptor are hatched and among in solution, convert it into two valency molecules (Figure 12).Forming correlated is that S537 does not combine with ACE2 when having antibody to exist, though the existence of antibody strengthens its bonding force (Figure 12).Thereby the dimer state of these results suggest S1 may improve fusion efficiencies by strengthening total bonding force.

Solubility S extracellular region is a tripolymer.The viral after birth glycoprotein of I class fusion rotein such as the hemagglutinin (HA) of influenza virus form tripolymer by striding the film district.Because SARS-CoV S glycoprotein is considered to belong to I class fusion rotein recently, S2 subunit may dominate the tripolymerization of whole S glycoprotein.Yet the Dimerized S1 that has the S2 of tripolymerization may cause the oligomer of higher category, their formation to depend on the Dimerized binding site of available on the natural S albumen.In order to detect this possibility, having removed the size of the extracellular soluble outskirt (Se) of striding film district and kytoplasm inner segment estimates with glue filtering method, as shown in Figure 13, size is detected near the mixture of tripolymer (MW 512kDa), does not have to find to have the oligomer of higher category.These results not only point out the Se fragment and perhaps the relevant S of film of total length be tripolymer at natural unbound state, and the Dimerized site that shows S1 is used to the interaction between tripolymer not too easily.

These results have following prompting: 1) the SU subunit of SARS-CoV S glycoprotein (S1) forms dimer, 2) Dimerized district is not overlapping, and upstream at receptor binding domain, 3) remove Dimerized district and promptly block fusion, 4) efficient of Dimerized S1 bind receptor molecule is strong far beyond the monomer fragment that contains receptor binding domain, 5) the S extracellular region of solubility forms tripolymer under the glue filtration condition.

Existing in the past report, the SU subunit of some I class fusion rotein (with acceptor molecule bonded albumen) can form dimer, comprises, for example the S1 of the gp120 of retroviral HIV-1 and S1 coronavirus MHV.J.Virol.74:4448-55 such as Center (2000); J.Biol.Chem.277:19727-34 such as Lewicki (2002).Up to this research, the Dimerized effect in the mediation film merges of S1 is unclear, and now, the gp140 of extracellular soluble outskirt such as HIV-1 and the membrane-associated protein of SIV (Env) form tripolymer and generally accepted, though also can be observed the dimer and the tetramer.Center et al.Proc.Nat′l Acad.Sci.U.S.A.98：14877-82(2001)。Similar, at least one comprises that possible fusion intermediate 4 level structures of coronavirus of the S2 of SARS-CoV are tripolymer.Liu et al.Lancet 363：938-947(2004)；Bosch et al.Proc.Nat′l Acad.Sci.U.S.A.101：8455-60(2004)。Form and correlatedly be, the S2 of some Notes of Key Data MHV be monomer after S1 separates.J.Biol.Chem.277:19727-34 such as Lewicki (2002).Dimer is being brought into play keying action to trimerical conversion in the fusion protein mediated fusion of II class.Like this, the someone thinks that the change of quaternary structure of some coronavirus may bring into play effect in merging mechanism.It should be noted that the Env of HIV-1 and the S glycoprotein of MHV all are cut, SU subunit can separate from striding film subunit, yet this separation may be inessential to merging.What form contrast is, the S of SARS-CoVd is not cut when relevant when being expressed as film, or soluble form and cutting be not merge essential.Like this, though the S glycoprotein of SARS-CoV is I class fusion rotein, infer for the Env of I class fusion rotein to be converted into metastable upper state to drive the rule of fusion reaction after being cut that not being cut is an exception.

About SU (S1) district of the S glycoprotein of SARS-CoV can form dimer and and the extracellular fragment (S2) of striding the film district form trimerical discovery and showed an interesting topology situation, if two monomers in the tripolymer also form dimer, so the 3rd monomer remain freely can and another tripolymer in free monomer reaction form dimer between two tripolymers.Another situation is that each the monomeric direction in the tripolymer does not allow to form dimer in tripolymer, and the monomer of reserving in free binding site and other tripolymers forms dimer.In this case, people can expect a netted trimerical formation.Last a kind of situation is that trimerical three-dimensional structure may not allow any monomeric Dimerized site to react to each other with monomer between the same or different tripolymer.The support of a kind of preliminary data that may be provided here in back promptly under described glue filtration condition, has not detected high level oligomer.Under those conditions, or form dimer in the tripolymer, but monomeric configuration do not allow to react to each other with other trimerical monomers, or be not enough to originally detect a little less than such reaction too, or trimerical three-dimensional structure does not allow the generation of Dimerized reaction.

Here the digital proof that provides can not take place in deletion Dimerized district back fusion, points out Dimerized district to work in fusion, though its mechanism may not be by Dimerized reaction.In addition, under natural condition, the concentration of surperficial S glycoprotein can be very high, as seen in micro-taking pictures, might Dimerizedly be reflected in " net " of stablizing interactional molecule and work, perhaps the albumen net that enters of some similar mediation II class fusion rotein.Such net, if any, may increase and the interactional avidity of acceptor molecule, and facilitate the formation of merging pore structure, or even under the situation that lacks the S cutting that produces the high energy metastable condition, provide the energy that drives fusion reaction by the after birth molecular network of set in advance.

Embodiment 20

The serum that obtains with the dna immunization mouse of coding RBD polypeptide suppresses the S cell fusion mediated

Present embodiment shows that the dna immunization Mammals with coding receptor binding domain polypeptide can prevent SARS to infect.

Material and method:

Mouse is divided into 3 groups: A group mouse contains the segmental plasmid pSecTag-SRBD of the S319-518 immunity of the receptor binding domain of spike protein with coding from 1# to 5#; B group 1# is to the pEAK-10-RBD-Fc immunity of 5# mouse with the segmental fusion rotein of RBD (S319-518) of coding and Fc fusion; C group 1# uses the control plasmid immunity to the 3# mouse.5 BALB/C mouse of each group are respectively 0 day, 14 days and immunity in 28 days.Mouse carries out immunity with particle gun, and each dosage is lower than 2 μ gDNA.Gathered serum on the 56th day.In Figure 14 A-B, first digit is represented the mouse individuality, letter representative immune group separately, the used extent of dilution of last digitized representation.

Cell (293T) with from being hatched by the antiserum(antisera) of mice immunized, then with express the proteic cytomixis of S.Merge the method for describing in the example with the front and measure (also seeing BBRC such as Xiao 2003).PC represents the not positive control of increase serum, and to the 2# mouse, used dilution factor is 10,100 and 1000 to the 1# of each group, and to the 3# mouse of A group, B group and control group, used dilution factor is 20 and 100.

The result

See Figure 14 A from the sero-fast antibody titers that mouse obtains.As shown in the figure, the antibody titers of the mouse of usefulness coding spike protein receptor area (S319-518, A group and B group) dna immunization is very high, and being diluted to 1: 7250 when being used for ELISA still has very strong reaction with antigen.

Just as shown in Figure 14B, the antiserum(antisera) that obtains from the mouse with the dna immunization of coding spike protein receptor binding domain suppresses to express the fusion of the proteic cell of S, and is dose-dependence.Like this, from 1A and 2A, promptly the antiserum(antisera) of the mouse of the receptor binding domain dna immunization of usefulness coding S when with dilution in 1: 10, obviously suppresses the protein mediated cytogamy of S.During greater dilution (1: 100 and 1: 1000), this sero-fast effect reduces.From mouse 3A (dilution in 1: 20), also observe similar results in the inhibition that the antiserum(antisera) pair cell of mouse 4A (dilution in 1: 20) and mouse 5A (dilution in 1: 20) merges always.

These Notes of Key Datas can induce the immune response of very strong anti-spike protein and can prevent SARS to infect with the dna immunization Mammals of the polypeptide of coding S protein receptor land.Just as described above, the S glycoprotein fragment that contains receptor binding domain of solubility suppresses S cell fusion mediated (seeing Figure 15).

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Here all patents of quoting or mentioning and publication are being represented those skilled in the art's relevant with the present invention state of the art, as a reference each patent or publication are attached to herein by reference, its degree be attached to this paper separately or list in this paper with its integral body by reference with its integral body identical.The applicant is retained in this explanation the citation patent mentioned of any of these or any and all material and the information right of publication.

Special methods described here and composition are representative preferred embodiment, are exemplary, are not intended to as limitation of the scope of the invention.When other purpose, direction and the embodiment of those skilled in the art,, contained by spirit of the present invention as defining in the authority of claim in this explanation of research time generation.Those skilled in the art should quite know clearly, to the various replacements of invention disclosed herein with revise and do not depart from scope and spirit of the present invention.Here the present invention of illustrative ground description may be suitable for not implementing under the situation of disclosed any element or some element or any restriction or some restriction as key element here in shortage.Here the method described of illustrative ground may be suitable for different steps in order enforcements with process, they needn't stick to mention here or claim in order of steps.As " one ", " one " and " described " that are used for here with additional claim,, comprise one or many implications unless specialize in the literary composition.For example, the content of " host cell " has comprised a plurality of this cells (for example culture and colony) and analogue.Under any circumstance this patent all can not be construed as limited to concrete disclosed specific example or embodiment here.Under any circumstance this patent all can not be construed as limited to any auditor, official or the employee's of patent and trade-mark administration office statement, unless such statement defending party to the application especially, unconditionally to the sky represents to adopt in writing.

Used term and expression are used as descriptive term and non-limiting term, here be not intended to these terms and express an equivalent of the feature of showing and describing or certain part of equivalent are foreclosed, but, as what advocated, should approve that various modifications within the scope of the present invention are possible.Like this, though the present invention is specifically open by embodiment preferred and washability feature quilt, those skilled in the art may seek the modification of notion disclosed herein and change, such modification and change are considered in covering scope of the present invention, in accessory claim defined.

Here the present invention has been carried out broadly, described prevailingly, various narrower and inferior general descriptions in the open scope of this generality also constitute a part of the present invention.This comprises of the present invention general description that has supplementary condition or remove the negativity restriction of any theme from upperseat concept, and whether the material that no matter picked-off has concrete narration at this.

Other embodiments are in following claim.In addition, organize when describing with Markush when feature of the present invention or aspect, those those skilled in the art are to be appreciated that the present invention also describes with any individual member or the subgroup member of Markush group.

Sequence table

＜110〉US Government (National Institutes of Health)

D.S. Stefan Dimitrow (Dimitrov, Dimiter S.)

X. Xiao (Xiao, Xiaodong)

＜120〉soluble fragments of sars coronavirus spike glycoprotein

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<150>US 60/524,642

<151>2003-11-25

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<170>FastSEQ for Windows Version 4.0

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＜213〉sars coronavirus

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Met Phe Ile Phe Leu Leu Phe Leu Thr Leu Thr Ser Gly Ser Asp Leu

1 5 10 15

Asp Arg Cys Thr Thr Phe Asp Asp Val Gln Ala Pro Asn Tyr Thr Gln

20 25 30

His Thr Ser Ser Met Arg Gly Val Tyr Tyr Pro Asp Glu Ile Phe Arg

35 40 45

Ser Asp Thr Leu Tyr Leu Thr Gln Asp Leu Phe Leu Pro Phe Tyr Ser

50 55 60

Asn Val Thr Gly Phe His Thr Ile Asn His Thr Phe Gly Asn Pro Val

65 70 75 80

Ile Pro Phe Lys Asp Gly Ile Tyr Phe Ala Ala Thr Glu Iys Ser Asn

85 90 95

Val Val Arg Gly Trp Val Phe Gly Ser Thr Met Asn Asn Lys Ser Gln

100 105 110

Ser Val Ile Ile Ile Asn Asn Ser Thr Asn Val Val Ile Arg Ala Cys

115 120 125

Asn Phe Glu Leu Cys Asp Asn Pro Phe Phe Ala Val Ser Lys Pro Met

130 135 140

Gly Thr Gln Thr His Thr Met Ile Phe Asp Asn Ala Phe Asn Cys Thr

145 150 155 160

Phe Glu Tyr Ile Ser Asp Ala Phe Ser Leu Asp Val Ser Glu Lys Ser

165 170 175

Gly Asn Phe Lys His Leu Arg Glu Phe Val Phe Lys Asn Lys Asp Gly

180 185 190

Phe Leu Tyr Val Tyr Lys Gly Tyr Gln Pro Ile Asp Val Val Arg Asp

195 200 205

Leu Pro Ser Gly Phe Asn Thr Leu Lys Pro Ile Phe Lys Leu Pro Leu

210 215 220

Gly Ile Asn Ile Thr Asn Phe Arg Ala Ile Leu Thr Ala Phe Ser Pro

225 230 235 240

Ala Gln Asp Ile Trp Gly Thr Ser Ala Ala Ala Tyr Phe Val Gly Tyr

245 250 255

Leu Lys Pro Thr Thr Phe Met Leu Lys Tyr Asp Glu Asn Gly Thr Ile

260 265 270

Thr Asp Ala Val Asp Cys Ser Gln Asn Pro Leu Ala Glu Leu Lys Cys

275 280 285

Ser Val Lys Ser Phe Glu Ile Asp Lys Gly Ile Tyr Gln Thr Ser Asn

290 295 300

Phe Arg Val Val Pro Ser Gly Asp Val Val Arg Phe Pro Asn Ile Thr

305 310 315 320

Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Lys Phe Pro Ser

325 330 335

Val Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn Cys Val Ala Asp Tyr

340 345 350

Ser Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr Phe Lys Cys Tyr Gly

355 360 365

Val Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe Ser Asn Val Tyr Ala

370 375 380

Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln Ile Ala Pro Gly

385 390 395 400

Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe

405 410 415

Met Gly Cys Val Leu Ala Trp Asn Thr Arg Asn Ile Asp Ala Thr Ser

420 425 430

Thr Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His Gly Lys Leu

435 440 445

Arg Pro Phe Glu Arg Asp Ile Ser Asn Val Pro Phe Ser Pro Asp Gly

450 455 460

Lys Pro Cys Thr Pro Pro Ala Leu Asn Cys Tyr Trp Pro Leu Asn Asp

465 470 475 480

Tyr Gly Phe Tyr Thr Thr Thr Gly Ile Gly Tyr Gln Pro Tyr Arg Val

485 490 495

Val Val Leu Ser Phe Glu Leu Leu Asn Ala Pro Ala Thr Val Cys Gly

500 505 510

Pro Lys Leu Ser Thr Asp Leu Ile Lys Asn Gln Cys Val Asn Phe Asn

515 520 525

Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Pro Ser Ser Lys Arg

530 535 540

Phe Gln Pro Phe Gln Gln Phe Gly Arg Asp Val Ser Asp Phe Thr Asp

545 550 555 560

Ser Val Arg Asp Pro Lys Thr Ser Glu Ile Leu Asp Ile Ser Pro Cys

565 570 575

Ala Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Ala Ser Ser

580 585 590

Glu Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Asp Val Ser Thr

595 600 605

Ala Ile His Ala Asp Gln Leu Thr Pro Ala Trp Arg Ile Tyr Ser Thr

610 615 620

Gly Asn Asn Val Phe Gln Thr Gln Ala Gly Cys Leu Ile Gly Ala Glu

625 630 635 640

His Val Asp Thr Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile

645 650 655

Cys Ala Ser Tyr His Thr Val Ser Leu Leu Arg Ser Thr Ser Gln Lys

660 665 670

Ser Ile Val Ala Tyr Thr Met Ser Leu Gly Ala Asp Ser Ser Ile Ala

675 680 685

Tyr Ser Asn Asn Thr Ile Ala Ile Pro Thr Asn Phe Ser Ile Ser Ile

690 695 700

Thr Thr Glu Val Met Pro Val Ser Met Ala Lys Thr Ser Val Asp Cys

705 710 715 720

Asn Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ala Asn Leu Leu Leu

725 730 735

Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Ser Gly Ile

740 745 750

Ala Ala Glu Gln Asp Arg Asn Thr Arg Glu Val Phe Ala Gln Val Lys

755 760 765

Gln Met Tyr Lys Thr Pro Thr Leu Lys Tyr Phe Gly Gly Phe Asn Phe

770 775 780

Ser Gln Ile Leu Pro Asp Pro Leu Lys Pro Thr Lys Arg Ser Phe Ile

785 790 795 800

Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly Phe Met

805 810 815

Lys Gln Tyr Gly Glu Cys Leu Gly Asp Ile Asn Ala Arg Asp Leu Ile

820 825 830

Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu Leu Thr

835 840 845

Asp Asp Met Ile Ala Ala Tyr Thr Ala Ala Leu Val Ser Gly Thr Ala

850 855 860

Thr Ala Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile Pro Phe

865 870 875 880

Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr Gln Asn

885 890 895

Val Leu Tyr Glu Asn Gln Lys Gln Ile Ala Asn Gln Phe Asn Lys Ala

900 905 910

Ile Ser Gln Ile Gln Glu Ser Leu Thr Thr Thr Ser Thr Ala Leu Gly

915 920 925

Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu

930 935 940

Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val Leu Asn

945 950 955 960

Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln Ile Asp

965 970 975

Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val Thr Gln

980 985 990

Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala

995 1000 1005

Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Val Asp Phe

1010 1015 1020

Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ala Ala Pro His

1025 1030 1035 1040

Gly Val Val Phe Leu His Val Thr Tyr Val Pro Ser Gln Glu Arg Asn

1045 1050 1055

Phe Thr Thr Ala Pro Ala Ile Cys His Glu Gly Lys Ala Tyr Phe Pro

1060 1065 1070

Arg Glu Gly Val Phe Val Phe Asn Gly Thr Ser Trp Phe Ile Thr Gln

1075 1080 1085

Arg Asn Phe Phe Ser Pro Gln Ile Ile Thr Thr Asp Asn Thr Phe Val

1090 1095 1100

Ser Gly Asn Cys Asp Val Val Ile Gly Ile Ile Asn Asn Thr Val Tyr

1105 1110 1115 1120

Asp Pro Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys

1125 1130 1135

Tyr Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser

1140 1145 1150

Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu

1155 1160 1165

Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu

1170 1175 1180

Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Val Trp Leu

1185 1190 1195 1200

Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Leu Leu

1205 1210 1215

Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Ala Cys Ser Cys

1220 1225 1230

Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro Val Leu Lys

1235 1240 1245

Gly Val Lys Leu His Tyr Thr

1250 1255

<210>2

<211>3768

<212>DNA

＜213〉sars coronavirus

<400>2

atgtttattt tcttattatt tcttactctc actagtggta gtgaccttga ccggtgcacc 60

acttttgatg atgttcaagc tcctaattac actcaacata cttcatctat gaggggggtt 120

tactatcctg atgaaatttt tagatcagac actctttatt taactcagga tttatttctt 180

ccattttatt ctaatgttac agggtttcat actattaatc atacgtttgg caaccctgtc 240

atacctttta aggatggtat ttattttgct gccacagaga aatcaaatgt tgtccgtggt 300

tgggtttttg gttctaccat gaacaacaag tcacagtcgg tgattattat taacaattct 360

actaatgttg ttatacgagc atgtaacttt gaattgtgtg acaacccttt ctttgctgtt 420

tctaaaccca tgggtacaca gacacatact atgatattcg ataatgcatt taattgcact 480

ttcgagtaca tatctgatgc cttttcgctt gatgtttcag aaaagtcagg taattttaaa 540

cacttacgag agtttgtgtt taaaaataaa gatgggtttc tctatgttta taagggctat 600

caacctatag atgtagttcg tgatctacct tctggtttta acactttgaa acctattttt 660

aagttgcctc ttggtattaa cattacaaat tttagagcca ttcttacagc cttttcacct 720

gctcaagaca tttggggcac gtcagctgca gcctattttg ttggctattt aaagccaact 780

acatttatgc tcaagtatga tgaaaatggt acaatcacag atgctgttga ttgttctcaa 840

aatccacttg ctgaactcaa atgctctgtt aagagctttg agattgacaa aggaatttac 900

cagacctcta atttcagggt tgttccctca ggagatgttg tgagattccc taatattaca 960

aacttgtgtc cttttggaga ggtttttaat gctactaaat tcccttctgt ctatgcatgg 1020

gagagaaaaa aaatttctaa ttgtgttgct gattactctg tgctctacaa ctcaacattt 1080

ttttcaacct ttaagtgcta tggcgtttct gccactaagt tgaatgatct ttgcttctcc 1140

aatgtctatg cagattcttt tgtagtcaag ggagatgatg taagacaaat agcgccagga 1200

caaactggtg ttattgctga ttataattat aaattgccag atgatttcat gggttgtgtc 1260

cttgcttgga atactaggaa cattgatgct acttcaactg gtaattataa ttataaatat 1320

aggtatctta gacatggcaa gcttaggccc tttgagagag acatatctaa tgtgcctttc 1380

tcccctgatg gcaaaccttg caccccacct gctcttaatt gttattggcc attaaatgat 1440

tatggttttt acaccactac tggcattggc taccaacctt acagagttgt agtactttct 1500

tttgaacttt taaatgcacc ggccacggtt tgtggaccaa aattatccac tgaccttatt 1560

aagaaccagt gtgtcaattt taattttaat ggactcactg gtactggtgt gttaactcct 1620

tcttcaaaga gatttcaacc atttcaacaa tttggccgtg atgtttctga tttcactgat 1680

tccgttcgag atcctaaaac atctgaaata ttagacattt caccttgcgc ttttgggggt 1740

gtaagtgtaa ttacacctgg aacaaatgct tcatctgaag ttgctgttct atatcaagat 1800

gttaactgca ctgatgtttc tacagcaatt catgcagatc aactcacacc agcttggcgc 1860

atatattcta ctggaaacaa tgtattccag actcaagcag gctgtcttat aggagctgag 1920

catgtcgaca cttcttatga gtgcgacatt cctattggag ctggcatttg tgctagttac 1980

catacagttt ctttattacg tagtactagc caaaaatcta ttgtggctta tactatgtct 2040

ttaggtgctg atagttcaat tgcttactct aataacacca ttgctatacc tactaacttt 2100

tcaattagca ttactacaga agtaatgcct gtttctatgg ctaaaacctc cgtagattgt 2160

aatatgtaca tctgcggaga ttctactgaa tgtgctaatt tgcttctcca atatggtagc 2220

ttttgcacac aactaaatcg tgcactctca ggtattgctg ctgaacagga tcgcaacaca 2280

cgtgaagtgt tcgctcaagt caaacaaatg tacaaaaccc caactttgaa atattttggt 2340

ggttttaatt tttcacaaat attacctgac cctctaaagc caactaagag gtcttttatt 2400

gaggacttgc tctttaataa ggtgacactc gctgatgctg gcttcatgaa gcaatatggc 2460

gaatgcctag gtgatattaa tgctagagat ctcatttgtg cgcagaagtt caatggactt 2520

acagtgttgc cacctctgct cactgatgat atgattgctg cctacactgc tgctctagtt 2580

agtggtactg ccactgctgg atggacattt ggtgctggcg ctgctcttca aatacctttt 2640

gctatgcaaa tggcatatag gttcaatggc attggagtta cccaaaatgt tctctatgag 2700

aaccaaaaac aaatcgccaa ccaatttaac aaggcgatta gtcaaattca agaatcactt 2760

acaacaacat caactgcatt gggcaagctg caagacgttg ttaaccagaa tgctcaagca 2820

ttaaacacac ttgttaaaca acttagctct aattttggtg caatttcaag tgtgctaaat 2880

gatatccttt cgcgacttga taaagtcgag gcggaggtac aaattgacag gttaattaca 2940

ggcagacttc aaagccttca aacctatgta acacaacaac taatcagggc tgctgaaatc 3000

agggcttctg ctaatcttgc tgctactaaa atgtctgagt gtgttcttgg acaatcaaaa 3060

agagttgact tttgtggaaa gggctaccac cttatgtcct tcccacaagc agccccgcat 3120

ggtgttgtct tcctacatgt cacgtatgtg ccatcccagg agaggaactt caccacagcg 3180

ccagcaattt gtcatgaagg caaagcatac ttccctcgtg aaggtgtttt tgtgtttaat 3240

ggcacttctt ggtttattac acagaggaac ttcttttctc cacaaataat tactacagac 3300

aatacatttg tctcaggaaa ttgtgatgtc gttattggca tcattaacaa cacagtttat 3360

gatcctctgc aacctgagct cgactcattc aaagaagagc tggacaagta cttcaaaaat 3420

catacatcac cagatgttga tcttggcgac atttcaggca ttaacgcttc tgtcgtcaac 3480

attcaaaaag aaattgaccg cctcaatgag gtcgctaaaa atttaaatga atcactcatt 3540

gaccttcaag aattgggaaa atatgagcaa tatattaaat ggccttggta tgtttggctc 3600

ggcttcattg ctggactaat tgccatcgtc atggttacaa tcttgctttg ttgcatgact 3660

agttgttgca gttgcctcaa gggtgcatgc tcttgtggtt cttgctgcaa gtttgatgag 3720

gatgactctg agccagttct caagggtgtc aaattacatt acacataa 3768

<210>3

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<400>3

agtcggatcc ggtaggctta tcattagag 29

<210>4

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<400>4

ccatcagggg agaaaggcac 20

<210>5

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<400>5

gtgcctttct cccctgatgg 20

<210>6

<211>19

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<400>6

gaagagcagc gccagcacc 19

<210>7

<211>19

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<400>7

ggtgctggcg ctgctcttc 19

<210>8

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<400>8

actgtctaga gttcgtttat gtgtaatg 28

<210>9

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<400>9

agtcggatcc gaccggtgca ccacttttg 29

<210>10

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<400>10

agtcgggccc ctgttcagca gcaatacc 28

<210>11

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<400>11

actgggatcc gaagtgttcg ctcaagtc 28

<210>12

<211>26

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<400>12

actgtctaga ttgctcatat tttccc 26

<210>13

<211>740

<212>PRT

＜213〉sars coronavirus

<400>13

Asp Arg Cys Thr Thr Phe Asp Asp Val Gln Ala Pro Asn Tyr Thr Gln

1 5 10 15

His Thr Ser Ser Met Arg Gly Val Tyr Tyr Pro Asp Glu Ile Phe Arg

20 25 30

Ser Asp Thr Leu Tyr Leu Thr Gln Asp Leu Phe Leu Pro Phe Tyr Ser

35 40 45

Asn Val Thr Gly Phe His Thr Ile Asn His Thr Phe Gly Asn Pro Val

50 55 60

Ile Pro Phe Lys Asp Gly Ile Tyr Phe Ala Ala Thr Glu Lys Ser Asn

65 70 75 80

Val Val Arg Gly Trp Val Phe Gly Ser Thr Met Asn Asn Lys Ser Gln

85 90 95

Ser Val Ile Ile Ile Asn Asn Ser Thr Asn Val Val Ile Arg Ala Cys

100 105 110

Asn Phe Glu Leu Cys Asp Asn Pro Phe Phe Ala Val Ser Lys Pro Met

115 120 125

Gly Thr Gln Thr His Thr Met Ile Phe Asp Asn Ala Phe Asn Cys Thr

130 135 140

Phe Glu Tyr Ile Ser Asp Ala Phe Ser Leu Asp Val Ser Glu Lys Ser

145 150 155 160

Gly Asn Phe Lys His Leu Arg Glu Phe Val Phe Lys Asn Lys Asp Gly

165 170 175

Phe Leu Tyr Val Tyr Lys Gly Tyr Gln Pro Ile Asp Val Val Arg Asp

180 185 190

Leu Pro Ser Gly Phe Asn Thr Leu Lys Pro Ile Phe Lys Leu Pro Leu

195 200 205

Gly Ile Asn Ile Thr Asn Phe Arg Ala Ile Leu Thr Ala Phe Ser Pro

210 215 220

Ala Gln Asp Ile Trp Gly Thr Ser Ala Ala Ala Tyr Phe Val Gly Tyr

225 230 235 240

Leu Lys Pro Thr Thr Phe Met Leu Lys Tyr Asp Glu Asn Gly Thr Ile

245 250 255

Thr Asp Ala Val Asp Cys Ser Gln Asn Pro Leu Ala Glu Leu Lys Cys

260 265 270

Ser Val Lys Ser Phe Glu Ile Asp Lys Gly Ile Tyr Gln Thr Ser Asn

275 280 285

Phe Arg Val Val Pro Ser Gly Asp Val Val Arg Phe Pro Asn Ile Thr

290 295 300

Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Lys Phe Pro Ser

305 310 315 320

Val Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn Cys Val Ala Asp Tyr

325 330 335

Ser Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr Phe Lys Cys Tyr Gly

340 345 350

Val Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe Ser Asn Val Tyr Ala

355 360 365

Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln Ile Ala Pro Gly

370 375 380

Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe

385 390 395 400

Met Gly Cys Val Leu Ala Trp Asn Thr Arg Asn Ile Asp Ala Thr Ser

405 410 415

Thr Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His Gly Lys Leu

420 425 430

Arg Pro Phe Glu Arg Asp Ile Ser Asn Val Pro Phe Ser Pro Asp Gly

435 440 445

Lys Pro Cys Thr Pro Pro Ala Leu Asn Cys Tyr Trp Pro Leu Asn Asp

450 455 460

Tyr Gly Phe Tyr Thr Thr Thr Gly Ile Gly Tyr Gln Pro Tyr Arg Val

465 470 475 480

Val Val Leu Ser Phe Glu Leu Leu Asn Ala Pro Ala Thr Val Cys Gly

485 490 495

Pro Lys Leu Ser Thr Asp Leu Ile Lys Asn Gln Cys Val Asn Phe Asn

500 505 510

Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Pro Ser Ser Lys Arg

515 520 525

Phe Gln Pro Phe Gln Gln Phe Gly Arg Asp Val Ser Asp Phe Thr Asp

530 535 540

Ser Val Arg Asp Pro Lys Thr Ser Glu Ile Leu Asp Ile Ser Pro Cys

545 550 555 560

Ala Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Ala Ser Ser

565 570 575

Glu Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Asp Val Ser Thr

580 585 590

Ala Ile His Ala Asp Gln Leu Thr Pro Ala Trp Arg Ile Tyr Ser Thr

595 600 605

Gly Asn Asn Val Phe Gln Thr Gln Ala Gly Cys Leu Ile Gly Ala Glu

610 615 620

His Val Asp Thr Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile

625 630 635 640

Cys Ala Ser Tyr His Thr Val Ser Leu Leu Arg Ser Thr Ser Gln Lys

645 650 655

Ser Ile Val Ala Tyr Thr Met Ser Leu Gly Ala Asp Ser Ser Ile Ala

660 665 670

Tyr Ser Asn Asn Thr Ile Ala Ile Pro Thr Asn Phe Ser Ile Ser Ile

675 680 685

Thr Thr Glu Val Met Pro Val Ser Met Ala Lys Thr Ser Val Asp Cys

690 695 700

Asn Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ala Asn Leu Leu Leu

705 710 715 720

Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Ser Gly Ile

725 730 735

Ala Ala Glu Gln

740

<210>14

<211>429

<212>PRT

＜213〉sars coronavirus

<400>14

Glu Val Phe Ala Gln Val Lys Gln Met Tyr Lys Thr Pro Thr Leu Lys

1 5 10 15

Tyr Phe Gly Gly Phe Asn Phe Ser Gln Ile Leu Pro Asp Pro Leu Lys

20 25 30

Pro Thr Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr

35 40 45

Leu Ala Asp Ala Gly Phe Met Lys Gln Tyr Gly Glu Cys Leu Gly Asp

50 55 60

Ile Asn Ala Arg Asp Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr

65 70 75 80

Val Leu Pro Pro Leu Leu Thr Asp Asp Met Ile Ala Ala Tyr Thr Ala

85 90 95

Ala Leu Val Ser Gly Thr Ala Thr Ala Gly Trp Thr Phe Gly Ala Gly

100 105 110

Ala Ala Leu Gln Ile Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn

115 120 125

Gly Ile Gly Val Thr Gln Asn Val Leu Tyr Glu Asn Gln Lys Gln Ile

130 135 140

Ala Asn Gln Phe Asn Lys Ala Ile Ser Gln Ile Gln Glu Ser Leu Thr

145 150 155 160

Thr Thr Ser Thr Ala Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn

165 170 175

Ala Gln Ala Leu Asn Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly

180 185 190

Ala Ile Ser Ser Val Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val

195 200 205

Glu Ala Glu Val Gln Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser

210 215 220

Leu Gln Thr Tyr Val Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg

225 230 235 240

Ala Ser Ala Asn Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly

245 250 255

Gln Ser Lys Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser

260 265 270

Phe Pro Gln Ala Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr

275 280 285

Val Pro Ser Gln Glu Arg Asn Phe Thr Thr Ala Pro Ala Ile Cys His

290 295 300

Glu Gly Lys Ala Tyr Phe Pro Arg Glu Gly Val Phe Val Phe Asn Gly

305 310 315 320

Thr Ser Trp Phe Ile Thr Gln Arg Asn Phe Phe Ser Pro Gln Ile Ile

325 330 335

Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val Val Ile Gly

340 345 350

Ile Ile Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro Glu Leu Asp Ser

355 360 365

Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn His Thr Ser Pro Asp

370 375 380

Val Asp Leu Gly Asp Ile Ser Gly Ile Asn Ala Ser Val Val Asn Ile

385 390 395 400

Gln Lys Glu Ile Asp Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu

405 410 415

Ser Leu Ile Asp Leu Gln Glu Leu Gly Lys Tyr Glu Gln

420 425

<210>15

<211>1170

<212>PRT

＜213〉sars coronavirus

<400>15

Asp Arg Cys Thr Thr Phe Asp Asp Val Gln Ala Pro Asn Tyr Thr Gln

1 5 10 15

His Thr Ser Ser Met Arg Gly Val Tyr Tyr Pro Asp Glu Ile Phe Arg

20 25 30

Ser Asp Thr Leu Tyr Leu Thr Gln Asp Leu Phe Leu Pro Phe Tyr Ser

35 40 45

Asn Val Thr Gly Phe His Thr Ile Asn His Thr Phe Gly Asn Pro Val

50 55 60

Ile Pro Phe Lys Asp Gly Ile Tyr Phe Ala Ala Thr Glu Lys Ser Asn

65 70 75 80

Val Val Arg Gly Trp Val Phe Gly Ser Thr Met Asn Asn Lys Ser Gln

85 90 95

Ser Val Ile Ile Ile Asn Asn Ser Thr Asn Val Val Ile Arg Ala Cys

100 105 110

Asn Phe Glu Leu Cys Asp Asn Pro Phe Phe Ala Val Ser Lys Pro Met

115 120 125

Gly Thr Gln Thr His Thr Met Ile Phe Asp Asn Ala Phe Asn Cys Thr

130 135 140

Phe Glu Tyr Ile Ser Asp Ala Phe Ser Leu Asp Val Ser Glu Lys Ser

145 150 155 160

Gly Asn Phe Lys His Leu Arg Glu Phe Val Phe Lys Asn Lys Asp Gly

165 170 175

Phe Leu Tyr Val Tyr Lys Gly Tyr Gln Pro Ile Asp Val Val Arg Asp

180 185 190

Leu Pro Ser Gly Phe Asn Thr Leu Lys Pro Ile Phe Lys Leu Pro Leu

195 200 205

Gly Ile Asn Ile Thr Asn Phe Arg Ala Ile Leu Thr Ala Phe Ser Pro

210 215 220

Ala Gln Asp Ile Trp Gly Thr Ser Ala Ala Ala Tyr Phe Val Gly Tyr

225 230 235 240

Leu Lys Pro Thr Thr Phe Met Leu Lys Tyr Asp Glu Asn Gly Thr Ile

245 250 255

Thr Asp Ala Val Asp Cys Ser Gln Asn Pro Leu Ala Glu Leu Lys Cys

260 265 270

Ser Val Lys Ser Phe Glu Ile Asp Lys Gly Ile Tyr Gln Thr Ser Asn

275 280 285

Phe Arg Val Val Pro Ser Gly Asp Val Val Arg Phe Pro Asn Ile Thr

290 295 300

Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Lys Phe Pro Ser

305 310 315 320

Val Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn Cys Val Ala Asp Tyr

325 330 335

Ser Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr Phe Lys Cys Tyr Gly

340 345 350

Val Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe Ser Asn Val Tyr Ala

355 360 365

Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln Ile Ala Pro Gly

370 375 380

Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe

385 390 395 400

Met Gly Cys Val Leu Ala Trp Asn Thr Arg Asn Ile Asp Ala Thr Ser

405 410 415

Thr Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His Gly Lys Leu

420 425 430

Arg Pro Phe Glu Arg Asp Ile Ser Asn Val Pro Phe Ser Pro Asp Gly

435 440 445

Lys Pro Cys Thr Pro Pro Ala Leu Asn Cys Tyr Trp Pro Leu Asn Asp

450 455 460

Tyr Gly Phe Tyr Thr Thr Thr Gly Ile Gly Tyr Gln Pro Tyr Arg Val

465 470 475 480

Val Val Leu Ser Phe Glu Leu Leu Asn Ala Pro Ala Thr Val Cys Gly

485 490 495

Pro Lys Leu Ser Thr Asp Leu Ile Lys Asn Gln Cys Val Asn Phe Asn

500 505 510

Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Pro Ser Ser Lys Arg

515 520 525

Phe Gln Pro Phe Gln Gln Phe Gly Arg Asp Val Ser Asp Phe Thr Asp

530 535 540

Ser Val Arg Asp Pro Lys Thr Ser Glu Ile Leu Asp Ile Ser Pro Cys

545 550 555 560

Ala Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Ala Ser Ser

565 570 575

Glu Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Asp Val Ser Thr

580 585 590

Ala Ile His Ala Asp Gln Leu Thr Pro Ala Trp Arg Ile Tyr Ser Thr

595 600 605

Gly Asn Asn Val Phe Gln Thr Gln Ala Gly Cys Leu Ile Gly Ala Glu

610 615 620

His Val Asp Thr Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile

625 630 635 640

Cys Ala Ser Tyr His Thr Val Ser Leu Leu Arg Ser Thr Ser Gln Lys

645 650 655

Ser Ile Val Ala Tyr Thr Met Ser Leu Gly Ala Asp Ser Ser Ile Ala

660 665 670

Tyr Ser Asn Asn Thr Ile Ala Ile Pro Thr Asn Phe Ser Ile Ser Ile

675 680 685

Thr Thr Glu Val Met Pro Val Ser Met Ala Lys Thr Ser Val Asp Cys

690 695 700

Asn Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ala Asn Leu Leu Leu

705 710 715 720

Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Ser Gly Ile

725 730 735

Ala Ala Glu Gln Asp Glu Val Phe Ala Gln Val Lys Gln Met Tyr Lys

740 745 750

Thr Pro Thr Leu Lys Tyr Phe Gly Gly Phe Asn Phe Ser Gln Ile Leu

755 760 765

Pro Asp Pro Leu Lys Pro Thr Lys Arg Ser Phe Ile Glu Asp Leu Leu

770 775 780

Phe Asn Lys Val Thr Leu Ala Asp Ala Gly Phe Met Lys Gln Tyr Gly

785 790 795 800

Glu Cys Leu Gly Asp Ile Asn Ala Arg Asp Leu Ile Cys Ala Gln Lys

805 810 815

Phe Asn Gly Leu Thr Val Leu Pro Pro Leu Leu Thr Asp Asp Met Ile

820 825 830

Ala Ala Tyr Thr Ala Ala Leu Val Ser Gly Thr Ala Thr Ala Gly Trp

835 840 845

Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile Pro Phe Ala Met Gln Met

850 855 860

Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr Gln Asn Val Leu Tyr Glu

865 870 875 880

Asn Gln Lys Gln Ile Ala Asn Gln Phe Asn Lys Ala Ile Ser Gln Ile

885 890 895

Gln Glu Ser Leu Thr Thr Thr Ser Thr Ala Leu Gly Lys Leu Gln Asp

900 905 910

Val Val Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu Val Lys Gln Leu

915 920 925

Ser Ser Asn Phe Gly Ala Ile Ser Ser Val Leu Asn Asp Ile Leu Ser

930 935 940

Arg Leu Asp Lys Val Glu Ala Glu Val Gln Ile Asp Arg Leu Ile Thr

945 950 955 960

Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val Thr Gln Gln Leu Ile Arg

965 970 975

Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala Thr Lys Met Ser

980 985 990

Glu Cys Val Leu Gly Gln Ser Lys Arg Val Asp Phe Cys Gly Lys Gly

995 1000 1005

Tyr His Leu Met Ser Phe Pro Gln Ala Ala Pro His Gly Val Val Phe

1010 1015 1020

Leu His Val Thr Tyr Val Pro Ser Gln Glu Arg Asn Phe Thr Thr Ala

1025 1030 1035 1040

Pro Ala Ile Cys His Glu Gly Lys Ala Tyr Phe Pro Arg Glu Gly Val

1045 1050 1055

Phe Val Phe Asn Gly Thr Ser Trp Phe Ile Thr Gln Arg Asn Phe Phe

1060 1065 1070

Ser Pro Gln Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys

1075 1080 1085

Asp Val Val Ile Gly Ile Ile Asn Asn Thr Val Tyr Asp Pro Leu Gln

1090 1095 1100

Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn

1105 1110 1115 1120

His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn Ala

1125 1130 1135

Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu Val Ala

1140 1145 1150

Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu Gly Lys Tyr

1155 1160 1165

Glu Gln

1170

<210>16

<211>21

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic k chain leader sequence

<400>16

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Asp

20

<210>17

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic myc epitope

<400>17

Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu

1 5 10

<210>18

<211>6

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic histidine mark thing

<400>18

His His His His His His

1 5

<210>19

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<400>19

ctagctcgag caacagcatc tgtg 24

<210>20

<211>100

<212>PRT

＜213〉sars coronavirus

<400>20

Met Phe Ile Phe Leu Leu Phe Leu Thr Leu Thr Ser Gly Ser Asp Leu

1 5 10 15

Asp Arg Cys Thr Thr Phe Asp Asp Val Gln Ala Pro Asn Tyr Thr Gln

20 25 30

His Thr Ser Ser Met Arg Gly Val Tyr Tyr Pro Asp Glu Ile Phe Arg

35 40 45

Ser Asp Thr Leu Tyr Leu Thr Gln Asp Leu Phe Leu Pro Phe Tyr Ser

50 55 60

Asn Val Thr Gly Phe His Thr Ile Asn His Thr Phe Gly Asn Pro Val

65 70 75 80

Ile Pro Phe Lys Asp Gly Ile Tyr Phe Ala Ala Thr Glu Lys Ser Asn

85 90 95

Val Val Arg Gly

100

<210>21

<211>100

<212>PRT

＜213〉sars coronavirus

<400>21

Trp Val Phe Gly Ser Thr Met Asn Asn Lys Ser Gln Ser Val Ile Ile

1 5 10 15

Ile Asn Asn Ser Thr Asn Val Val Ile Arg Ala Cys Asn Phe Glu Leu

20 25 30

Cys Asp Asn Pro Phe Phe Ala Val Ser Lys Pro Met Gly Thr Gln Thr

35 40 45

His Thr Met Ile Phe Asp Asn Ala Phe Asn Cys Thr Phe Glu Tyr Ile

50 55 60

Ser Asp Ala Phe Ser Leu Asp Val Ser Glu Lys Ser Gly Asn Phe Lys

65 70 75 80

His Leu Arg Glu Phe Val Phe Lys Asn Lys Asp Gly Phe Leu Tyr Val

85 90 95

Tyr Lys Gly Tyr

100

<210>22

<211>100

<212>PRT

＜213〉sars coronavirus

<400>22

Gln Pro Ile Asp Val Val Arg Asp Leu Pro Ser Gly Phe Asn Thr Leu

1 5 10 15

Lys Pro Ile Phe Lys Leu Pro Leu Gly Ile Asn Ile Thr Asn Phe Arg

20 25 30

Ala Ile Leu Thr Ala Phe Ser Pro Ala Gln Asp Ile Trp Gly Thr Ser

35 40 45

Ala Ala Ala Tyr Phe Val Gly Tyr Leu Lys Pro Thr Thr Phe Met Leu

50 55 60

Lys Tyr Asp Glu Asn Gly Thr Ile Thr Asp Ala Val Asp Cys Ser Gln

65 70 75 80

Asn Pro Leu Ala Glu Leu Lys Cys Ser Val Lys Ser Phe Glu Ile Asp

85 90 95

Lys Gly Ile Tyr

100

<210>23

<211>100

<212>PRT

＜213〉sars coronavirus

<400>23

Gln Thr Ser Asn Phe Arg Val Val Pro Ser Gly Asp Val Val Arg Phe

1 5 10 15

Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr

20 25 30

Lys Phe Pro Ser Val Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn Cys

35 40 45

Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr Phe

50 55 60

Lys Cys Tyr Gly Val Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe Ser

65 70 75 80

Asn Val Tyr Ala Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln

85 90 95

Ile Ala Pro Gly

100

<210>24

<211>100

<212>PRT

＜213〉sars coronavirus

<400>24

Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe

1 5 10 15

Met Gly Cys Val Leu Ala Trp Asn Thr Arg Asn Ile Asp Ala Thr Ser

20 25 30

Thr Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His Gly Lys Leu

35 40 45

Arg Pro Phe Glu Arg Asp Ile Ser Asn Val Pro Phe Ser Pro Asp Gly

50 55 60

Lys Pro Cys Thr Pro Pro Ala Leu Asn Cys Tyr Trp Pro Leu Asn Asp

65 70 75 80

Tyr Gly Phe Tyr Thr Thr Thr Gly Ile Gly Tyr Gln Pro Tyr Arg Val

85 90 95

Val Val Leu Ser

100

<210>25

<211>100

<212>PRT

＜213〉sars coronavirus

<400>25

Phe Glu Leu Leu Asn Ala Pro Ala Thr Val Cys Gly Pro Lys Leu Ser

1 5 10 15

Thr Asp Leu Ile Lys Asn Gln Cys Val Asn Phe Asn Phe Asn Gly Leu

20 25 30

Thr Gly Thr Gly Val Leu Thr Pro Ser Ser Lys Arg Phe Gln Pro Phe

35 40 45

Gln Gln Phe Gly Arg Asp Val Ser Asp Phe Thr Asp Ser Val Arg Asp

50 55 60

Pro Lys Thr Ser Glu Ile Leu Asp Ile Ser Pro Cys Ala Phe Gly Gly

65 70 75 80

Val Ser Val Ile Thr Pro Gly Thr Asn Ala Ser Ser Glu Val Ala Val

85 90 95

Leu Tyr Gln Asp

100

<210>26

<211>100

<212>PRT

＜213〉sars coronavirus

<400>26

Val Asn Cys Thr Asp Val Ser Thr Ala Ile His Ala Asp Gln Leu Thr

1 5 10 15

Pro Ala Trp Arg Ile Tyr Ser Thr Gly Asn Asn Val Phe Gln Thr Gln

20 25 30

Ala Gly Cys Leu Ile Gly Ala Glu His Val Asp Thr Ser Tyr Glu Cys

35 40 45

Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala Ser Tyr His Thr Val Ser

50 55 60

Leu Leu Arg Ser Thr Ser Gln Lys Ser Ile Val Ala Tyr Thr Met Ser

65 70 75 80

Leu Gly Ala Asp Ser Ser Ile Ala Tyr Ser Asn Asn Thr Ile Ala Ile

85 90 95

Pro Thr Asn Phe

100

<210>27

<211>100

<212>PRT

＜213〉sars coronavirus

<400>27

Ser Ile Ser Ile Thr Thr Glu Val Met Pro Val Ser Met Ala Lys Thr

1 5 10 15

Ser Val Asp Cys Asn Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ala

20 25 30

Asn Leu Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala

35 40 45

Leu Ser Gly Ile Ala Ala Glu Gln Asp Arg Asn Thr Arg Glu Val Phe

50 55 60

Ala Gln Val Lys Gln Met Tyr Lys Thr Pro Thr Leu Lys Tyr Phe Gly

65 70 75 80

Gly Phe Asn Phe Ser Gln Ile Leu Pro Asp Pro Leu Lys Pro Thr Lys

85 90 95

Arg Ser Phe Ile

100

<210>28

<211>100

<212>PRT

＜213〉sars coronavirus

<400>28

Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly Phe Met

1 5 10 15

Lys Gln Tyr Gly Glu Cys Leu Gly Asp Ile Asn Ala Arg Asp Leu Ile

20 25 30

Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu Leu Thr

35 40 45

Asp Asp Met Ile Ala Ala Tyr Thr Ala Ala Leu Val Ser Gly Thr Ala

50 55 60

Thr Ala Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile Pro Phe

65 70 75 80

Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr Gln Asn

85 90 95

Val Leu Tyr Glu

100

<210>29

<211>100

<212>PRT

＜213〉sars coronavirus

<400>29

Asn Gln Lys Gln Ile Ala Asn Gln Phe Asn Lys Ala Ile Ser Gln Ile

1 5 10 15

Gln Glu Ser Leu Thr Thr Thr Ser Thr Ala Leu Gly Lys Leu Gln Asp

20 25 30

Val Val Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu Val Lys Gln Leu

35 40 45

Ser Ser Asn Phe Gly Ala Ile Ser Ser Val Leu Asn Asp Ile Leu Ser

50 55 60

Arg Leu Asp Lys Val Glu Ala Glu Val Gln Ile Asp Arg Leu Ile Thr

65 70 75 80

Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val Thr Gln Gln Leu Ile Arg

85 90 95

Ala Ala Glu Ile

100

<210>30

<21l>100

<212>PRT

＜213〉sars coronavirus

<400>30

Arg Ala Ser Ala Asn Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu

1 5 10 15

Gly Gln Ser Lys Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met

20 25 30

Ser Phe Pro Gln Ala Ala Pro His Gly Val Val Phe Leu His Val Thr

35 40 45

Tyr Val Pro Ser Gln Glu Arg Asn Phe Thr Thr Ala Pro Ala Ile Cys

50 55 60

His Glu Gly Lys Ala Tyr Phe Pro Arg Glu Gly Val Phe Val Phe Asn

65 70 75 80

Gly Thr Ser Trp Phe Ile Thr Gln Arg Asn Phe Phe Ser Pro Gln Ile

85 90 95

Ile Thr Thr Asp

100

<210>31

<211>90

<212>PRT

＜213〉sars coronavirus

<400>31

Asn Thr Phe Val Ser Gly Asn Cys Asp Val Val Ile Gly Ile Ile Asn

l 5 10 15

Asn Thr Val Tyr Asp Pro Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu

20 25 30

Glu Leu Asp Lys Tyr Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu

35 40 45

Gly Asp Ile Ser Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu

50 55 60

Ile Asp Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile

65 70 75 80

Asp Leu Gln Glu Leu Gly Lys Tyr Glu Gln

85 90

<210>32

<211>200

<212>PRT

＜213〉sars coronavirus

<400>32

Met Phe Ile Phe Leu Leu Phe Leu Thr Leu Thr Ser Gly Ser Asp Leu

1 5 10 15

Asp Arg Cys Thr Thr Phe Asp Asp Val Gln Ala Pro Asn Tyr Thr Gln

20 25 30

His Thr Ser Ser Met Arg Gly Val Tyr Tyr Pro Asp Glu Ile Phe Arg

35 40 45

Ser Asp Thr Leu Tyr Leu Thr Gln Asp Leu Phe Leu Pro Phe Tyr Ser

50 55 60

Asn Val Thr Gly Phe His Thr Ile Asn His Thr Phe Gly Asn Pro Val

65 70 75 80

Ile Pro Phe Lys Asp Gly Ile Tyr Phe Ala Ala Thr Glu Lys Ser Asn

85 90 95

Val Val Arg Gly Trp Val Phe Gly Ser Thr Met Asn Asn Lys Ser Gln

100 105 110

Ser Val Ile Ile Ile Asn Asn Ser Thr Asn Val Val Ile Arg Ala Cys

115 120 125

Asn Phe Glu Leu Cys Asp Asn Pro Phe Phe Ala Val Ser Lys Pro Met

130 135 140

Gly Thr Gln Thr His Thr Met Ile Phe Asp Asn Ala Phe Asn Cys Thr

145 150 155 160

Phe Glu Tyr Ile Ser Asp Ala Phe Ser Leu Asp Val Ser Glu Lys Ser

165 170 175

Gly Asn Phe Lys His Leu Arg Glu Phe Val Phe Lys Asn Lys Asp Gly

180 185 190

Phe Leu Tyr Val Tyr Lys Gly Tyr

195 200

<210>33

<211>200

<212>PRT

＜213〉sars coronavirus

<400>33

Gln Pro Ile Asp Val Val Arg Asp Leu Pro Ser Gly Phe Asn Thr Leu

1 5 10 15

Lys Pro Ile Phe Lys Leu Pro Leu Gly Ile Asn Ile Thr Asn Phe Arg

20 25 30

Ala Ile Leu Thr Ala Phe Ser Pro Ala Gln Asp Ile Trp Gly Thr Ser

35 40 45

Ala Ala Ala Tyr Phe Val Gly Tyr Leu Lys Pro Thr Thr Phe Met Leu

50 55 60

Lys Tyr Asp Glu Asn Gly Thr Ile Thr Asp Ala Val Asp Cys Ser Gln

65 70 75 80

Asn Pro Leu Ala Glu Leu Lys Cys Ser Val Lys Ser Phe Glu Ile Asp

85 90 95

Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val Val Pro Ser Gly Asp

100 105 110

Val Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val

115 120 125

Phe Asn Ala Thr Lys Phe Pro Ser Val Tyr Ala Trp Glu Arg Lys Lys

130 135 140

Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Thr Phe

145 150 155 160

Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Ala Thr Lys Leu Asn Asp

165 170 175

Leu Cys Phe Ser Asn Val Tyr Ala Asp Ser Phe Val Val Lys Gly Asp

180 185 190

Asp Val Arg Gln IleAla Pro Gly

195 200

<210>34

<211>200

<212>PRT

＜213〉sars coronavirus

<400>34

Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe

1 5 10 15

Met Gly Cys Val Leu Ala Trp Asn Thr Arg Asn Ile Asp Ala Thr Ser

20 25 30

Thr Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His Gly Lys Leu

35 40 45

Arg Pro Phe Glu Arg Asp Ile Ser Asn Val Pro Phe Ser Pro Asp Gly

50 55 60

Lys Pro Cys Thr Pro Pro Ala Leu Asn Cys Tyr Trp Pro Leu Asn Asp

65 70 75 80

Tyr Gly Phe Tyr Thr Thr Thr Gly Ile Gly Tyr Gln Pro Tyr Arg Val

85 90 95

Val Val Leu Ser Phe Glu Leu Leu Asn Ala Pro Ala Thr Val Cys Gly

100 105 110

Pro Lys Leu Ser Thr Asp Leu Ile Lys Asn Gln Cys Val Asn Phe Asn

115 120 125

Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Pro Ser Ser Lys Arg

130 135 140

Phe Gln Pro Phe Gln Gln Phe Gly Arg Asp Val Ser Asp Phe Thr Asp

145 150 155 160

Ser Val Arg Asp Pro Lys Thr Ser Glu Ile Leu Asp Ile Ser Pro Cys

165 170 175

Ala Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Ala Ser Ser

180 185 190

Glu Val Ala Val Leu Tyr Gln Asp

195 200

<210>35

<211>200

<212>PRT

＜213〉sars coronavirus

<400>35

Val Asn Cys Thr Asp Val Ser Thr Ala Ile His Ala Asp Gln Leu Thr

1 5 10 15

Pro Ala Trp Arg Ile Tyr Ser Thr Gly Asn Asn Val Phe Gln Thr Gln

20 25 30

Ala Gly Cys Leu Ile Gly Ala Glu His Val Asp Thr Ser Tyr Glu Cys

35 40 45

Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala Ser Tyr His Thr Val Ser

50 55 60

Leu Leu Arg Ser Thr Ser Gln Lys Ser Ile Val Ala Tyr Thr Met Ser

65 70 75 80

Leu Gly Ala Asp Ser Ser Ile Ala Tyr Ser Asn Asn Thr Ile Ala Ile

85 90 95

Pro Thr Asn Phe Ser Ile Ser Ile Thr Thr Glu Val Met Pro Val Ser

100 105 110

Met Ala Lys Thr Ser Val Asp Cys Asn Met Tyr Ile Cys Gly Asp Ser

115 120 125

Thr Glu Cys Ala Asn Leu Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln

130 135 140

Leu Asn Arg Ala Leu Ser Gly Ile Ala Ala Glu Gln Asp Arg Asn Thr

145 150 155 160

Arg Glu Val Phe Ala Gln Val Lys Gln Met Tyr Lys Thr Pro Thr Leu

165 170 175

Lys Tyr Phe Gly Gly Phe Asn Phe Ser Gln Ile Leu Pro Asp Pro Leu

180 185 190

Lys Pro Thr Lys Arg Ser Phe Ile

195 200

<210>36

<211>200

<212>PRT

＜213〉sars coronavirus

<400>36

Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly Phe Met

1 5 10 15

Lys Gln Tyr Gly Glu Cys Leu Gly Asp Ile Asn Ala Arg Asp Leu Ile

20 25 30

Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu Leu Thr

35 40 45

Asp Asp Met Ile Ala Ala Tyr Thr Ala Ala Leu Val Ser Gly Thr Ala

50 55 60

Thr Ala Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile Pro Phe

65 70 75 80

Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr Gln Asn

85 90 95

Val Leu Tyr Glu Asn Gln Lys Gln Ile Ala Asn Gln Phe Asn Lys Ala

100 105 110

Ile Ser Gln Ile Gln Glu Ser Leu Thr Thr Thr Ser Thr Ala Leu Gly

115 120 125

Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu

130 135 140

Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val Leu Asn

145 150 155 160

Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln Ile Asp

165 170 175

Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val Thr Gln

180 185 190

Gln Leu Ile Arg Ala Ala Glu Ile

195 200

<210>37

<211>190

<212>PRT

＜213〉sars coronavirus

<400>37

Arg Ala Ser Ala Asn Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu

1 5 10 15

Gly Gln Ser Lys Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met

20 25 30

Ser Phe Pro Gln Ala Ala Pro His Gly Val Val Phe Leu His Val Thr

35 40 45

Tyr Val Pro Ser Gln Glu Arg Asn Phe Thr Thr Ala Pro Ala Ile Cys

50 55 60

His Glu Gly Lys Ala Tyr Phe Pro Arg Glu Gly Val Phe Val Phe Asn

65 70 75 80

Gly Thr Ser Trp Phe Ile Thr Gln Arg Asn Phe Phe Ser Pro Gln Ile

85 90 95

Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val Val Ile

100 105 110

Gly Ile Ile Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro Glu Leu Asp

115 120 125

Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn His Thr Ser Pro

130 135 140

Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn Ala Ser Val Val Asn

145 150 155 160

Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu Val Ala Lys Asn Leu Asn

165 170 175

Glu Ser Leu Ile Asp Leu Gln Glu Leu Gly Lys Tyr Glu Gln

180 185 190

<210>38

<211>400

<212>PRT

＜213〉sars coronavirus

<400>38

Met Phe Ile Phe Leu Leu Phe Leu Thr Leu Thr Ser Gly Ser Asp Leu

1 5 10 15

Asp Arg Cys Thr Thr Phe Asp Asp Val Gln Ala Pro Asn Tyr Thr Gln

20 25 30

His Thr Ser Ser Met Arg Gly Val Tyr Tyr Pro Asp Glu Ile Phe Arg

35 40 45

Ser Asp Thr Leu Tyr Leu Thr Gln Asp Leu Phe Leu Pro Phe Tyr Ser

50 55 60

Asn Val Thr Gly Phe His Thr Ile Asn His Thr Phe Gly Asn Pro Val

65 70 75 80

Ile Pro Phe Lys Asp Gly Ile Tyr Phe Ala Ala Thr Glu Lys Ser Asn

85 90 95

Val Val Arg Gly Trp Val Phe Gly Ser Thr Met Asn Asn Lys Ser Gln

100 105 110

Ser Val Ile Ile Ile Asn Asn Ser Thr Asn Val Val Ile Arg Ala Cys

115 120 125

Asn Phe Glu Leu Cys Asp Asn Pro Phe Phe Ala Val Ser Lys Pro Met

130 135 140

Gly Thr Gln Thr His Thr Met Ile Phe Asp Asn Ala Phe Asn Cys Thr

145 150 155 160

Phe Glu Tyr Ile Ser Asp Ala Phe Ser Leu Asp Val Ser Glu Lys Ser

165 170 175

Gly Asn Phe Lys His Leu Arg Glu Phe Val Phe Lys Asn Lys Asp Gly

180 185 190

Phe Leu Tyr Val Tyr Lys Gly Tyr Gln Pro Ile Asp Val Val Arg Asp

195 200 205

Leu Pro Ser Gly Phe Asn Thr Leu Lys Pro Ile Phe Lys Leu Pro Leu

210 215 220

Gly Ile Asn Ile Thr Asn Phe Arg Ala Ile Leu Thr Ala Phe Ser Pro

225 230 235 240

Ala Gln Asp Ile Trp Gly Thr Ser Ala Ala Ala Tyr Phe Val Gly Tyr

245 250 255

Leu Lys Pro Thr Thr Phe Met Leu Lys Tyr Asp Glu Asn Gly Thr Ile

260 265 270

Thr Asp Ala Val Asp Cys Ser Gln Asn Pro Leu Ala Glu Leu Lys Cys

275 280 285

Ser Val Lys Ser Phe Glu Ile Asp Lys Gly Ile Tyr Gln Thr Ser Asn

290 295 300

Phe Arg Val Val Pro Ser Gly Asp Val Val Arg Phe Pro Asn Ile Thr

305 310 315 320

Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Lys Phe Pro Ser

325 330 335

Val Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn Cys Val Ala Asp Tyr

340 345 350

Ser Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr Phe Lys Cys Tyr Gly

355 360 365

Val Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe Ser Asn Val Tyr Ala

370 375 380

Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln Ile Ala Pro Gly

385 390 395 400

<210>39

<211>600

<212>PRT

＜213〉sars coronavirus

<400>39

Met Phe Ile Phe Leu Leu Phe Leu Thr Leu Thr Ser Gly Ser Asp Leu

1 5 10 15

Asp Arg Cys Thr Thr Phe Asp Asp Val Gln Ala Pro Asn Tyr Thr Gln

20 25 30

His Thr Ser Ser Met Arg Gly Val Tyr Tyr Pro Asp Glu Ile Phe Arg

35 40 45

Ser Asp Thr Leu Tyr Leu Thr Gln Asp Leu Phe Leu Pro Phe Tyr Ser

50 55 60

Asn Val Thr Gly Phe His Thr Ile Asn His Thr Phe Gly Asn Pro Val

65 70 75 80

Ile Pro Phe Lys Asp Gly Ile Tyr Phe Ala Ala Thr Glu Lys Ser Asn

85 90 95

Val Val Arg Gly Trp Val Phe Gly Ser Thr Met Asn Asn Lys Ser Gln

100 105 110

Ser Val Ile Ile Ile Asn Asn Ser Thr Asn Val Val Ile Arg Ala Cys

115 120 125

Asn Phe Glu Leu Cys Asp Asn Pro Phe Phe Ala Val Ser Lys Pro Met

130 135 140

Gly Thr Gln Thr His Thr Met Ile Phe Asp Asn Ala Phe Asn Cys Thr

145 150 155 160

Phe Glu Tyr Ile Ser Asp Ala Phe Ser Leu Asp Val Ser Glu Lys Ser

165 170 175

Gly Asn Phe Lys His Leu Arg Glu Phe Val Phe Lys Asn Lys Asp Gly

180 185 190

Phe Leu Tyr Val Tyr Lys Gly Tyr Gln Pro Ile Asp Val Val Arg Asp

195 200 205

Leu Pro Ser Gly Phe Asn Thr Leu Lys Pro Ile Phe Lys Leu Pro Leu

210 215 220

Gly Ile Asn Ile Thr Asn Phe Arg Ala Ile Leu Thr Ala Phe Ser Pro

225 230 235 240

Ala Gln Asp Ile Trp Gly Thr Ser Ala Ala Ala Tyr Phe Val Gly Tyr

245 250 255

Leu Lys Pro Thr Thr Phe Met Leu Lys Tyr Asp Glu Asn Gly Thr Ile

260 265 270

Thr Asp Ala Val Asp Cys Ser Gln Asn Pro Leu Ala Glu Leu Lys Cys

275 280 285

Ser Val Lys Ser Phe Glu Ile Asp Lys Gly Ile Tyr Gln Thr Ser Asn

290 295 300

Phe Arg Val Val Pro Ser Gly Asp Val Val Arg Phe Pro Asn Ile Thr

305 310 315 320

Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Lys Phe Pro Ser

325 330 335

Val Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn Cys Val Ala Asp Tyr

340 345 350

Ser Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr Phe Lys Cys Tyr Gly

355 360 365

Val Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe Ser Asn Val Tyr Ala

370 375 380

Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln Ile Ala Pro Gly

385 390 395 400

Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe

405 410 415

Met Gly Cys Val Leu Ala Trp Asn Thr Arg Asn Ile Asp Ala Thr Ser

420 425 430

Thr Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His Gly Lys Leu

435 440 445

Arg Pro Phe Glu Arg Asp Ile Ser Asn Val Pro Phe Ser Pro Asp Gly

450 455 460

Lys Pro Cys Thr Pro Pro Ala Leu Asn Cys Tyr Trp Pro Leu Asn Asp

465 470 475 480

Tyr Gly Phe Tyr Thr Thr Thr Gly Ile Gly Tyr Gln Pro Tyr Arg Val

485 490 495

Val Val Leu Ser Phe Glu Leu Leu Asn Ala Pro Ala Thr Val Cys Gly

500 505 510

Pro Lys Leu Ser Thr Asp Leu Ile Lys Asn Gln Cys Val Asn Phe Asn

515 520 525

Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Pro Ser Ser Lys Arg

530 535 540

Phe Gln Pro Phe Gln Gln Phe Gly Arg Asp Val Ser Asp Phe Thr Asp

545 550 555 560

Ser Val Arg Asp Pro Lys Thr Ser Glu Ile Leu Asp Ile Ser Pro Cys

565 570 575

Ala Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Ala Ser Ser

580 585 590

Glu Val Ala Val Leu Tyr Gln Asp

595 600

<210>40

<211>800

<212>PRT

＜213〉sars coronavirus

<400>40

Met Phe Ile Phe Leu Leu Phe Leu Thr Leu Thr Ser Gly Ser Asp Leu

1 5 10 15

Asp Arg Cys Thr Thr Phe Asp Asp Val Gln Ala Pro Asn Tyr Thr Gln

20 25 30

His Thr Ser Ser Met Arg Gly Val Tyr Tyr Pro Asp Glu Ile Phe Arg

35 40 45

Ser Asp Thr Leu Tyr Leu Thr Gln Asp Leu Phe Leu Pro Phe Tyr Ser

50 55 60

Asn Val Thr Gly Phe His Thr Ile Asn His Thr Phe Gly Asn Pro Val

65 70 75 80

Ile Pro Phe Lys Asp Gly Ile Tyr Phe Ala Ala Thr Glu Lys Ser Asn

85 90 95

Val Val Arg Gly Trp Val Phe Gly Ser Thr Met Asn Asn Lys Ser Gln

100 105 110

Ser Val Ile Ile Ile Asn Asn Ser Thr Asn Val Val Ile Arg Ala Cys

115 120 125

Asn Phe Glu Leu Cys Asp Asn Pro Phe Phe Ala Val Ser Lys Pro Met

130 135 140

Gly Thr Gln Thr His Thr Met Ile Phe Asp Asn Ala Phe Asn Cys Thr

145 150 155 160

Phe Glu Tyr Ile Ser Asp Ala Phe Ser Leu Asp Val Ser Glu Lys Ser

165 170 175

Gly Asn Phe Lys His Leu Arg Glu Phe Val Phe Lys Asn Lys Asp Gly

180 185 190

Phe Leu Tyr Val Tyr Lys Gly Tyr Gln Pro Ile Asp Val Val Arg Asp

195 200 205

Leu Pro Ser Gly Phe Asn Thr Leu Lys Pro Ile Phe Lys Leu Pro Leu

210 215 220

Gly Ile Asn Ile Thr Asn Phe Arg Ala Ile Leu Thr Ala Phe Ser Pro

225 230 235 240

Ala Gln Asp Ile Trp Gly Thr Ser Ala Ala Ala Tyr Phe Val Gly Tyr

245 250 255

Leu Lys Pro Thr Thr Phe Met Leu Lys Tyr Asp Glu Asn Gly Thr Ile

260 265 270

Thr Asp Ala Val Asp Cys Ser Gln Asn Pro Leu Ala Glu Leu Lys Cys

275 280 285

Ser Val Lys Ser Phe Glu Ile Asp Lys Gly Ile Tyr Gln Thr Ser Asn

290 295 300

Phe Arg Val Val Pro Ser Gly Asp Val Val Arg Phe Pro Asn Ile Thr

305 310 315 320

Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Lys Phe Pro Ser

325 330 335

Val Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn Cys Val Ala Asp Tyr

340 345 350

Ser Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr Phe Lys Cys Tyr Gly

355 360 365

Val Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe Ser Asn Val Tyr Ala

370 375 380

Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln Ile Ala Pro Gly

385 390 395 400

Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe

405 410 415

Met Gly Cys Val Leu Ala Trp Asn Thr Arg Asn Ile Asp Ala Thr Ser

420 425 430

Thr Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His Gly Lys Leu

435 440 445

Arg Pro Phe Glu Arg Asp Ile Ser Asn Val Pro Phe Ser Pro Asp Gly

450 455 460

Lys Pro Cys Thr Pro Pro Ala Leu Asn Cys Tyr Trp Pro Leu Asn Asp

465 470 475 480

Tyr Gly Phe Tyr Thr Thr Thr Gly Ile Gly Tyr Gln Pro Tyr Arg Val

485 490 495

Val Val Leu Ser Phe Glu Leu Leu Asn Ala Pro Ala Thr Val Cys Gly

500 505 510

Pro Lys Leu Ser Thr Asp Leu Ile Lys Asn Gln Cys Val Asn Phe Asn

515 520 525

Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Pro Ser Ser Lys Arg

530 535 540

Phe Gln Pro Phe Gln Gln Phe Gly Arg Asp Val Ser Asp Phe Thr Asp

545 550 555 560

Ser Val Arg Asp Pro Lys Thr Ser Glu Ile Leu Asp Ile Ser Pro Cys

565 570 575

Ala Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Ala Ser Ser

580 585 590

Glu Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Asp Val Ser Thr

595 600 605

Ala Ile His Ala Asp Gln Leu Thr Pro Ala Trp Arg Ile Tyr Ser Thr

610 615 620

Gly Asn Asn Val Phe Gln Thr Gln Ala Gly Cys Leu Ile Gly Ala Glu

625 630 635 640

His Val Asp Thr Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile

645 650 655

Cys Ala Ser Tyr His Thr Val Ser Leu Leu Arg Ser Thr Ser Gln Lys

660 665 670

Ser Ile Val Ala Tyr Thr Met Ser Leu Gly Ala Asp Ser Ser Ile Ala

675 680 685

Tyr Ser Asn Asn Thr Ile Ala Ile Pro Thr Asn Phe Ser Ile Ser Ile

690 695 700

Thr Thr Glu Val Met Pro Val Ser Met Ala Lys Thr Ser Val Asp Cys

705 710 715 720

Asn Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ala Asn Leu Leu Leu

725 730 735

Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Ser Gly Ile

740 745 750

Ala Ala Glu Gln Asp Arg Asn Thr Arg Glu Val Phe Ala Gln Val Lys

755 760 765

Gln Met Tyr Lys Thr Pro Thr Leu Lys Tyr Phe Gly Gly Phe Asn Phe

770 775 780

Ser Gln Ile Leu Pro Asp Pro Leu Lys Pro Thr Lys Arg Ser Phe Ile

785 790 795 800

<210>41

<211>1000

<212>PRT

＜213〉sars coronavirus

<400>41

Met Phe Ile Phe Leu Leu Phe Leu Thr Leu Thr Ser Gly Ser Asp Leu

1 5 10 15

Asp Arg Cys Thr Thr Phe Asp Asp Val Gln Ala Pro Asn Tyr Thr Gln

20 25 30

His Thr Ser Ser Met Arg Gly Val Tyr Tyr Pro Asp Glu Ile Phe Arg

35 40 45

Ser Asp Thr Leu Tyr Leu Thr Gln Asp Leu Phe Leu Pro Phe Tyr Ser

50 55 60

Asn Val Thr Gly Phe His Thr Ile Asn His Thr Phe Gly Asn Pro Val

65 70 75 80

Ile Pro Phe Lys Asp Gly Ile Tyr Phe Ala Ala Thr Glu Lys Ser Asn

85 90 95

Val Val Arg Gly Trp Val Phe Gly Ser Thr Met Asn Asn Lys Ser Gln

100 105 110

Ser Val Ile Ile Ile Asn Asn Ser Thr Asn Val Val Ile Arg Ala Cys

115 120 125

Asn Phe Glu Leu Cys Asp Asn Pro Phe Phe Ala Val Ser Lys Pro Met

130 135 140

Gly Thr Gln Thr His Thr Met Ile Phe Asp Asn Ala Phe Asn Cys Thr

145 150 155 160

Phe Glu Tyr Ile Ser Asp Ala Phe Ser Leu Asp Val Ser Glu Lys Ser

165 170 175

Gly Asn Phe Lys His Leu Arg Glu Phe Val Phe Lys Asn Lys Asp Gly

180 185 190

Phe Leu Tyr Val Tyr Lys Gly Tyr Gln Pro Ile Asp Val Val Arg Asp

195 200 205

Leu Pro Ser Gly Phe Asn Thr Leu Lys Pro Ile Phe Lys Leu Pro Leu

210 215 220

Gly Ile Asn Ile Thr Asn Phe Arg Ala Ile Leu Thr Ala Phe Ser Pro

225 230 235 240

Ala Gln Asp Ile Trp Gly Thr Ser Ala Ala Ala Tyr Phe Val Gly Tyr

245 250 255

Leu Lys Pro Thr Thr Phe Met Leu Lys Tyr Asp Glu Asn Gly Thr Ile

260 265 270

Thr Asp Ala Val Asp Cys Ser Gln Asn Pro Leu Ala Glu Leu Lys Cys

275 280 285

Ser Val Lys Ser Phe Glu Ile Asp Lys Gly Ile Tyr Gln Thr Ser Asn

290 295 300

Phe Arg Val Val Pro Ser Gly Asp Val Val Arg Phe Pro AsnIle Thr

305 310 315 320

Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Lys Phe Pro Ser

325 330 335

Val Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn Cys Val Ala Asp Tyr

340 345 350

Ser Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr Phe Lys Cys Tyr Gly

355 360 365

Val Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe Ser Asn Val Tyr Ala

370 375 380

Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln Ile Ala Pro Gly

385 390 395 400

Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe

405 410 415

Met Gly Cys Val Leu Ala Trp Asn Thr Arg Asn Ile Asp Ala Thr Ser

420 425 430

Thr Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His Gly Lys Leu

435 440 445

Arg Pro Phe Glu Arg Asp Ile Ser Asn Val Pro Phe Ser Pro Asp Gly

450 455 460

Lys Pro Cys Thr Pro Pro Ala Leu Asn Cys Tyr Trp Pro Leu Asn Asp

465 470 475 480

Tyr Gly Phe Tyr Thr Thr Thr Gly Ile Gly Tyr Gln Pro Tyr Arg Val

485 490 495

Val Val Leu Ser Phe Glu Leu Leu Asn Ala Pro Ala Thr Val Cys Gly

500 505 510

Pro Lys Leu Ser Thr Asp Leu Ile Lys Asn Gln Cys Val Asn Phe Asn

515 520 525

Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Pro Ser Ser Lys Arg

530 535 540

Phe Gln Pro Phe Gln Gln Phe Gly Arg Asp Val Ser Asp Phe Thr Asp

545 550 555 560

Ser Val Arg Asp Pro Lys Thr Ser Glu Ile Leu Asp Ile Ser Pro Cys

565 570 575

Ala Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Ala Ser Ser

580 585 590

Glu Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Asp Val Ser Thr

595 600 605

Ala Ile His Ala Asp Gln Leu Thr Pro Ala Trp Arg Ile Tyr Ser Thr

610 615 620

Gly Asn Asn Val Phe Gln Thr Gln Ala Gly Cys Leu Ile Gly Ala Glu

625 630 635 640

His Val Asp Thr Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile

645 650 655

Cys Ala Ser Tyr His Thr Val Ser Leu Leu Arg Ser Thr Ser Gln Lys

660 665 670

Ser Ile Val Ala Tyr Thr Met Ser Leu Gly Ala Asp Ser Ser Ile Ala

675 680 685

Tyr Ser Asn Asn Thr Ile Ala Ile Pro Thr Asn Phe Ser Ile Ser Ile

690 695 700

Thr Thr Glu Val Met Pro Val Ser Met Ala Lys Thr Ser Val Asp Cys

705 710 715 720

Asn Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ala Asn Leu Leu Leu

725 730 735

Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Ser Gly Ile

740 745 750

Ala Ala Glu Gln Asp Arg Asn Thr Arg Glu Val Phe Ala Gln Val Lys

755 760 765

Gln Met Tyr Lys Thr Pro Thr Leu Lys Tyr Phe Gly Gly Phe Asn Phe

770 775 780

Ser Gln Ile Leu Pro Asp Pro Leu Lys Pro Thr Lys Arg Ser Phe Ile

785 790 795 800

Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly Phe Met

805 810 815

Lys Gln Tyr Gly Glu Cys Leu Gly Asp Ile Asn Ala Arg Asp Leu Ile

820 825 830

Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu Leu Thr

835 840 845

Asp Asp Met Ile Ala Ala Tyr Thr Ala Ala Leu Val Ser Gly Thr Ala

850 855 860

Thr Ala Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile Pro Phe

865 870 875 880

Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr Gln Asn

885 890 895

Val Leu Tyr Glu Asn Gln Lys Gln Ile Ala Asn Gln Phe Asn Lys Ala

900 905 910

Ile Ser Gln Ile Gln Glu Ser Leu Thr Thr Thr Ser Thr Ala Leu Gly

915 920 925

Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu

930 935 940

Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val Leu Asn

945 950 955 960

Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln Ile Asp

965 970 975

Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val Thr Gln

980 985 990

Gln Leu Ile Arg Ala Ala Glu Ile

995 1000

<210>42

<211>1190

<212>PRT

＜213〉sars coronavirus

<400>42

Met Phe Ile Phe Leu Leu Phe Leu Thr Leu Thr Ser Gly Ser Asp Leu

1 5 10 15

Asp Arg Cys Thr Thr Phe Asp Asp Val Gln Ala Pro Asn Tyr Thr Gln

20 25 30

His Thr Ser Ser Met Arg Gly Val Tyr Tyr Pro Asp Glu Ile Phe Arg

35 40 45

Ser Asp Thr Leu Tyr Leu Thr Gln Asp Leu Phe Leu Pro Phe Tyr Ser

50 55 60

Asn Val Thr Gly Phe His Thr Ile Asn His Thr Phe Gly Asn Pro Val

65 70 75 80

Ile Pro Phe Lys Asp Gly Ile Tyr Phe Ala Ala Thr Glu Lys Ser Asn

85 90 95

Val Val Arg Gly Trp Val Phe Gly Ser Thr Met Asn Asn Lys Ser Gln

100 105 110

Ser Val Ile Ile Ile Asn Asn Ser Thr Asn Val Val Ile Arg Ala Cys

115 120 125

Asn Phe Glu Leu Cys Asp Asn Pro Phe Phe Ala Val Ser Lys Pro Met

130 135 140

Gly Thr Gln Thr His Thr Met Ile Phe Asp Asn Ala Phe Asn Cys Thr

145 150 155 160

Phe Glu Tyr Ile Ser Asp Ala Phe Ser Leu Asp Val Ser Glu Lys Ser

165 170 175

Gly Asn Phe Lys His Leu Arg Glu Phe Val Phe Lys Asn Lys Asp Gly

180 185 190

Phe Leu Tyr Val Tyr Lys Gly Tyr Gln Pro Ile Asp Val Val Arg Asp

195 200 205

Leu Pro Ser Gly Phe Asn Thr Leu Lys Pro Ile Phe Lys Leu Pro Leu

210 215 220

Gly Ile Asn Ile Thr Asn Phe Arg Ala Ile Leu Thr Ala Phe Ser Pro

225 230 235 240

Ala Gln Asp Ile Trp Gly Thr Ser Ala Ala Ala Tyr Phe Val Gly Tyr

245 250 255

Leu Lys Pro Thr Thr Phe Met Leu Lys Tyr Asp Glu Asn Gly Thr Ile

260 265 270

Thr Asp Ala Val Asp Cys Ser Gln Asn Pro Leu Ala Glu Leu Lys Cys

275 280 285

Ser Val Lys Ser Phe Glu Ile Asp Lys Gly Ile Tyr Gln Thr Ser Asn

290 295 300

Phe Arg Val Val Pro Ser Gly Asp Val Val Arg Phe Pro Asn Ile Thr

305 310 315 320

Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Lys Phe Pro Ser

325 330 335

Val Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn Cys Val Ala Asp Tyr

340 345 350

Ser Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr Phe Lys Cys Tyr Gly

355 360 365

Val Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe Ser Asn Val Tyr Ala

370 375 380

Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln Ile Ala Pro Gly

385 390 395 400

Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe

405 410 415

Met Gly Cys Val Leu Ala Trp Asn Thr Arg Asn Ile Asp Ala Thr Ser

420 425 430

Thr Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His Gly Lys Leu

435 440 445

Arg Pro Phe Glu Arg Asp Ile Ser Asn Val Pro Phe Ser Pro Asp Gly

450 455 460

Lys Pro Cys Thr Pro Pro Ala Leu Asn Cys Tyr Trp Pro Leu Asn Asp

465 470 475 480

Tyr Gly Phe Tyr Thr Thr Thr Gly Ile Gly Tyr Gln Pro Tyr Arg Val

485 490 495

Val Val Leu Ser Phe Glu Leu Leu Asn Ala Pro Ala Thr Val Cys Gly

500 505 510

Pro Lys Leu Ser Thr Asp Leu Ile Lys Asn Gln Cys Val Asn Phe Asn

515 520 525

Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Pro Ser Ser Lys Arg

530 535 540

Phe Gln Pro Phe Gln Gln Phe Gly Arg Asp Val Ser Asp Phe Thr Asp

545 550 555 560

Ser Val Arg Asp Pro Lys Thr Ser Glu Ile Leu Asp Ile Ser Pro Cys

565 570 575

Ala Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Ala Ser Ser

580 585 590

Glu Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Asp Val Ser Thr

595 600 605

Ala Ile His Ala Asp Gln Leu Thr Pro Ala Trp Arg Ile Tyr Ser Thr

610 615 620

Gly Asn Asn Val Phe Gln Thr Gln Ala Gly Cys Leu Ile Gly Ala Glu

625 630 635 640

His Val Asp Thr Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile

645 650 655

Cys Ala Ser Tyr His Thr Val Ser Leu Leu Arg Ser Thr Ser Gln Lys

660 665 670

Ser Ile Val Ala Tyr Thr Met Ser Leu Gly Ala Asp Ser Ser Ile Ala

675 680 685

Tyr Ser Asn Asn Thr Ile Ala Ile Pro Thr Asn Phe Ser Ile Ser Ile

690 695 700

Thr Thr Glu Val Met Pro Val Ser Met Ala Lys Thr Ser Val Asp Cys

705 710 715 720

Asn Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ala Asn Leu Leu Leu

725 730 735

Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Ser Gly Ile

740 745 750

Ala Ala Glu Gln Asp Arg Asn Thr Arg Glu Val Phe Ala Gln Val Lys

755 760 765

Gln Met Tyr Lys Thr Pro Thr Leu Lys Tyr Phe Gly Gly Phe Asn Phe

770 775 780

Ser Gln Ile Leu Pro Asp Pro Leu Lys Pro Thr Lys Arg Ser Phe Ile

785 790 795 800

Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly Phe Met

805 810 815

Lys Gln Tyr Gly Glu Cys Leu Gly Asp Ile Asn Ala Arg Asp Leu Ile

820 825 830

Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu Leu Thr

835 840 845

Asp Asp Met Ile Ala Ala Tyr Thr Ala Ala Leu Val Ser Gly Thr Ala

850 855 860

Thr Ala Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile Pro Phe

865 870 875 880

Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr Gln Asn

885 890 895

Val Leu Tyr Glu Asn Gln Lys Gln Ile Ala Asn Gln Phe Asn Lys Ala

900 905 910

Ile Ser Gln Ile Gln Glu Ser Leu Thr Thr Thr Ser Thr Ala Leu Gly

915 920 925

Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu

930 935 940

Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val Leu Asn

945 950 955 960

Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln Ile Asp

965 970 975

Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val Thr Gln

980 985 990

Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala

995 1000 1005

Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Val Asp Phe

1010 1015 1020

Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ala Ala Pro His

1025 1030 1035 1040

Gly Val Val Phe Leu His Val Thr Tyr Val Pro Ser Gln Glu Arg Asn

1045 1050 1055

Phe Thr Thr Ala Pro Ala Ile Cys His Glu Gly Lys Ala Tyr Phe Pro

1060 1065 1070

Arg Glu Gly Val Phe Val Phe Asn Gly Thr Ser Trp Phe Ile Thr Gln

1075 1080 1085

Arg Asn Phe Phe Ser Pro Gln Ile Ile Thr Thr Asp Asn Thr Phe Val

1090 1095 1100

Ser Gly Asn Cys Asp Val Val Ile Gly Ile Ile Asn Asn Thr Val Tyr

1105 1110 1115 1120

Asp Pro Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys

1125 1130 1135

Tyr Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser

1140 1145 1150

Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu

1155 1160 1165

Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu

1170 1175 1180

Leu Gly Lys Tyr Glu Gln

1185 1190

<210>43

<211>84

<212>PRT

＜213〉sars coronavirus

<400>43

Asp Arg Cys Thr Thr Phe Asp Asp Val Gln Ala Pro Asn Tyr Thr Gln

1 5 10 15

His Thr Ser Ser Met Arg Gly Val Tyr Tyr Pro Asp Glu Ile Phe Arg

20 25 30

Ser Asp Thr Leu Tyr Leu Thr Gln Asp Leu Phe Leu Pro Phe Tyr Ser

35 40 45

Asn Val Thr Gly Phe His Thr Ile Asn His Thr Phe Gly Asn Pro Val

50 55 60

Ile Pro Phe Lys Asp Gly Ile Tyr Phe Ala Ala Thr Glu Lys Ser Asn

65 70 75 80

Val Val Arg Gly

<210>44

<211>184

<212>PRT

＜213〉sars coronavirus

<400>44

Asp Arg Cys Thr Thr Phe Asp Asp Val Gln Ala Pro Asn Tyr Thr Gln

1 5 10 15

His Thr Ser Ser Met Arg Gly Val Tyr Tyr Pro Asp Glu Ile Phe Arg

20 25 30

Ser Asp Thr Leu Tyr Leu Thr Gln Asp Leu Phe Leu Pro Phe Tyr Ser

35 40 45

Asn Val Thr Gly Phe His Thr Ile Asn His Thr Phe Gly Asn Pro Val

50 55 60

Ile Pro Phe Lys Asp Gly Ile Tyr Phe Ala Ala Thr Glu Lys Ser Asn

65 70 75 80

Val Val Arg Gly Trp Val Phe Gly Ser Thr Met Asn Asn Lys Ser Gln

85 90 95

Ser Val Ile Ile Ile Asn Asn Ser Thr Asn Val Val Ile Arg Ala Cys

100 105 110

Asn Phe Glu Leu Cys Asp Asn Pro Phe Phe Ala Val Ser Lys Pro Met

115 120 125

Gly Thr Gln Thr His Thr Met Ile Phe Asp Asn Ala Phe Asn Cys Thr

130 135 140

Phe Glu Tyr Ile Ser Asp Ala Phe Ser Leu Asp Val Ser Glu Lys Ser

145 150 155 160

Gly Asn Phe Lys His Leu Arg Glu Phe Val Phe Lys Asn Lys Asp Gly

165 170 175

Phe Leu Tyr Val Tyr Lys Gly Tyr

180

<210>45

<211>384

<212>PRT

＜213〉sars coronavirus

<400>45

Asp Arg Cys Thr Thr Phe Asp Asp Val Gln Ala Pro Asn Tyr Thr Gln

1 5 10 15

His Thr Ser Ser Met Arg Gly Val Tyr Tyr Pro Asp Glu Ile Phe Arg

20 25 30

Ser Asp Thr Leu Tyr Leu Thr Gln Asp Leu Phe Leu Pro Phe Tyr Ser

35 40 45

Asn Val Thr Gly Phe His Thr Ile Asn His Thr Phe Gly Asn Pro Val

50 55 60

Ile Pro Phe Lys Asp Gly Ile Tyr Phe Ala Ala Thr Glu Lys Ser Asn

65 70 75 80

Val Val Arg Gly Trp Val Phe Gly Ser Thr Met Asn Asn Lys Ser Gln

85 90 95

Ser Val Ile Ile Ile Asn Asn Ser Thr Asn Val Val Ile Arg Ala Cys

100 105 110

Asn Phe Glu Leu Cys Asp Asn Pro Phe Phe Ala Val Ser Lys Pro Met

115 120 125

Gly Thr Gln Thr His Thr Met Ile Phe Asp Asn Ala Phe Asn Cys Thr

130 135 140

Phe Glu Tyr Ile Ser Asp Ala Phe Ser Leu Asp Val Ser Glu Lys Ser

145 150 155 160

Gly Asn Phe Lys His Leu Arg Glu Phe Val Phe Lys Asn Lys Asp Gly

165 170 175

Phe Leu Tyr Val Tyr Lys Gly Tyr Gln Pro Ile Asp Val Val Arg Asp

180 185 190

Leu Pro Ser Gly Phe Asn Thr Leu Lys Pro Ile Phe Lys Leu Pro Leu

195 200 205

Gly Ile Asn Ile Thr Asn Phe Arg Ala Ile Leu Thr Ala Phe Ser Pro

210 215 220

Ala Gln Asp Ile Trp Gly Thr Ser Ala Ala Ala Tyr Phe Val Gly Tyr

225 230 235 240

Leu Lys Pro Thr Thr Phe Met Leu Lys Tyr Asp Glu Asn Gly Thr Ile

245 250 255

Thr Asp Ala Val Asp Cys Ser Gln Asn Pro Leu Ala Glu Leu Lys Cys

260 265 270

Ser Val Lys Ser Phe Glu Ile Asp Lys Gly Ile Tyr Gln Thr Ser Asn

275 280 285

Phe Arg Val Val Pro Ser Gly Asp Val Val Arg Phe Pro Asn Ile Thr

290 295 300

Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Lys Phe Pro Ser

305 310 315 320

Val Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn Cys Val Ala Asp Tyr

325 330 335

Ser Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr Phe Lys Cys Tyr Gly

340 345 350

Val Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe Ser Asn Val Tyr Ala

355 360 365

Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln Ile Ala Pro Gly

370 375 380

<210>46

<211>584

<212>PRT

＜213〉sars coronavirus

<400>46

Asp Arg Cys Thr Thr Phe Asp Asp Val Gln Ala Pro Asn Tyr Thr Gln

1 5 10 15

His Thr Ser Ser Met Arg Gly Val Tyr Tyr Pro Asp Glu Ile Phe Arg

20 25 30

Ser Asp Thr Leu Tyr Leu Thr Gln Asp Leu Phe Leu Pro Phe Tyr Ser

35 40 45

Asn Val Thr Gly Phe His Thr Ile Asn His Thr Phe Gly Asn Pro Val

50 55 60

Ile Pro Phe Lys Asp Gly Ile Tyr Phe Ala Ala Thr Glu Lys Ser Asn

65 70 75 80

Val Val Arg Gly Trp Val Phe Gly Ser Thr Met Asn Asn Lys Ser Gln

85 90 95

Ser Val Ile Ile Ile Asn Asn Ser Thr Asn Val Val Ile Arg Ala Cys

100 105 110

Asn Phe Glu Leu Cys Asp Asn Pro Phe Phe Ala Val Ser Lys Pro Met

115 120 125

Gly Thr Gln Thr His Thr Met Ile Phe Asp Asn Ala Phe Asn Cys Thr

130 135 140

Phe Glu Tyr Ile Ser Asp Ala Phe Ser Leu Asp Val Ser Glu Lys Ser

145 150 155 160

Gly Asn Phe Lys His Leu Arg Glu Phe Val Phe Lys Asn Lys Asp Gly

165 170 175

Phe Leu Tyr Val Tyr Lys Gly Tyr Gln Pro Ile Asp Val Val Arg Asp

180 185 190

Leu Pro Ser Gly Phe Asn Thr Leu Lys Pro Ile Phe Lys Leu Pro Leu

195 200 205

Gly Ile Asn Ile Thr Asn Phe Arg Ala Ile Leu Thr Ala Phe Ser Pro

210 215 220

Ala Gln Asp Ile Trp Gly Thr Ser Ala Ala Ala Tyr Phe Val Gly Tyr

225 230 235 240

Leu Lys Pro Thr Thr Phe Met Leu Lys Tyr Asp Glu Asn Gly Thr Ile

245 250 255

Thr Asp Ala Val Asp Cys Ser Gln Asn Pro Leu Ala Glu Leu Lys Cys

260 265 270

Ser Val Lys Ser Phe Glu Ile Asp Lys Gly Ile Tyr Gln Thr Ser Asn

275 280 285

Phe Arg Val Val Pro Ser Gly Asp Val Val Arg Phe Pro Asn Ile Thr

290 295 300

Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Lys Phe Pro Ser

305 310 315 320

Val Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn Cys Val Ala Asp Tyr

325 330 335

Ser Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr Phe Lys Cys Tyr Gly

340 345 350

Val Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe Ser Asn Val Tyr Ala

355 360 365

Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln Ile Ala Pro Gly

370 375 380

Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe

385 390 395 400

Met Gly Cys Val Leu Ala Trp Asn Thr Arg Asn Ile Asp Ala Thr Ser

405 410 415

Thr Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His Gly Lys Leu

420 425 430

Arg Pro Phe Glu Arg Asp Ile Ser Asn Val Pro Phe Ser Pro Asp Gly

435 440 445

Lys Pro Cys Thr Pro Pro Ala Leu Asn Cys Tyr Trp Pro Leu Asn Asp

450 455 460

Tyr Gly Phe Tyr Thr Thr Thr Gly Ile Gly Tyr Gln Pro Tyr Arg Val

465 470 475 480

Val Val Leu Ser Phe Glu Leu Leu Asn Ala Pro Ala Thr Val Cys Gly

485 490 495

Pro Lys Leu Ser Thr Asp Leu Ile Lys Asn Gln Cys Val Asn Phe Asn

500 505 510

Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Pro Ser Ser Lys Arg

515 520 525

Phe Gln Pro Phe Gln Gln Phe Gly Arg Asp Val Ser Asp Phe Thr Asp

530 535 540

Ser Val Arg Asp Pro Lys Thr Ser Glu Ile Leu Asp Ile Ser Pro Cys

545 550 555 560

Ala Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Ala Ser Ser

565 570 575

Glu Val Ala Val Leu Tyr Gln Asp

580

<210>47

<211>784

<212>PRT

＜213〉sars coronavirus

<400>47

Asp Arg Cys Thr Thr Phe Asp Asp Val Gln Ala Pro Asn Tyr Thr Gln

1 5 10 15

His Thr Ser Ser Met Arg Gly Val Tyr Tyr Pro Asp Glu Ile Phe Arg

20 25 30

Ser Asp Thr Leu Tyr Leu Thr Gln Asp Leu Phe Leu Pro Phe Tyr Ser

35 40 45

Asn Val Thr Gly Phe His Thr Ile Ash His Thr Phe Gly Asn Pro Val

50 55 60

Ile Pro Phe Lys Asp Gly Ile Tyr Phe Ala Ala Thr Glu Lys Ser Asn

65 70 75 80

Val Val Arg Gly Trp Val Phe Gly Ser Thr Met Asn Asn Lys Ser Gln

85 90 95

Ser Val Ile Ile Ile Asn Asn Ser Thr Asn Val Val Ile Arg Ala Cys

100 105 110

Asn Phe Glu Leu Cys Asp Asn Pro Phe Phe Ala Val Ser Lys Pro Met

115 120 125

Gly Thr Gln Thr His Thr Met Ile Phe Asp Asn Ala Phe Asn Cys Thr

130 135 140

Phe Glu Tyr Ile Ser Asp Ala Phe Ser Leu Asp Val Ser Glu Lys Ser

145 150 155 160

Gly Asn Phe Lys His Leu Arg Glu Phe Val Phe Lys Asn Lys Asp Gly

165 170 175

Phe Leu Tyr Val Tyr Lys Gly Tyr Gln Pro Ile Asp Val Val Arg Asp

180 185 190

Leu Pro Ser Gly Phe Asn Thr Leu Lys Pro Ile Phe Lys Leu Pro Leu

195 200 205

Gly Ile Asn Ile Thr Asn Phe Arg Ala Ile Leu Thr Ala Phe Ser Pro

210 215 220

Ala Gln Asp Ile Trp Gly Thr Ser Ala Ala Ala Tyr Phe Val Gly Tyr

225 230 235 240

Leu Lys Pro Thr Thr Phe Met Leu Lys Tyr Asp Glu Asn Gly Thr Ile

245 250 255

Thr Asp Ala Val Asp Cys Ser Gln Asn Pro Leu Ala Glu Leu Lys Cys

260 265 270

Ser Val Lys Ser Phe Glu Ile Asp Lys Gly Ile Tyr Gln Thr Ser Asn

275 280 285

Phe Arg Val Val Pro Ser Gly Asp Val Val Arg Phe Pro Asn Ile Thr

290 295 300

Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Lys Phe Pro Ser

305 310 315 320

Val Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn Cys Val Ala Asp Tyr

325 330 335

Ser Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr Phe Lys Cys Tyr Gly

340 345 350

Val Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe Ser Asn Val Tyr Ala

355 360 365

Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln Ile Ala Pro Gly

370 375 380

Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe

385 390 395 400

Met Gly Cys Val Leu Ala Trp Asn Thr Arg Asn Ile Asp Ala Thr Ser

405 410 415

Thr Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His Gly Lys Leu

420 425 430

Arg Pro Phe Glu Arg Asp Ile Ser Asn Val Pro Phe Ser Pro Asp Gly

435 440 445

Lys Pro Cys Thr Pro Pro Ala Leu Asn Cys Tyr Trp Pro Leu Asn Asp

450 455 460

Tyr Gly Phe Tyr Thr Thr Thr Gly Ile Gly Tyr Gln Pro Tyr Arg Val

465 470 475 480

Val Val Leu Ser Phe Glu Leu Leu Asn Ala Pro Ala Thr Val Cys Gly

485 490 495

Pro Lys Leu Ser Thr Asp Leu Ile Lys Asn Gln Cys Val Asn Phe Asn

500 505 510

Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Pro Ser Ser Lys Arg

515 520 525

Phe Gln Pro Phe Gln Gln Phe Gly Arg Asp Val Ser Asp Phe Thr Asp

530 535 540

Ser Val Arg Asp Pro Lys Thr Ser Glu Ile Leu Asp Ile Ser Pro Cys

545 550 555 560

Ala Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Ala Ser Ser

565 570 575

Glu Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Asp Val Ser Thr

580 585 590

Ala Ile His Ala Asp Gln Leu Thr Pro Ala Trp Arg Ile Tyr Ser Thr

595 600 605

Gly Asn Asn Val Phe Gln Thr Gln Ala Gly Cys Leu Ile Gly Ala Glu

610 615 620

His Val Asp Thr Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile

625 630 635 640

Cys Ala Ser Tyr His Thr Val Ser Leu Leu Arg Ser Thr Ser Gln Lys

645 650 655

Ser Ile Val Ala Tyr Thr Met Ser Leu Gly Ala Asp Ser Ser Ile Ala

660 665 670

Tyr Ser Asn Asn Thr Ile Ala Ile Pro Thr Asn Phe Ser Ile Ser Ile

675 680 685

Thr Thr Glu Val Met Pro Val Ser Met Ala Lys Thr Ser Val Asp Cys

690 695 700

Ash Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ala Asn Leu Leu Leu

705 710 715 720

Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Ser Gly Ile

725 730 735

Ala Ala Glu Gln Asp Arg Asn Thr Arg Glu Val Phe Ala Gln Val Lys

740 745 750

Gln Met Tyr Lys Thr Pro Thr Leu Lys Tyr Phe Gly Gly Phe Asn Phe

755 760 765

Ser Gln Ile Leu Pro Asp Pro Leu Lys Pro Thr Lys Arg Ser Phe Ile

770 775 780

<210>48

<211>984

<212>PRT

＜213〉sars coronavirus

<400>48

Asp Arg Cys Thr Thr Phe Asp Asp Val Gln Ala Pro Asn Tyr Thr Gln

1 5 10 15

His Thr Ser Ser Met Arg Gly Val Tyr Tyr Pro Asp Glu Ile Phe Arg

20 25 30

Ser Asp Thr Leu Tyr Leu Thr Gln Asp Leu Phe Leu Pro Phe Tyr Ser

35 40 45

Asn Val Thr Gly Phe His Thr Ile Asn His Thr Phe Gly Asn Pro Val

50 55 60

Ile Pro Phe Lys Asp Gly Ile Tyr Phe Ala Ala Thr Glu Lys Ser Asn

65 70 75 80

Val Val Arg Gly Trp Val Phe Gly Ser Thr Met Asn Asn Lys Ser Gln

85 90 95

Ser Val Ile Ile Ile Asn Asn Ser Thr Asn Val Val Ile Arg Ala Cys

100 105 110

Asn Phe Glu Leu Cys Asp Asn Pro Phe Phe Ala Val Ser Lys Pro Met

115 120 125

Gly Thr Gln Thr His Thr Met Ile Phe Asp Asn Ala Phe Asn Cys Thr

130 135 140

Phe Glu Tyr Ile Ser Asp Ala Phe Ser Leu Asp Val Ser Glu Lys Ser

145 150 155 160

Gly Asn Phe Lys His Leu Arg Glu Phe Val Phe Lys Asn Lys Asp Gly

165 170 175

Phe Leu Tyr Val Tyr Lys Gly Tyr Gln Pro Ile Asp Val Val Arg Asp

180 185 190

Leu Pro Ser Gly Phe Asn Thr Leu Lys Pro Ile Phe Lys Leu Pro Leu

195 200 205

Gly Ile Asn Ile Thr Asn Phe Arg Ala Ile Leu Thr Ala Phe Ser Pro

210 215 220

Ala Gln Asp Ile Trp Gly Thr Ser Ala Ala Ala Tyr Phe Val Gly Tyr

225 230 235 240

Leu Lys Pro Thr Thr Phe Met Leu Lys Tyr Asp Glu Asn Gly Thr Ile

245 250 255

Thr Asp Ala Val Asp Cys Ser Gln Asn Pro Leu Ala Glu Leu Lys Cys

260 265 270

Ser Val Lys Ser Phe Glu Ile Asp Lys Gly Ile Tyr Gln Thr Ser Asn

275 280 285

Phe Arg Val Val Pro Ser Gly Asp Val Val Arg Phe Pro Asn Ile Thr

290 295 300

Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Lys Phe Pro Ser

305 310 315 320

Val Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn Cys Val Ala Asp Tyr

325 330 335

Ser Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr Phe Lys Cys Tyr Gly

340 345 350

Val Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe Ser Asn Val Tyr Ala

355 360 365

Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln Ile Ala Pro Gly

370 375 380

Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe

385 390 395 400

Met Gly Cys Val Leu Ala Trp Asn Thr Arg Asn Ile Asp Ala Thr Ser

405 410 415

Thr Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His Gly Lys Leu

420 425 430

Arg Pro Phe Glu Arg Asp Ile Ser Asn Val Pro Phe Ser Pro Asp Gly

435 440 445

Lys Pro Cys Thr Pro Pro Ala Leu Asn Cys Tyr Trp Pro Leu Asn Asp

450 455 460

Tyr Gly Phe Tyr Thr Thr Thr Gly Ile Gly Tyr Gln Pro Tyr Arg Val

465 470 475 480

Val Val Leu Ser Phe Glu Leu Leu Asn Ala Pro Ala Thr Val Cys Gly

485 490 495

Pro Lys Leu Ser Thr Asp Leu Ile Lys Asn Gln Cys Val Asn Phe Asn

500 505 510

Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Pro Ser Ser Lys Arg

515 520 525

Phe Gln Pro Phe Gln Gln Phe Gly Arg Asp Val Ser Asp Phe Thr Asp

530 535 540

Ser Val Arg Asp Pro Lys Thr Ser Glu Ile Leu Asp Ile Ser Pro Cys

545 550 555 560

Ala Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Ala Ser Ser

565 570 575

Glu Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Asp Val Ser Thr

580 585 590

Ala Ile His Ala Asp Gln Leu Thr Pro Ala Trp Arg Ile Tyr Ser Thr

595 600 605

Gly Asn Asn Val Phe Gln Thr Gln Ala Gly Cys Leu Ile Gly Ala Glu

610 615 620

His Val Asp Thr Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile

625 630 635 640

Cys Ala Ser Tyr His Thr Val Ser Leu Leu Arg Ser Thr Ser Gln Lys

645 650 655

Ser Ile Val Ala Tyr Thr Met Ser Leu Gly Ala Asp Ser Ser Ile Ala

660 665 670

Tyr Ser Asn Asn Thr Ile Ala Ile Pro Thr Asn Phe Ser Ile Ser Ile

675 680 685

Thr Thr Glu Val Met Pro Val Ser Met Ala Lys Thr Ser Val Asp Cys

690 695 700

Asn Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ala Asn Leu Leu Leu

705 710 715 720

Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Ser Gly Ile

725 730 735

Ala Ala Glu Gln Asp Arg Asn Thr Arg Glu Val Phe Ala Gln Val Lys

740 745 750

Gln Met Tyr Lys Thr Pro Thr Leu Lys Tyr Phe Gly Gly Phe Asn Phe

755 760 765

Ser Gln Ile Leu Pro Asp Pro Leu Lys Pro Thr Lys Arg Ser Phe Ile

770 775 780

Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly Phe Met

785 790 795 800

Lys Gln Tyr Gly Glu Cys Leu Gly Asp Ile Asn Ala Arg Asp Leu Ile

805 810 815

Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu Leu Thr

820 825 830

Asp Asp Met Ile Ala Ala Tyr Thr Ala Ala Leu Val Ser Gly Thr Ala

835 840 845

Thr Ala Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile Pro Phe

850 855 860

Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr Gln Asn

865 870 875 880

Val Leu Tyr Glu Asn Gln Lys Gln Ile Ala Asn Gln Phe Asn Lys Ala

885 890 895

Ile Ser Gln Ile Gln Glu Ser Leu Thr Thr Thr Ser Thr Ala Leu Gly

900 905 910

Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu

915 920 925

Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val Leu Ash

930 935 940

Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln Ile Asp

945 950 955 960

Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val Thr Gln

965 970 975

Gln Leu Ile Arg Ala Ala Glu Ile

980

<210>49

<211>1174

<212>PRT

＜213〉sars coronavirus

<400>49

Asp Arg Cys Thr Thr Phe Asp Asp Val Gln Ala Pro Asn Tyr Thr Gln

1 5 10 15

His Thr Ser Ser Met Arg Gly Val Tyr Tyr Pro Asp Glu Ile Phe Arg

20 25 30

Ser Asp Thr Leu Tyr Leu Thr Gln Asp Leu Phe Leu Pro Phe Tyr Ser

35 40 45

Asn Val Thr Gly Phe His Thr Ile Asn His Thr Phe Gly Asn Pro Val

50 55 60

Ile Pro Phe Lys Asp Gly Ile Tyr Phe Ala Ala Thr Glu Lys Ser Asn

65 70 75 80

Val Val Arg Gly Trp Val Phe Gly Ser Thr Met Asn Asn Lys Ser Gln

85 90 95

Ser Val Ile Ile Ile Asn Asn Ser Thr Asn Val Val Ile Arg Ala Cys

100 105 110

Asn Phe Glu Leu Cys Asp Asn Pro Phe Phe Ala Val Ser Lys Pro Met

115 120 125

Gly Thr Gln Thr His Thr Met Ile Phe Asp Asn Ala Phe Asn Cys Thr

130 135 140

Phe Glu Tyr Ile Ser Asp Ala Phe Ser Leu Asp Val Ser Glu Lys Ser

145 150 155 160

Gly Asn Phe Lys His Leu Arg Glu Phe Val Phe Lys Asn Lys Asp Gly

165 170 175

Phe Leu Tyr Val Tyr Lys Gly Tyr Gln Pro Ile Asp Val Val Arg Asp

180 185 190

Leu Pro Ser Gly Phe Asn Thr Leu Lys Pro Ile Phe Lys Leu Pro Leu

195 200 205

Gly Ile Asn Ile Thr Asn Phe Arg Ala Ile Leu Thr Ala Phe Ser Pro

210 215 220

Ala Gln Asp Ile Trp Gly Thr Ser Ala Ala Ala Tyr Phe Val Gly Tyr

225 230 235 240

Leu Lys Pro Thr Thr Phe Met Leu Lys Tyr Asp Glu Asn Gly Thr Ile

245 250 255

Thr Asp Ala Val Asp Cys Ser Gln Asn Pro Leu Ala Glu Leu Lys Cys

260 265 270

Ser Val Lys Ser Phe Glu Ile Asp Lys Gly Ile Tyr Gln Thr Ser Asn

275 280 285

Phe Arg Val Val Pro Ser Gly Asp Val Val Arg Phe Pro Asn Ile Thr

290 295 300

Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Lys Phe Pro Ser

305 310 315 320

Val Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn Cys Val Ala Asp Tyr

325 330 335

Ser Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr Phe Lys Cys Tyr Gly

340 345 350

Val Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe Ser Asn Val Tyr Ala

355 360 365

Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln Ile Ala Pro Gly

370 375 380

Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe

385 390 395 400

Met Gly Cys Val Leu Ala Trp Asn Thr Arg Asn Ile Asp Ala Thr Ser

405 410 415

Thr Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His Gly Lys Leu

420 425 430

Arg Pro Phe Glu Arg Asp Ile Ser Asn Val Pro Phe Ser Pro Asp Gly

435 440 445

Lys Pro Cys Thr Pro Pro Ala Leu Asn Cys Tyr Trp Pro Leu Asn Asp

450 455 460

Tyr Gly Phe Tyr Thr Thr Thr Gly Ile Gly Tyr Gln Pro Tyr Arg Val

465 470 475 480

Val Val Leu Ser Phe Glu Leu Leu Asn Ala Pro Ala Thr Val Cys Gly

485 490 495

Pro Lys Leu Ser Thr Asp Leu Ile Lys Asn Gln Cys Val Asn Phe Asn

500 505 5l0

Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Pro Ser Ser Lys Arg

515 520 525

Phe Gln Pro Phe Gln Gln Phe Gly Arg Asp Val Ser Asp Phe Thr Asp

530 535 540

Ser Val Arg Asp Pro Lys Thr Ser Glu Ile Leu Asp Ile Ser Pro Cys

545 550 555 560

Ala Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Ala Ser Ser

565 570 575

Glu Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Asp Val Ser Thr

580 585 590

Ala Ile His Ala Asp Gln Leu Thr Pro Ala Trp Arg Ile Tyr Ser Thr

595 600 605

Gly Asn Asn Val Phe Gln Thr Gln Ala Gly Cys Leu Ile Gly Ala Glu

610 615 620

His Val Asp Thr Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile

625 630 635 640

Cys Ala Ser Tyr His Thr Val Ser Leu Leu Arg Ser Thr Ser Gln Lys

645 650 655

Ser Ile Val Ala Tyr Thr Met Ser Leu Gly Ala Asp Ser Ser Ile Ala

660 665 670

Tyr Ser Asn Asn Thr Ile Ala Ile Pro Thr Asn Phe Ser Ile Ser Ile

675 680 685

Thr Thr Glu Val Met Pro Val Ser Met Ala Lys Thr Ser Val Asp Cys

690 695 700

Asn Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ala Asn Leu Leu Leu

705 710 715 720

Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Ser Gly Ile

725 730 735

Ala Ala Glu Gln Asp Arg Asn Thr Arg Glu Val Phe Ala Gln Val Lys

740 745 750

Gln Met Tyr Lys Thr Pro Thr Leu Lys Tyr Phe Gly Gly Phe Asn Phe

755 760 765

Ser Gln Ile Leu Pro Asp Pro Leu Lys Pro Thr Lys Arg Ser Phe Ile

770 775 780

Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly Phe Met

785 790 795 800

Lys Gln Tyr Gly Glu Cys Leu Gly Asp Ile Asn Ala Arg Asp Leu Ile

805 810 815

Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu Leu Thr

820 825 830

Asp Asp Met Ile Ala Ala Tyr Thr Ala Ala Leu Val Ser Gly Thr Ala

835 840 845

Thr Ala Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile Pro Phe

850 855 860

Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr Gln Asn

865 870 875 880

Val Leu Tyr Glu Asn Gln Lys Gln Ile Ala Asn Gln Phe Asn Lys Ala

885 890 895

Ile Ser Gln Ile Gln Glu Ser Leu Thr Thr Thr Ser Thr Ala Leu Gly

900 905 910

Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu

915 920 925

Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val Leu Asn

930 935 940

Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln Ile Asp

945 950 955 960

Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val Thr Gln

965 970 975

Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala

980 985 990

Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Val Asp Phe

995 1000 1005

Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ala Ala Pro His

1010 1015 1020

Gly Val Val Phe Leu His Val Thr Tyr Val Pro Ser Gln Glu Arg Asn

1025 1030 1035 1040

Phe Thr Thr Ala Pro Ala Ile Cys His Glu Gly Lys Ala Tyr Phe Pro

1045 1050 1055

Arg Glu Gly Val Phe Val Phe Asn Gly Thr Ser Trp Phe Ile Thr Gln

1060 1065 1070

Arg Asn Phe Phe Ser Pro Gln Ile Ile Thr Thr Asp Asn Thr Phe Val

1075 1080 1085

Ser Gly Asn Cys Asp Val Val Ile Gly Ile Ile Asn Asn Thr Val Tyr

1090 1095 1100

Asp Pro Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys

1105 1110 1115 1120

Tyr Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser

1125 1130 1135

Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu

1140 1145 1150

Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu

1155 1160 1165

Leu Gly Lys Tyr Glu Gln

1170

<210>50

<211>260

<212>PRT

＜213〉sars coronavirus

<400>50

Asp Arg Cys Thr Thr Phe Asp Asp Val Gln Ala Pro Asn Tyr Thr Gln

1 5 10 15

His Thr Ser Ser Met Arg Gly Val Tyr Tyr Pro Asp Glu Ile Phe Arg

20 25 30

Ser Asp Thr Leu Tyr Leu Thr Gln Asp Leu Phe Leu Pro Phe Tyr Ser

35 40 45

Asn Val Thr Gly Phe His Thr Ile Asn His Thr Phe Gly Asn Pro Val

50 55 60

Ile Pro Phe Lys Asp Gly Ile Tyr Phe Ala Ala Thr Glu Lys Ser Asn

65 70 75 80

Val Val Arg Gly Trp Val Phe Gly Ser Thr Met Asn Asn Lys Ser Gln

85 90 95

Ser Val Ile Ile Ile Asn Asn Ser Thr Asn Val Val Ile Arg Ala Cys

100 105 110

Asn Phe Glu Leu Cys Asp Asn Pro Phe Phe Ala Val Ser Lys Pro Met

115 120 125

Gly Thr Gln Thr His Thr Met Ile Phe Asp Asn Ala Phe Asn Cys Thr

130 135 140

Phe Glu Tyr Ile Ser Asp Ala Phe Ser Leu Asp Val Ser Glu Lys Ser

145 150 155 160

Gly Asn Phe Lys His Leu Arg Glu Phe Val Phe Lys Asn Lys Asp Gly

165 170 175

Phe Leu Tyr Val Tyr Lys Gly Tyr Gln Pro Ile Asp Val Val Arg Asp

180 185 190

Leu Pro Ser Gly Phe Asn Thr Leu Lys Pro Ile Phe Lys Leu Pro Leu

195 200 205

Gly Ile Asn Ile Thr Asn Phe Arg Ala Ile Leu Thr Ala Phe Ser Pro

210 215 220

Ala Gln Asp Ile Trp Gly Thr Ser Ala Ala Ala Tyr Phe Val Gly Tyr

225 230 235 240

Leu Lys Pro Thr Thr Phe Met Leu Lys Tyr Asp Glu Asn Gly Thr Ile

245 250 255

Thr Asp Ala Val

260

<210>51

<211>430

<212>PRT

＜213〉sars coronavirus

<400>51

Asp Arg Cys Thr Thr Phe Asp Asp Val Gln Ala Pro Asn Tyr Thr Gln

1 5 10 15

His Thr Ser Ser Met Arg Gly Val Tyr Tyr Pro Asp Glu Ile Phe Arg

20 25 30

Ser Asp Thr Leu Tyr Leu Thr Gln Asp Leu Phe Leu Pro Phe Tyr Ser

35 40 45

Asn Val Thr Gly Phe His Thr Ile Asn His Thr Phe Gly Asn Pro Val

50 55 60

Ile Pro Phe Lys Asp Gly Ile Tyr Phe Ala Ala Thr Glu Lys Ser Asn

65 70 75 80

Val Val Arg Gly Trp Val Phe Gly Ser Thr Met Asn Asn Lys Ser Gln

85 90 95

Ser Val Ile Ile Ile Asn Asn Ser Thr Asn Val Val Ile Arg Ala Cys

100 105 110

Asn Phe Glu Leu Cys Asp Asn Pro Phe Phe Ala Val Ser Lys Pro Met

115 120 125

Gly Thr Gln Thr His Thr Met Ile Phe Asp Asn Ala Phe Asn Cys Thr

130 135 140

Phe Glu Tyr Ile Ser Asp Ala Phe Ser Leu Asp Val Ser Glu Lys Ser

145 150 155 160

Gly Asn Phe Lys His Leu Arg Glu Phe Val Phe Lys Asn Lys Asp Gly

165 170 175

Phe Leu Tyr Val Tyr Lys Gly Tyr Gln Pro Ile Asp Val Val Arg Asp

180 185 190

Leu Pro Ser Gly Phe Asn Thr Leu Lys Pro Ile Phe Lys Leu Pro Leu

195 200 205

Gly Ile Asn Ile Thr Asn Phe Arg Ala Ile Leu Thr Ala Phe Ser Pro

210 215 220

Ala Gln Asp Ile Trp Gly Thr Ser Ala Ala Ala Tyr Phe Val Gly Tyr

225 230 235 240

Leu Lys Pro Thr Thr Phe Met Leu Lys Tyr Asp Glu Asn Gly Thr Ile

245 250 255

Thr Asp Ala Val Asp Cys Ser Gln Asn Pro Leu Ala Glu Leu Lys Cys

260 265 270

Ser Val Lys Ser Phe Glu Ile Asp Lys Gly Ile Tyr Gln Thr Ser Asn

275 280 285

Phe Arg Val Val Pro Ser Gly Asp Val Val Arg Phe Pro Asn Ile Thr

290 295 300

Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Lys Phe Pro Ser

305 310 315 320

Val Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn Cys Val Ala Asp Tyr

325 330 335

Ser Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr Phe Lys Cys Tyr Gly

340 345 350

Val Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe Ser Asn Val Tyr Ala

355 360 365

Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln Ile Ala Pro Gly

370 375 380

Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe

385 390 395 400

Met Gly Cys Val Leu Ala Trp Asn Thr Arg Asn Ile Asp Ala Thr Ser

405 410 415

Thr Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His Gly

420 425 430

<210>52

<211>521

<212>PRT

＜213〉sars coronavirus

<400>52

Asp Arg Cys Thr Thr Phe Asp Asp Val Gln Ala Pro Asn Tyr Thr Gln

1 5 10 15

His Thr Ser Ser Met Arg Gly Val Tyr Tyr Pro Asp Glu Ile Phe Arg

20 25 30

Ser Asp Thr Leu Tyr Leu Thr Gln Asp Leu Phe Leu Pro Phe Tyr Ser

35 40 45

Asn Val Thr Gly Phe His Thr Ile Asn His Thr Phe Gly Asn Pro Val

50 55 60

Ile Pro Phe Lys Asp Gly Ile Tyr Phe Ala Ala Thr Glu Lys Ser Asn

65 70 75 80

Val Val Arg Gly Trp Val Phe Gly Ser Thr Met Asn Asn Lys Ser Gln

85 90 95

Ser Val Ile Ile Ile Asn Asn Ser Thr Asn Val Val Ile Arg Ala Cys

100 105 110

Asn Phe Glu Leu Cys Asp Asn Pro Phe Phe Ala Val Ser Lys Pro Met

115 120 125

Gly Thr Gln Thr His Thr Met Ile Phe Asp Asn Ala Phe Asn Cys Thr

130 135 140

Phe Glu Tyr Ile Ser Asp Ala Phe Ser Leu Asp Val Ser Glu Lys Ser

145 150 155 160

Gly Asn Phe Lys His Leu Arg Glu Phe Val Phe Lys Asn Lys Asp Gly

165 170 175

Phe Leu Tyr Val Tyr Lys Gly Tyr Gln Pro Ile Asp Val Val Arg Asp

180 185 190

Leu Pro Ser Gly Phe Asn Thr Leu Lys Pro Ile Phe Lys Leu Pro Leu

195 200 205

Gly Ile Asn Ile Thr Asn Phe Arg Ala Ile Leu Thr Ala Phe Ser Pro

210 215 220

Ala Gln Asp Ile Trp Gly Thr Ser Ala Ala Ala Tyr Phe Val Gly Tyr

225 230 235 240

Leu Lys Pro Thr Thr Phe Met Leu Lys Tyr Asp Glu Asn Gly Thr Ile

245 250 255

Thr Asp Ala Val Asp Cys Ser Gln Asn Pro Leu Ala Glu Leu Lys Cys

260 265 270

Ser Val Lys Ser Phe Glu Ile Asp Lys Gly Ile Tyr Gln Thr Ser Asn

275 280 285

Phe Arg Val Val Pro Ser Gly Asp Val Val Arg Phe Pro Asn Ile Thr

290 295 300

Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Lys Phe Pro Ser

305 310 315 320

Val Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn Cys Val Ala Asp Tyr

325 330 335

Ser Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr Phe Lys Cys Tyr Gly

340 345 350

Val Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe Ser Asn Val Tyr Ala

355 360 365

Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln Ile Ala Pro Gly

370 375 380

Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe

385 390 395 400

Met Gly Cys Val Leu Ala Trp Asn Thr Arg Asn Ile Asp Ala Thr Ser

405 410 415

Thr Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His Gly Lys Leu

420 425 430

Arg Pro Phe Glu Arg Asp Ile Ser Asn Val Pro Phe Ser Pro Asp Gly

435 440 445

Lys Pro Cys Thr Pro Pro Ala Leu Asn Cys Tyr Trp Pro Leu Asn Asp

450 455 460

Tyr Gly Phe Tyr Thr Thr Thr Gly Ile Gly Tyr Gln Pro Tyr Arg Val

465 470 475 480

Val Val Leu Ser Phe Glu Leu Leu Asn Ala Pro Ala Thr Val Cys Gly

485 490 495

Pro Lys Leu Ser Thr Asp Leu Ile Lys Asn Gln Cys Val Asn Phe Asn

500 505 510

Phe Asn Gly Leu Thr Gly Thr Gly Val

515 520

<210>53

<211>777

<212>PRT

＜213〉artificial sequence

<220>

＜223〉the 17-157 amino acids sequence of synthetic SEQ ID NO:1 adds mouse K chain leader sequence N end and myc epitope C end and poly histidine mark thing

<400>53

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Asp Asp Arg Cys Thr Thr Phe Asp Asp Val Gln Ala

20 25 30

Pro Asn Tyr Thr Gln His Thr Ser Ser Met Arg Gly Val Tyr Tyr Pro

35 40 45

Asp Glu Ile Phe Arg Ser Asp Thr Leu Tyr Leu Thr Gln Asp Leu Phe

50 55 60

Leu Pro Phe Tyr Ser Asn Val Thr Gly Phe His Thr Ile Asn His Thr

65 70 75 80

Phe Gly Asn Pro Val Ile Pro Phe Lys Asp Gly Ile Tyr Phe Ala Ala

85 90 95

Thr Glu Lys Ser Asn Val Val Arg Gly Trp Val Phe Gly Ser Thr Met

100 105 110

Asn Asn Lys Ser Gln Ser Val Ile Ile Ile Asn Asn Ser Thr Asn Val

115 120 125

Val Ile Arg Ala Cys Asn Phe Glu Leu Cys Asp Asn Pro Phe Phe Ala

130 135 140

Val Ser Lys Pro Met Gly Thr Gln Thr His Thr Met Ile Phe Asp Asn

145 150 155 160

Ala Phe Asn Cys Thr Phe Glu Tyr Ile Ser Asp Ala Phe Ser Leu Asp

165 170 175

VaI Ser Glu Lys Ser Gly Asn Phe Lys His Leu Arg Glu Phe Val Phe

180 185 190

Lys Asn Lys Asp Gly Phe Leu Tyr Val Tyr Lys Gly Tyr Gln Pro Ile

195 200 205

Asp Val Val Arg Asp Leu Pro Ser Gly Phe Asn Thr Leu Lys Pro Ile

210 215 220

Phe Lys Leu Pro Leu Gly Ile Asn Ile Thr Asn Phe Arg Ala Ile Leu

225 230 235 240

Thr Ala Phe Ser Pro Ala Gln Asp Ile Trp Gly Thr Ser Ala Ala Ala

245 250 255

Tyr Phe Val Gly Tyr Leu Lys Pro Thr Thr Phe Met Leu Lys Tyr Asp

260 265 270

Glu Asn Gly Thr Ile Thr Asp Ala Val Asp Cys Ser Gln Asn Pro Leu

275 280 285

Ala Glu Leu Lys Cys Ser Val Lys Ser Phe Glu Ile Asp Lys Gly Ile

290 295 300

Tyr Gln Thr Ser Asn Phe Arg Val Val Pro Ser Gly Asp Val Val Arg

305 310 315 320

Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala

325 330 335

Thr Lys Phe Pro Ser Val Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn

340 345 350

Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr

355 360 365

Phe Lys Cys Tyr Gly Val Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe

370 375 380

Ser Asn Val Tyr Ala Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg

385 390 395 400

Gln Ile Ala Pro Gly Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys

405 410 415

Leu Pro Asp Asp Phe Met Gly Cys Val Leu Ala Trp Asn Thr Arg Asn

420 425 430

Ile Asp Ala Thr Ser Thr Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu

435 440 445

Arg His Gly Lys Leu Arg Pro Phe Glu Arg Asp Ile Ser Asn Val Pro

450 455 460

Phe Ser Pro Asp Gly Lys Pro Cys Thr Pro Pro Ala Leu Asn Cys Tyr

465 470 475 480

Trp Pro Leu Asn Asp Tyr Gly Phe Tyr Thr Thr Thr Gly Ile Gly Tyr

485 490 495

Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu Asn Ala Pro

500 505 510

Ala Thr Val Cys Gly Pro Lys Leu Ser Thr Asp Leu Ile Lys Asn Gln

515 520 525

Cys Val Asn Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr

530 535 540

Pro Ser Ser Lys Arg Phe Gln Pro Phe Gln Gln Phe Gly Arg Asp Val

545 550 555 560

Ser Asp Phe Thr Asp Ser Val Arg Asp Pro Lys Thr Ser Glu Ile Leu

565 570 575

Asp Ile Ser Pro Cys Ala Phe Gly Gly Val Ser Val Ile Thr Pro Gly

580 585 590

Thr Asn Ala Ser Ser Glu Val Ala Val Leu Tyr Gln Asp Val Asn Cys

595 600 605

Thr Asp Val Ser Thr Ala Ile His Ala Asp Gln Leu Thr Pro Ala Trp

610 615 620

Arg Ile Tyr Ser Thr Gly Asn Asn Val Phe Gln Thr Gln Ala Gly Cys

625 630 635 640

Leu Ile Gly Ala Glu His Val Asp Thr Ser Tyr Glu Cys Asp Ile Pro

645 650 655

Ile Gly Ala Gly Ile Cys Ala Ser Tyr His Thr Val Ser Leu Leu Arg

660 665 670

Ser Thr Ser Gln Lys Ser Ile Val Ala Tyr Thr Met Ser Leu Gly Ala

675 680 685

Asp Ser Ser Ile Ala Tyr Ser Asn Asn Thr Ile Ala Ile Pro Thr Asn

690 695 700

Phe Ser Ile Ser Ile Thr Thr Glu Val Met Pro Val Ser Met Ala Lys

705 710 715 720

Thr Ser Val Asp Cys Asn Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys

725 730 735

Ala Asn Leu Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg

740 745 750

Ala Leu Ser Gly Ile Ala Ala Glu Gln Glu Gln Lys Leu Ile Ser Glu

755 760 765

Glu Asp Leu His His His His His His

770 775

<210>54

<211>297

<212>PRT

＜213〉artificial sequence

<220>

＜223〉the 17-276 amino acids sequence of synthetic SEQ ID NO:1 adds mouse K chain leader sequence N end and myc epitope C end and poly histidine mark thing

<400>54

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Asp Asp Arg Cys Thr Thr Phe Asp Asp Val Gln Ala

20 25 30

Pro Asn Tyr Thr Gln His Thr Ser Ser Met Arg Gly Val Tyr Tyr Pro

35 40 45

Asp Glu Ile Phe Arg Ser Asp Thr Leu Tyr Leu Thr Gln Asp Leu Phe

50 55 60

Leu Pro Phe Tyr Ser Asn Val Thr Gly Phe His Thr Ile Asn His Thr

65 70 75 80

Phe Gly Asn Pro Val Ile Pro Phe Lys Asp Gly Ile Tyr Phe Ala Ala

85 90 95

Thr Glu Lys Ser Asn Val Val Arg Gly Trp Val Phe Gly Ser Thr Met

100 105 110

Asn Asn Lys Ser Gln Ser Val Ile Ile Ile Asn Asn Ser Thr Asn Val

115 120 125

Val Ile Arg Ala Cys Asn Phe Glu Leu Cys Asp Asn Pro Phe Phe Ala

130 135 140

Val Ser Lys Pro Met Gly Thr Gln Thr His Thr Met Ile Phe Asp Asn

145 150 155 160

Ala Phe Asn Cys Thr Phe Glu Tyr Ile Ser Asp Ala Phe Ser Leu Asp

165 170 175

Val Ser Glu Lys Ser Gly Asn Phe Lys His Leu Arg Glu Phe Val Phe

180 185 190

Lys Asn Lys Asp Gly Phe Leu Tyr Val Tyr Lys Gly Tyr Gln Pro Ile

195 200 205

Asp Val Val Arg Asp Leu Pro Ser Gly Phe Asn Thr Leu Lys Pro Ile

210 215 220

Phe Lys Leu Pro Leu Gly Ile Asn Ile Thr Asn Phe Arg Ala Ile Leu

225 230 235 240

Thr Ala Phe Ser Pro Ala Gln Asp Ile Trp Gly Thr Ser Ala Ala Ala

245 250 255

Tyr Phe Val Gly Tyr Leu Lys Pro Thr Thr Phe Met Leu Lys Tyr Asp

260 265 270

Glu Asn Gly Thr Ile Thr Asp Ala Val Glu Gln Lys Leu Ile Ser Glu

275 280 285

Glu Asp Leu His His His His His His

290 295

<210>55

<211>558

<212>PRT

＜213〉artificial sequence

<220>

＜223〉the 17-537 amino acids sequence of synthetic SEQ ID NO:1 adds mouse K chain leader sequence N end and myc epitope C end and poly histidine mark thing

<400>55

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Asp Asp Arg Cys Thr Thr Phe Asp Asp Val Gln Ala

20 25 30

Pro Asn Tyr Thr Gln His Thr Ser Ser Met Arg Gly Val Tyr Tyr Pro

35 40 45

Asp Glu Ile Phe Arg Ser Asp Thr Leu Tyr Leu Thr Gln Asp Leu Phe

50 55 60

Leu Pro Phe Tyr Ser Asn Val Thr Gly Phe His Thr Ile Asn His Thr

65 70 75 80

Phe Gly Asn Pro Val Ile Pro Phe Lys Asp Gly Ile Tyr Phe Ala Ala

85 90 95

Thr Glu Lys Ser Asn Val Val Arg Gly Trp Val Phe Gly Ser Thr Met

100 105 110

Asn Asn Lys Ser Gln Ser Val Ile Ile Ile Asn Asn Ser Thr Asn Val

115 120 125

Val Ile Arg Ala Cys Asn Phe Glu Leu Cys Asp Asn Pro Phe Phe Ala

130 135 140

Val Ser Lys Pro Met Gly Thr Gln Thr His Thr Met Ile Phe Asp Asn

145 150 155 160

Ala Phe Asn Cys Thr Phe Glu Tyr Ile Ser Asp Ala Phe Ser Leu Asp

165 170 175

Val Ser Glu Lys Ser Gly Asn Phe Lys His Leu Arg Glu Phe Val Phe

180 185 190

Lys Asn Lys Asp Gly Phe Leu Tyr Val Tyr Lys Gly Tyr Gln Pro Ile

195 200 205

Asp Val Val Arg Asp Leu Pro Ser Gly Phe Asn Thr Leu Lys Pro Ile

210 215 220

Phe Lys Leu Pro Leu Gly Ile Asn Ile Thr Asn Phe Arg Ala Ile Leu

225 230 235 240

Thr Ala Phe Ser Pro Ala Gln Asp Ile Trp Gly Thr Ser Ala Ala Ala

245 250 255

Tyr Phe Val Gly Tyr Leu Lys Pro Thr Thr Phe Met Leu Lys Tyr Asp

260 265 270

Glu Asn Gly Thr Ile Thr Asp Ala Val Asp Cys Ser Gln Asn Pro Leu

275 280 285

Ala Glu Leu Lys Cys Ser Val Lys Ser Phe Glu Ile Asp Lys Gly Ile

290 295 300

Tyr Gln Thr Ser Asn Phe Arg Val Val Pro Ser Gly Asp Val Val Arg

305 310 315 320

Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala

325 330 335

Thr Lys Phe Pro Ser Val Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn

340 345 350

Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr

355 360 365

Phe Lys Cys Tyr Gly Val Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe

370 375 380

Ser Asn Val Tyr Ala Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg

385 390 395 400

Gln Ile Ala Pro Gly Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys

405 410 415

Leu Pro Asp Asp Phe Met Gly Cys Val Leu Ala Trp Asn Thr Arg Asn

420 425 430

Ile Asp Ala Thr Ser Thr Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu

435 440 445

Arg His Gly Lys Leu Arg Pro Phe Glu Arg Asp Ile Ser Asn Val Pro

450 455 460

Phe Ser Pro Asp Gly Lys Pro Cys Thr Pro Pro Ala Leu Asn Cys Tyr

465 470 475 480

Trp Pro Leu Asn Asp Tyr Gly Phe Tyr Thr Thr Thr Gly Ile Gly Tyr

485 490 495

Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu Asn Ala Pro

500 505 510

Ala Thr Val Cys Gly Pro Lys Leu Ser Thr Asp Leu Ile Lys Asn Gln

515 520 525

Cys Val Asn Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Glu Gln

530 535 540

Lys Leu Ile Ser Glu Glu Asp Leu His His His His His His

545 550 555

<210>56

<211>739

<212>PRT

＜213〉artificial sequence

<220>

＜223〉the 17-756 amino acids sequence of synthetic SEQ ID NO:1, the N end does not have signal peptide

<400>56

Asp Arg Cys Thr Thr Phe Asp Asp Val Gln Ala Pro Asn Tyr Thr Gln

1 5 10 15

His Thr Ser Ser Met Arg Gly Val Tyr Tyr Pro Asp Glu Ile Phe Arg

20 25 30

Ser Asp Thr Leu Tyr Leu Thr Gln Asp Leu Phe Leu Pro Phe Tyr Ser

35 40 45

Asn Val Thr Gly Phe His Thr Ile Asn His Thr Phe Gly Asn Pro Val

50 55 60

Ile Pro Phe Lys Asp Gly Ile Tyr Phe Ala Ala Thr Glu Lys Ser Asn

65 70 75 80

Val Val Arg Gly Trp Val Phe Gly Ser Thr Met Asn Asn Lys Ser Gln

85 90 95

Ser Val Ile Ile Ile Asn Asn Ser Thr Asn Val Val Ile Arg Ala Cys

100 105 110

Asn Phe Glu Leu Cys Asp Asn Pro Phe Phe Ala Val Ser Lys Pro Met

115 120 125

Gly Thr Gln Thr His Thr Met Ile Phe Asp Asn Ala Phe Asn Cys Thr

130 135 140

Phe Glu Tyr Ile Ser Asp Ala Phe Ser Leu Asp Val Ser Glu Lys Ser

145 150 155 160

Gly Asn Phe Lys His Leu Arg Glu Phe Val Phe Lys Asn Lys Asp Gly

165 170 175

Phe Leu Tyr Val Tyr Lys Gly Tyr Gln Pro Ile Asp Val Val Arg Asp

180 185 190

Leu Pro Ser Gly Phe Asn Thr Leu Lys Pro Ile Phe Lys Leu Pro Leu

195 200 205

Gly Ile Asn Ile Thr Asn Phe Arg Ala Ile Leu Thr Ala Phe Ser Pro

210 215 220

Ala Gln Asp Ile Trp Gly Thr Ser Ala Ala Ala Tyr Phe Val Gly Tyr

225 230 235 240

Leu Lys Pro Thr Thr Phe Met Leu Lys Tyr Asp Glu Asn Gly Thr Ile

245 250 255

Thr Asp Ala Val Asp Cys Ser Gln Asn Pro Leu Ala Glu Leu Lys Cys

260 265 270

Ser Val Lys Ser Phe Glu Ile Asp Lys Gly Ile Tyr Gln Thr Ser Asn

275 280 285

Phe Arg Val Val Pro Ser Gly Asp Val Val Arg Phe Pro Asn Ile Thr

290 295 300

Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Lys Phe Pro Ser

305 310 315 320

Val Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn Cys Val Ala Asp Tyr

325 330 335

Ser Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr Phe Lys Cys Tyr Gly

340 345 350

Val Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe Ser Asn Val Tyr Ala

355 360 365

Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln Ile Ala Pro Gly

370 375 380

Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe

385 390 395 400

Met Gly Cys Val Leu Ala Trp Asn Thr Arg Asn Ile Asp Ala Thr Ser

405 410 415

Thr Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His Gly Lys Leu

420 425 430

Arg Pro Phe Glu Arg Asp Ile Ser Asn Val Pro Phe Ser Pro Asp Gly

435 440 445

Lys Pro Cys Thr Pro Pro Ala Leu Asn Cys Tyr Trp Pro Leu Asn Asp

450 455 460

Tyr Gly Phe Tyr Thr Thr Thr Gly Ile Gly Tyr Gln Pro Tyr Arg Val

465 470 475 480

Val Val Leu Ser Phe Glu Leu Leu Asn Ala Pro Ala Thr Val Cys Gly

485 490 495

Pro Lys Leu Ser Thr Asp Leu Ile Lys Asn Gln Cys Val Asn Phe Asn

500 505 510

Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Pro Ser Ser Lys Arg

515 520 525

Phe Gln Pro Phe Gln Gln Phe Gly Arg Asp Val Ser Asp Phe Thr Asp

530 535 540

Ser Val Arg Asp Pro Lys Thr Ser Glu Ile Leu Asp Ile Ser Pro Cys

545 550 555 560

Ala Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Ala Ser Ser

565 570 575

Glu Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Asp Val Ser Thr

580 585 590

Ala Ile His Ala Asp Gln Leu Thr Pro Ala Trp Arg Ile Tyr Ser Thr

595 600 605

Gly Asn Asn Val Phe Gln Thr Gln Ala Gly Cys Leu Ile Gly Ala Glu

610 615 620

His Val Asp Thr Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile

625 630 635 640

Cys Ala Ser Tyr His Thr Val Ser Leu Leu Arg Ser Thr Ser Gln Lys

645 650 655

Ser Ile Val Ala Tyr Thr Met Ser Leu Gly Ala Asp Ser Ser Ile Ala

660 665 670

Tyr Ser Asn Asn Thr Ile Ala Ile Pro Thr Asn Phe Ser Ile Ser Ile

675 680 685

Thr Thr Glu Val Met Pro Val Ser Met Ala Lys Thr Ser Val Asp Cys

690 695 700

Asn Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ala Asn Leu Leu Leu

705 710 715 720

Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Ser Gly Ile

725 730 735

Ala Ala Glu

<210>57

<211>265

<212>PRT

＜213〉artificial sequence

<220>

＜223〉the 272-537 amino acids sequence of synthetic SEQ ID NO:1

<400>57

Ile Thr Asp Ala Val Asp Cys Ser Gln Asn Pro Leu Ala Glu Leu Lys

1 5 10 15

Cys Ser Val Lys Ser Phe Glu Ile Asp Lys Gly Ile Tyr Gln Thr Ser

20 25 30

Asn Phe Arg Val Val Pro Ser Gly Asp Val Val Arg Phe Asn Ile Thr

35 40 45

Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Lys Phe Pro Ser

50 55 60

Val Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn Cys Val Ala Asp Tyr

65 70 75 80

Ser Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr Phe Lys Cys Tyr Gly

85 90 95

Val Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe Ser Asn Val Tyr Ala

100 105 110

Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln Ile Ala Pro Gly

115 120 125

Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe

130 135 140

Met Gly Cys Val Leu Ala Trp Asn Thr Arg Asn Ile Asp Ala Thr Ser

145 150 155 160

Thr Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His Gly Lys Leu

165 170 175

Arg Pro Phe Glu Arg Asp Ile Ser Asn Val Pro Phe Ser Pro Asp Gly

180 185 190

Lys Pro Cys Thr Pro Pro Ala Leu Asn Cys Tyr Trp Pro Leu Asn Asp

195 200 205

Tyr Gly Phe Tyr Thr Thr Thr Gly Ile Gly Tyr Gln Pro Tyr Arg Val

210 215 220

Val Val Leu Ser Phe Glu Leu Leu Asn Ala Pro Ala Thr Val Cys Gly

225 230 235 240

Pro Lys Leu Ser Thr Asp Leu Ile Lys Asn Gln Cys Val Asn Phe Asn

245 250 255

Phe Asn Gly Leu Thr Gly Thr Gly Val

260 265

<210>58

<211>17

<212>PRT

＜213〉sars coronavirus

<400>58

Asp Val Gln Ala Pro Asn Tyr Thr Gln His Thr Ser Ser Met Arg Gly

1 5 10 15

Cys

<210>59

<211>15

<212>PRT

＜213〉sars coronavirus

<400>59

Pro Ser Ser Lys Arg Phe Gln Pro Gln Gln Phe Gly Arg Asp Cys

1 5 10 15

<210>60

<211>16

<212>PRT

＜213〉sars coronavirus

<400>60

Met Phe Ile Phe Leu Leu Phe Leu Thr Leu Thr Ser Gly Ser Asp Leu

1 5 10 15

<210>61

<211>235

<212>PRT

＜213〉artificial sequence

<220>

＜223〉the 303-537 amino acids sequence of synthetic SEQ ID NO:1 contains receptor binding domains

<400>61

Ser Asn Phe Arg Val Val Pro Ser Gly Asp Val Val Arg Phe Pro Asn

1 5 10 15

Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Lys Phe

20 25 30

Pro Ser Val Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn Cys Val Ala

35 40 45

Asp Tyr Ser Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr Phe Lys Cys

50 55 60

Tyr Gly Val Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe Ser Asn Val

65 70 75 80

Tyr Ala Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln Ile Ala

85 90 95

Pro Gly Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp

100 105 110

Asp Phe Met Gly Cys Val Leu Ala Trp Asn Thr Arg Asn Ile Asp Ala

115 120 125

Thr Ser Thr Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His Gly

130 135 140

Lys Leu Arg Pro Phe Glu Arg Asp Ile Ser Asn Val Pro Phe Ser Pro

145 150 155 160

Asp Gly Lys Pro Cys Thr Pro Pro Ala Leu Asn Cys Tyr Trp Pro Leu

165 170 175

Asn Asp Tyr Gly Phe Tyr Thr Thr Thr Gly Ile Gly Tyr Gln Pro Tyr

180 185 190

Arg Val Val Val Leu Ser Phe Glu Leu Leu Asn Ala Pro Ala Thr Val

195 200 205

Cys Gly Pro Lys Leu Ser Thr Asp Leu Ile Lys Asn Gln Cys Val Asn

210 215 220

Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val

225 230 235

<210>62

<211>199

<212>PRT

＜213〉artificial sequence

<220>

＜223〉the 319-517 amino acids sequence of synthetic SEQ ID NO:1 contains receptor binding domains

<400>62

Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn AIa Thr Lys Phe

1 5 10 15

Pro Ser Val Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn Cys Val Ala

20 25 30

Asp Tyr Ser Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr Phe Lys Cys

35 40 45

Tyr Gly Val Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe Ser Asn Val

50 55 60

Tyr Ala Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln Ile Ala

65 70 75 80

Pro Gly Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp

85 90 95

Asp Phe Met Gly Cys Val Leu Ala Trp Asn Thr Arg Asn Ile Asp Ala

100 105 110

Thr Ser Thr Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His Gly

115 120 125

Lys Leu Arg Pro Phe Glu Arg Asp Ile Ser Asn Val Pro Phe Ser Pro

130 135 140

Asp Gly Lys Pro Cys Thr Pro Pro Ala Leu Asn Cys Tyr Trp Pro Leu

145 150 155 160

Asn Asp Tyr Gly Phe Tyr Thr Thr Thr Gly Ile Gly Tyr Gln Pro Tyr

165 170 175

Arg Val Val Val Leu Ser Phe Glu Leu Leu Asn Ala Pro Ala Thr Val

180 185 190

Cys Gly Pro Lys Leu Ser Thr

195

<210>63

<211>200

<212>PRT

＜213〉artificial sequence

<220>

＜223〉the 319-518 amino acids sequence of synthetic SEQ ID NO:1 contains receptor binding domains

<400>63

Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Lys Phe

1 5 10 15

Pro Ser Val Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn Cys Val Ala

20 25 30

Asp Tyr Ser Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr Phe Lys Cys

35 40 45

Tyr Gly Val Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe Ser Asn Val

50 55 60

Tyr Ala Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln Ile Ala

65 70 75 80

Pro Gly Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp

85 90 95

Asp Phe Met Gly Cys Val Leu Ala Trp Asn Thr Arg Asn Ile Asp Ala

100 105 110

Thr Ser Thr Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His Gly

115 120 125

Lys Leu Arg Pro Phe Glu Arg Asp Ile Ser Asn Val Pro Phe Ser Pro

130 135 140

Asp Gly Lys Pro Cys Thr Pro Pro Ala Leu Asn Cys Tyr Trp Pro Leu

145 150 155 160

Asn Asp Tyr Gly Phe Tyr Thr Thr Thr Gly Ile Gly Tyr Gln Pro Tyr

165 170 175

Arg Val Val Val Leu Ser Phe Glu Leu Leu Asn Ala Pro Ala Thr Val

180 185 190

Cys Gly Pro Lys Leu Ser Thr Asp

195 200

<210>64

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<400>64

gatcggatcc ggtacaatca cag 23

<210>65

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<400>65

gatcgggccc gacacactgg ttc 23

Claims (84)

1. the polypeptide fragment of a SEQ ID NO:1 or its conservative property varient, wherein said polypeptide can produce body fluid or cell immune response when being used to inoculate animal.

2. one kind has any one polypeptide among SEQ ID NO:13,14,15,20-59, the 61-63, and wherein said polypeptide can produce body fluid or cell immune response when being used to inoculate animal.

3. one kind has any one polypeptide among the SEQ ID NO:13,14,15,25,34,46,51,52,56,57,58,59,61,62 or 63, and wherein said polypeptide can produce body fluid or cell immune response when being used to inoculate animal.

4. claim 1,2 or 3 polypeptide, wherein said polypeptide is solvable in the aqueous solution.

5. claim 1,2 or 3 polypeptide, wherein said animal is a Mammals.

6. the polypeptide of claim 5, wherein said Mammals is behaved.

7. claim 1,2 or 3 polypeptide, wherein said polypeptide is aminoterminal or carboxyl terminal sealing.

8. coupling protein, described coupling protein contain carrier proteins and with it link coupled have (a) SEQ ID NO:13,14,15,20-59,61-63; (b) peptide fragment of SEQ ID NO:1; Or any one second polypeptide (a) or in the conservative property varient (b).

9. the coupling protein of claim 8, wherein said carrier proteins is solvable in the aqueous solution.

10. the coupling protein of claim 9, wherein said carrier proteins is selected from bovine serum albumin, keyhole  hemocyanin, Protalbinic acid, mice serum albumin, albumin rabbit serum.

11. the coupling protein of claim 8, wherein said coupling protein can produce body fluid or cell immune response when being used to inoculate animal.

12. the coupling protein of claim 11, wherein said animal are Mammals.

13. the coupling protein of claim 12, wherein said Mammals is behaved.

14. containing, an immune peptide, described immune peptide have (a) SEQ ID NO:13,14,15,20-59,61-63; Or (b) any one polypeptide and link coupled arsanilic acid, Sulphanilic Acid, ethanoyl or picryl with it in the fragment of SEQ ID NO:1.

15. the immune peptide of claim 14, wherein said immune peptide can produce body fluid or cell immune response when being used to inoculate animal.

16. the immune peptide of claim 15, wherein said animal are Mammals.

17. the immune peptide of claim 16, wherein said Mammals is behaved.

18. an immune composition, described immune composition comprise adjuvant and have SEQ IDNO:13,14,15,20-59,61-63; Or any one polypeptide in the fragment of SEQ ID NO:1.

19. the immune composition of claim 18, wherein said adjuvant are selected from the bacterium, polysaccharide, mineral oil, Freund's incomplete adjuvant, Freund's complete adjuvant, aluminum phosphate, molysite, zinc salt, calcium salt, acetylize tyrosine, acetylize sugar, the polysaccharide of positively charged ion derivatize, polysaccharide, polyphosphonitrile, biodegradable microspheres, monophosphoryl lipid A and the quil A of anionic derivativeization of aluminium hydroxide, lipid A, deactivation.

20. the immune composition of claim 18, wherein said polypeptide are aminoterminal or carboxyl terminal sealing.

21. the peptide mimics of an aminoacid sequence, described aminoacid sequence have (a) SEQID NO:13,14,15,20-59,61-63; (b) fragment of SEQ ID NO:1, or (a) or in the conservative property varient (b) any one.

22. an immune composition, described immune composition comprise adjuvant and have (a) SEQID NO:13,14,15,20-59,61-63; (b) fragment of SEQ ID NO:1, or the peptide mimics of any one aminoacid sequence (a) or in the conservative property varient (b).

23. the immune composition of claim 22, wherein said adjuvant are selected from the bacterium, polysaccharide, mineral oil, Freund's incomplete adjuvant, Freund's complete adjuvant, aluminum phosphate, molysite, zinc salt, calcium salt, acetylize tyrosine, acetylize sugar, the polysaccharide of positively charged ion derivatize, polysaccharide, polyphosphonitrile, biodegradable microspheres, monophosphoryl lipid A and the quil A of anionic derivativeization of aluminium hydroxide, lipid A, deactivation.

24. a nucleic acid fragment, described nucleic acid fragment coding has (a) SEQ ID NO:13,14,15,20-59,61-63; (b) the segmental peptide of SEQ ID NO:1, or any one polypeptide (a) or in the conservative property varient (b).

25. being connected to coding with comprising being operated property, an expression cassette, described expression cassette have (a) SEQ ID NO:13,14,15,20-59,61-63; (b) the segmental peptide of SEQ ID NO:1, or the promotor of the nucleic acid fragment of any one polypeptide (a) or in the conservative property varient (b).

26. the expression cassette of claim 25, wherein said promotor are constitutive promoter or adjustment type promotor.

27. a nucleic acid construct, described nucleic acid construct contain carrier and coding (a) has any one polypeptide among SEQ ID NO:13,14,15,20-59, the 61-63; (b) the segmental peptide of SEQ IDNO:1; (c) (a) or the nucleic acid fragment of conservative property varient (b); Or the expression cassette of claim 25.

28. the nucleic acid construct of claim 27, wherein said carrier are selected from plasmid, glutinous grain, yeast artificial chromosome, bacterial artificial chromosome, F-factor, virus, expression vector and phasmid.

29. a recombinant virus, described recombinant virus contain virus vector and coding (a) has any one polypeptide among SEQ ID NO:13,14,15,20-59, the 61-63; (b) the segmental peptide of SEQ IDNO:1; (c) (a) or the nucleic acid fragment of conservative property varient (b); Or the expression cassette of claim 25.

30. the recombinant virus of claim 29, wherein said virus vector is selected from vaccinia virus, canary pox virus, adenovirus and simplexvirus.

31. a composition, described composition comprise pharmaceutical carrier and (a) have any one polypeptide among SEQ IDNO:13,14,15,20-59, the 61-63; (b) the segmental peptide of SEQ ID NO:1; Or (c) (a) or conservative property varient (b).

32. the composition of claim 31, wherein said composition is made into to treat the preparation of SARS-CoV.

33. the preparation that the composition of claim 31, wherein said composition are made into to suppress SARS-CoV and mammalian cell fusion or enter mammalian cell.

34. a composition, described composition comprise pharmaceutical carrier and coding (a) has any one polypeptide among SEQID NO:13,14,15,20-59, the 61-63; (b) the segmental peptide of SEQ ID NO:1; (c) (a) or the nucleic acid fragment of conservative property varient (b); Or the expression cassette of claim 30.

35. the composition of claim 34, wherein said composition is made into to treat the preparation of SARS-CoV.

36. the composition of claim 34, wherein said composition are made into to prevent SARS-CoV and mammalian cell to merge or enter the preparation of mammalian cell.

37. comprising pharmaceutical carrier, virus vector and coding (a), a viral vaccine, described viral vaccine have any one polypeptide among SEQ ID NO:13,14,15,20-59, the 61-63; (b) the segmental peptide of SEQ ID NO:1; (c) (a) or the nucleic acid fragment of conservative property varient (b); Or the expression cassette of claim 30.

38. the viral vaccine of claim 34, wherein said viral vaccine is made into unit dosage form.

39. a peptide vaccine, described peptide vaccine comprise pharmaceutical carrier and (a) have any one peptide among SEQ IDNO:13,14,15,20-59, the 61-63; (b) fragment of SEQ ID NO:1; (c) (a) or peptide mimics (b); Or (d) (a) or conservative property varient (b).

40. the peptide vaccine of claim 39, wherein said peptide vaccine is made into unit dosage form.

41. a microorganism vaccine, described microorganism vaccine comprise pharmaceutical carrier and expression (a) has any one peptide among SEQ ID NO:13,14,15,20-59, the 61-63; (b) fragment of SEQID NO:1; Or (c) microorganism of (a) or conservative property varient (b).

42. the microorganism vaccine of claim 41, wherein said microorganism are selected from salmonella and monocyte hyperplasia listeria spp.

43. the microorganism vaccine of claim 42, wherein said microorganism vaccine is made into unit dosage form.

44. a dna vaccination, described dna vaccination comprise pharmaceutical carrier and have inserted the aminoacid sequence shown in any one among coding (a) SEQ ID NO:13,14,15,20-59, the 61-63; (b) fragment of SEQ ID NO:1; Or (c) carrier of the nucleic acid fragment of (a) or conservative property varient (b).

45. the dna vaccination of claim 44, wherein said carrier are selected from plasmid, glutinous grain, yeast artificial chromosome, bacterial artificial chromosome, F-factor, virus and phasmid.

46. the dna vaccination of claim 44, wherein said dna vaccination is made into the form of unitary dose.

47. the dna vaccination of claim 46, wherein said dna vaccination further contains the muscular death agent.

48. the dna vaccination of claim 47, wherein said muscular death agent is bupivacaine or cardiotoxin.

49. an antibody, the aminoacid sequence shown in any one among described antibody and SEQ ID NO:13,14,15,20-59, the 61-63; Or the fragment combination of SEQ ID NO:1.

50. the antibody of claim 49, the aminoacid sequence shown in any one among wherein said antibodies specific ground and SEQ ID NO:13,14,15,20-59, the 61-63; Or the fragment combination of SEQ IDNO:1.

51. the antibody of claim 49, wherein said antibodies specific ground combines with S protein receptor land.

52. the antibody of claim 49, wherein said antibody are the antibody fragment or the humanized antibody of monoclonal antibody, polyclonal antibody, single-chain antibody, antigen-binding.

53. the antibody of claim 52, wherein said antigen-binding antibody fragment are scFv, Fv, Fab ', Fab, double-stranded antibody, linear antibody or F (ab ') ₂

54. the antibody of claim 49, wherein said antibody and the coupling of certification mark thing.

55. the antibody of claim 54, wherein said certification mark thing is fluorescin, fluorescent marker, radioactively labelled substance, enzyme or affinity tag.

56. the antibody of claim 49, wherein said antibody and toxin conjugated.

57. the antibody of claim 56, wherein said toxin are A streptomycin, ribosome inactivating protein, α-broom aspergillin, set toxalbumin, aspergillin, limitation toxin, rnase, epipodophyllotoxin, diphtheria toxin, Pseudomonas aeruginosa extracellular toxin, Ricin, Dx, daunorubicin, taxol, ethidium bromide, mitomycin, Etoposide, table Podophyllum emodi var chinense thiophene glucosides, vincristine(VCR), vinealeucoblastine(VLB), colchicine, dihydroxy anthrax rhzomorph diketone, dactinomycin, PE40, toxalbumin or glucocorticosteroid in vain.

58. a medicinal compositions, described medicinal compositions comprise pharmaceutical carrier and with SEQID NO:13,14,15,20-59,61-63 in the aminoacid sequence shown in any one; Or the fragment bonded antibody of SEQ ID NO:1.

59. the method that immune Mammals is levied with anti-serious acute respiratory system synthesis, described method comprise give described Mammals treatment significant quantity with SEQ ID NO:13,14,15,20-59,61-63 in the aminoacid sequence shown in any one; Or the fragment bonded antibody of SEQ ID NO:1.

60. the method for claim 59, the aminoacid sequence shown in any one among wherein said antibodies specific ground and SEQ ID NO:13,14,15,20-59, the 61-63; Or the fragment combination of SEQ IDNO:1.

61. the method for claim 59, wherein said Mammals is behaved.

62. the mammiferous serious acute respiratory system synthesis of a treatment method of levying, described method comprise give described Mammals treatment significant quantity with SEQ ID NO:13,14,15,20-59,61-63 in the aminoacid sequence shown in any one; Or the fragment bonded antibody of SEQ ID NO:1.

63. the method for claim 62, the aminoacid sequence shown in any one among wherein said antibodies specific ground and SEQ ID NO:13,14,15,20-59, the 61-63; Or the fragment combination of SEQ IDNO:1.

64. the method for claim 62, wherein said Mammals is behaved.

65. the method for claim 59 or 62, wherein said antibody and pharmaceutical carrier or thinner are made preparation.

66. treat or suppress the method that Mammals serious acute respiratory system synthesis is levied for one kind, described method comprises the aminoacid sequence shown in any one among SEQ ID NO:13,14,15,20-59, the 61-63 that contains that gives described Mammals treatment significant quantity; Or the segmental S polypeptide of SEQ IDNO:1.

67. an immunoreactive method of inducing the SARS coronary virus resistant spike protein in mammalian body, described method comprise the aminoacid sequence shown in any one among SEQ ID NO:13,14,15,20-59, the 61-63 that contains for the treatment of significant quantity; Or the segmental polypeptide of SEQ IDNO:1.

68. the method for claim 67, wherein said polypeptide contain the aminoacid sequence shown in any one among the SEQ ID NO:13,14,15,25,34,51,52,56,57,58,59,61,62,63; Or the fragment of SEQ ID NO:1.

69. the method for claim 67, wherein said Mammals is behaved.

70. a method of diagnosing animal serious acute respiratory system synthesis to levy, described method comprises:

(a) make the aminoacid sequence shown in any one among the biological sample that is obtained from animal and SEQ ID NO:13,14,15,20-59, the 61-63; Or the fragment bonded antibody of SEQ ID NO:1 contact;

(b) determine whether described antibody combines with described biological sample.

71. the method for claim 70, wherein said animal are Mammals.

72. the method for claim 70, wherein said Mammals is behaved.

73. a method for preparing antibody, described method comprises: the peptide fragment that obtains to be had by (a) polypeptide of the aminoacid sequence shown in the SEQ IDNO:1; (b) has the polypeptide of the aminoacid sequence shown in any one among SEQ ID NO:13,14,15,20-59, the 61-63; (c) (a) or peptide mimics (b), or (d) animal of (a) or conservative property varient immunity (b); And separate and (a) bonded antibody.

74. a method for preparing antibody, described method comprise acquisition by the animal of coupling protein immunity, described coupling protein have carrier proteins and with it link coupled (a) have the peptide fragment of the polypeptide of the aminoacid sequence shown in the SEQ IDNO:1; (b) has the polypeptide of the aminoacid sequence shown in any one among SEQ ID NO:1,13,14,15, the 20-55; (c) (a) or peptide mimics (b), or (d) (a) or conservative property varient (b); And separate and have the polypeptide bonded antibody of the aminoacid sequence shown in the SEQ ID NO:1.

75. a test kit, described test kit contain wrapping material and with SEQ ID NO:1,13,14,15,20-59,61-63 in the aminoacid sequence shown in any one; Or SEQ IDNO:1 fragment bonded antibody or fit.

76. the test kit of claim 75, wherein said antibody and pharmaceutical carrier or thinner are made preparation.

77. the test kit of claim 75 further comprises syringe.

78. a test kit, described test kit contain the aminoacid sequence shown in any one among SEQ ID NO:13,14,15,20-59, the 61-63 that contains of wrapping material and treatment significant quantity; Or the segmental S polypeptide of SEQ ID NO:1.

79. the test kit of claim 78, wherein said S polypeptide and pharmaceutical carrier or thinner are made preparation.

80. the test kit of claim 78, described test kit further comprises syringe.

81. a monoclonal antibody, described monoclonal antibody combine with the aminoacid sequence shown in any one among SEQ IDNO:1,13,14,15,20-59, the 61-63 specifically.

82. an isolating polyclonal antibody, described polyclonal antibody combine with the aminoacid sequence shown in any one among SEQID NO:1,13,14,15,20-59, the 61-63 specifically.

83. one kind fit, any one described aminoacid sequence among described fit and SEQ ID NO:1,13,14,15,20-59, the 61-63; Or the fragment combination of SEQ ID NO:1.

84. a medicinal compositions, described medicinal compositions comprise pharmaceutical carrier and with SEQID NO:1,13,14,15,20-59,61-63 in the aminoacid sequence shown in any one; Or the fragment bonded of SEQ ID NO:1 is fit.

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