CN1160370C - A novel human cell cysle control related protein and a sequence encoding the same - Google Patents

Abstract

The present invention discloses a new human protein with a cancer suppressing function, polynucleotide for coding polypeptide, a method for generating the polypeptide by a recombination technique and a method of the polypeptide for the treatment of various diseases, such as cancer, etc., and also discloses an antagonist for resisting the polypeptide, the treating action of the antagonist and an application of the polyribonucleotide for coding the new human protein with a cancer suppressing function.

Description

New human cell cysle control related protein and encoding sequence thereof

Invention field

The invention belongs to biological technical field, specifically, the present invention relates to human cell cycle control genes involved (crn-like gene, the polypeptide of CLG) proteic polynucleotide, and this polynucleotide encoding that new coding has cancer suppressing function.The invention still further relates to the purposes and the preparation of these polynucleotide and polypeptide.

Background of invention

Cell fission and propagation are one of essential characteristics of cell activities.When human body and other multicellular organisms grow, the increase of cell number, the renewal of aging, dead cell, the continuity of life all needs finish by the propagation of cell.Be that cell is by incident in a series of cells cell generation cycle, realize the process of cell growth and propagation, there is strict succession between each incident, its regulatory mechanism relates to the many levels and the multiple factor, not only relate to somatomedin, cyclic nucleotide, hormone, proto-oncogene etc., the more important thing is the control that also is subjected to the cell cycle regulating system, as the regulation and control of cell division cycle gene, cyclin, cyclin dependent kinase and arrestin thereof etc.

The regulation and control of cell cycle are the key problems in the RESEARCH ON CELL-BIOLOGY, and it relates to the orderly growth of embryonic cell, the normal hyperplasia of body cell, the regeneration of cell and differentiation, and problem such as aging of cell, apoptosis.The normal cell cycle must be carried out strict orderly regulation and control, and cell cycle regulating will make unusually that cell is undergone mutation, distortion or canceration.

The cell cycle regulator control system is furtherd investigate in recent years, this system mainly comprises cell division cycle (celldivision cycle, cdc) gene, cyclin (cyclin), cyclin dependent kinase (cyclin-dependent kinase, CDK) and the arrestin (CKI) of cyclin dependent kinase etc.People have been separated to the cell cycle control gene of various human according to yeast (yeast), fruit bat (drosophila) and nematode (c.elegans) isotype biology.As Lieberman etc. according to the homology analytical procedure, be separated to one and yeast rad9+ homologous human cell cycle check point (checkpoint) controlling gene (Lieberman, H.B., Hopkins, K.M., et al.Proc.Natl.Acad.Sci.USA 93:13890-13895; 1996).

Cancer is one of principal disease of harm humans health.In order to treat effectively and prophylaxis of tumours, people more and more pay close attention to genetic treatment of tumor at present.Therefore, this area presses for people's albumen and the agonist/inhibitor thereof that development research has cancer suppressing function, especially participates in the albumen of cell cycle regulating effect.

Summary of the invention

The purpose of this invention is to provide the new people's protein polypeptide of a class with cancer suppressing function with and fragment, analogue and derivative.

Another object of the present invention provides the polynucleotide of these polypeptide of coding.

Another object of the present invention provides the method for these polypeptide of production and the purposes of this polypeptide and encoding sequence.

In a first aspect of the present invention, novel isolated protein polypeptide with cancer suppressing function is provided, it comprises the polypeptide with SEQ ID NO:2 aminoacid sequence; Or its conservative property variation polypeptide or its active fragments or its reactive derivative.Preferably, this polypeptide is the polypeptide that comprises SEQ ID NO:2 aminoacid sequence.

In a second aspect of the present invention, a kind of isolating polynucleotide are provided, it comprises a nucleotide sequence, and this nucleotide sequence is shown at least 85% homogeny with a kind of nucleotides sequence that is selected from down group: the polynucleotide of the above-mentioned CLG protein polypeptide of (a) encoding; (b) with polynucleotide (a) complementary polynucleotide.Preferably, the polypeptide of this polynucleotide encoding has the aminoacid sequence of SEQ IDNO:2.More preferably, the sequence of these polynucleotide is selected from down group: the coding region sequence of SEQ ID NO:3 (22-2586 position) or full length sequence (1-2659 position).

In a third aspect of the present invention, the carrier that contains above-mentioned polynucleotide is provided, and has been transformed or host cell of transduceing or the host cell that is directly transformed or transduce by above-mentioned polynucleotide by this carrier.

In a fourth aspect of the present invention, provide preparation to have the preparation method of the polypeptide of CLG protein-active, this method comprises: (a) be fit to cultivate the above-mentioned host cell that is transformed or transduce under the proteic condition of expression CLG; (b) from culture, isolate polypeptide with CLG protein-active.

In a fifth aspect of the present invention, provide and above-mentioned CLG protein polypeptide specificity bonded antibody.The nucleic acid molecule that can be used for detecting also is provided, and it contains a successive 10-800 Nucleotide in the above-mentioned polynucleotide.

In a sixth aspect of the present invention, a kind of pharmaceutical composition is provided, it contains the CLG protein polypeptide of the present invention and the pharmaceutically acceptable carrier of safe and effective amount.These pharmaceutical compositions can be treated illnesss such as cancer and cellular abnormality propagation.

Others of the present invention are because the disclosure of this paper is conspicuous to those skilled in the art.

Fig. 1 is the rna expression spectrum of CLG.

Fig. 2 has shown the restraining effect that the CLG full-length cDNA forms human liver cancer cell SMMC-7721 colony.Wherein Fig. 2 A is vehicle Control (pCMV-Script), and the colony number is 56; Fig. 2 B is CLG (pCMV-Script/CLG), and the colony number only is 3.

Fig. 3 is the paraffin section photo of the tumor tissues that forms behind the SMMC-7721 cell inoculation nude mice with the CLG transfection.Fig. 3 A is in the necrosis region, visible more positive apoptotic cells; Fig. 3 B is necrotic area not, the apoptotic cell that as seen is dispersed in.

Fig. 4 has shown " trapezoidal " DNA (DNA ladder) electrophorogram behind the transfection CLG gene.Wherein each swimming lane is: 1. transfection empty carrier; 2. transfection p53; 3. transfection CLG; 4. untransfected control point; 5. molecular weight standard.

The SDS-PAGE electrophorogram of Fig. 5 CLG protein expression.Wherein, each swimming lane is: 1. molecular weight of albumen standard; 2.pET32a-CLG the ultrasonic supernatant of thalline; 3.pET32a the ultrasonic supernatant of thalline; 4.pET32a-CLG thalline ultrasound precipitation; 5.pET32a thalline ultrasound precipitation.

Detailed Description Of The Invention

The present invention adopts large-scale cDNA clone transfection cancer cell, has on the basis of cancer suppressing action in acquisition, proves new gene through order-checking, further obtains full length cDNA clone. DNA transfection evidence, the albumen with cancer suppressing function of the present invention has the effect that suppresses clone's formation, its inhibiting rate 〉=50% to cancer cell (HCC).

In an example, from the human placenta cDNA library, be separated to a new gene with the cell cycle regulating protein height homology of nematode, fruit bat and yeast, the cell cycle regulating protein of it and nematode has 66% (557/837) homology, cell cycle regulating protein (crooked neck with fruit bat, crn) 41% (157/375) homology is arranged, controlling albumen with the cell cycle of yeast has 37% (228/599) homology. Can judge people CLG (crn-like gene from bioinformatics; Former sequence number is PP3898, GenBank accession number AF258567, log date on April 24th, 2000) be a new human cell cycle related gene. Find that through preliminary functional study CLG can be in the growth of external inhibition human liver cancer cell SMMC-7721; The original position apoptosis of HCC SMMC-7721 transplanted tumor in nude mice tissue detects and the DNA electrophoretic analysis shows, energy induced tumor Apoptosis behind the CLG gene transfection SMMC-7721 cell, and the growth of inhibition tumor cell.

In this article, " cell cycle control related gene albumen ", " CLG albumen " and " PP3898 albumen " are used interchangeably, and all refer to have the polypeptide that human cell cycle is controlled the amino acid sequence (SEQ ID NO:2) of related gene albumen. They comprise the cell cycle control related gene albumen that contains or do not contain initial methionine.

As used herein, " separation " refers to that material separates (if crude, primal environment namely is natural surroundings) from its primal environment. Do not have separation and purification such as the polynucleotide under the native state in the active somatic cell and polypeptide, but same polynucleotide or polypeptide as from native state with in other materials that exist separately, then for separation and purification.

As used herein, " CLG albumen or the polypeptide of separation " refers to that the CLG polypeptide is substantially free of natural relative other albumen, lipid, carbohydrate or other material. Those skilled in the art can use the purified technology of protein purifying CLG albumen of standard. Basically pure polypeptide can produce single master tape on non-reduced polyacrylamide gel. The purity of CLG polypeptide can be used amino acid sequence analysis.

Polypeptide of the present invention can be restructuring polypeptide, natural polypeptides, synthetic polypeptide, the polypeptide of preferably recombinating. Polypeptide of the present invention can be the product of natural purifying, or the product of chemical synthesis, or uses the restructuring technology to produce from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell). According to the used host of restructuring production decision, polypeptide of the present invention can be glycosylated, maybe can be nonglycosylated. Polypeptide of the present invention also can comprise or not comprise initial methionine residues.

The present invention also comprises fragment, derivative and the analog of people CLG albumen. As used herein, term " fragment ", " derivative " refer to basically keep the identical biological function of natural human CLG albumen of the present invention or active polypeptide with " analog ". Polypeptide fragment of the present invention, derivative or analog can be that (i) has one or more conservative or substituted polypeptide of non-conservation amino acid residue (preferred conservative amino acid residue), and the amino acid residue of such replacement can be also can not encoded by genetic code, or (ii) in one or more amino acid residues, has a polypeptide of substituted radical, or (iii) mature polypeptide and another compound (such as the compound that prolongs the polypeptide half-life, polyethylene glycol for example) merge formed polypeptide, or (iv) additional amino acid sequence is fused to this peptide sequence and the polypeptide that forms (such as targeting sequencing or secretion sequence or be used for sequence or the proteinogen sequence of this polypeptide of purifying). According to the instruction of this paper, these fragments, derivative and analog belong to the known scope of those skilled in the art.

Polynucleotides of the present invention can be dna form or rna form. Dna form comprises cDNA, genomic DNA or artificial synthetic DNA. DNA can be strand or double-stranded. DNA can be coding strand or noncoding strand. Take CLG albumen as example, the coding region sequence of encoding mature polypeptide can be identical with the coding region sequence shown in the SEQ ID NO:3 or the variant of degeneracy. As used herein, " variant of degeneracy " refers in the present invention encode and has the protein of SEQ ID NO:2, but with the differentiated nucleotide sequence of coding region sequence shown in the SEQ ID NO:3.

The polynucleotides of encoding mature polypeptide comprise: the coded sequence of an encoding mature polypeptide; The coded sequence of mature polypeptide+various additional code sequences; The coded sequence of mature polypeptide (+optional additional code sequence)+non-coding sequence.

Term " polynucleotides of coded polypeptide " can be the polynucleotides that comprise this polypeptide of encoding, and also can be the polynucleotides that also comprise additional code and/or non-coding sequence.

The invention still further relates to the varient of above-mentioned polynucleotide, its coding has the polypeptide of identical aminoacid sequence or fragment, analogue and the derivative of polypeptide with the present invention.The varient of these polynucleotide can be the allelic variant of natural generation or the varient that non-natural takes place.These nucleotide diversity bodies comprise and replace varient, deletion mutation body and insert varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it may be replacement, disappearance or the insertion of one or more Nucleotide, but can be from not changing the function of its encoded polypeptides in fact.

The invention still further relates to and above-mentioned sequence hybridization and two sequences between have at least 50%, preferably at least 70%, the polynucleotide of at least 80% homogeny more preferably.The present invention be more particularly directed under stringent condition and the interfertile polynucleotide of polynucleotide of the present invention.In the present invention, " stringent condition " is meant: (1) than hybridization under low ionic strength and the comparatively high temps and wash-out, as 0.2 * SSC, and 0.1%SDS, 60 ℃; Or (2) hybridization the time is added with denaturing agent, as 50% (v/v) methane amide, 0.1% calf serum/0.1%Ficoll, 42 ℃ etc.; Or (3) only at the homogeny between the two sequences at least more than 95%, be more preferably 97% and just hybridize when above.And the polypeptide of interfertile polynucleotide encoding has identical biological function and activity with the mature polypeptide shown in the SEQ ID NO:2.

The invention still further relates to nucleic acid fragment with above-mentioned sequence hybridization.As used herein, the length of " nucleic acid fragment " contains 15 Nucleotide at least, better is at least 30 Nucleotide, is more preferably at least 50 Nucleotide, preferably more than at least 100 Nucleotide.Nucleic acid fragment can be used for the amplification technique (as PCR) of nucleic acid to determine and/or the proteic polynucleotide of separation coding CLG.

Dna sequence dna of the present invention can obtain with several method.For example, with hybridization technique DNA isolation well known in the art.These technology including, but not limited to: 1) with probe and genome or the hybridization of cDNA library to detect homology nucleotide sequence and 2) antibody screening of expression library to be to detect the dna fragmentation of the clone with common structure feature.

The proteic specific DNA fragment sequence of coding CLG produces also and can obtain with following method: 1) separate double chain DNA sequence from genomic dna; 2) the chemical synthesising DNA sequence is to obtain the double-stranded DNA of required polypeptide.

In the above-mentioned method of mentioning, isolation of genomic DNA is least commonly used.When the whole aminoacid sequence of the polypeptide product of needs was known, the direct chemical of dna sequence dna is synthetic to be the method for often selecting for use.When if required amino acid whose whole sequence is not known, the direct chemical of dna sequence dna is synthetic to be impossible, and the method for selecting for use is the separation of cDNA sequence.The standard method that separates interested cDNA is from the donorcells separating mRNA of this gene of high expression level and carries out reverse transcription, forms plasmid or phage cDNA library.Extract the existing multiple proven technique of method of mRNA, test kit also can obtain (Qiagene) from commercial channels.And the construction cDNA library also is usual method (Sambrook, et al., MolecularCloning, A Laboratory Manual, Cold Spring Harbor Laboratory.New York, 1989).Also can obtain the cDNA library of commercial offers, as the different cDNA library of Clontech company.When being used in combination the polymeric enzyme reaction technology, even few expression product also can be cloned.

Available ordinary method is screened gene of the present invention from these cDNA libraries.These methods include, but is not limited to: (1) DNA-DNA or DNA-RNA hybridization: the function of (2) marker gene occurs or forfeiture; (3) level of the proteic transcript of mensuration CLG; (4), detect the protein product of genetic expression by immunological technique or mensuration biologic activity.Aforesaid method can singly be used, but also several different methods combined utilization.

In (1) kind method, hybridizing used probe is and any a part of homology of polynucleotide of the present invention that at least 15 Nucleotide of its length better are at least 30 Nucleotide, are more preferably at least 50 Nucleotide, preferably at least 100 Nucleotide.In addition, the length of probe within 2kb, preferably is within the 1kb usually.Probe used herein is the dna sequence dna of chemosynthesis on the basis of gene DNA sequence information of the present invention normally.Gene of the present invention itself or fragment are certainly as probe.The mark of dna probe can be used radio isotope, fluorescein or enzyme (as alkaline phosphatase) etc.

In (4) kind method, detect the protein product of CLG protein gene expression and can use immunological technique such as Western blotting, radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA) etc.

Use method (Saiki, the et al.Science 1985 of round pcr DNA amplification/RNA; 230:1350-1354) be optimized for acquisition gene of the present invention.When particularly being difficult to obtain the cDNA of total length from the library, can preferably use RACE method (the terminal rapid amplifying method of RACE-cDNA), the primer that is used for PCR can suitably be selected according to sequence information of the present invention disclosed herein, and available ordinary method is synthetic.Available ordinary method is as the DNA/RNA fragment by gel electrophoresis separation and purifying amplification.

The gene of the present invention that obtains as mentioned above, the perhaps available ordinary method of mensuration such as dideoxy chain termination (Sanger et al.PNAS, 1977,74 of the nucleotide sequence of various dna fragmentations etc.; 5463-5467).This class nucleotide sequencing is available commercial sequencing kit etc. also.In order to obtain the cDNA sequence of total length, order-checking need be carried out repeatedly.Sometimes need to measure a plurality of clones' cDNA sequence, just can be spliced into the cDNA sequence of total length.

The present invention also relates to comprise the carrier of polynucleotide of the present invention, and the host cell that produces through genetically engineered with carrier of the present invention or CLG albumen coded sequence, and the method that produces polypeptide of the present invention through recombinant technology.

By the recombinant DNA technology of routine, can utilize polymerized nucleoside acid sequence of the present invention to can be used to express or produce CLG protein polypeptide (Science, 1984 of reorganization; 224:1431).In general following steps are arranged:

(1). with the proteic polynucleotide of coding people CLG of the present invention (or varient), or transform or the transduction proper host cell with the recombinant expression vector that contains these polynucleotide;

(2). the host cell of in suitable medium, cultivating;

(3). separation, protein purification from substratum or cell.

Among the present invention, people CLG albumen polynucleotide sequence can be inserted in the recombinant expression vector.Term " recombinant expression vector " refers to that bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell virus, mammalian cell virus are as adenovirus, retrovirus or other carriers.The carrier of Shi Yonging includes but not limited in the present invention: and the expression vector based on T7 of in bacterium, expressing (Rosenberg, et al.Gene, 1987,56:125); The pMSXND expression vector of in mammalian cell, expressing (Lee and Nathans, J Bio Chem.263:3521,1988) and at the carrier that derives from baculovirus of expressed in insect cells.In a word, as long as can duplicate in host and stablize, any plasmid and carrier can be used.A key character of expression vector is to contain replication orgin, promotor, marker gene and translation controlling elements usually.

Method well-known to those having ordinary skill in the art can be used to make up and contains people CLG encoding histone dna sequence dna and suitable transcribing/the translate expression vector of control signal.These methods comprise (Sambroook, et al) such as extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technologys of body.Described dna sequence dna can effectively be connected on the suitable promotor in the expression vector, and is synthetic to instruct mRNA.The representative example of these promotors has: colibacillary lac or trp promotor; Lambda particles phage PL promotor; Eukaryotic promoter comprises LTRs and some other known may command gene expression promoter in protokaryon or eukaryotic cell or its virus of CMV immediate early promoter, HSV thymidine kinase promoter, early stage and late period SV40 promotor, retrovirus.Expression vector also comprises ribosome bind site and the transcription terminator that translation initiation is used.

In addition, expression vector preferably comprises one or more selected markers, to be provided for selecting the phenotypic character of transformed host cells, cultivate Tetrahydrofolate dehydrogenase, neomycin resistance and the green fluorescent protein (GFP) of usefulness as eukaryotic cell, or be used for colibacillary tsiklomitsin or amicillin resistance.

Comprise the carrier of above-mentioned suitable dna sequence dna and suitable promotor or control sequence, can be used to transform appropriate host cell, so that it can marking protein.

Host cell can be a prokaryotic cell prokaryocyte, as bacterial cell; Or eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as mammalian cell.Representative example has: intestinal bacteria, streptomyces; The bacterial cell of Salmonella typhimurium; Fungal cell such as yeast; Vegetable cell; The zooblast of the insect cell of fruit bat S2 or Sf9: CHO, COS or Bowes melanoma cells etc.

When polynucleotide of the present invention are expressed in higher eucaryotic cells, be enhanced if will make to transcribe when in carrier, inserting enhancer sequence.Enhanser is the cis acting factor of DNA, and nearly 10 to 300 base pairs act on promotor transcribing with enhancing gene usually.Can for example be included in the SV40 enhanser of 100 to 270 base pairs of replication origin side in late period one, at the polyoma enhanser of replication origin side in late period one and adenovirus enhanser etc.

Persons skilled in the art all know how to select appropriate carriers, promotor, enhanser and host cell.

Can carry out with routine techniques well known to those skilled in the art with the recombinant DNA transformed host cell.When the host was prokaryotic organism such as intestinal bacteria, the competent cell that can absorb DNA can be used CaCl in exponential growth after date results ₂Method is handled, and used step is well-known in this area.Alternative is to use MgCl ₂If desired, transforming also the method for available electroporation carries out.When the host is an eukaryote, can select following DNA transfection method for use: coprecipitation of calcium phosphate method, conventional mechanical method such as microinjection, electroporation, liposome packing etc.

The transformant that obtains can be cultivated with ordinary method, expresses the polypeptide of coded by said gene of the present invention.According to used host cell, used substratum can be selected from various conventional substratum in the cultivation.Under the condition that is suitable for the host cell growth, cultivate.After host cell grows into suitable cell density, induce the promotor of selection with suitable method (as temperature transition or chemical induction), cell is cultivated for some time again.

Recombinant polypeptide in the above methods can wrap by in cell, extracellular or on cytolemma, express or be secreted into the extracellular.If desired, can utilize its physics, the separating by various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation handles, with protein precipitant handle (salt analysis method), centrifugal, the broken bacterium of infiltration, superly handle, the combination of super centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods.

The people CLG albumen or the polypeptide of reorganization are of use in many ways.These purposes include, but is not limited to: the direct disease due to the low or forfeiture and be used to screen and promote or antibody, polypeptide or other part of antagonism CLG protein function as pharmacological agent CLG protein function.For example, antibody can be used for activating or suppressing the proteic function of people CLG.The peptide molecule that can suppress or stimulate people CLG protein function that can be used for seeking therapeutic value with the recombinant human CLG protein screening peptide library of expressing.

The present invention also provides screening of medicaments to identify the method that improves (agonist) or check the proteic medicament of (antagonist) people CLG.Agonist improves people CLG albumen biological function such as stimulate cellular proliferation, and antagonist prevention disorder such as the various cancer relevant with cell hyperproliferation with treatment.For example, can in the presence of medicine, mammalian cell or the proteic film preparation of expressing human CLG be cultivated with the people CLG albumen of mark.Measure the medicine raising then or check this interactional ability.

The proteic antagonist of people CLG comprises antibody, compound, acceptor disappearance thing and the analogue etc. that filter out.The proteic antagonist of people CLG can and be eliminated its function with people CLG protein binding, or suppresses the proteic generation of people CLG, or combines with the avtive spot of polypeptide and to make polypeptide can not bring into play biological function.The proteic antagonist of people CLG can be used for therepic use.

In screening during as the compound of antagonist, CLG albumen can be added during bioanalysis measures, determine by the interaction of measuring between compounds affect CLG albumen and its acceptor whether compound is antagonist.With the same quadrat method of above-mentioned SCREENED COMPOUND, can filter out the acceptor disappearance thing and the analogue of antagonist action.

Polypeptide of the present invention can be directly used in disease treatment, for example, and various malignant tumours and cellular abnormality propagation etc.

Polypeptide of the present invention, and fragment, derivative, analogue or their cell can be used as antigen to produce antibody.These antibody can be polyclone or monoclonal antibody.Polyclonal antibody can obtain by the method with this polypeptide direct injection animal.The technology of preparation monoclonal antibody comprises hybridoma technology, three knurl technology, people B-quadroma technology, EBV-hybridoma technology etc.

Can be with polypeptide of the present invention and antagonist and suitable pharmaceutical carrier combination back use.These carriers can be water, glucose, ethanol, salt, damping fluid, glycerine and their combination.Composition comprises the polypeptide or the antagonist of safe and effective amount and carrier and the vehicle that does not influence effect of drugs.These compositions can be used as medicine and are used for disease treatment.

The present invention also provides medicine box or the test kit that contains one or more containers, and one or more medicinal compositions compositions of the present invention are housed in the container.With these containers, can have by the given indicative prompting of government authorities of making, using or selling medicine or biological products, the government authorities that this prompting reflects production, uses or sells permits it to use on human body.In addition, polypeptide of the present invention can be used in combination with other treatment compound.

Pharmaceutical composition can be with mode administration easily, as by in part, intravenously, intraperitoneal, intramuscular, subcutaneous, the nose or the route of administration of intracutaneous.CLG albumen comes administration with the amount that treats and/or prevents concrete indication effectively.The proteic amount of CLG and the dosage range that are applied to the patient will depend on many factors, as administering mode, person's to be treated healthiness condition and diagnostician's judgement.

The proteic polynucleotide of people CLG also can be used for multiple therapeutic purpose.Gene therapy technology can be used for treating because cell proliferation, growth or the metabolic disturbance due to the proteic expression of CLG of the proteic nothing expression of CLG or unusual/non-activity.The CLG albumen that the gene therapy vector (as virus vector) of reorganization can be designed to express variation is to suppress endogenic CLG protein-active.For example, a kind of CLG albumen of variation can be the CLG albumen that shortens, lacked signal conduction function territory, though can combine with the substrate in downstream, lacks signaling activity.Therefore the gene therapy vector of reorganization can be used for treating the disease of CLG protein expression or active caused by abnormal.Deriving from viral expression vector such as retrovirus, adenovirus, adeno-associated virus (AAV), hsv, parvovirus etc. can be used for the CLG protein gene is transferred in the cell.The method that structure carries the recombinant viral vector of CLG protein gene is found in existing document (Sambrook, et al.).Recombinant human CLG protein gene can be packaged in the liposome and be transferred in the cell in addition.

Suppress the oligonucleotide (comprising sense-rna and DNA) of people CLG protein mRNA and ribozyme also within the scope of the invention.Ribozyme is the enzyme sample RNA molecule that a kind of energy specificity is decomposed specific RNA, and its mechanism of action is to carry out the endonuclease effect after ribozyme molecule and the hybridization of complementary target RNA-specific.The RNA of antisense and DNA and ribozyme can obtain with existing any RNA or DNA synthetic technology, as the technology widespread use of solid phase phosphoamide chemical synthesis synthetic oligonucleotide.Antisense rna molecule can be transcribed acquisition by the dna sequence dna of this RNA that encodes in external or body.This dna sequence dna has been incorporated into the downstream of rna polymerase promoter of carrier.In order to increase the stability of nucleic acid molecule, available several different methods is modified it, and as increasing the sequence length of both sides, the connection between the ribonucleoside is used phosphoric acid thioester bond or peptide bond but not phosphodiester bond.

Polynucleotide imports tissue or intracellular method comprises: directly be injected into polynucleotide in the in-vivo tissue; Or external by carrier (as virus, phage or plasmid etc.) earlier with the polynucleotide transfered cell in, again cell is transplanted in the body etc.

The present invention also provides the antibody at people CLG proteantigen determinant.These antibody include, but is not limited to: the fragment that polyclonal antibody, monoclonal antibody, chimeric antibody, single-chain antibody, Fab fragment and Fab expression library produce.The proteic antibody of anti-people CLG can be used in the immunohistochemistry technology, detects the people CLG albumen in the biopsy specimen.

With the also available labelled with radioisotope of the protein bound monoclonal antibody of people CLG, inject in the body and can follow the tracks of its position and distribution.This radiolabeled antibody can be used as a kind of atraumatic diagnostic method and is used for the location of tumour cell and has judged whether transfer.

Antibody among the present invention can be used for treating or prevention and the relevant disease of people CLG albumen.The antibody that gives suitable dosage can stimulate or block proteic generation of people CLG or activity.

Antibody also can be used for designing the immunotoxin at a certain privileged sites in the body.As the monoclonal antibody of people CLG albumen high-affinity can with bacterium or plant poison (as diphtheria toxin, ricin, abrine etc.) covalent attachment.A kind of usual method is with sulfydryl linking agent such as SPDP, attacks the amino of antibody, by the exchange of disulfide linkage, toxin is incorporated on the antibody, and this hybrid antibody can be used for killing the cell of people CLG protein positive.

The production of polyclonal antibody can choose CLG albumen or polypeptide immune animal, as rabbit, mouse, rat etc.Multiple adjuvant can be used for the enhancing immunity reaction, includes but not limited to freund's adjuvant etc.

People CLG protein monoclonal antibody can with hybridoma technology production (Kohler and Milstein.Nature, 1975,256:495-497).With the variable region bonded chimeric antibody in human constant region and inhuman source can with existing technology production (Morrison et al, PNAS, 1985,81:6851).And the technology of existing manufacture order chain antibody (U.S.PatNo.4946778) also can be used for producing the proteic single-chain antibody of anti-people CLG.

Can be incorporated into the rondom polypeptide storehouse that solid formation forms by the various amino acid that may make up by screening with the protein bound peptide molecule of people CLG obtains.During screening, must carry out mark to people CLG protein molecular.

The invention still further relates to the diagnostic testing process of quantitative and detection and localization people CLG protein level.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.The people CLG protein level that is detected in the test can be with laying down a definition the importance of people CLG albumen in various diseases and be used to the disease of diagnosing CLG albumen to work.

The proteic polynucleotide of CLG can be used for the diagnosis and the treatment of CLG protein related diseases.Aspect diagnosis, the proteic polynucleotide of CLG can be used for detecting the proteic expression of CLG CLG abnormal exprssion whether or under morbid state.Can be used for the hybridization of biopsy specimen to judge the proteic abnormal expression of CLG as CLG protein D NA sequence.Hybridization technique comprises the Southern blotting, Northern blotting, in situ hybridization etc.These technological methods all are disclosed mature technologies, and relevant test kit all can obtain from commercial channels.Part or all of polynucleotide of the present invention can be used as probe stationary on microarray (Microarray) or DNA chip (being called " gene chip " again), is used for analyzing the differential expression analysis and the gene diagnosis of tissue gene.Carry out RNA-polymerase chain reaction (RT-PCR) amplification in vitro with the special primer of CLG albumen and also can detect the proteic transcription product of CLG.

The sudden change that detects the CLG protein gene also can be used for the disease of diagnosing CLG albumen relevant.The form of CLG protein mutation comprises that the point mutation compared with normal wild type CLG protein D NA sequence, transposition, disappearance, reorganization and other are any unusual etc.Available existing technology such as Southern blotting, dna sequence analysis, PCR and in situ hybridization detect sudden change.In addition, sudden change might influence proteic expression, therefore can judge indirectly that with Northern blotting, Western blotting gene has or not sudden change.

Sequence of the present invention identifies it also is valuable to karyomit(e).This sequence can be specifically at certain bar human chromosome particular location and and can with its hybridization.At present, need to identify the concrete site of each gene on the karyomit(e).Now, have only chromosomal marker thing seldom to can be used for the marker chromosomes position based on actual sequence data (repetition polymorphism).According to the present invention, for these sequences are associated with disease related gene, its important the first step is positioned these dna sequence dnas on the karyomit(e) exactly.

In brief, prepare PCR primer (preferred 15-35bp), sequence can be positioned on the karyomit(e) according to cDNA.Then, these primers are used for the somatocyte hybrid cell that the PCR screening contains each bar human chromosome.Have only those hybrid cells that contain corresponding to the people's gene of primer can produce the fragment of amplification.

The PCR localization method of somatocyte hybrid cell is that DNA is navigated to concrete chromosomal quick method.Use Oligonucleolide primers of the present invention,, can utilize one group to realize inferior location from specific chromosomal fragment or a large amount of genomic clone by similar approach.Localized other the similar strategy of basis that can be used for dyeing comprises in situ hybridization, uses the karyomit(e) prescreen and the hybridization preliminary election of the airflow classification of mark, thereby makes up the special cDNA storehouse of karyomit(e).

The cDNA clone is carried out fluorescence in situ hybridization (FISH) with Metaphase Chromosome, can in a step, accurately carry out chromosomal localization.The summary of this technology is referring to Verma etc., Human Chromosomes:a Manual of BasicTechniques, Pergamon Press, New York (1988).

In case sequence is positioned to chromosome position accurately, the physical location of this sequence on karyomit(e) just can be associated with the gene map data.These data for example are found in, V.Mckusick, Mendelian Inheritance in Man (can by with the online acquisition of Johns Hopkins University Welch Medical Library).Can pass through linkage analysis then, determine gene and navigated to relation between the disease on the chromosomal region already.

Then, need to measure ill and not cDNA between diseased individuals or genome sequence difference.If observe certain sudden change in some or all of diseased individuals, and this sudden change is not observed in any normal individual, then this sudden change may be the cause of disease of disease.More ill and diseased individuals not is usually directed at first seek the variation of structure in the karyomit(e), as from the horizontal visible of karyomit(e) or use based on detectable disappearance of the PCR of cDNA sequence or transposition.Resolving power according to present physical mapping and assignment of genes gene mapping technology, being accurately positioned to the cDNA of the chromosomal region relevant with disease, can be a kind of (the supposing that 1 megabasse mapping resolving power and every 20kb are corresponding to a gene) between 50 to 500 potential Disease-causing genes.

CLG pyrenoids thuja acid full length sequence of the present invention or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.When sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.

In case obtained relevant sequence, just can obtain this sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.

In addition, also the method for available synthetic is synthesized relevant sequence, especially fragment length more in short-term.Usually, by first synthetic a plurality of small segments, and then connect and to obtain the very long fragment of sequence.

At present, can be fully come the dna sequence dna of code book invention albumen (or its fragment, or derivatives thereof) by chemosynthesis.This dna sequence dna can be introduced then in the various dna moleculars (as carrier) and cell in this area.In addition, also can will suddenly change and introduce in the protein sequence of the present invention by chemosynthesis.

In addition, because CLG albumen of the present invention has the natural acid sequence that is derived from the people, therefore, compare, estimate to have higher active and/or lower side effect (for example in the intravital immunogenicity of people lower or do not have) being applied to man-hour with the albumen of the same clan that derives from other species.

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions among the embodiment, usually according to people such as normal condition such as Sambrook, molecular cloning: the condition described in the laboratory manual (New York:Cold SpringHarbor Laboratory Press, 1989), or the condition of advising according to manufacturer.

The acquisition of embodiment 1:cDNA gene fragment and the restraining effect that the cancer cells clone is formed

PP3898 (being CLG) comes from ordinary method and makes up the human placenta cDNA library.Get the placenta tissue at 3,6,10 monthly ages, (GIBCO BRL company) extracts total RNA by manufacturer's specification sheets with Trizol reagent, extracts mRNA with the mRNA test kit (Pharmacia company) of purifying.Make up the cDNA library of above-mentioned mRNA with pCMV-script TMXR cDNA library construction test kit (Seratagene company).Wherein ThermoScript II is used MMLV-RT-Superscript II (GIBCOBRL) instead, and reverse transcription reaction carries out at 42 ℃.Transform XL 10-Gold recipient cell, obtained 1 * 10 ⁶The cDNA library of cfu/ μ g cDNA titre.The first round is picking cDNA clone at random, is probe with high abundance cDNA clone with the cDNA clone who has proved cancer inhibitor cell growth function thereafter, screening by hybridization cDNA library, weak positive and negative clone of picking.With Qiagen 96 orifice plate plasmid extraction test kits, carry out the extraction of plasmid DNA by shop instruction.Plasmid DNA and empty carrier transfection simultaneously hepatoma cell line 7721.After the 100ng DNA alcohol precipitation drying, add 6 μ l H2O dissolving, treat transfection.Add 0.74 μ l liposome and 9.3 μ l serum-free mediums in every part of DNA sample, behind the mixing, room temperature was placed 10 minutes.Add 150 μ l serum-free mediums in every pipe, divide equally and add 3 holes and grow in 7721 cells of 96 orifice plates, placed 2 hours for 37 ℃, every hole adds 50 μ l serum-free mediums again, 37 ℃ 24 hours.Every hole is changed 100 μ l and is trained liquid entirely, 37 ℃ 24 hours, change the full training liquid 100 μ l that contain G418,37 ℃ 24-48 hour, the limit is observed, the training liquid that G418 concentration does not wait is changed on the limit.After about 2-3 time, there is the clone to form up to the microscopy cell, counting.Find that PP3898 (being CLG) has the cell clone formation effect (inhibiting rate is more than 50% or 50%) of inhibition, the result is as shown in the table.

Table 1 cDNA clone's transfectional cell (7721) clone formation situation

CDNA clone title C DNA cloning number (three repetitions) empty carrier clone number (three repetitions)

PP3898 (being CLG) 200 33 34 38

The cDNA clone is adopted two deoxidation cessation method, on the ABI377 automatic dna sequencer, measure the nucleotide sequence of the nearly 500bp of one end.After the analysis, be defined as novel gene cloning, check order fully again, find that the CLG fragment is non-full length sequence, need to obtain full length cDNA clone with the RACE method.

Embodiment 2:RACE method obtains full length cDNA clone and the RT-PCR method obtains the CLC gene

1.RACE method obtains full length cDNA clone

To finding after the analysis of PP3898 cDNA cloned sequence that gene is still imperfect, adopt the Clontech SMART RACEcDNA of company amplification kit (Cat.No.K1811-1), design gene specific primer (as shown in table 2 below), by specification is operated, and obtains full-length clone.

Table 2 CLG gene specific primer

Clone's title	Special primer 1 (pp3898-NB)	Special primer 2 (pp3898-B)
			PP3898	5’TCATCCAGCCGGTCACTTGACTTGA 3’	5’GCCACAGCTGGTAGTTGGACTTGCC 3’

Particularly, the PP3898 clone is used following primer:

Universal primer mix (UPM) Long 5 ' CTAATACGACTCACTATAGGGCAAGCAGTGGTAACAACGCAGAGT3 '

Nido universal primer (NUP) 5 ' AAGCAGTGGTAACAACGCAGAGT 3 '

pp3898-NB 5’TCATCCAGCCGGTCACTTGACTTGA 3’

pp3898-B 5’GCCACAGCTGGTAGTTGGACTTGCC 3’

With human placenta mRNA is parent material, obtain cDNA. by Clontech company SMART RACE cDNA amplification kit (Cat#1811-1) specification sheets and carry out first round PCR with the pp3898-B primer of UPM primer and gene specific respectively then, carry out second with the pp3898-NB primer of NUP primer and gene specific again and take turns PCR, obtain gene fragment.

Reaction conditions is as follows: 94 ℃ 1 minute, a circulation; 94 ℃ 30 seconds, 72 ℃ 4 minutes, 5 circulations; 94 ℃ 30 seconds, 70 ℃ 4 minutes, 5 circulations; 94 ℃ 20 seconds, 65 ℃ 30 seconds, 68 ℃ 4 minutes, 27 circulations.

SMART RACE reaction obtains 5 ' of CLG gene and extends fragment, connects through reorganization, obtains the cDNA clone of total length CLG gene, shown in SEQ ID NO:1.

2.RT-PCR method obtains the CLG gene

Synthetic primer CLG-F (5 ' ATGGTGGTGATGGCGCGACTCTCG 3 ', bp 22-45 position) and CLG-R (5 ' GGTCAGTCTTCCTTCAGGCTCCC 3 ', bp 2569-2591 position), adopt the method (wherein the RT-PCR reaction conditions is identical with above-mentioned SMART RACE reaction conditions) of RT-PCR, thereby obtain to contain the CLG gene of complete coding region, RT-PCR product size is 2570bp.

Embodiment 3:CLG cDNA clone's sequential analysis

A: nucleotide sequence (SEQ ID NO:1) length: 2659

GGTACCTGGG CATCCAGAAA AATGGTGGTG ATGGCGCGAC TCTCGCGGCC CGAGCGGCCG 60

GACCTTGTCT TCGAGGAAGA GGACCTCCCC TATGAGGAGG AAATCATGCG GAACCAATTC 120

TCTGTCAAAT GCTGGCTTCG CTACATCGAG TTCAAACAGG GCGCCCCGAA GCCCAGGCTC 180

AATCAGCTAT ACGAGCGGGC ACTCAAGCTG CTGCCCTGCA GCTACAAACT CTGGTACCGA 240

TACCTGAAGG CGCGTCGGGC ACAGGTGAAG CATCGCTGTG TGACCGACCC TGCCTATGAA 300

GATGTCAACA ACTGTCATGA GAGGGCCTTT GTGTTCATGC ACAAGATGCC TCGTCTGTGG 360

CTAGATTACT GCCAGTTCCT CATGGACCAG GGGCGCGTCA CACACACCCG CCGCACCTTC 420

GACCGTGCCC TCCGGGCACT GCCCATCACG CAGCACTCTC GAATTTGGCC CCTGTATCTG 480

CGCTTCCTGC GCTCACACCC ACTGCCTGAG ACAGCTGTGC GAGGCTATCG GCGCTTCCTC 540

AAGCTGAGTC CTGAGAGTGC AGAGGAGTAC ATTGAGTACC TCAAGTCAAG TGACCGGCTG 600

GATGAGGCCG CCCAGCGCCT GGCCACCGTG GTGAACGACG AGCGTTTCGT GTCTAAGGCC 660

GGCAAGTCCA ACTACCAGCT GTGGCACGAG CTGTGCGACC TCATCTCCCA GAATCCGGAC 720

AAGGTACAGT CCCTCAATGT GGACGCCATC ATCCGCGGGG GCCTCACCCG CTTCACCGAC 780

CAGCTGGGCA AGCTCTGGTG TTCTCTCGCC GACTACTACA TCCGCAGCGG CCATTTCGAG 840

AAGGCTCGGG ACGTGTACGA GGAGGCCATC CGGACAGTGA TGACCGTGCG GGACTTCACA 900

CAGGTGTTTG ACAGCTACGC CCAGTTCGAG GAGAGCATGA TCGCTGCAAA GATGGAGACC 960

GCCTCGGAGC TGGGGCGCGA GGAGGAGGAT GATGTGGACC TGGAGCTGCG CCTGGCCCGC 1020

TTCGAGCAGC TCATCAGCCG GCGGCCCCTG CTCCTCAACA GCGTCTTGCT GCGCCAAAAC 1080

CCACACCACG TGCACGAGTG GCACAAGCGT GTCGCCCTGC ACCAGGGCCG CCCCCGGGAG 1140

ATCATCAACA CCTACACAGA GGCTGTGCAG ACGGTGGACC CCTTCAAGGC CACAGGCAAG 1200

CCCCACACTC TGTGGGTGGC GTTTGCCAAG TTTTATGAGG ACAACGGACA GCTGGACGAT 1260

GCCCGTGTCA TCCTGGAGAA GGCCACCAAG GTGAACTTCA AGCAGGTGGA TGACCTGGCA 1320

AGCGTGTGGT GTCAGTGCGG AGAGCTGGAG CTCCGACACG AGAACTACGA TGAGGCCTTG 1380

CGGCTGCTGC GAAAGGCCAC GGCGCTGCCT GCCCGCCGGG CCGAGTACTT TGATGGTTCA 1440

GAGCCCGTGC AGAACCGCGT GTACAAGTCA CTGAAGGTCT GGTCCATGCT CGCCGACCTG 1500

GAGGAGAGCC TCGGCACCTT CCAGTCCACC AAGGCCGTGT ACGACCGCAT CCTGGACCTG 1560

CGTATCGCAA CACCCCAGAT CGTCATCAAC TATGCCATGT TCCTGGAGGA GCACAAGTAC 1620

TTCGAGGAGA GCTTCAAGGC GTACGAGCGC GGCATCTCGC TGTTCAAGTG GCCCAACGTG 1680

TCCGACATCT GGAGCACCTA CCTGACCAAA TTCATTGCCC GCTATGGGGG CCGCAAGCTG 1740

GAGCGGGCAC GGGACCTGTT TGAACAGGCT CTGGACGGCT GCCCCCCAAA ATATGCCAAG 1800

ACCTTGTACC TGCTGTACGC ACAGCTGGAG GAGGAGTGGG GCCTGGCCCG GCATGCCATG 1860

GCCGTGTACG AGCGTGCCAC CAGGGCCGTG GAGCCCGCCC AGCAGTATGA CATGTTCAAC 1920

ATCTACATCA AGCGGGCGGC CGAGATCTAT GGGGTCACCC ACACCCGCGG CATCTACCAG 1980

AAGGCCATTG AGGTGCTGTC GGACGAGCAC GCGCGTGAGA TGTGCCTGCG GTTTGCAGAC 2040

ATGGAGTGCA AGCTCGGGGA GATTGACCGC GCCCGGGCCA TCTACAGCTT CTGCTCCCAG 2100

ATCTGTGACC CCCGGACGAC CGGCGCGTTC TGGCAGACGT GGAAGGACTT TGAGGTCCGG 2160

CATGGCAATG AGGACACCAT CAAGGAAATG CTGCGTATCC GGCGCAGCGT GCAGGCCACG 2220

TACAACACGC AGGTCAACTT CATGGCCTCG CAGATGCTCA AGGTCTCGGG CAGTGCCACG 2280

GGCACCGTGT CTGACCTGGC CCCTGGGCAG AGTGGCATGG ACGACATGAA GCTGCTGGAA 2340

CAGCGGGCAG AGCAGCTGGC GGCTGAGGCG GAGCGTGACC AGCCCTTGCG CGCCCAGAGC 2400

AAGATCCTGT TCGTGAGGAG TGACGCCTCC CGGGAGGAGC TGGCAGAGCT GGCACAGCAG 2460

GTCAACCCCG AGGAGATCCA GCTGGGCGAG GACGAGGACG AGGACGAGAT GGACCTGGAG 2520

CCCAACGAGG TTCGGCTGGA GCAGCAGAGC GTGCCAGCCG CAGTGTTTGG GAGCCTGAAG 2580

GAAGACTGAC CCGTCCCTCC CCCCTCCCCA CCCCCTCCCC AATACAGCTA CGTTTGTAAA 2640

AAAAAAAAAA AAAAAAAAA 2659

B: aminoacid sequence (SEQ ID NO:2) length: 855

1 MVVMARLSRP ERPDLVFEEE DLPYEEEIMR NQFSVKCWLR YIEFKQGAPK

51 PRLNQLYERA LKLLPCSYKL WYRYLKARRA QVKHRCVTDP AYEDVNNCHE

101 RAFVFMHKMP RLWLDYCQFL MDQGRVTHTR RTFDRALRAL PITQHSRIWP

151 LYLRFLRSHP LPETAVRGYR RFLKLSPESA EEYIEYLKSS DRLDEAAQRL

201 ATVVNDERFV SKAGKSNYQL WHELCDLISQ NPDKVQSLNV DAIIRGGLTR

251 FTDQLGKLWC SLADYYIRSG HFEKARDVYE EAIRTVMTVR DFTQVFDSYA

301 QFEESMIAAK METASELGRE EEDDVDLELR LARFEQLISR RPLLLNSVLL

351 RQNPHHVHEW HKRVALHQGR PREIINTYTE AVQTVDPFKA TGKPHTLWVA

401 FAKFYEDNGQ LDDARVILEK ATKVNFKQVD DLASVWCQCG ELELRHENYD

451 EALRLLRKAT ALPARRAEYF DGSEPVQNRV YKSLKVWSML ADLEESLGTF

501 QSTKAVYDRI LDLRIATPQI VINYAMFLEE HKYFEESFKA YERGISLFKW

551 PNVSDIWSTY LTKFIARYGG RKLERARDLF EQALDGCPPK YAKTLYLLYA

601 QLEEEWGLAR HAMAVYERAT RAVEPAQQYD MFNIYIKRAA EIYGVTHTRG

651 IYQKAIEVLS DEHAREMCLR FADMECKLGE IDRARAIYSF CSQICDPRTT

701 GAFWQTWKDF EVRHGNEDTI KEMLRIRRSV QATYNTQVNF MASQMLKVSG

751 SATGTVSDLA PGQSGMDDMK LLEQRAEQLA AEAERDQPLR AQSKILFVRS

801 DASREELAEL AQQVNPEEIQ LGEDEDEDEM DLEPNEVRLE QQSVPAAVFG

851 SLKED

C. Nucleotide and amino acid composite sequence: (SEQ ID NO:3) clone number: CLG (being PP3898) start code: 22 ATG stop coding: 2589 TGA protein molecular weights: 100004.44

1 GGT ACC TGG GCA TCC AGA AAA ATG GTG GTG ATG GCG CGA CTC TCG CGG 48

1 Met Val Val Met Ala Arg Leu Ser Arg 9

49 CCC GAG CGG CCG GAC CTT GTC TTC GAG GAA GAG GAC CTC CCC TAT GAG 96

10 Pro Glu Arg Pro Asp Leu Val Phe Glu Glu Glu Asp Leu Pro Tyr Glu 25

97 GAG GAA ATC ATG CGG AAC CAA TTC TCT GTC AAA TGC TGG CTT CGC TAC 144

26 Glu Glu Ile Met Arg Asn Gln Phe Ser Val Lys Cys Trp Leu Arg Tyr 41

145 ATC GAG TTC AAA CAG GGC GCC CCG AAG CCC AGG CTC AAT CAG CTA TAC 192

42 Ile Glu Phe Lys Gln Gly Ala Pro Lys Pro Arg Leu Asn Gln Leu Tyr 57

193 GAG CGG GCA CTC AAG CTG CTG CCC TGC AGC TAC AAA CTC TGG TAC CGA 240

58 Glu Arg Ala Leu Lys Leu Leu Pro Cys Ser Tyr Lys Leu Trp Tyr Arg 73

241 TAC CTG AAG GCG CGT CGG GCA CAG GTG AAG CAT CGC TGT GTG ACC GAC 288

74 Tyr Leu Lys Ala Arg Arg Ala Gln Val Lys His Arg Cys Val Thr Asp 89

289 CCT GCC TAT GAA GAT GTC AAC AAC TGT CAT GAG AGG GCC TTT GTG TTC 336

90 Pro Ala Tyr Glu Asp Val Asn Asn Cys His Glu Arg Ala Phe Val Phe 105

337 ATG CAC AAG ATG CCT CGT CTG TGG CTA GAT TAC TGC CAG TTC CTC ATG 384

106 Met His Lys Met Pro Arg Leu Trp Leu Asp Tyr Cys Gln Phe Leu Met 121

385 GAC CAG GGG CGC GTC ACA CAC ACC CGC CGC ACC TTC GAC CGT GCC CTC 432

122 Asp Gln Gly Arg Val Thr His Thr Arg Arg Thr Phe Asp Arg Ala Leu 137

433 CGG GCA CTG CCC ATC ACG CAG CAC TCT CGA ATT TGG CCC CTG TAT CTG 480

138 Arg Ala Leu Pro Ile Thr Gln His Ser Arg Ile Trp Pro Leu Tyr Leu 153

481 CGC TTC CTG CGC TCA CAC CCA CTG CCT GAG ACA GCT GTG CGA GGC TAT 528

154 Arg Phe Leu Arg Ser His Pro Leu Pro Glu Thr Ala Val Arg Gly Tyr 169

529 CGG CGC TTC CTC AAG CTG AGT CCT GAG AGT GCA GAG GAG TAC ATT GAG 576

170 Arg Arg Phe Leu Lys Leu Ser Pro Glu Ser Ala Glu Glu Tyr Ile Glu 185

577 TAC CTC AAG TCA AGT GAC CGG CTG GAT GAG GCC GCC CAG CGC CTG GCC 624

186 Tyr Leu Lys Ser Ser Asp Arg Leu Asp Glu Ala Ala Gln Arg Leu Ala 201

625 ACC GTG GTG AAC GAC GAG CGT TTC GTG TCT AAG GCC GGC AAG TCC AAC 672

202 Thr Val Val Asn Asp Glu Arg Phe Val Ser Lys Ala Gly Lys Ser Asn 217

673 TAC CAG CTG TGG CAC GAG CTG TGC GAC CTC ATC TCC CAG AAT CCG GAC 720

218 Tyr Gln Leu Trp His Glu Leu Cys Asp Leu Ile Ser Gln Asn Pro Asp 233

721 AAG GTA CAG TCC CTC AAT GTG GAC GCC ATC ATC CGC GGG GGC CTC ACC 768

234 Lys Val Gln Ser Leu Asn Val Asp Ala Ile Ile Arg Gly Gly Leu Thr 249

769 CGC TTC ACC GAC CAG CTG GGC AAG CTC TGG TGT TCT CTC GCC GAC TAC 816

250 Arg Phe Thr Asp Gln Leu Gly Lys Leu Trp Cys Ser Leu Ala Asp Tyr 265

817 TAC ATC CGC AGC GGC CAT TTC GAG AAG GCT CGG GAC GTG TAC GAG GAG 864

266 Tyr Ile Arg Ser Gly His Phe Glu Lys Ala Arg Asp Val Tyr Glu Glu 281

865 GCC ATC CGG ACA GTG ATG ACC GTG CGG GAC TTC ACA CAG GTG TTT GAC 912

282 Ala Ile Arg Thr Val Met Thr Val Arg Asp Phe Thr Gln Val Phe Asp 297

913 AGC TAC GCC CAG TTC GAG GAG AGC ATG ATC GCT GCA AAG ATG GAG ACC 960

298 Ser Tyr Ala Gln Phe Glu Glu Ser Met Ile Ala Ala Lys Met Glu Thr 313

961 GCC TCG GAG CTG GGG CGC GAG GAG GAG GAT GAT GTG GAC CTG GAG CTG 1008

314 Ala Ser Glu Leu Gly Arg Glu Glu Glu Asp Asp Val Asp Leu Glu Leu 329

1009 CGC CTG GCC CGC TTC GAG CAG CTC ATC AGC CGG CGG CCC CTG CTC CTC 1056

330 Arg Leu Ala Arg Phe Glu Gln Leu Ile Ser Arg Arg Pro Leu Leu Leu 345

1057 AAC AGC GTC TTG CTG CGC CAA AAC CCA CAC CAC GTG CAC GAG TGG CAC 1104

346 Asn Ser Val Leu Leu Arg Gln Asn Pro His His Val His Glu Trp His 361

1105 AAG CGT GTC GCC CTG CAC CAG GGC CGC CCC CGG GAG ATC ATC AAC ACC 1152

362 Lys Arg Val Ala Leu His Gln Gly Arg Pro Arg Glu Ile Ile Asn Thr 377

1153 TAC ACA GAG GCT GTG CAG ACG GTG GAC CCC TTC AAG GCC ACA GGC AAG 1200

378 Tyr Thr Glu Ala Val Gln Thr Val Asp Pro Phe Lys Ala Thr Gly Lys 393

1201 CCC CAC ACT CTG TGG GTG GCG TTT GCC AAG TTT TAT GAG GAC AAC GGA 1248

394 Pro His Thr Leu Trp Val Ala Phe Ala Lys Phe Tyr Glu Asp Asn Gly 409

1249 CAG CTG GAC GAT GCC CGT GTC ATC CTG GAG AAG GCC ACC AAG GTG AAC 1296

410 Gln Leu Asp Asp Ala Arg Val Ile Leu Glu Lys Ala Thr Lys Val Asn 425

1297 TTC AAG CAG GTG GAT GAC CTG GCA AGC GTG TGG TGT CAG TGC GGA GAG 1344

426 Phe Lys Gln Val Asp Asp Leu Ala Ser Val Trp Cys Gln Cys Gly Glu 441

1345 CTG GAG CTC CGA CAC GAG AAC TAC GAT GAG GCC TTG CGG CTG CTG CGA 1392

442 Leu Glu Leu Arg His Glu Asn Tyr Asp Glu Ala Leu Arg Leu Leu Arg 457

1393 AAG GCC ACG GCG CTG CCT GCC CGC CGG GCC GAG TAC TTT GAT GGT TCA 1440

458 Lys Ala Thr Ala Leu Pro Ala Arg Arg Ala Glu Tyr Phe Asp Gly Ser 473

1441 GAG CCC GTG CAG AAC CGC GTG TAC AAG TCA CTG AAG GTC TGG TCC ATG 1488

474 Glu Pro Val Gln Asn Arg Val Tyr Lys Ser Leu Lys Val Trp Ser Met 489

1489 CTC GCC GAC CTG GAG GAG AGC CTC GGC ACC TTC CAG TCC ACC AAG GCC 1536

490 Leu Ala Asp Leu Glu Glu Ser Leu Gly Thr Phe Gln Ser Thr Lys Ala 505

1537 GTG TAC GAC CGC ATC CTG GAC CTG CGT ATC GCA ACA CCC CAG ATC GTC 1584

506 Val Tyr Asp Arg Ile Leu Asp Leu Arg Ile Ala Thr Pro Gln Ile Val 521

1585 ATC AAC TAT GCC ATG TTC CTG GAG GAG CAC AAG TAC TTC GAG GAG AGC 1632

522 Ile Asn Tyr Ala Met Phe Leu Glu Glu His Lys Tyr Phe Glu Glu Ser 537

1633 TTC AAG GCG TAC GAG CGC GGC ATC TCG CTG TTC AAG TGG CCC AAC GTG 1680

538 Phe Lys Ala Tyr Glu Arg Gly Ile Ser Leu Phe Lys Trp Pro Asn Val 553

1681 TCC GAC ATC TGG AGC ACC TAC CTG ACC AAA TTC ATT GCC CGC TAT GGG 1728

554 Ser Asp Ile Trp Ser Thr Tyr Leu Thr Lys Phe Ile Ala Arg Tyr Gly 569

1729 GGC CGC AAG CTG GAG CGG GCA CGG GAC CTG TTT GAA CAG GCT CTG GAC 1776

570 Gly Arg Lys Leu Glu Arg Ala Arg Asp Leu Phe Glu Gln Ala Leu Asp 585

1777 GGC TGC CCC CCA AAA TAT GCC AAG ACC TTG TAC CTG CTG TAC GCA CAG 1824

586 Gly Cys Pro Pro Lys Tyr Ala Lys Thr Leu Tyr Leu Leu Tyr Ala Gln 601

1825 CTG GAG GAG GAG TGG GGC CTG GCC CGG CAT GCC ATG GCC GTG TAC GAG 1872

602 Leu Glu Glu Glu Trp Gly Leu Ala Arg His Ala Met Ala Val Tyr Glu 617

1873 CGT GCC ACC AGG GCC GTG GAG CCC GCC CAG CAG TAT GAC ATG TTC AAC 1920

618 Arg Ala Thr Arg Ala Val Glu Pro Ala Gln Gln Tyr Asp Met Phe Asn 633

1921 ATC TAC ATC AAG CGG GCG GCC GAG ATC TAT GGG GTC ACC CAC ACC CGC 1968

634 Ile Tyr Ile Lys Arg Ala Ala Glu Ile Tyr Gly Val Thr His Thr Arg 649

1969 GGC ATC TAC CAG AAG GCC ATT GAG GTG CTG TCG GAC GAG CAC GCG CGT 2016

650 Gly Ile Tyr Gln Lys Ala Ile Glu Val Leu Ser Asp Glu His Ala Arg 665

2017 GAG ATG TGC GTG CGG TTT GCA GAC ATG GAG TGC AAG CTC GGG GAG ATT 2064

666 Glu Met Cys Leu Arg Phe Ala Asp Met Glu Cys Lys Leu Gly Glu Ile 681

2065 GAC CGC GCC CGG GCC ATC TAC AGC TTC TGC TCC CAG ATC TGT GAC CCC 2112

682 Asp Arg Ala Arg Ala Ile Tyr Ser Phe Cys Ser Gln Ile Cys Asp Pro 697

2113 CGG ACG ACC GGC GCG TTC TGG CAG ACG TGG AAG GAC TTT GAG GTC CGG 2160

698 Arg Thr Thr Gly Ala Phe Trp Gln Thr Trp Lys Asp Phe Glu Val Arg 713

2161 CAT GGC AAT GAG GAC ACC ATC AAG GAA ATG CTG CGT ATC CGG CGC AGC 2208

714 His Gly Asn Glu Asp Thr Ile Lys Glu Met Leu Arg Ile Arg Arg Ser 729

2209 GTG CAG GCC ACG TAC AAC ACG CAG GTC AAC TTC ATG GCC TCG CAG ATG 2256

730 Val Gln Ala Thr Tyr Asn Thr Gln Val Asn Phe Met Ala Ser Gln Met 745

2257 CTC AAG GTC TCG GGC AGT GCC ACG GGC ACC GTG TCT GAC CTG GCC CCT 2304

746 Leu Lys Val Ser Gly Ser Ala Thr Gly Thr Val Ser Asp Leu Ala Pro 761

2305 GGG CAG AGT GGC ATG GAC GAC ATG AAG CTG CTG GAA CAG CGG GCA GAG 2352

762 Gly Gln Ser Gly Met Asp Asp Met Lys Leu Leu Glu Gln Arg Ala Glu 777

2353 CAG CTG GCG GCT GAG GCG GAG CGT GAC CAG CCC TTG CGC GCC CAG AGC 2400

778 Gln Leu Ala Ala Glu Ala Glu Arg Asp Gln Pro Leu Arg Ala Gln Ser 793

2401 AAG ATC CTG TTC GTG AGG AGT GAC GCC TCC CGG GAG GAG CTG GCA GAG 2448

794 Lys Ile Leu Phe Val Arg Ser Asp Ala Ser Arg Glu Glu Leu Ala Glu 809

2449 CTG GCA CAG CAG GTC AAC CCC GAG GAG ATC CAG CTG GGC GAG GAC GAG 2496

810 Leu Ala Gln Gln Val Asn Pro Glu Glu Ile Gln Leu Gly Glu Asp Glu 825

2497 GAC GAG GAC GAG ATG GAC CTG GAG CCC AAC GAG GTT CGG CTG GAG CAG 2544

826 Asp Glu Asp Glu Met Asp Leu Glu Pro Asn Glu Val Arg Leu Glu Gln 841

2545 CAG AGC GTG CCA GCC GCA GTG TTT GGG AGC CTG AAG GAA GAC TGA CCC 2592

842 Gln Ser Val Pro Ala Ala Val Phe Gly Ser Leu Lys Glu Asp *** 856

2593 GTC CCT CCC CCC TCC CCA CCC CCT CCC CAA TAC AGC TAC GTT TGT AAA 2640

2641 AAA AAA AAA AAA AAA AAA A 2659

Embodiment 4: homology comparison and structural analysis

Use Blast software, aminoacid sequence to CLG carries out homology analysis, discovery and fruit bat, nematode and zymic cell cycle regulating protein height homology: with cell cycle regulating protein (the crooked neck of fruit bat, crn) 41% (157/375) homology is arranged, have 66% (557/837) homology with the cell cycle regulating protein of nematode, 37% (228/599) homology is arranged with zymic cell cycle control albumen.

Query=CLG Sbjct=crn (fruit bat)

＞crn mrna length=702

Score value=69.9bits (168), predicated value=6e-11

Homogeny=88/375 (23%), similarity=157/375 (41%), breach=33/375 (8%)

Query：398 WVAFAKFYEDNGQLDDARVILEKATKVNFKQVDDLAS-VWCQCGELELRHENYDEALRLL 456

W+ FA+F E +G + +R + E+A V F D + ++ E + +D A +

Sbjct：213 WIKFARFEESHGFIHGSRRVFERA--VEFFGDDYIEERLFIAFARFEEGQKEHDRARIIY 270

Query：457 RKATA-LPARRAEYFDGSEPVQNRVYKSLKVWSMLADLEESLGTFQSTKAVYDRILDLRI 515

+ A LP D ++ + K K + A +E+ + K Y++ +

Sbjct：271 KYALDHLPK------DRTQELFKAYTKHEKKYGDRAGIEDVI--VSKRKYQYEQEVAANP 322

Query：516 ATPQIVINYAMFLEEHKYFEESFKAYERGISLFKWPNVSDIWSTYLTKFI-----ARYGG 570

+Y +E ++ + YER IS N + W Y+ +I

Sbjct：323 TNYDAWFDYLRLIEAEGDRDQIRETYERAISNVPPANEKNFWRRYIYLWINYALYEELEA 382

Query：571 RKLERARDLFEQALDGCPPKYA--KTLYLLYAQLE---EEWGLARHAMAVYERATRAVEP 625

ER R +++ L+ P K L+LLYAQ E +E AR A+ + A+

Sbjct：383 EDAERTRQIYKTCLELIPHKQFTFSKLWLLYAQFEIRCKELQRARKALGL------AIGM 436

Query：626 AQQYDMFNIYIKRAAEIYGVTHTRGIYQKAIEVLSDEHAREMCLRFADMECKLGEIDRAR 685

+ +F YI ++ R +Y+K +E + M +FA++E LG+ DRAR

Sbjct：437 CPRDKLFRGYIDLEIQLREFERCRMLYEKFLEFGPENCVTWM--KFAELENLLGDTDRAR 494

Query：686 AIYSFCSQICDPRTTGAFWQTWKDFEVRHGNEDTIKEMLRIRRSVQATYNTQV-NFMASQ 744

AI+ Q W+ + DFEV G + +++ R ++ T + +V N A

Sbjct：495 AIFELAVQQPRLDMPEELWKAYIDFEVALGETELARQL--YERLLERTQHVKVWNSFAKF 552

Query：745 MLKVSGSATGTVSDL 759

+ +S +G ++L

Sbjct：553 EMGLSHGDSGPDAEL 567

Query=CLG Sbjct=and drosophila cell cycle regulatory protein homologous nematode (C.Elegans) albumen＞with drosophila cell cycle regulatory protein homologous nematode (C.Elegans) albumen. length=855 score values=792 bits (2023), predicated value=0.0 homogeny=395/837 (47%), similarity=557/837 (66%), breach=17/837 (2%)

Query：20 EDLPYEEEIMRNQFSVKCWLRYIEFK--QGAPKPRLNQLYERALKLLPCSYKLWYRYLKA 77

ED+P+EE+I+RN SV CW RYI+ K +P ++ +YERAL + SYKLWY YLK

Sbjct：25 EDVPFEEDIIRNPTSVNCWQRYIDHKLQNKSPAKQMFLIYERALAVFERSYKLWYHYLKY 84

Query：78 RRAQVKHRCVTDPAYEDVNNCHERAFVFMHKMPRLWLDYCQFLMDQGRVTHTRRTFDRAL 137

R + + ++C TD ++ + + +ER + +HKMPR+W+ YC+ ++ +G +T TRR FDRAL

Sbjct：85 RESTIVNKCPTDNSWRALCDTYERCLMRLHKMPRIWICYCEVMIKRGLITETRRVFDRAL 144

Query：138 RALPITQHSRIWPLYLRFLRSHPLPETAVRGYRRFLKLSPESAEEYIEYLKSSDRLDEAA 197

R+LP+TQH RIW LY+ FL SH LPET +R YRR+LK++P++ E+Y+EYL D++DEAA

Sbjct：145 RSLPVTQHMRIWTLYIGFLTSHDLPETTIRVYRRYLKMNPKAREDYVEYLIERDQIDEAA 204

Query：198 QRLATVVNDERFVSKAGKSNYQLWHELCDLISQNPDKVQSLNVDAIIRGGLTRFTDQLGK 257

+L T+VN ++ VS+ G++ +QLW +LCDLIS+NP K+ SLNVDAIIR G+ R+TDQ+G

Sbjct：205 KELTTLVNQDQNVSEKGRTAHQLWTQLCDLISKNPVKIFSLNVDAIIRQGIYRYTDQVGF 264

Query：258 LWCSLADYYIRSGHFEKARDVYEEAIRTVMTVRDFTQVFDSYAQFEESMIAAKMETASXX 317

LWCSLADYYIRS FE+ARDVYEEAI V TVRDF QV+D+YA FEE ++ M+

Sbjct：265 LWCSLADYYIRSAEFERARDVYEEAIAKVSTVRDFAQVYDAYAAFEEREVSIMMQEVEQS 324

Query：318 XXXXXXXXXXXXXXXXFEQLISRRPLLLNSVLLRQNPHHVHEWHKRVALHQGRPREIINT 377

++ L+ R+ L+NSVLLRQNPH+V EW RV +++G + I T

Sbjct：325 GDPEEEVDLEWMFQR-YQHLMERKNELMNSVLLRQNPHNVGEWLNRVNIYEGNYNKQIET 383

Query：378 YTEAVQTVDPFKATGKPHTLWVAFAKFYEDNGQLDDARVILEKATKVNFKQVDDLASVWC 437

+EAV++V+P GK LW+ AK YEDNG LD AR E A F V +LA+VWC

Sbjct：384 FKEAVKSVNPKIQVGKVRDLWIGLAKLYEDNGDLDAARKTFETAVISQFGGVSELANVWC 443

Query：438 QCGELELRHENYDEALRLLRKATALPARRAEYFDGSEPVQNRVYKSLKVWSMLADLEESL 497

E+E++H+ AL ++++A +P + ++ + VQ RV++S +W+M AD EE

Sbjct：444 AYAEMEMKHKRAKAALTVMQRACVVP--KPGDYENMQSVQARVHRSPILWAMYADYEECC 501

Query：498 GTFQSTKAVYDRILDLRIATPQIVINYAMFLEEHKYFEESFKAYERGISLFKWPNVSDIW 557

GT +S + VYD++++LR+A+PQ+++NYAMFLEE++YFE +F+AYE+GI+LFKWP V DIW

SbjCt：502 GTVESCRKVYDKMIELRVASPQMIMNYAMFLEENEYFELAFQAYEKGIALFKWPGVFDIW 561

Query：558 STYLTKFIARYGGRKLERARDLFEQALDGCPPKYAKTLYLLYAQLEEEWGLARHAMAVYE 617

+TYL KFI RYGG+KLERARDLFEQ L+ CPP +AK ++LLYA+LEEE GLARHA+++Y

Sbjct：562 NTYLVKFIKRYGGKKLERARDLFEQCLENCPPTHAKYIFLLYAKLEEEHGLARHALSIYN 621

Query：618 RATRAVEPAQQYDMFNIYIKRAAEIYGVTHTRGIYQKAIEVLSDEHAREMCLRFADMECK 677

RA V+ A + M+NIYIK+ E+YG+ R I+++AI L ++ +R M LR+A +E

Sbjct：622 RACSGVDRADMHSMYNIYIKKVQEMYGIAQCRPIFERAISELPEDKSRAMSLRYAQLETT 681

Query：678 LGEIDRARAIYSFCSQICDPRTTGAFWQTWKDFEVRHGNEDTIKEMLRIRRSVQATYNTQ 737

+GEIDRARAIY+ ++I DP+ FW TWK+FEV HGNE T+++MLR+RRSV+A+YN

Sbjct：682 VGEIDRARAIYAHAAEISDPKVHVKFWDTWKNFEVAHGNEATVRDMLRVRRSVEASYNVN 741

Query：738 VNFMASQM-LKVSGSATGTVSDLAPGQSGMDDMKXXXXXXXXXXXXXXXDQPLRAQSKIL 796

V + QM + A T + P S +D + Q + I

Sbjct：742 VTLTSVQMRVDAERKAQETTTSSNPMDS-LDQQQQQPSDGAGSIT-----QVSMNKGNIS 795

Query：797 FVRSDASREELAELAQQVNPEEIQLGXXXXXXXXXXXXXXVRLEQQSVPAAVFGSLK 853

FVR + + NP+EI L + + + VPA +FG+LK

Sbjct：796 FVRGAG---KTVQQNTTENPDEIDLDEDDDDEEDDGGDADISV--KVVPAQIFGNLK 847

The cell cycle regulating protein that Query=CLG Sbjct=infers (yeast)

＞cell cycle regulating protein length=674 of inferring

Score value=78.4bits (190), predicated value=1e-13

Homogeny=125/599 (20%), similarity=228/599 (37%), breach=115/599 (19%)

Query：24 YEEEIMRNQFSVKCWLRYIEFKQGAPK-PRLNQLYERALKLLPCSYKLWYRYLKARRAQV 82

+E+ I RN+ ++ W+RY +++ + R ++ERAL + LW +Y++ ++

Sbjct：59 FEDAIRRNRLAMGHWMRYGQWELDQKEFARARSVFERALDVDSTYIPLWLKYIEC---EM 115

Query：83 KHRCVTDPAYEDVNNCHERAFVFMHKMPRLWLDYCQFLMDQGRVTHTRRTFDRALRALPI 142

K+R+ N +RA + ++ +LW Y G +T R+ F+R L+ P

Sbjct：116 KNRNINH-----ARNLFDRAVTQLPRVDKLWYKYVYMEEMLGNITGCRQVFERWLKWEP- 169

Query：143 TQHSRIWPLYLRFLRSHPLPETAVRGYRRFLKLSPESAEEYIEYLKSSDRLDEAAQRLAT 202

W Y+R R+ E A Y RF+ +PE ++ + + + AA

Sbjct：170 --DENCWMSYIRMERRYHENERARGIYERFVVVHPE-VTNWLRWARFEEECGNAA----- 221

Query：203 VVNDERFVSKAGKSNYQLWHELCDLISQNPDKVQSLNVDAIIRGGLTRFTDQLGKLWCSL 262

N +V +DA+ + L + + +

Sbjct：222 ----------------------------NVRQVYLAAIDALGQEFLNE------RFFIAF 247

Query：263 ADYYIRSGHFEKARDVYEEAIRTVMTVRDFTQVFDSYAQFEESMIAAKMETASXXXXXXX 322

A + IR +E+AR +++ AI M +++ Y FE+

Sbjct：248 AKFEIRQKEYERARTIFKYAI-DFMPRSKSMELYKEYTHFEKQF----------------- 290

Query：323 XXXXXXXXXXXFEQLISRRPLLLNSVLLRQNPHHVHEWHKRVALHQ--GRPREIINTYTE 380

E + + L LL+ +P+ W + L + G I TY +

Sbjct：291 ------GDHLGVESTVLDKRRLQYEKLLKDSPYDYDTWLDLLKLEESAGDINTIRETYEK 344

Query：381 AVQTVDPF---KATGKPHTLWVAFAKFYE-DNGQLDDARVILEKATKVNFKQVDDLASVW 436

A+ V A + +W+ + F E D +D AR + ++A K+ + A +W

Sbjct：345 AIAKVPEVVEKNAWRRYVYIWLNYCLFEEIDVKDVDRARKVYQEALKLIPHKKFTFAKLW 404

Query：437 CQCGELELRHENYDEALRLLRKATAL---PARRAEYFDGSEPVQN----RVY-------- 481

ELR D A + L +A + P Y + + ++ R+

Sbjct：405 LMYAMFELRQRKIDVARKTLGRALGMCPKPKLFRGYIEFEDAIKQFDRCRILYEKWILYD 464

Query：482 -KSLKVWSMLADLEESLGTFQSTKAVYDRILDLRI-ATPQIVIN-YAMFLEEHKYFEESF 538

++ W A LE LG +A+Y+ ++ I TP++V Y F E + ++

Sbjct：465 PEACAPWLGYAALETKLGDSDRARALYNLAVNQPILETPELVWKAYIDFEFEEMEYGKAR 524

Query：539 KAYERGISLFKWPNVSDIWSTYLTKFIARYGGRKLE-------------RARDLFEQAL 584

Y++ L P+V +W ++ IA E RAR++FE AL

Sbjct：525 SIYQQ--LLRTAPHVK-VWISFANFEIAHLEDDDEEPPNEEVASPTAVVRARNVFENAL 580

The proteic aminoacid sequence of CLG is carried out structural analysis, discovery contains and crn consensus sequence (crn consensus) like the fruit bat crn protein, and the crn consensus sequence in this fruit bat is similar to the TPR motif (tetratrico peptide repeat motif) in the yeast related gene albumen.The gene family that has the TPR motif in the existing known yeast is a series of cell cycle controlling genes, as CDCl6, CDC23, nuc2+ and bimA etc.In addition, the TPR gene family member in the yeast also has the negative regulatory factor SSN6 of sucrose induction gene, the negative regulatory factor SKI3 and the albumen of yeast killer toxin (yeast killer toxin) to import relevant mitochondrial membrane albumen MAS70 etc.The CLG gene is the same with these fruit bats and yeast genes, and this conservative motif is series connection straight line repeated arrangement in the protein sequence of CLG, occur repeatedly, has general character.

The crn albumen conserved sequence that contains in the CLG gene

1- VKCWLRYIEFKQGAPK-PRLNQLYERALKLLPCS (35-67) 13

|| |::| :|:: : | ::|||||::||

2- PRLWLDYCQFLMDQGRVTHTRRTFDRALRALPIT (110-143) 10

:||: | :| :: ::|: ::|||: ||

3- SRIWPLYLRFLRSHPLPETAVRGYRRFLKLSPES (146-179) 9

::| | || : : | : |:| |:: |:

4- GKLWCSLADYYIRSGHFEKARDVYEEAIRTVMTV (256-289) 9

||| : | : ::::||::||:|::

5- TQVFDSYAQFEESMIAAKMETASELGREEEDD-- (293-324) 7

||:||| : ::: | |:

6- VDLELRLARFEQLISRRPLLLNSVLLRQNPHHVH (325-358) 7

| | :: ||||:|::

7- HTLWVAFAKFYEDNGQLDDARVILEKATKVNFKQ (395-428) 11

||: :|:| | ::| || | |:|

8- LKVWSMLADLEESLGTFQSTKAVYDRILDLRIAT (484-517) 9

:|:| | :|| | :: :: :|:| |::

9- PQIVINYAMFLEEHKYFEESFKAYERGISLFKWP (518-551) 9

:: |:|| | | | :::: : |||:: ::

10- KTLYLLYAQLEEEWGLARHAMAVYERATRAVEPA (593-626) 10

|:: ||::|| :| :||||

11- YDMFNIYIKRAAEIYGVTHTRGIYQKAIEVLSDE (629-662) 6

: ::| ||::|:| |

12- REMCLRFADMECKLGEIDRARAIYSFCSQICDPR (665-698) 11

:::| :| | |||||| ||

VKLWIKYARFEELLKEIDRAREIYERALEFLPRD crn consensus sequence

VKLWIKYARFEELLKEIDRAREIYERALEFLPRD crn consensus sequence

|: ::: | | ::::| : :::|||: | :

AEAWFGLGHIYEKLGDLEKALDAFQKALELDPNN TPR motif

The rna expression spectrum of embodiment 5:CLG in people's tissue

With CLG is probe, hybridizes with the mRNA diaphragm (Clontech) of the multiple tissue of people.The result as shown in Figure 1, find CLG wide expression in the popular feeling (H), brain (B), placenta (P), lung (Lu), liver (Li), muscle (SM), kidney (K) and pancreas (Pa) tissue, and expression amount is homogeneous comparatively, wherein the transcript size in human brain, placenta, lung, liver, kidney and pancreatic tissue is about 2.6kb, consistent with the full-length cDNA size of the CLG that is obtained among the embodiment 2, the transcript that a size is about 3.0kb then appears in the heart and muscle tissue.

Embodiment 6:CLG forms the restraining effect of (Colony Formation) to the human liver cancer cell colony

The CLG gene that contains complete coding region (embodiment 2) that RT-PCR obtains adopts AdvanTAge ^TMPCR Cloning reagent closes (Clontech, Cat#:K1901-1) clone in pT-Adv carrier (Clontech), EcoR I enzyme is cut then, recovery contains the CLG gene fragment of complete coding region, (Stratagene Cat#:212220), obtains plasmid pCMV-Script/CLG to subclone to eukaryotic expression vector pCMV-Script again, carry out the extraction of plasmid DNA with Qiagen plasmid extraction test kit, use for transfection.

Get 5 μ g CLG plasmid DNA (carrier is pCMV-Script), the DMEM that adds the serum-free antibiotic-free is to 600 μ l, and blank replaces plasmid DNA with sterilization milli-Q water.Other gets 25 μ l liposome Lipofectamine (GIBCOBRL), adds antibiotic DMEM to 600 μ of serum-free l, and above-mentioned plasmid DNA and liposome are mixed, softly shake up, room temperature was placed 30-45 minute, added the DMEM 1800 μ l of serum-free antibiotic-free again, and cumulative volume is 3ml.The 3ml transfection composite is added (1-2 * 10 in the SMMC-7721 cell that grows in the 6cm culture dish ⁵Cell/every culture dish), cultivate after 6 hours for 37 ℃, use full nutrient solution instead, after 24 hours, use the full training liquid of G418 again instead, colony occurs about about two weeks, painted colony number is observed in Viola crystallina (crystal violet) dyeing.

The result shows that CLG full-length cDNA transfection SMMC-7721 cell can significantly suppress colony and form.The SMMC-7721 cell only forms 3 colonies behind the transfection CLG, and the SMMC-7721 cell of transfection empty carrier then forms 56 colonies, and CLG gene pairs human liver cancer cell SMMC-7721 has an obvious suppression effect (Fig. 2 A and 2B) external.

The experiment of one-tenth knurl and the apoptosis of embodiment 7:CLG gene transformation cell detect

By the identical method of embodiment 6, with CLG gene transfection human liver cancer cell SMMC-7721, then with the cellular control unit enlarged culturing of transformant and untransfected CLG gene, the subcutaneous vaccination nude mice, observe the influence that the CLG gene pairs becomes the knurl effect, experiment is divided into CLG transfection group and control group, every group of 6 nude mices, every nude inoculation 2 * 10 ⁶Cell, the observation period was 6 weeks, became the knurl experimental result to see Table 3.

Table 3 SMMC-7721 cell becomes the knurl experimental result

Heavy (g) T assay of the average knurl of group knurl heavy (g)

1 2 3 4 5 6

Contrast 0.24 0.11 0.15 0.16 0.12 0.18 0.16 P＜0.05

CLG 0.14 0.07 0.01 0.03 0.10 0.07 0.07

In becoming the knurl experiment, behind the SMMC-7721 cell of 6 experiment nude inoculation CLG transfection, the average knurl of tumour that forms heavily is 0.07 gram, and 6 formed knurls of inoculation SMMC-7721 cell self-controlled group heavily are 0.16 gram, paired two sample analysis T check through mean value, the two has significant difference, p＜0.05, and tumour inhibiting rate is 50%.

The SMMC-7721 cell of CLG transfection, the tumor tissues that forms behind the inoculation nude mice, make paraffin section, carry out apoptosis in situ detection (Roche company) with the Tunel method, as seen hepatic positive apoptotic cells, visible more positive apoptotic cells (shown in Fig. 3 A) in necrosis region wherein, the apoptotic cell (shown in Fig. 3 B) that as seen is dispersed in necrotic area not.

After-80 ℃ of refrigerated became tumor tissue to pulverize, with the DNA extracted solution digestion that contains RNA enzyme and Proteinase K, 50 ℃ digested 2 hours, and the phenol extracting is 2 times then, alcohol precipitation DNA, and 1.5% agarose gel electrophoresis analysis, whether observe has " trapezoidal " DNA band.The plastidogenetic tumor tissues DNA of the SMMC-7721 of transfection CLG full-length gene is through 1.5% agarose gel electrophoresis analysis, and visible significantly " trapezoidal " DNA band is similar to the electrophoresis behavior of the plastidogenetic tumor tissues DNA of SMMC-7721 of injection transfection p53.Single large fragment DNA band only appears in the plastidogenetic tumor tissues DNA of SMMC-7721 without plastidogenetic tumor tissues DNA of the SMMC-7721 of transfection and transfection pCMV-Script empty carrier plasmid DNA, does not have " trapezoidal " DNA band.After showing the full-length cDNA transfection SMMC-7721 cell of CLG gene, has the SMMC-7721 of inducing cell generation effect of apoptosis.

The protein expression of embodiment 8:CLG gene

According to the CLG full length cDNA sequence, design primer 3 (5 ' TCG GAATTCGTGGTGATGGCGCGACTT 3 ', 5 ' end contains EcoR I site) and primer 4 (5 ' TCT AAGCTTTCAGTCTTCCTTCAGGCT 3 ', 5 ' end contains Hind III site), pcr amplification CLG coding region sequence.The PCR reaction conditions is: 94 ℃, and sex change 5 min; Press 94 ℃ of sex change 1min then, 55 ℃ of annealing 1min, 72 ℃ are extended 2min, carry out 40 circulations; Last 72 ℃ are extended 10min.Carrying out enzyme after the reaction product electrophoresis detection again cuts back to close.To insert the EcoR I-Hind III site of carrier pET32a (Novagen) through the CLG coding region segment that pcr amplification and enzyme cut back to close, obtain the pET32a-CLG recombinant plasmid.

With the recombinant plasmid transformed e. coli bl21 (DE3) that builds, choose single colony inoculation in LB substratum (containing acillin 50mg/L), 37 ℃ of overnight incubation, 2% inoculum size is transferred in 5ml LB substratum, be cultured to OD600≤0.5 o'clock, be transferred among the 150ml LB (containing acillin 50mg/L) by 1% amount again, during to OD600=0.5-1.0, IPTG induces (1mM), continue to cultivate centrifugal collection thalline, ultrasonication thalline about 3 hours, behind the high speed centrifugation, the CLG albumen (Fig. 5) than high expression level all appears in discovery in supernatant and precipitation.The proteic molecular weight of CLG is 100kDa, and is consistent with predicted molecular weight.

Claims (10)

1. isolating people CLG albumen is characterized in that it is selected from down group:

(a) has the polypeptide of SEQ ID NO:2 aminoacid sequence;

(b) SEQ ID NO:2 aminoacid sequence is formed through replacement, disappearance or the insertion of one or more amino-acid residues, and have suppress function that the liver cancer cell colony forms by (a) polypeptides derived.

2. polypeptide as claimed in claim 1 is characterized in that, this amino acid sequence of polypeptide is shown in SEQ ID NO:2.

3. isolating polynucleotide is characterized in that, it contains a nucleotide sequence, and this nucleotide sequence is selected from down group:

(a) polynucleotide of polypeptide according to claim 1 of encoding;

(b) with polynucleotide (a) complementary polynucleotide.

4. polynucleotide as claimed in claim 3 is characterized in that the polypeptide of this polynucleotide encoding has the aminoacid sequence of SEQ IDNO:2.

5. polynucleotide as claimed in claim 3 is characterized in that, the sequence of these polynucleotide is selected from down group:

The coding region sequence of 22-2258 position among the SEQ ID NO:3, or 1-2659 position full length sequence.

6. a carrier is characterized in that, it contains the described polynucleotide of claim 3.

7. a host cell is characterized in that, it is a kind of host cell that is selected from down group:

(a) host cell that transforms or transduce with the described carrier of claim 6;

(b) host cell that transforms or transduce with the described polynucleotide of claim 3.

8. preparation method with polypeptide of people CLG protein-active is characterized in that this method comprises:

(a) under the proteic condition of suitable expressing human CLG, cultivate the described host cell of claim 7;

(b) from culture, isolate polypeptide with people CLG protein-active.

9. energy and the described people CLG of claim 1 protein-specific bonded antibody.

10. a pharmaceutical composition is characterized in that, it contains the described polypeptide of claim 1 and the pharmaceutically acceptable carrier of safe and effective amount.

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