CN1468962A - Expression and purification of small envelope protein of soluble SARS virus - Google Patents
Expression and purification of small envelope protein of soluble SARS virus Download PDFInfo
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- CN1468962A CN1468962A CNA031291481A CN03129148A CN1468962A CN 1468962 A CN1468962 A CN 1468962A CN A031291481 A CNA031291481 A CN A031291481A CN 03129148 A CN03129148 A CN 03129148A CN 1468962 A CN1468962 A CN 1468962A
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Abstract
The present invention provides the expression plasmid pGEX2T-E of soluble SARS virus virus E protein capable of being expressed in host cell with the preservation number of CCTCC No. M203047, and the its expression method. The expression method includes obtaining SARS virus E protein DNA segment (TR02) from SARS virus cDNA library via PCR process; constructing SARS virus E protein expression plasmid pGEX2T-E with pGEX2T vector; and transforming pGEX2T-E plasmid into host cell and expressing in the host cell. The purifying process of soluble SARS virus E protein expression product includes dialysis of cell lysate liquid, Glutathione Sepharose 4B column chromatography and molecular sieve purification. The process of the present invention is simple and can obtain protein with high purity.
Description
Technical field
The present invention relates to molecule and RESEARCH ON CELL-BIOLOGY field, be specifically related to expression and the purifying of the little envelop protein of soluble SARS virus in host cell (SARS small envelope E protein).
Background technology
At the beginning of the end of the year to 2,003 2002, " atypical pneumonia (severe acuterespiratory syndrome; SARS) " case is found first in China Guangdong Province of a kind of being called as, more than 30 countries and regions outburst in the world subsequently, 25 provinces, cities and autonomous regions such as the Beijing on SARS ground in China, Shanxi, Inner Mongol " are walked crosswise " wantonly, and China people's lives and properties are constituted a serious threat.SARS is the new virus that a kind of infectivity is strong, existence is strong, lethality rate is high, and by the end of on May 19th, 2003, whole world accumulative total reported cases reached 7864 people, death 643 people.SARS by universally acknowledged be human common enemy.After the SARS outburst, caused the great attention of national governments and research institution, The World Health Organization's technician goes to the epidemic-stricken area to instruct preventing and controlling, and various countries scientific research personnel combined efforts is sought the pathogenic agent of SARS jointly and prevented and treated method.Through multinational scientific research personnel's effort, find that finally sars coronavirus (coronavirus) is that SARS infects, patient falls ill and lethal arch-criminal.The full genome of SARS virus is measured by the scientist of Canada, the U.S. and China respectively, and this pathogenesis for the research SARS virus is significant to design anti-SARS medicine.
Sars coronavirus (SARS-Cov) belongs to nido virales (Order:Nidovirales), coronaviridae (Family:Coronaviridae), coronavirus genus (Genus:Coronavirus).According to its genome structure classification, it belongs to strand justice virus [(+) sense, ssRNA virus], to discovering of other known coronavirus, its peplos (envelop) mainly comprises three kinds of glycoprotein, respectively called after S albumen (Spike Protein), M albumen (Membrane Protein), E albumen (Envelope Protein).In the part virus strain, can also find a kind of HE albumen (Haemagglutinin-esterase).Wherein E albumen is a kind of less relatively protein, mainly is distributed on the peplos.The E albumen of SARS virus is minimum structural protein, and 76 amino acid are only arranged.Its aminoacid sequence is as follows: MYSFVSEETGTLIVNSVLLFLAFVVFLLVTLAILTALRLCAYCCNIVNVSLVKPTV YVYSRVKNLNSSEGVPDLLV.
Consistent with other coronavirus, it is charged residue that SARS-CoV E albumen comprises its side of a hydrophobic section (residue 12-37), and then is the zone of halfcystine enrichment.Applying biological information science analysis tool BLAST and FASTA analyze E (Envelop) albumen that shows E albumen and other coronavirus and mate significantly.The E albumen of pfam analysis announcement SARS has showed the characteristic of a member in the NS3_EnvE protein family preferably.InterProScan analyzes and shows that E albumen is the integral part of peplos (Envelop), and conservative sequence is also found in other virus, as gastroenteritis virus (gastroenteritis) and murine hepatitis virus (murinehepatitis).SignalP analyzes expectation, and it contains an anchor (transmembrane anchor) (possibility reaches 0.939) of striding film.Tmpred analyze to show the E albumen of SARS contain with other known viruse coating on this protein like stride the film section, be positioned between residue 17-34.TMHMM analyze to estimate a kind of membranin (a type IImembrane protein) of the II type of being made up of most hydrophilic (hydrophilic domain) section (46 residues) and the surface that one of carbon tip is positioned at virion.
Perhaps can helpful and guidance to other coronavirus E protein function result of study to the proteic function understanding of SARS virus E.General Study thinks that all the E albumen of coronavirus and M albumen are essential and fully to the assembling of virion.Be the proteic effect of understanding murine hepatitis virus (MHV) E, someone incites somebody to action wherein, and the proteic charged residue of E has carried out alanine mutation, the virus of sudden change demonstrates responsive to temperature type, even the form of most mutated viruses also becomes to tighten and elongates under the temperature that is fit to, can't form normal wild-type (wild-type), visible E albumen has play a part important or even crucial in the form of coronavirus forms.It is that virus replication is necessary that the E albumen that studies show that some coronavirus such as heritable gastroenteritis coronavirus (TGEV) is also arranged.Also have test to show, the proteic expression of E also can cause the apoptosis of DBT cell in the DBT cell that murine hepatitis virus (MHV) infects, and S albumen, M albumen and the proteic expression of N can not cause the apoptosis of cell.Studies show that recently make the reproduction speed of murine hepatitis virus (MHV) reduce by 10,000 times though remove the E gene, virus still can be duplicated, this prompting E albumen plays an important role in virus replication, but perhaps is not this quality factor of virus replication.
Therefore, will have important theory and realistic meaning in external great expression and purification of soluble SARS virus E albumen with its important biological function of further exploration.Unique correlation technique experience that the present inventor utilizes in the experimentation to be accumulated through experiment repeatedly, in intestinal bacteria successful expression and purifying soluble SARS virus E albumen, thereby finished the present invention.
An object of the present invention is to provide and in host cell, to express the proteic expression plasmid of SARS virus E;
Another object of the present invention provides that soluble SARS virus E albumen is expressed and the method for purifying in host cell.
Summary of the invention
One aspect of the present invention provides can express the proteic expression plasmid pGEX2T-E of soluble SARS virus E in host cell, be preserved in Chinese typical culture collection center (CCTCC), specific name is intestinal bacteria M15/pGEX2T-SARS-E, May 30 2003 preservation day, preserving number CCTCCNo.M203047.
Another aspect of the present invention provides the transformed host cells with plasmid pGEX2T-E.Described host cell can be prokaryotic cell prokaryocyte or eukaryotic cell.
Another aspect of the present invention is provided at expresses the proteic method of soluble SARS virus E in the host cell, may further comprise the steps:
1) adopt round pcr from SARS virus cDNA library, to obtain SARS virus E protein D NA fragment (TRO2);
2) with pGEX2T vector construction SARS virus E protein expressing plasmid pGEX2T-E;
3) the pGEX2T-E plasmid is transformed in host cell, and in host cell, express.
The purification process that also has an aspect that the soluble SARS virus E protein expressioning product is provided of the present invention comprises lysis, lysate dialysis, Glutathione Sepharose 4B column chromatography and molecular sieve purification.
The simple declaration of accompanying drawing
Figure 1 shows that soluble SARS virus E protein expressing plasmid (pGEX2T-E) makes up synoptic diagram.
Figure 2 shows that SARS virus E albumen PCR product D NA agarose electrophoretic analysis collection of illustrative plates.
Figure 3 shows that the pGEX2T-E sequencer address.
Figure 4 shows that SARS virus E protein purification product polyacrylamide gel electrophoresis analysis collection of illustrative plates.Among the figure, the GST-E on the 1.Glutathione Sepharose 4B post; 2. the zymoplasm enzyme is cut the GST that leaves on the rear pillar; 3. the zymoplasm enzyme is cut behind the wash-out of back and the E albumen behind the FPLC purifying; 4. marker (Marker).
Embodiment
Test materials:
The pQE30 carrier, coli strain M15 is preserved by this chamber.Various restriction enzymes, T
4Toolenzyme such as dna ligase, high-fidelity DNA polymerase is available from TaKaRa company.The dna marker thing, protein label is available from Invitrogen company.IPTG, Ampicillin Trihydrate and kantlex are available from Sigma company.Glue reclaims test kit available from Hua Shun company.
The structure of embodiment 1 SARS virus E protein expressing plasmid (pGEX2T-E)
Utilize round pcr from SARS virus cDNA library, to obtain SARS virus E protein D NA fragment (gene order is according to TRO2), by shown in Figure 1, the proteic expression plasmid of the SARS virus E of construction expression solubility (pGEX2T-E).
1) design primer, the proteic sequence of amplification E
Primer sequence:
FW：5’ATTA
GGATCC?ATGTACTCATTCGTTTCG3’
RV：5’TTAA
GAATTCTTAGGACCAGAAGATCAGG?3’
Pcr amplification reaction (100 μ l):
Template: 2 μ l; 10 * Pyrobest
TMBuffer:10 μ l; DNTP (2.5mM): 8 μ l; FW:4 μ l; RV:4 μ l; High-fidelity DNA polymerase (pyrobest DNA polymerase): 1 μ l; Water: 71 μ l.
Reaction conditions: 95 ℃ 8 minutes;
(95 ℃ 30 seconds; 60 ℃ 30 seconds; 72 ℃ 30 seconds) 30 the circulation;
72 ℃ 10 minutes.
Figure 2 shows that SARS virus E albumen PCR product D NA agarose electrophoretic analysis collection of illustrative plates.
2) with the proteic PCR product cloning of E to the pGEX2T carrier
Proteic PCR product of E and pGEX2T cut (experimental technique is with reference to " molecular cloning experiment guide ", Science Press, second edition in 1999) with BamHI and EcoRI enzyme respectively; (the PCR enzyme that reclaims E is cut the nearly 250bp of product to cut product with test kit (Hua Shun company) recovery enzyme; The pGEX2T enzyme is cut the nearly 4.9kb of product); Connect with the T4DNA ligase enzyme; Connect product and be transformed into competent cell BL21 (DE3); Select correct clone (enzyme is cut evaluation).
Enzyme is cut evaluation(experimental technique is with reference to " molecular cloning experiment guide ", Science Press, second edition in 1999): BamHI ﹠amp; The EcoRI enzyme is cut evaluation, and the clone who cuts out 228 bands is correct.
The dna sequencing report: pGEX2T-E expression vector sequencer address as shown in Figure 3, pGEX2T-E dna sequencing result and E proteinogen dna sequence dna relatively, the result is in full accord.
The proteic expression of embodiment 2 soluble SARS virus E
The pGEX2T-E plasmid is transformed in e. coli bl21 (DE3), and IPTG (concentration 0.2mM) induces, and the Ampicillin Trihydrate is a microbiotic, 18 ℃ of temperature, 6 hours expression times.Under above-mentioned expression condition, 6 liters of LB substratum are through 4 ℃, 4000rpm after centrifugal 30 minutes, with the 10 gram bacterial precipitation things that obtain with the packing of every part 3 gram ,-80 ℃ of preservations.
The purifying of embodiment 3 expression products
Under 4 ℃, 3 gram bacterial precipitation things are suspended in the 15mL cracking buffered soln (50mM Tris-HCl, pH8.0,300mM NaCl, 1mM DDT, 5mM EDTA, 1mM PMSF) ultrasonic disruption (ultrasound intensity 200～300W; Ultrasound environments: mixture of ice and water; Total ultrasonic time 15 minutes, every ultrasonic 1.5 minutes, gap 1 minute).Through 4 ℃, 15, the 000rpm ultracentrifugation discarded throw out after 1 hour.With 14mL supernatant liquor dialysis (the dialysis buffer liquid: PBS under 4 ℃ that obtain, 1mM DDT, 5mMEDTA, pH7.4) spend the night after, with Glutathione Sepharose 4B post (Pharmacia company product) purifying protein, wherein: lavation buffer solution is the PBS (pH7.4) that contains 1mM DDT, 5mM EDTA; The enzyme system of cutting is PBS+80 unit's zymoplasm; E albumen elution buffer is the PBS (pH7.4) that contains 1mM DDT, 5mM EDTA; The GST elution buffer is the 50mM Tris-HCl (pH8.0) that contains the 50mM reductive glutathione.The 10mL protein crude extract administration that obtains is further purified through protein purification system FPLC (Pharmacia company product) molecular sieve obtains pure SARS virus E albumen (as shown in Figure 4).
The possibility of utilizing on the industry
Utilize the present invention can in host cell, obtain a large amount of soluble SARS virus E albumen (as, In the Escherichia coli, every liter of LB culture medium can be produced 10 milligrams of E albumen), utilize Glutathione Sepharose 4B column chromatography and molecular sieve method of purification can obtain the albumen of purifying. The present invention has behaviour Make simple, purity of protein high.
The SARS small envelope E protein that utilizes the present invention to obtain can be used for:
1) research of structure biology aspect
A. cultivate the SARS small envelope E protein crystal, resolve SARS by X-ray crystal diffraction technology The virus E protein crystal structure;
B. carry out NMR research, to seek the three-dimensional structure under the SARS small envelope E protein solution state.
2) want protein-interacting research with body weight for humans
The SARS small envelope E protein and the biophysical technology that utilize the present invention to obtain can be carried out and body weight for humans Want albumen (such as immune system) repercussion study, infect the human normal cell to explore SARS virus Approach, for anti-SARS drug design provides foundation.
3) SARS diagnosis antibody research
The SARS virus E albumen that utilizes the present invention to obtain combines with the other biological engineering, can carry out the research of SARS diagnosis antibody.
ACGTTATCGACTGCACGGTGCACCAATGCTTCTGGCGTCAGGCAGCCATCGGAAGCTGTGGTATGGCTGTGCAGGTCGTAAATCACTGCATAATTCGTGTCGCTCAAGGCGCACTCCCGTTCTGGATAATGTTTTTTGCGCCGACATCATAACGGTTCTGGCAAATATTCTGAAATGAGCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGTATTCATGTCCCCTATACTAGGTTATTGGAAAATTAAGGGCCTTGTGCAACCCACTCGACTTCTTTTGGAATATCTTGAAGAAAAATATGAAGAGCATTTGTATGAGCGCGATGAAGGTGATAAATGGCGAAACAAAAAGTTTGAATTGGGTTTGGAGTTTCCCAATCTTCCTTATTATATTGATGGTGATGTTAAATTAACACAGTCTATGGCCATCATACGTTATATAGCTGACAAGCACAACATGTTGGGTGGTTGTCCAAAAGAGCGTGCAGAGATTTCAATGCTTGAAGGAGCGGTTTTGGATATTAGATACGGTGTTTCGAGAATTGCATATAGTAAAGACTTTGAAACTCTCAAAGTTGATTTTCTTAGCAAGCTACCTGAAATGCTGAAAATGTTCGAAGATCGTTTATGTCATAAAACATATTTAAATGGTGATCATGTAACCCATCCTGACTTCATGTTGTATGACGCTCTTGATGTTGTTTTATACATGGACCCAATGTGCCTGGATGCGTTC CCAAAATTAGTTTGTTTTAAAAAACGTATTGAAGCTATCCCACAAATTGATAAGTACTTGAAATCCAGCAAGTATATAGCATGGCCTTTGCAGGGCTGGCAAGCCACGTTTGGTGGTGGCGACCATCCTCCAAAATCGGATCTGGTTCCGCGTzGGATCCATGTACTCATTCGTTTCGGAAGAAACAGGTACGTTAATAGTTAATAGCGTACTTCT TTTTCTTGCTTTCGTGGTATTCTTGCTAGTCACACTAGCCATCCTTACTGCGCTTC GATTGTGTGCGTACTGCTGCAATATTGTTAACGTGAGTTTAGTAAAACCAACGGTT TACGTCTACTCGCGTGTTAAAAATCTGAACTCTTCTGAAGGAGTTCCTGATCTTCT GGTCCTAA
GAATTCATCGTGACTGACTGACGATCTGCCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGACCCAGTCACGTAGCGATAGCGGAGTGTATAATTCTTGAAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGC CAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATAAATTCCGACACCATCGAATGGTGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGGAAGAGAGTCAATTCAGGGTGGTGAATGTGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGGATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCTTTGCCTGGTTTCCGGCACCAGAAGCGGTGCCGGAAAGCTGGCTGGAGTGCGATCTTCCTGAGGCCGATACTGTCGTCGTCCCCTCAAACTGGCAGATGCACGGTTACGATGCGCCCATCTACACCAACGTAACCTATCCCATTACGGTCAATCCGCCGTTTGTTCCCACGGAGAATCCGACGGGTTGTTACTCGCTCACATTTAATGTTGATGAAAGCTGGCTACAGGAAGGCCAGACGCGAATTATTTTTGATGGCGTTGGAATT
Claims (7)
1. can in host cell, express the proteic expression plasmid of soluble SARS virus E, be preserved in Chinese typical culture collection center, May 30 2003 preservation day, preserving number M203047.
2.1,：ACGTTATCGACTGCACGGTGCACCAATGCTTCTGGCGTCAGGCAGCCATCGGAAGCTGTGGTATGGCTGTGCAGGTCGTAAATCACTGCATAATTCGTGTCGCTCAAGGCGCACTCCCGTTCTGGATAATGTTTTTTGCGCCGACATCATAACGGTTCTGGCAAATATTCTGAAATGAGCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGTATTCATGTCCCCTATACTAGGTTATTGGAAAATTAAGGGCCTTGTGCAACCCACTCGACTTCTTTTGGAATATCTTGAAGAAAAATATGAAGAGCATTTGTATGAGCGCGATGAAGGTGATAAATGGCGAAACAAAAAGTTTGAATTGGGTTTGGAGTTTCCCAATCTTCCTTATTATATTGATGGTGATGTTAAATTAACACAGTCTATGGCCATCATACGTTATATAGCTGACAAGCACAACATGTTGGGTGGTTGTCCAAAAGAGCGTGCAGAGATTTCAATGCTTGAAGGAGCGGTTTTGGATATTAGATACGGTGTTTCGAGAATTGCATATAGTAAAGACTTTGAAACTCTCAAAGTTGATTTTCTTAGCAAGCTACCTGAAATGCTGAAAATGTTCGAAGATCGTTTATGTCATAAAACATATTTAAATGGTGATCATGTAACCCATCCTGACTTCATGTTGTATGACGCTCTTGATGTTGTTTTATACATGGACCCAATGTGCCTGGATGCGTTCCCAAAATTAGTTTGTTTTAAAAAACGTATTGAAGCTATCCCACAAATTGATAAGTACTTGAAATCCAGCAAGTATATAGCATGGCCTTTGCAGGGCTGGCAAGCCACGTTTGGTGGTGGCGACCATCCTCCAAAATCGGATCTGGTTCCGCGTGGATCCATGTACTCATTCGTTTCGGAAGAAACAGGTACGTTAATAGTTAATAGCGTACTTCT TTTTCTTGCTTTCGTGGTATTCTTGCTAGTCACACTAGCCATCCTTACTGCGCTTC GATTGTGTGCGTACTGCTGCAATATTGTTAACGTGAGTTTAGTAAAACCAACGGTT TACGTCTACTCGCGTGTTAAAAATCTGAACTCTTCTGAAGGAGTTCCTGATCTTCT GGTCCTAA
GAATTCATCGTGACTGACTGACGATCTGCCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGGCGCAGCCATGACCCAGTCACGTAGCGATAGCGGAGTGTATAATTCTTGAAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCGCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACAC CGCATAAATTCCGACACCATCGAATGGTGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGGAAGAGAGTCAATTCAGGGTGGTGAATGTGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGGATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCTTTGCCTGGTTTCCGGCACCAGAAGCGGTGCCGGAAAGCTGGCTGGAGTGCGATCTTCCTGAGGCCGATACTGTCGTCGTCCCCTCAAACTGGCAGATGCACGGTTACGATGCGCCCATCTACACCAACGTAACCTATCCCATTACGGTCAATCCGCCGTTTGTTCCCACGGAGAATCCGACGGGTTGTTACTCGCTCACATTTAATGTTGATGAAAGCTGGCTACAGGAAGGCCAGACGCGAATTATTTTTGATGGCGTTGGAATT
3. one kind with the described expression plasmid pGEX2T-E of claim 1 transformed host cells.
4. host cell as claimed in claim 3, described host cell are prokaryotic cell prokaryocyte or eukaryotic cell.
5. host cell as claimed in claim 4, described host cell are intestinal bacteria.
6. in host cell, express the proteic method of soluble SARS virus E, may further comprise the steps:
1) from SARS virus cDNA library, obtains SARS virus E protein D NA fragment with round pcr;
2) with pGEX2T vector construction SARS virus E protein expressing plasmid pGEX2T;
3) the pGEX2T plasmid is transformed in host cell, and in host cell, express.
7. the purification process of soluble SARS virus E protein expressioning product comprises dialysis of cell lysate liquid, Glutathione Sepharose 4B column chromatography and molecular sieve purification.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA031291481A CN1468962A (en) | 2003-06-05 | 2003-06-05 | Expression and purification of small envelope protein of soluble SARS virus |
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| Application Number | Priority Date | Filing Date | Title |
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| CNA031291481A CN1468962A (en) | 2003-06-05 | 2003-06-05 | Expression and purification of small envelope protein of soluble SARS virus |
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| CN1468962A true CN1468962A (en) | 2004-01-21 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1820020B (en) * | 2003-05-06 | 2010-04-28 | 技术持有有限公司 | SARS-coronavirus virus-like particles and methods of use |
| CN111978377A (en) * | 2020-07-09 | 2020-11-24 | 苏州和锐生物科技有限公司 | COVID-19 antigen, preparation method and application |
-
2003
- 2003-06-05 CN CNA031291481A patent/CN1468962A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1820020B (en) * | 2003-05-06 | 2010-04-28 | 技术持有有限公司 | SARS-coronavirus virus-like particles and methods of use |
| CN111978377A (en) * | 2020-07-09 | 2020-11-24 | 苏州和锐生物科技有限公司 | COVID-19 antigen, preparation method and application |
| CN111978377B (en) * | 2020-07-09 | 2022-05-31 | 苏州和锐生物科技有限公司 | COVID-19 antigen, preparation method and application |
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