CN1952135A - Polynucleotide related to cell multiplication and its encoded polypeptide and application - Google Patents

Abstract

The invention discloses a novel polynucleotide and its coding polypeptide and antibody of the polypeptide. The polynucleotide codes protein with the function of promoting cell multiplication. The invention also discloses the application of such novel polynucleotide exogenous expressing in host cells promoting cell multiplication. The invention also discloses the use of the said polypeptide, antibodies in the drug preparation for the prevention and treatment of human cell multiplication interrelated diseases, especially in developing drugs for treating autoallergic disease, cancer and traumatic injury recovery.

Description

Polynucleotide and coded polypeptide and purposes that cell proliferation is relevant

Technical field

The invention belongs to biological technical field, relate to the gene expression regulation field, specifically, the present invention relates to the new coding of a class and have the proteic polynucleotide of people that promote the cell proliferation function, and encoded polypeptides, the antibody of polypeptide.The invention still further relates to the new polynucleotide of this class external source expressing promoting in host cell and advance the application of cell proliferation.The invention still further relates to described polypeptide, antibody in the preparation prevention disease relevant with body cell multiplication, the especially purposes on the medicine of the injury repairing of developing autoimmune disease, tumour or disease and wound with treatment.

Background technology

Human body belongs to high multicellular organism, but the people is grown, divides, differentiated by a zygote simultaneously; Human body nearly tens million of necrocytosis every day, simultaneously there is the cell fission of respective numbers to breed the balance that realizes cell number again, so the division of cell and propagation are one of the most basic features of survivaling cell, play decisive role for the existence of life entity.Cell proliferation can be divided into cell growth and cell fission, and its meaning is 1. to grow and is multicellular organisms; 2. trauma repair; 3. cell replenishes.It is the fissional main mode of higher organism that silk (indirectly) division (mitosis) is arranged, and its main feature is to form MA, and reduction division (meiosis) is special mitotic division.

The speed of cell proliferation is called for short the cell cycle by decision cell generation cycle (cell division cycle), is meant that the parental cell division finishes the successive processes that is experienced to daughter cell division end.Cell cycle is divided into interval and division stage, according to the difference of cell cycle, can be divided into continuous splitted cell, rest cell and somatoblast not to cell.In continuous splitted cell, sexual cell, stem cell, epidermic cell have the mitotic activity of height, and in various diseases, tumour cell has the activity of malignant proliferation.

The regulation and control of cell proliferation are positioned at core status in biological and medical research field, and the multiplication regulatory disorder will cause cell chromosome unusual.In brief, multiplication regulatory can be divided into following six classes: the 1. regulation and control of gene level: comprise cell division cycle gene cdc family, oncogene, cancer suppressor gene, the adjusting of the check position of cell cycle; 2. the regulation and control of protein level: comprise cyclin cyclin, cyclin deopendent protein kinase CDK, cyclin deopendent protein kinase arrestin CDKI etc.; 3. the adjusting of cytokine: comprise S phase activation factor SPF and maturation promoting factor MPF etc.; 4. the adjusting of somatomedin and acceptor thereof: by with cytolemma on single-minded receptors bind, activate a series of signal conduction path, proliferation signal is delivered in the nuclear the most at last, trigger cell propagation, isolating now somatomedin comprises: Urogastron EGF, platelet derived growth factor PDGF, fibroblast growth factor FGF, nerve growth factor NGF, insulin-like growth factor I GF etc.; 5. the adjusting of hormone: the intravital many hormones all propagation of pair cell have promoter action, as tethelin, sexual hormoue etc.: 6. Ca ²⁺The adjusting of-calmodulin: the regulatory site of this system's on cell proliferation synthesizes, induces nuclear membrane to disintegrate, participate in chromosome movement etc. for starting DNA.

The propagation of cell relates to the multiple physiology of body and the generation of pathologic process, and the research of cells involved propagation is a focus of biology, medical research always.The breeding that is known that different cells now has different physiological pathology meanings, and the breeding of cell and meaning thereof have time and spatial specificity, and cell proliferation has participated in the generation and the evolution of the multiple disease of body unusually.Research cell proliferation regulation and control and with the relation of disease, will play directive function to prevention and clinical treatment, the prognosis of multiple disease, now be exemplified below:

1) from the cell proliferation angle, tumour be since the malignant proliferation of cell due to

Tumour cell that it is generally acknowledged vicious transformation is because growth out of control, hyper-proliferative, see that from apoptotic angle what think tumour is that the apoptosis mechanism of tumour is suppressed and can not normally carries out the result that necrocytosis is removed, but the cell that generally believes vicious transformation now growth out of control, hyper-proliferative are various tumorigenic main causes.Above mention, the intravital normal cell of machine propagation is subjected to the regulation and control of number of ways, complexity of formation and exquisite regulated and control network, and normal thus body cell propagation has had the dependency of contact inhibition, cell matrix and intercellular substance; In case and certain link imbalance of this regulated and control network just might cause the generation of tumour, as all there being the persistence activatory phenomenon of the MAPK path relevant in the kinds of tumor cells system with propagation.Therefore, designing the tumor treatment method from the cell proliferation angle is exactly to revise the propagation regulation system of tumour cell, makes it regain the dependency of contact inhibition, cell matrix and intercellular substance.

2) research of cell proliferation will bring real breakthrough to the treatment of some autoimmune disease

Autoimmune disease comprises an intractable immunologic derangement of big class and the disease of the cellular abnormality that causes propagation, as squama disease, polycyth(a)emia etc.In a broad sense, the abnormality proliferation of the bone-marrow-derived lymphocyte of autoreactivity T lymphocyte and generation antibody is the main immunopathogenesis mechanism that causes autoimmune disease, under the normal circumstances, the activation and proliferation of immunocyte and apoptosis are subjected to extremely complicated and accurate regulation and control, but in pathologic process, under the hormesis of autoantigen, the immunocyte of identification autoantigen is activated propagation and apoptosis does not take place, and then causes the generation of autoimmune disease.

3) research of cell proliferation will bring breakthrough for various diseases and post-traumatic injury repairing

The recovery of a lot of diseases and the wound function of damaged tissue and organ after healing is a difficult problem that troubles clinical treatment circle always.As the microbial rheumatic heart disease of haemolysis type hammer, the damage of the irreversibility that normal body can not repair has taken place at the back valve that controls inflammation, other multiple viruses and bacterium also can cause the valvular heart disease that body can not be repaired in addition; Chronic diseases such as and for example various types of sacroiliitis, uremia, all the body injury decompensation that exists in various degree causes irreversible prognosis; Heal for the above formed scar of burn of two degree now for another example and can only take dermatoplastic alternative medicine or the like.Research for cell proliferation and regulation and control thereof will help to disclose the relevant Normocellular propagation of startup, will bring breakthrough for various diseases and post-traumatic injury repairing, as the report of paid on probation fox-3A gene therapy valvular heart disease now.

4) research of cell proliferation will help to solve an a series of medical difficult problem

Along with growth in the living standard, cardiovascular and cerebrovascular disease, senile dementia, nerve injury newly occur and caused numerous medical difficult problems such as vegetative patient, these treatment of diseases also depend on the propagation of relevant cell to a great extent, rebuild the irreplaceable effect that plays as the proliferation research of vascular endothelial cell for the blood vessel configuration in the cardiovascular and cerebrovascular patient process.

5) cell proliferation Research advances in regulation will help further to set forth the essence of life, disclose the rule of organism operation

Since the generation of a lot of diseases is relevant unusually with cell proliferation and regulation and control thereof with development, it is logical so some disease being carried out therapeutic cell proliferation intervention.Many genes are relevant with promotion or inhibition tumor cell proliferation, and it is the new trial of research oncotherapy that therefore transgenosis by all means, signal transduction pathway intervention wait the malignant proliferation of closure body inner tumour cell, and therapeutic value is immeasurable.On the contrary, can be with promoting Normocellular propagation to carry out various diseases and post-traumatic injury repairing, therefore, propagation discovery of genes involved and Regulation Mechanism research thereof for further discussion cell proliferation and regulatory mechanism thereof for much treatment of diseases are significant from now on.

Summary of the invention

The research of people's gene group is international focus at present, except that the method for large scale sequencing, also lacks the high flux screening that begins from functional study and has the method for the gene of certain function.Deficiency at this present situation and existing medicine or reagent the purpose of this invention is to provide the new coding of a class and has the proteic polynucleotide of people that promote cel l proliferation.

Another object of the present invention provides this class polynucleotide encoded polypeptide.

Another object of the present invention provides carrier and this class polynucleotide that contain these class polynucleotide and carrier transforms or the host cell of transduction.

Another object of the present invention provides the antibody of this class polynucleotide encoded polypeptide and the nucleic acid molecule that is used to detect.

Another object of the present invention provides the application that the new polynucleotide of this class external source expressing promoting in host cell is advanced cell proliferation.

Another object of the present invention provides produces these polynucleotide and the method for its encoded polypeptides and the purposes of this polynucleotide and encoded polypeptides thereof.

For achieving the above object, the present invention is by the following technical solutions:

In a first aspect of the present invention, novel isolating polynucleotide are provided, it comprises coding and has the proteic nucleotide sequence that promotes the cell proliferation function, and this nucleotide sequence is selected from: (a) polynucleotide of polypeptide that contain the aminoacid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14 with coding have the polynucleotide of at least 70% similarity; (b) coding contains the polynucleotide of polypeptide that aminoacid sequence with SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14 has the aminoacid sequence of at least 70% similarity; (c) with (a) or polynucleotide complementary polynucleotide (b).

Preferably, the polypeptide of this polynucleotide encoding has the aminoacid sequence of the group of being selected from down: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14.

Preferably, the sequence of these polynucleotide is shown at least 85% similarity with the nucleotides sequence that is selected from down group: (a) coding region sequence or the full length sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13; (b) at least one sequence of the sequence of in genetic code degeneracy scope, mentioning in corresponding to (a); (c) with (a) or at least one sequence of the sequence complementary sequence hybridization of mentioning (b).

More preferably, the sequence of these polynucleotide is selected from coding region sequence or the full length sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13.

In a second aspect of the present invention, above-mentioned Nucleotide encoded polypeptide is provided, and it comprises the polypeptide with the aminoacid sequence in the group of being selected from down: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQID NO:12, SEQ ID NO:14; Or the polypeptide that has similarity more than at least 90% with above arbitrary aminoacid sequence, or its conservative property variation polypeptide or its active fragments or its reactive derivative.

Preferably, this polypeptide is the polypeptide with aminoacid sequence of the group of being selected from down: SEQ ID NO:2, SEQ ID NO:4, SEQ IDNO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14.

In a third aspect of the present invention, the carrier that contains above-mentioned polynucleotide is provided, and, also provides the host cell that is transformed or transduce by above-mentioned polynucleotide by the host cell that this carrier transforms or transduces.

In a fourth aspect of the present invention, provide and aforementioned polypeptides specificity bonded antibody, the nucleic acid molecule that can be used for detecting also is provided, it contains 8-100 successive Nucleotide in above-mentioned arbitrary polynucleotide.

In a fifth aspect of the present invention, provide above-mentioned polynucleotide external source expressing promoting in host cell to advance the application of cell proliferation.

In a sixth aspect of the present invention, provide above-mentioned polynucleotide and polypeptide to prevent and/or treat purposes in the medicine with the body cell multiplication diseases associated in preparation.Preferably, prevent and/or treat purposes in the medicine of injury repairing of the autoimmune disease relevant, tumour or disease and wound in preparation with body cell multiplication.

Aspect the present invention ground the 7th, a kind of pharmaceutical composition is provided, it contains polypeptide and the pharmaceutically acceptable carrier that promotes the body cell multiplication function that have among the present invention of safe and effective amount.

In a eighth aspect of the present invention, the external detection method of a kind of autoimmune disease or tumour is provided, utilize above-mentioned antibody or nucleic acid fragment to detect the existence or the level of the polypeptide in host's sample.

Other aspects of the present invention since disclosing of the technology of this paper will be apparent to those skilled in the art.

As used herein, " isolating " is meant that material separates (if natural substance, primal environment promptly is a natural surroundings) from its primal environment.Do not have separation and purification as polynucleotide under the native state in the active somatic cell and polypeptide, if but same polynucleotide or polypeptide from native state, separate with common other materials that exist, then be separation and purification.Such polynucleotide may be the parts of a certain carrier, the part that also possible such polynucleotide or polypeptide are a certain composition, since carrier or composition are not the compositions of their natural surroundings, these polynucleotide or polypeptide remain isolating.

As used herein, " similarity " is meant and is used for describing the height that detects same DNA base between sequence and the target sequence or amino-acid residue order proportion in Nucleotide or the peptide sequence comparison process, it is a kind of direct quantitative relation, recently measure degree similar between nucleotide sequence or the peptide sequence by the same or analogous percentage of part, this similarity per-cent can calculate by the existing comparison method in this area, example has the comparison method FASTA program (Pearson between sequence in twos, W.R.and Lipman, D.J.1988.Improved tools for biological sequence comparison.Proc.Natl.Acad.Sci.85:2444-2448), blast program (Altschul, S.F., et al.1990 Basic local alignment search tool.J.Mol.Biol.215:403-410) etc., or Multiple Sequence Alignment Method CLUSTAL W (CORPET, F.1998 Multiple sequencealignment with hierarchical clustering.Nucleic Acids Res., 16:10881-10890) etc.Homologous sequence is meant the different sequences that form through divergent evolution from a certain common ancestor, can judge homology between aligned sequences according to similarity per-cent.When similarity degree is very high between gene or protein, represents that they have one section common evolution course, thereby judge that they can have similar biological function.When similarity degree, detect sequence and target sequence may be a homologous sequence than being easier to infer with at least 50%.Preferably, has at least 70% similarity degree; More preferably, has at least 85% similarity degree; Best, has at least 90% similarity degree.And when the similarity degree is lower than 20%, just be difficult to determine or can't determine at all whether it has homology.

Polynucleotide of the present invention comprise that its complementary strand can be dna form or rna form.Dna form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or double-stranded.DNA can be coding strand or noncoding strand.As used herein, " coding has the polynucleotide of the polypeptide that promotes the cell proliferation function " can be the polynucleotide that comprise this polypeptide of encoding, and also can be the polynucleotide that also comprise additional code sequence and/or non-coding sequence.With gene TRAF3IP3 encoded polypeptide is example, and the coding region sequence of encoding mature polypeptide can be identical with the coding region sequence shown in the SEQ ID NO:1 or the varient of genetic code degeneracy.As used herein, " genetic code degeneracy " is meant that an amino acid has the phenomenon of several codons.The varient of the genetic code degeneracy of TRAF3IP3 encoded polypeptide refer to the encode Nucleotide of polypeptide in the present invention for example with SEQ ID NO:2, and the coding region sequence shown in this Nucleotide and the SEQ ID NO:1 has difference.Have the polypeptide that promotes the body cell multiplication function for other, can the rest may be inferred.

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 the complementary sequence hybridization of polynucleotide sequence of the present invention 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 the interfertile polynucleotide of the complementary sequence of polynucleotide sequence therewith.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 polypeptide of the present invention.

Polynucleotide sequence of the present invention can obtain with this area existent method.These technology including, but not limited to: (1) is by hybridization technique DNA isolation sequence; (2) artificial chemical synthesising DNA sequence; (3) by the required polynucleotide of the extensive acquisition in construction cDNA library; (4) pcr amplification technology.

First method is to make up genomic library or cDNA library earlier, filters out goal gene or sequence by technology such as molecular hybridizations from genomic library or cDNA library then.When the biological gene group was smaller, this method is success easily; When the biological gene group is very big, make up difficulty of its complete genomic library, the quantities of removing to clone goal gene again from huge library is also very big.

Second method is by the long dna fragmentation of the once synthetic 100-200bp of the sequence that designs, and connects into complete gene with these synthetic fragment combination again.The price of the method for the gene order of this synthetic length is very expensive.This method be mainly used in synthetic as primer, connect the first grade nucleic acid fragment.

The third method is with usual method construction cDNA library, this area, repeatedly after the order-checking, in conjunction with bioinformatic analysis technology (Ota et al.Nat Genet.2004 Jan; 36 (1): 40-5), obtain purpose cDNA clone on a large scale.The bioinformatic analysis technology includes but not limited to BLAST or BLAT and the comparison of existing public database, as the refseq database etc.; With Phred algorithm assessment sequencing quality; ATGpr algorithm with the probability of occurrence that calculates transcription initiation codon ATG screens full length cDNA sequence etc.

The 4th kind of method method (Saiki, the et al.Science 1985 of round pcr DNA amplification/RNA; 230:1350-1354).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 method of advantageous applications of the present invention is that the amplification in mixing the cDNA library of two-step approach flux RT-PCR technology obtains a large amount of cDNA clones.Mix the cDNA library and comprise existing cDNA library and tumour library.

Gene of the present invention, the perhaps available ordinary method of mensuration of nucleotide sequence such as various dna fragmentations, as dideoxy chain termination (Sanger et al.PNAS, 1977,74:5463-5467); Also available commercial sequencing kit etc.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.

Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthetic polypeptide, preferred recombinant polypeptide.Polypeptide of the present invention can be the product of natural purifying, or the product of chemosynthesis, or uses recombinant technology to produce from protokaryon or eucaryon host (as bacterium, yeast, higher plant, insect and mammalian cell).Polypeptide of the present invention can be glycosylated, also can be nonglycosylated.Polypeptide of the present invention can comprise or not comprise initial methionine residues.

The present invention also comprises fragment, derivative and the analogue of the people's protein polypeptide with the polynucleotide encoding that promotes the body cell multiplication function.Term " fragment ", " derivative " are meant with " analogue " and keep basically and natural identical biological function or the active polypeptide of people's protein polypeptide that promotes the body cell multiplication function that have of the present invention.Polypeptide fragment of the present invention, derivative and analogue can be: one or more conservative or substituted polypeptide of non-conservation amino-acid residue (preferential conservative amino acid residue) (a) are arranged, and the amino-acid residue that replaces like this can be also can not encoded by genetic code, or (b) in one or more amino-acid residues, has a polypeptide of substituted radical, or (c) mature polypeptide and another compound (such as the compound that prolongs the polypeptide transformation period) merge formed polypeptide, or (d) additional aminoacid sequence is fused to this peptide sequence and the polypeptide that forms (as leader sequence or secretion sequence or be used for the sequence or the proteinogen sequence of this polypeptide of purifying).

Polypeptide of the present invention can utilize polynucleotide sequence of the present invention to express or produce protein polypeptide (Science, 1984 that promote the body cell multiplication function that have of reorganization by conventional recombinant DNA technology; 224:1431).May further comprise the steps:

(1), or transforms or the transduction proper host cell with the expression vector that contains these polynucleotide with polynucleotide of the present invention (or its varient);

(2) host cell that culturing step (1) obtains in suitable medium;

(3) separation, the required protein polypeptide of purifying from substratum or cell.

Polynucleotide among the present invention and polypeptide preferably provide with isolating form, more preferably are purified to homogeneous.

The present invention also relates to comprise the carrier of polynucleotide of the present invention.Among the present invention, the polynucleotide sequence that coding has the people's protein polypeptide that promotes the body cell multiplication function can be inserted in the recombinant expression vector.Term " recombinant expression vector " refers to bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell virus, mammalian cell virus, as adenovirus, retrovirus, and perhaps other carriers.The carrier of Shi Yonging can be a prokaryotic expression carrier in the present invention, also can be carrier for expression of eukaryon, as the expression vector (Rosenberg based on T7 that expresses in bacterium, et al.Gene, 1987,56:125), the carrier for expression of eukaryon pcDNA of high expression level in mammalian cell ^TM3.1/myc-hisB (-) (Invitrogen), pcDNA3.1/V5-His-TOPO (Invitrogen below is abbreviated as pcDT).The preferred pcDT of the present invention, it can directly be connected with the PCR product and makes up carrier for expression of eukaryon, has improved the efficient of large-scale production greatly.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.Making up the expression vector that contains polynucleotide sequence of the present invention and transcribe/translate control signal with method well-known to those having ordinary skill in the art gets final product.These methods comprise (Sambrook such as extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant DNA technology of body, et al.Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory.New York, 1989).

Polynucleotide sequence of the present invention can be connected to effectively and instruct mRNA synthetic on the suitable promotor in the expression vector.The representative example of these promotors has: colibacillary lac or trp promotor; The P of lambda particles phage _LPromotor; 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.Expression vector preferably comprises one or more selected markers, being provided for selecting the phenotypic character of transformed host cells, as being used for colibacillary tsiklomitsin or amicillin resistance or eukaryotic cell and cultivating green fluorescent protein (GFP), neomycin resistance and the Tetrahydrofolate dehydrogenase of usefulness.

The invention still further relates to the host cell that produces through genetically engineered with above-mentioned carrier or polynucleotide of the present invention.Carrier of the present invention and polynucleotide can be used to transform appropriate host cell, have the protein that promotes the body cell multiplication function so that it can be expressed.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; Vegetable cell; The insect cell of fruit bat S2 or Sf9; The zooblast of CHO, COS or Bowes melanoma cells; 293 T, Hela cell etc.

When polynucleotide of the present invention are expressed, transcribe enhancing if will make when in carrier, inserting enhancer sequence in higher eucaryotic cells.Enhanser is the cis acting factor of DNA, and 10-300 base pair arranged usually, acts on promotor transcribing with enhancing gene.Example has: at the SV40 enhanser of 100-270 the base pair in replication origin downstream, at the polyoma enhanser in replication origin downstream and adenovirus enhanser etc.

Those of ordinary skill in the art knows 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 host cell was prokaryotic cell prokaryocyte such as intestinal bacteria, the competent cell that can absorb DNA can be collected at the exponential growth after date, uses CaCl ₂Method is handled, and used step is well-known in the art.Alternative is MgCl ₂Handle, also the method for available electroporation is handled.When the host is eukaryotic cell, can select following transfection method: 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 polynucleotide encoded polypeptide of the present invention.Select suitable conventional substratum according to selected host cell, under the condition that is suitable for the host cell growth, cultivate.After host cell grows into suitable cell density, with appropriate means such as temperature inversion or chemical induction, induce the promotor of selection, cell is cultivated for some time again.

Recombinant polypeptide in the aforesaid method can wrap by in cell, extracellular or on cytolemma, express or be secreted into the extracellular.If desired, can utilize its physics, chemical separating and the purification of Recombinant polypeptide by various separation methods with other characteristics.These methods are well-known to those skilled in the art, handle as the renaturation of routine, handle the combination of (salt analysis method), centrifugal, the broken bacterium of infiltration, ultrasonication, super centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography and other various liquid chromatography (LC) technology or these methods with protein precipitant.

The invention still further relates to any a part of homologous nucleic acid fragment with polynucleotide of the present invention.As used herein, the length of " nucleic acid fragment " contains 15 Nucleotide at least, is preferably at least 30 Nucleotide, is more preferably at least 50 Nucleotide, and best is at least 100 Nucleotide.This nucleic acid fragment is the dna sequence dna of chemosynthesis on the basis of nucleotide sequence information of the present invention normally.Above-mentioned nucleic acid fragment can be used for pcr amplification technology (as primer) and have the polynucleotide that promote the body cell multiplication function to determine and/or to separate coding; Also can be used as the used probe of hybridization.Also can be used for the RNA perturbation technique.Part or all of polynucleotide of the present invention also can be used as probe stationary on microarray (Microarray) or DNA chip, is used for analyzing the differential expression and the gene diagnosis of tissue gene.The mark of probe can be used radio isotope, fluorescein or enzyme (as alkaline phosphatase) etc.

Polypeptide of the present invention can be directly as the pharmacological agent disease, as injury repairing of autoimmune disease, tumour or disease and wound etc.; Also can be used for screening the proteic antibody, polypeptide or other part that promote or resist the function with promotion cell proliferation, for example, screening can be used for promoting or suppressing the antibody of proteic function of the present invention.Albumen of the present invention with the reorganization of expressing screens peptide library, is used to seek the peptide molecule that can promote or suppress proteic function of the present invention of therapeutic value.

Polypeptide of the present invention can use separately or use with suitable pharmaceutical carrier combination back.Composition comprises the polypeptide or the antagonist of safe and effective amount and does not influence the carrier and the excipient of effect of drugs.These carriers can be water, glucose, ethanol, salt, damping fluid, glycerine and their combination.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.The consumption that delivers medicine to the patient depends on many factors, as administering mode, person's to be treated healthiness condition and diagnostician's judgement.

Polypeptide of the present invention also can use by express these polypeptide at live body.For example patient's cell can carry out the genetically engineered operation by the gene at external use code book invention polypeptide, then engineering cell is offered the patient, makes engineering cell this peptide species of high expression level in vivo, thereby reaches the purpose of treatment.

Have and promote the proteic polynucleotide of people of cell proliferation function also to can be used for multiple therapeutic purpose.Can be used on and treat in the gene therapy technology owing to have the people's protein abnormal expression or the active disease that causes unusually of the function that promotes cell proliferation.The gene therapy vector (as virus vector) of reorganization can be designed to express the people's albumen that promotes the cell proliferation function that has of variation, suppresses the endogenic proteic activity of people that promotes the cell proliferation function that has.People's protein gene with promotion cell proliferation function of reorganization also can be packaged in the liposome and be transferred in the cell.

Suppress the oligonucleotide (comprising sense-rna and DNA) of polypeptide mRNA of the present invention and nucleic acid also within the scope of the invention.Sense-rna and DNA and nucleic acid can be synthetic with this area existent method.In order to increase the stability of nucleic acid molecule, available several different methods is modified it, as increasing the sequence length of both sides, the connection between ribonucleoside phosphoric acid thioester bond or peptide bond.

Polypeptide of the present invention and fragment thereof, derivative, analogue or the cell of expressing them can be used as antigen and produce antibody.These antibody include but not limited to the antibody that monoclonal antibody, polyclonal antibody, chimeric antibody, single-chain antibody, Fab fragment and Fab expression library produce.The antibody of polypeptide of the present invention can be produced with preparation method for antibody well known in the art.Example has: monoclonal antibody can with hybridoma technology production (Kohler and Milstein.Nature, 1975,256:495-497).The available polypeptide immune animal of the present invention of the production of polyclonal antibody is as rabbit, mouse, rat etc.Multiple adjuvant can be used for the enhancing immunity reaction, includes but not limited to freund's adjuvant.The variable region bonded chimeric antibody in human constant region and inhuman source can be produced with existing technology (Morrison etal.PNAS, 1985,81:6851).The also available existing technology production of single-chain antibody (U.S.Pat No.4946778).

Antibody of the present invention can be used in the immunohistochemistry technology, detects the albumen that promotes the cell proliferation function that has in the living specimen.Can also be used for the clinical diagnosis, treatment, therapeutic evaluation of the disease relevant etc. clinically with having people's albumen of promoting the cell proliferation function.For example use labelled with radioisotope and polypeptide bonded monoclonal antibody of the present invention, inject then and follow the tracks of its position and distribution in the body, can be used as a kind of atraumatic diagnostic method and come the positioning tumor cell, or judge whether tumour cell shifts.Antibody among the present invention can also be used for the treatment of or prevent and have the relevant disease of people's albumen that promotes the cell proliferation function.The antibody that gives suitable dosage can stimulate or block and has proteic generation of people or the activity that promotes the cell proliferation function.

The invention still further relates to quantitatively and detection and localization has the diagnostic testing process of the protein level of promotion cell proliferation function.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.That detects in the experiment has a protein level that promotes the cell proliferation function, can have the importance of albumen in various diseases that promotes the cell proliferation function with laying down a definition and be used to diagnose to have the disease that the albumen that promotes the cell proliferation function works.

Proteic polynucleotide with promotion cell proliferation function can be used for having the diagnosis and the treatment of the protein related diseases that promotes the cell proliferation function.Aspect diagnosis, have the proteic polynucleotide that promote the cell proliferation function can be used for detecting have promote the cell proliferation function proteic expression whether, or under morbid state, have the abnormal exprssion that promotes the cell proliferation function.As the proteic dna sequence dna with promotion cell proliferation function can be used for that the hybridization of biopsy specimen is had the proteic abnormal expression that promotes the cell proliferation function with judgement.Hybridization technique is the disclosed mature technology in this area, comprises Southern blotting, Northern blotting, in situ hybridization etc., and relevant test kit 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, is used for analyzing the differential expression and the gene diagnosis of tissue gene.Carry out RNA-polymerase chain reaction (RT-PCR) amplification in vitro with the special primer of albumen and also can detect proteic transcription product with promotion cell proliferation function with promotion cell proliferation function.

The sudden change that detection has the protein gene that promotes the cell proliferation function also can be used for diagnosing the relevant disease of albumen with promotion cell proliferation function.Mutant form with the protein gene that promotes the cell proliferation function comprises that point mutation that the proteic dna sequence dna that promotes the cell proliferation function with having of normal wild type compares, transposition, disappearance, reorganization and other are any unusual etc.Existing technology in available this area 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, Westen blotting gene has or not sudden change.

In gene among the present invention healthy tissues that all are used in experiment, fetal tissue, the tumor tissues expression is arranged all, illustrate that it is the important propagation genes involved of human body self; Expression amount just has difference in different tissues, and the degree difference of its performance function in different tissues is described.

Below embodiment of the present invention are further described.

The present invention carries out the retrieval of people's Unknown Function predicted gene by the refseq database to NCBI, obtain people's unknown function gene order, further utilizing the Human_est database to carry out sequence by the BLASTn method proofreaies and correct, according to the sequences Design gene specific primer that obtains after proofreading and correct, from mix people's tissue cDNA library, obtain the coding region cDNA fragment of goal gene by the amplification of two-step approach flux RT-PCR technology.This coding region cDNA fragment and pcDT recombination to construct carrier for expression of eukaryon.Adopt pRL family renilla luciferase reporter gene method to detect the function of the gene promotion cell proliferation among the present invention.The pRL vehicle group is expressed renilla luciferase with becoming second nature, and under the identical situation of other transfection conditions, the active power of renilla luciferase can reflect the influence of gene to be checked for cell state, as cell number, cell survival rate etc.To compare the active gene that obviously reduces of renilla luciferase with the pcDB empty plasmid picks out.Behind the independent transfectional cell of these genes, utilize LIVE/DEAD ^Viability/Cytotoxicity Kit (L-3224) dyestuff pair cell dyes, and further observes and verify the cell survival state.Evidence in this test kit, Calcein-AM is a kind of endochylema fluorescent marker, no fluorescence own, the water-soluble green fluorescence material that the catalysis of cell lactonase generates behind the infiltration cell is difficult for appearing cell, is used for viable cell dyeing.And then detect the activity of gene induced cell proliferation of the present invention by mtt assay, and the fluidic cell experiment detects the influence of selected gene pairs cell cycle, all obtains positive findings, and what all degree was different has the effect of the propagation of inducing to institute's transfectional cell.Experiment shows that polypeptide of the present invention has remarkable, stable promotion cel l proliferation.

Owing to adopted above technical scheme, the present invention has following advantage:

1, provide mass-producing to clone and screen the technology platform of new gene.

2, cDNA sequence and the coded polypeptide thereof of human new functional gene TRAF3IP3, ZNF306, MGC4368, TINP1, SGOL1, ELF3, URP2 are provided;

3, find that first human new functional gene TRAF3IP3, ZNF306, MGC4368, TINP1, SGOL1, ELF3, URP2 have the effect that promotes cell proliferation, and efficient, stable;

4, TRAF3IP3, ZNF306, MGC4368, TINP1, SGOL1, ELF3, URP2 express at the most normal cells of body, illustrate that it is the relevant regulatory molecule of self important cell proliferation;

5, based on 4 above-mentioned advantages, the present invention is the cell proliferation mechanism of further studying, and the novel drugs of the injury repairing of exploitation treatment autoimmune disease, tumour or disease and wound etc., establish necessary base for starting new clinical diagnosis, therapeutic evaluation and prognostic indicator.

Description of drawings

The structure synoptic diagram of Fig. 1, carrier for expression of eukaryon pcDT-X (X is selected from one of TRAF3IP3, ZNF306, MGC4368, TINP1, SGOL1, ELF3, URP2).

Fig. 2, pRL family renilla luciferase gene reporter plasmid structure iron.

Fig. 3, TRAF3IP3, ZNF306, MGC4368, TINP1, SGOL1, ELF3, URP2 heterogenous expression are to the influence of pRL luciferase expression

The influence of Fig. 4, TRAF3IP3, ZNF306, MGC4368, TINP1, SGOL1, ELF3, the survival of URP2 heterogenous expression pair cell (light field: 10 *)

The influence of Fig. 5, TRAF3IP3, ZNF306, MGC4368, TINP1, SGOL1, ELF3, the survival of URP2 heterogenous expression pair cell (CalceinAM dyeing: 10 *)

Fig. 6, TRAF3IP3, ZNF306, MGC4368, TINP1, SGOL1, ELF3, URP2 heterogenous expression promote cell proliferation (MTT experiment)

Fig. 7, TRAF3IP3, ZNF306, MGC4368, TINP1, SG0L1, ELF3, URP2 heterogenous expression promote cell proliferation (flow cytometry experiment)

Embodiment

Below in conjunction with specific embodiment, further set forth the present invention.These embodiment only are used to the present invention is described and are not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, condition described in " molecular cloning experiment guide " (chopsticks such as the third edition [U.S.] Sa nurse Brooker in 2002, Science Press), or the condition of advising according to manufacturer.

Embodiment 1, two-step approach flux RT-PCR technology amplifying target genes

(1) the refseq database to NCBI carries out the retrieval of people's Unknown Function predicted gene, obtain people's unknown function gene order, and utilize the Human_est database to carry out sequence and proofread and correct by the BLASTn method, the sequence that finally obtains is set at down the group sequence: SEQ IDNO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, called after TRAF3IP3, ZNF306, MGC4368, TINP1, SGOL1, ELF3, URP2 respectively.Special primer according to these type of these genes of sequences Design:

The gene title	Upstream primer (5 '-3 ')	Downstream primer (5 '-3 ')
			TRAF3IP3 ZNF306 MGC4368 TINP1 SGOL1 ELF3 URP2	aacaggtgcttggaggtcatcatg cgctggaagctagagtgaggc ggcttgcgacgggcttct ctccagcggctgctcctg aggaagatagctgttgcagaagtagtg atggctgcaacctgtgagattagc tgtagcccgagacccacctg	ccaaggcagttgtcacaaattattc ctggcatgtatggcatgggtc cttcagcgtcacagccaggtag attgctgtcaaaccagtaagactgc ggactttacctcaagcagatgtgg atagttccaaccctcagttccgac agcagggcaggggcaatcag

(2) use above-mentioned primer, in existing cDNA library and tumour library, select template, carry out just expanding by the express spectra of goal gene.Existing library comprises 12 kinds of human normal tissues (heart, pancreas, testis, ovary, prostate gland, colon, small intestine, skeletal muscle, thymus gland, lymphoglandula, tonsilla, white corpuscle); 6 kinds of people's tumor tissues (lung cancer, carcinoma of the pancreas, ovarian cancer, prostate cancer, colorectal carcinoma, mammary cancer); With the cDNA library of 8 kinds of fetuses group long-pending (tire lung, fetal rhythm, tire liver, tire spleen, tire kidney, tire brain, tire skeletal muscle, tire thymus gland) (Clonetch, K1420-1,1241-1).It is as follows just to expand reaction conditions:

50 μ l PCR reaction:

CDNA mixing storehouse 5 ', 3 ' primer, 10 * Pyrobest buffer 2.5mM dNTPs Pyrobest distilled water

Each 1.0 μ l final concentration is that 2pmol/ μ l 5 μ l 4 μ l 1 μ l complements to 50 μ l

PCR extends the long segment of time according to the expansion goal gene, increases by the principle of 50sec/Kb:

The thing of just expanding production is purified to 30 μ l, with primers a large amount of in the removal PCR reaction system and dNTPs etc., and concentrates whole system, obtains the secondary amplification bank of corresponding target gene sequences, as two templates that expand (the big expansion).

(3) with the purified product in (2) as template, respectively each goal gene is carried out two expansions, reaction conditions is as follows:

50 μ l PCR (each gene) reaction:

One expands purified product 5 ', 3 ' primer 10 * Ex-Taq buffer 2.5mM dNTP Ex-Taq distilled water

1.6 being 2pmol/ μ l 5 μ l 4 μ l 0.5 μ l, μ l final concentration complements to 50 μ l

PCR extends the long segment of time according to the expansion goal gene, increases by the principle of 50sec/Kb:

The PCR product that obtains is got sample electrophoresis on the 10 μ l, selects the PCR product of amplified band, carries out equal-volume purifying (40 μ l).The gene that amplifies non-single band by the two-step pcr reaction reclaims test kit with Qiagen glue and cuts glue recovery purpose fragment.The result of amplification shows, the cDNA that all has gene TRAF3IP3, ZNF306, MGC4368, TINP1, SGOL1, ELF3, URP2 in the cell of these tissues, illustrate that TRAF3IP3, ZNF306, MGC4368, TINP1, SGOL1, ELF3, URP2 have produced the transcription product of gene TRAF3IP3, ZNF306, MGC4368, TINP1, SGOL1, ELF3, URP2 in the cell of these tissues, wider expression map is arranged, in multiple tissue, participate in the adjusting of transcription factor.

Embodiment 2, goal gene Construction of eukaryotic

With two expansion purified product and carrier for expression of eukaryon pcDNA3.1/V5-His-TOPO (Invitrogen is abbreviated as pcDT), carry out ligation according to the condition of test kit manufacturer suggestion.Connect product electric shocking method transformed into escherichia coli DH5 α, conversion product is grown containing on the solid LB plate culture medium of penbritin, select the monospecific polyclonal bacterium colony of growth, extract plasmid, cut with the EcoRI enzyme, enzyme is cut product and is identified with agarose gel electrophoresis, selected and inserted segmental positive colony, select correct forward by order-checking (ABI PRISM 3700 DNA analysis instrument) and insert clone, called after pcDT-TRAF3IP3, pcDT-ZNF306, pcDT-MGC4368, pcDT-TINP1, pcDT-SGOL1, pcDT-ELF3, pcDT-URP2 separately.

Collect nutrient solution simultaneously, analyze protein precipitation, obtain TRAF3IP3, ZNF306, MGC4368, TINP1, SGOL1, ELF3, URP2 polypeptide with SDS-PAGE.

TRAF3IP3, ZNF306, MGC4368, TINP1, SGOL1, ELF3, URP2 analysis of protein result show: TRAF3IP3, ZNF306, MGC4368, TINP1, SGOL1, ELF3, following group of sequence of URP2 protein sequence: shown in SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO:10, SEQ ID NO:12, the SEQ ID NO:14.

Embodiment 3, renilla luciferase reporter gene method are measured the effect of goal gene inducing cell propagation

With goal gene and the renilla luciferase reporter gene carrier cotransfection human embryo kidney 293T of pRL family cell, measure the influence of goal gene by the activity that detects renilla luciferase, as cell number, cytoactive etc. for cell state.To compare the active genescreen that obviously reduces of renilla luciferase with the pcDB empty plasmid comes out.The concrete operations step is as follows:

(1) cell cultures: with 1.2 * 10 ⁴Individual 293T cell (ATCC Number:CRL-11268) is with DMEM (Dulbecco ' the s modified Eagle ' s medium) substratum (Hyclone that contains 10% foetal calf serum, SH0022.02) be layered on 96 porocyte culture plate (Costar, 3599) go up (100 μ l nutrient solution/hole), at 5%CO ₂, 37 ℃ cell culture incubator (SANYO, MCO-15AC) the middle cultivation 24 hours.

(2) preparation transfection working fluid: with 2.5 μ l physiological saline dilution 20ng pRL family's renilla luciferase reporter gene carrier and 80ng gene to be checked, mixing gently, room temperature placement; Dilute 0.04 μ l VigoFect transfection reagent (prestige lattice Lars biotechnology (Beijing) company limited) with 2.5 μ l physiological saline equally, mixing gently, room temperature was placed 5 minutes; The VigoFect transfection reagent of dilution is dropwise added in the gene to be checked and reporter gene mixed solution of dilution, and mixing is to be the transfection working fluid gently, and room temperature was placed 15 minutes.

(3) transfection: with transfection working fluid mixing gently, dropwise be added in the 96 porocyte culture plates of completing cell in (1), place 37 ℃, 5%CO ₂Cell culture incubator in cultivated 24 hours.

(4) detect: transfection is after 24 hours, and (Eppendorf Centrifuge 5810R) centrifugal 5 minutes, abandons supernatant with 800 rev/mins with the cell of 96 orifice plates.Every hole adds 40 μ l cell pyrolysis liquids, places-80 ℃ of refrigerators more than 1 hour 96 orifice plates behind the mixing.Return to room temperature after 96 orifice plates are thawed from-80 ℃, every hole is drawn 10 μ l cell pyrolysis liquids and is moved to white enzyme plate, and (Genios Pro Tecan) detects the renilla luciferase activity with the microwell plate microplate reader.Renilla luciferase substrate consumption is every hole 25 μ l.

The pRL carrier is with renilla luciferase gene and basic expression promoter coupling, and constructive expression's renilla luciferase is provided.The change of condition can influence the expression of renilla luciferase in the experimentation, and these factors comprise cell survival situation (cell number, cell health situation etc.), transfection efficiency, lysis efficient and detection efficiency etc.Under the prerequisite of experimental system error, the transfection of same 96 orifice plate and detection efficiency are approximate substantially, therefore, the active obvious rising of renilla luciferase can reflect the increase of cell quantity or the enhancing of cytoactive, we with the pcDB plasmid in contrast, will with the pRL plasmid co-transfection after cause that the active genescreen that obviously reduces of renilla luciferase comes out.

The result is as shown in Figure 3: gene TRAF3IP3, ZNF306, MGC4368, TINP1, SGOL1, ELF3, URP2 and pcDB have relatively obviously raised the renilla luciferase activity.Show that gene TRAF3IP3, ZNF306, MGC4368, TINP1, SGOL1, ELF3, URP2 can change the survival condition of transfectional cell.

Embodiment 4, utilize dyestuff observation of cell propagation situation

Behind the independent transfection 293T of the gene that filters out among the embodiment 3 cell, utilize LIVE/DEAD ^Viability/Cytotoxicity Kit (Molecular Probe, L-3224) pair cell dyes, operate observation of cell survival condition (comprising light field and fluorescence excitation state) under inverted fluorescence microscope (Zeiss, Axiovert 200M) by product description is described.The concrete operations step is as follows:

This experiment is with 96 porocyte culture plates (Costar, 3599) operations, and each gene to be checked is provided with 3 parallel repeating holes, respectively with pcDB empty plasmid and K-ras plasmid as empty carrier and positive control.Consumption in the following step is the single hole consumption.

(1) cell cultures: with 1.2 * 10 ³Individual 293T cell is layered on (100 μ l nutrient solution/hole) on the 96 porocyte culture plates with the DMEM substratum that contains 10% foetal calf serum, puts 37 ℃, 5%CO ₂Cell culture incubator in cultivated 24 hours.

(2) preparation transfection working fluid: with 2.5 μ l physiological saline dilution 100ng gene to be checked, mixing gently, room temperature placement; Dilute 0.04 μ l VigoFect transfection reagent with 2.5 μ l physiological saline equally, mixing gently, room temperature was placed 5 minutes; The VigoFect transfection reagent of dilution dropwise is added in the cdna solution to be checked of dilution, and mixing is to be the transfection working fluid gently, and room temperature was placed 15 minutes.

(3) transfection: with transfection working fluid mixing gently, dropwise be added in the 96 porocyte culture plates of completing cell in (1), mixing is put 37 ℃, 5%CO gently ₂Cultivated 48 hours in the cell culture incubator.

(4) microscopy: transfection changed with Zeiss microscope observing cell form after 48 hours.Plasmid to be checked compares with pcDB and K-ras respectively.The result as shown in Figure 4, transfection TRAF3IP3, ZNF306, MGC4368, TINP1, SGOL1, ELF3, URP2 gene are after 24 hours, institute's cells transfected upgrowth situation is good, cell number obviously increases.

(5) dyeing: with 5ml PBS solution dilution 2.5 μ l 4mM Calcein AM (viable cell dyestuff) storage liquid, fully mixing; Final concentration is 1 μ M Calcein AM.Discard the nutrient solution of transfectional cell, use the PBS (37 ℃) of incubation to wash twice.Add 50 μ l and dilute good dyestuff, put 37 ℃, CO ₂Hatched in the incubator 30 minutes to 1 hour.

(6) microscopy: discard dyestuff,, add 100 μ l PBS, observe with the Zeiss inverted fluorescence microscope with PBS washing one time.Calcein AM excitation wavelength is 485 ± 10nm, and emission light is 530 ± 12.5nm.

The result as shown in Figure 5, gene TRAF3IP3, ZNF306, MGC4368, TINP1, SGOL1, ELF3, URP2 and empty carrier are relatively, the basic existing state of institute's transfectional cell is good in the same field of view, and green transfect cell (Calcein AM staining cell, green-emitting fluorescence) than empty carrier pcDB showed increased, approaching with positive control K-ras effect, TRAF3IP3, ZNF306, MGC4368, TINP1, SGOL1, ELF3 are described, URP2 can be in the propagation of inducing institute's transfectional cell in varying degrees.

Embodiment 5, MTT detect cell-proliferation activity

Behind the independent transfection 293T of the gene that filters out among the embodiment 3 cell, utilize CellTiter 96 ^Non-radioactiveCell Proliferation Assay Kit (Promega, G-4100) pair cell is handled, operate by product description is described, (BIO-RAD Model550) detects cell down and does growth curve at the absorption peak of 570nm in MCROPLATE READER.The concrete operations step is as follows:

This experiment is with 96 porocyte culture plates (Costar, 3599) operations, and each gene to be checked is provided with 6 parallel repeating holes, simultaneously parallelly does 6 blocks of plates to draw growth curve.Respectively with pcDB empty plasmid and K-ras plasmid as empty carrier and positive control.Consumption in the following step is the single hole consumption.

(1) cell cultures: with 1.2 * 10 ³Individual 293T cell is layered on (100 μ l nutrient solution/hole) on the 96 porocyte culture plates with the DMEM substratum that contains 10% foetal calf serum, puts 37 ℃, 5%CO ₂Cell culture incubator in cultivated 24 hours.

(2) preparation transfection working fluid: with 2.5 μ l physiological saline dilution 100ng gene to be checked, mixing gently, room temperature placement; Dilute 0.04 μ l VigoFect transfection reagent with 2.5 μ l physiological saline equally, mixing gently, room temperature was placed 5 minutes; The VigoFect transfection reagent of dilution dropwise is added in the cdna solution to be checked of dilution, and mixing is to be the transfection working fluid gently, and room temperature was placed 15 minutes.

(3) transfection: with transfection working fluid mixing gently, dropwise be added in the 96 porocyte culture plates of completing cell in (1), mixing is put 37 ℃, 5%CO gently ₂Cultivate in the cell culture incubator.

(4) add MTT: after the transfection per 24 hours, change with the microscope observing cell form.Every then hole adding 15ul MTT solution (5mM, Promega, Madison, WI, USA), and 37 ℃, 5%CO ₂Hatched in the cell culture incubator 4 hours, and added 100ulSolubilization/Stop Solution then at 37 ℃, 5%CO ₂Hatched again in the cell culture incubator 1 hour.

(5) detect: after hatching 1 hour, plate put into MCROPLATE READER (BIO-RAD, Model550) in, detect the absorption peak of every porocyte at 570nm, deduct absorption peak simultaneously as the 620nm of background.

(6) draw growth curve: each gene is an ordinate zou with six days measuring result OD570 all, is X-coordinate with time, draws the cell proliferation curve.

The result as shown in Figure 6, gene TRAF3IP3, ZNF306, MGC4368, TINP1, SGOL1, ELF3, URP2 and empty carrier are relatively, its growth curve slope all obviously increases, approaching with positive control K-ras effect, TRAF3IP3, ZNF306, MGC4368, TINP1, SGOL1, ELF3 are described, URP2 can be in the propagation of inducing institute's transfectional cell in varying degrees.

Embodiment 6, flow cytometer detect the cell cycle

Behind the independent transfection 293T of the gene that filters out among the embodiment 3 cell, utilize flow cytometer to detect the function of the cell cycle of plasmid to be checked with reflection corresponding gene inducing cell propagation.The concrete operations step is as follows:

(1) cell cultures: with 293T cell (1 * 10 ⁵) be layered on the 24 porocyte culture plates (Costar, 3599) with the DMEM substratum that contains 10% foetal calf serum, put 37 ℃, 5%CO ₂Cell culture incubator in cultivated 24 hours.

(2) preparation transfection working fluid: with 8 μ l physiological saline dilution 500ng plasmid to be checked, mixing gently, room temperature placement; Dilute 0.16 μ l VigoFect transfection reagent with 8 μ l physiological saline equally, mixing gently, room temperature was placed 5 minutes; The VigoFect transfection reagent of dilution dropwise is added in the plasmid solution to be checked of dilution, mixing gently, room temperature was placed 15 minutes.

(3) transfection: with transfection working fluid mixing gently, dropwise be added in the Tissue Culture Plate (500 μ l nutrient solution/hole), mixing is put 37 ℃, 5%CO gently ₂Cultivated 24 hours in the incubator.

(4) gather in the crops cell to be measured, each sample about 5 * 10 ⁵To 1 * 10 ⁶, wash 3 times with the PBS of precooling, 1,500rpm, 5min;

(5) with washing the phegma that cell abandons behind the supernatant for the last time cell is hanged, on Vortex, dropwise add 70% ethanol 3ml of precooling, mixing cell gently, 4 ℃ are fixedly spent the night;

(6) the fixed cell is washed once with PBS, and 1,500rpm, 5min adds the 500ulPBS mixing, adds 10ul (10mg/ml) Rnase, 37 ℃ of digestion 30min;

(7) with nylon membrane cell filtration is measured pipe (FALCON to FACS, 352052) in, every pipe adds PI-Triton-X100 (Beijing Bao Sai Bioisystech Co., Ltd of a 500ug/ml, CX1001-2) dye liquor, mixing, (FACSCalibur, BD USA) survey the cell cycle upflowing cell instrument behind the 10min.Analysis of cells M+G in the cycle ₂/ S accounts for the ratio of viable cell, and its height is the power of the cell-proliferation activity of representative just.

The result as shown in Figure 7, TRAF3IP3, ZNF306, MGC4368, TINP1, SG0L1, ELF3, URP2 and empty carrier more all have positive findings, and be similar to positive control K-ras effect, just each gene action degree varies.TRAF3IP3, ZNF306, MGC4368, TINP1, SGOL1, ELF3, URP2 inducing cell propagation in varying degrees is described.

Sequence table

＜110〉Sinogenomax Co., Ltd.

＜120〉relevant polynucleotide and coded polypeptide and the purposes of cell proliferation

<130>

<160>14

<170>PatentIn version 3.3

<210>1

<211>2058

<212>DNA

＜213〉people

<220>

<221>CDS

<222>(291)..(1886)

<400>1

gcccagacct atggattgaa agcgtgtgct ttacccatct gctgtcttgc tccatctgag 60

accagagcca agatctgccc aggactggaa tgctttcccg agtggcttga gttggagcct 120

gggactagga gcagccttat tgaggtacaa ttcatgtgct tgtgggtttg gatggcacaa 180

tctgtgtctg ggctaaggaa agcagacttg gcaccaacat taaccctgac agatccaggc 240

atctattcca ggaactggaa gccaagcgca acaggtgctt ggaggtcatc atg atc 296

Met Ile

1

agc cca gac ccc agg ccc tcc cct ggc ttg gcc cgg tgg gct gag agc 344

Ser Pro Asp Pro Arg Pro Ser Pro Gly Leu Ala Arg Trp Ala Glu Set

5 10 15

tat gag gcc aag tgt gag cgc agg caa gag atc cgt gaa agc cgc cgc 392

Tyr Glu Ala Lys Cys Glu Arg Arg Gln Glu Ile Arg Glu Ser Arg Arg

20 25 30

tgc cgt ccc aat gtg acc act tgc cgc cag gtg ggg aag acg ctg agg 440

Cys Arg Pro Asn Val Thr Thr Cys Arg Gln Val Gly Lys Thr Leu Arg

35 40 45 50

atc caa cag aga gag cag ctc cag aga gct cga ctg cag cag ttc ttc 488

Ile Gln Gln Arg Glu Gln Leu Gln Arg Ala Arg Leu Gln Gln Phe Phe

55 60 65

agg agg agg aac ctg gag cta gag gag aag ggc aaa gcg cag cat ccc 536

Arg Arg Arg Asn Leu Glu Leu Glu Glu Lys Gly Lys Ala Gln His Pro

70 75 80

cag gcc agg gag caa ggg ccc tcc agg cgg cca gga cag gtg aca ggc 584

Gln Ala Arg Glu Gln Gly Pro Ser Arg Arg Pro Gly Gln Val Thr Gly

85 90 95

acc agc tct gaa gtc ttt cca gcc cag cat cct cct ccc tca ggc atc 632

Thr Ser Ser Glu Val Phe Pro Ala Gln His Pro Pro Pro Ser Gly Ile

100 105 110

tgc agg gat ctg tct gac cac ctc tcc tca cag gct ggg ggc ctt cct 680

Cys Arg Asp Leu Ser Asp His Leu Ser Ser Gln Ala Gly Gly Leu Pro

115 120 125 130

cca cag gac act ccc atc aag aag cca ccc aaa cac cac cgt ggt act 728

Pro Gln Asp Thr Pro Ile Lys Lys Pro Pro Lys His His Arg Gly Thr

135 140 145

cag aca aag gca gaa gga cca aca att aag aac gat gcc agt cag caa 776

Gln Thr Lys Ala Glu Gly Pro Thr Ile Lys Asn Asp Ala Ser Gln Gln

150 155 160

acc aat tac gga gtt gca gtt ctg gat aag gaa atc atc cag ctt tct 824

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

165 170 175

gat tac ctc aaa gag gcc cta caa agg gag ctg gtc cta aaa cag aaa 872

Asp Tyr Leu Lys Glu Ala Leu Gln Arg Glu Leu Val Leu Lys Gln Lys

180 185 190

atg gtg att ctc caa gac cta ctg tcc act ctg att cag gcc tct gac 920

Met Val Ile Leu Gln Asp Leu Leu Ser Thr Leu Ile Gln Ala Ser Asp

195 200 205 210

agc tct tgg aag gga cag ctt aat gaa gac aaa ctg aag ggg aaa ctg 968

Ser Ser Trp Lys Gly Gln Leu Asn Glu Asp Lys Leu Lys Gly Lys Leu

215 220 225

aga tcc tta gaa aac cag cta tac acc tgt acc cag aaa tac tcc cct 1016

Arg Ser Leu Glu Asn Gln Leu Tyr Thr Cys Thr Gln Lys Tyr Ser Pro

230 235 240

tgg gga atg aaa aaa gta cta ctg gag atg gaa gac cag aaa aac agc 1064

Trp Gly Met Lys Lys Val Leu Leu Glu Met Glu Asp Gln Lys Asn Ser

245 250 255

tat gag cag aag gcc aag gag tca ctg cag aaa gtg ctg gag gag aaa 1112

Tyr Glu Gln Lys Ala Lys Glu Ser Leu Gln Lys Val Leu Glu Glu Lys

260 265 270

atg aat gca gag cag caa cta cag agc aca cag cga tcc ctg gcc ctg 1160

Met Asn Ala Glu Gln Gln Leu Gln Ser Thr Gln Arg Ser Leu Ala Leu

275 280 285 290

gca gag cag aag tgt gaa gag tgg agg agc cag tat gag gct ctg aag 1208

Ala Glu Gln Lys Cys Glu Glu Trp Arg Ser Gln Tyr Glu Ala Leu Lys

295 300 305

gag gac tgg agg acc ctt ggg acc cag cac agg gag ctg gag agc caa 1256

Glu Asp Trp Arg Thr Leu Gly Thr Gln His Arg Glu Leu Glu Ser Gln

310 315 320

ctc cac gtg ctt cag tcc aaa ctg cag gga gca gat agc agg gac tta 1304

Leu His Val Leu Gln Ser Lys Leu Gln Gly Ala Asp Ser Arg Asp Leu

325 330 335

cag atg aac cag gcc ctg cga ttt ttg gaa aat gag cac cag gaa ctg 1352

Gln Met Asn Gln Ala Leu Arg Phe Leu Glu Asn Glu His Gln Glu Leu

340 345 350

cag gcc aag att gaa tgc ctg caa ggg gac aga gac ctg tgc agc ttg 1400

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

355 360 365 370

gat acc cag gac cta caa gat caa cta aaa agg tca gag gca gag aaa 1448

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

375 380 385

ctc acc ctg gtg acc aga gta cag cag ttg cag ggt ttg ctt caa aat 1496

Leu Thr Leu Val Thr Arg Val Gln Gln Leu Gln Gly Leu Leu Gln Asn

390 395 400

caa tcc tta cag ctt caa gaa cag gag aaa ctc tta aca aag aaa gat 1544

Gln Ser Leu Gln Leu Gln Glu Gln Glu Lys Leu Leu Thr Lys Lys Asp

405 410 415

cag gct ttg ccc gtg tgg agt cca aag tcc ttc cct aac gaa gtg gag 1592

Gln Ala Leu Pro Val Trp Ser Pro Lys Ser Phe Pro Asn Glu Val Glu

420 425 430

cct gag ggt aca ggg aag gag aaa gac tgg gat ctc aga gac cag ctg 1640

Pro Glu Gly Thr Gly Lys Glu Lys Asp Trp Asp Leu Arg Asp Gln Leu

435 440 445 450

caa aag aag act ttg cag ctc cag gcc aag gaa aag gag tgc aga gaa 1688

Gln Lys Lys Thr Leu Gln Leu Gln Ala Lys Glu Lys Glu Cys Arg Glu

455 460 465

ctg cat tca gaa tta gac aac ctc agt gac gag tat ctc tcc tgc ctg 1736

Leu His Ser Glu Leu Asp Asn Leu Ser Asp Glu Tyr Leu Ser Cys Leu

470 475 480

cgt aag ctg cag cac tgt cga gaa gag ctg aac cag agc cag cag ctg 1784

Arg Lys Leu Gln His Cys Arg Glu Glu Leu Asn Gln Ser Gln Gln Leu

485 490 495

cct ccc aga agg caa tgt ggg cga tgg ctc cca gtg ctg atg gtg gtg 1832

Pro Pro Arg Arg Gln Cys Gly Arg Trp Leu Pro Val Leu Met Val Val

500 505 510

att gct gca gca ctg gca gtg ttc ctg gcc aat aaa gac aac ctg atg 1880

Ile Ala Ala Ala Leu Ala Val Phe Leu Ala Asn Lys Asp Asn Leu Met

515 520 525 530

atc tga ataatttgtg acaactgcct tgggtgaaaa tcagaagcaa gcaactcagc 1936

Ile

gaaaaactca gaaggtttgg gtacattaca gcttgggttt tccaactgac ttaggatttc 1996

tgacttttta ttaatttctt aacctactgt aaataaactt cacctgacca gattgttcct 2056

ca 2058

<210>2

<211>531

<212>PRT

＜213〉people

<400>2

Met Ile Ser Pro Asp Pro Arg Pro Ser Pro Gly Leu Ala Arg Trp Ala

1 5 10 15

Glu Ser Tyr Glu Ala Lys Cys Glu Arg Arg Gln Glu Ile Arg Glu Ser

20 25 30

Arg Arg Cys Arg Pro Asn Val Thr Thr Cys Arg Gln Val Gly Lys Thr

35 40 45

Leu Arg Ile Gln Gln Arg Glu Gln Leu Gln Arg Ala Arg Leu Gln Gln

50 55 60

Phe Phe Arg Arg Arg Asn Leu Glu Leu Glu Glu Lys Gly Lys Ala Gln

65 70 75 80

His Pro Gln Ala Arg Glu Gln Gly Pro Ser Arg Arg Pro Gly Gln Val

85 90 95

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

100 105 110

Gly Ile Cys Arg Asp Leu Ser Asp His Leu Ser Ser Gln Ala Gly Gly

115 120 125

Leu Pro Pro Gln Asp Thr Pro Ile Lys Lys Pro Pro Lys His His Arg

130 135 140

Gly Thr Gln Thr Lys Ala Glu Gly Pro Thr Ile Lys Asn Asp Ala Ser

145 150 155 160

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

165 170 175

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

180 185 190

Gln Lys Met Val Ile Leu Gln Asp Leu Leu Ser Thr Leu Ile Gln Ala

195 200 205

Ser Asp Ser Ser Trp Lys Gly Gln Leu Asn Glu Asp Lys Leu Lys Gly

210 215 220

Lys Leu Arg Ser Leu Glu Asn Gln Leu Tyr Thr Cys Thr Gln Lys Tyr

225 230 235 240

Ser Pro Trp Gly Met Lys Lys Val Leu Leu Glu Met Glu Asp Gln Lys

245 250 255

Asn Ser Tyr Glu Gln Lys Ala Lys Glu Ser Leu Gln Lys Val Leu Glu

260 265 270

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

275 280 285

Ala Leu Ala Glu Gln Lys Cys Glu Glu Trp Arg Ser Gln Tyr Glu Ala

290 295 300

Leu Lys Glu Asp Trp Arg Thr Leu Gly Thr Gln His Arg Glu Leu Glu

305 310 315 320

Ser Gln Leu His Val Leu Gln Ser Lys Leu Gln Gly Ala Asp Ser Arg

325 330 335

Asp Leu Gln Met Asn Gln Ala Leu Arg Phe Leu Glu Asn Glu His Gln

340 345 350

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

355 360 365

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

370 375 380

Glu Lys Leu Thr Leu Val Thr Arg Val Gln Gln Leu Gln Gly Leu Leu

385 390 395 400

Gln Asn Gln Ser Leu Gln Leu Gln Glu Gln Glu Lys Leu Leu Thr Lys

405 410 415

Lys Asp Gln Ala Leu Pro Val Trp Ser Pro Lys Ser Phe Pro Asn Glu

420 425 430

Val Glu Pro Glu Gly Thr Gly Lys Glu Lys Asp Trp Asp Leu Arg Asp

435 440 445

Gln Leu Gln Lys Lys Thr Leu Gln Leu Gln Ala Lys Glu Lys Glu Cys

450 455 460

Arg Glu Leu His Ser Glu Leu Asp Asn Leu Ser Asp Glu Tyr Leu Ser

465 470 475 480

Cys Leu Arg Lys Leu Gln His Cys Arg Glu Glu Leu Asn Gln Ser Gln

485 490 495

Gln Leu Pro Pro Arg Arg Gln Cys Gly Arg Trp Leu Pro Val Leu Met

500 505 510

Val Val Ile Ala Ala Ala Leu Ala Val Phe Leu Ala Asn Lys Asp Asn

515 520 525

Leu Met Ile

530

<210>3

<211>2250

<212>DNA

＜213〉people

<220>

<221>CDS

<222>(173)..(1789)

<400>3

gcactttgtg agtatggggt gaatcggcgt cggccttcca ctgtggggtt aaatctcatc 60

ccgcggctct cctcctgtcg gtcctgcagt tcttttgtcc ccgggtagag ggatcttctg 120

cagaaatagc gctggaagct agagtgaggc ctgagtactg ccttggccta gg atg gct 178

Met Ala

1

aga gaa tta agt gaa agc aca gcc ctg gat gcc cag tct aca gaa gac 226

Arg Glu Leu Ser Glu Ser Thr Ala Leu Asp Ala Gln Ser Thr Glu Asp

5 10 15

cag atg gag ctt ctg gtc ata aag gtg gag gaa gaa gaa gcc ggt ttt 274

Gln Met Glu Leu Leu Val Ile Lys Val Glu Glu Glu Glu Ala Gly Phe

20 25 30

ccc agt agc cca gat ctg ggt tct gag ggc tcc cgc gag cgc ttc cga 322

Pro Ser Ser Pro Asp Leu Gly Ser Glu Gly Ser Arg Glu Arg Phe Arg

35 40 45 50

ggc ttc cgc tac ccg gag gct gca ggc ccc cgc gag gcg ctg agt cgg 370

Gly Phe Arg Tyr Pro Glu Ala Ala Gly Pro Arg Glu Ala Leu Ser Arg

55 60 65

ctc cga gag ctc tgc cga cag tgg ctg cag cct gag atg cac agc aag 418

Leu Arg Glu Leu Cys Arg Gln Trp Leu Gln Pro Glu Met His Ser Lys

70 75 80

gag cag atc ctg gag ctg ctg gtg ctg gag cag ttc ctg acc atc ctg 466

Glu Gln Ile Leu Glu Leu Leu Val Leu Glu Gln Phe Leu Thr Ile Leu

85 90 95

ccg ggg aat ctg cag agc tgg gtg cgg gag cag cat cca gag agc ggg 514

Pro Gly Asn Leu Gln Ser Trp Val Arg Glu Gln His Pro Glu Ser Gly

100 105 110

gag gag gtg gtg gtg cta ttg gag tat ttg gag agg cag ctg gat gag 562

Glu Glu Val Val Val Leu Leu Glu Tyr Leu Glu Arg Gln Leu Asp Glu

115 120 125 130

ccg gcg ccg cag gtt tca ggt gtt gac cag ggg caa gaa ctg ctc tgt 610

Pro Ala Pro Gln Val Ser Gly Val Asp Gln Gly Gln Glu Leu Leu Cys

135 140 145

tgc aag atg gca cta ttg aca cca gcc cca ggg tca caa agt agc caa 658

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

150 155 160

ttt cag cta atg aag gct ctg ctc aag cat gaa tct gtg gga tcc cag 706

Phe Gln Leu Met Lys Ala Leu Leu Lys His Glu Ser Val Gly Ser Gln

165 170 175

cct tta caa gat aga gtt ctc cag gtc ccc gtg ctt gcc cat gga gga 754

Pro Leu Gln Asp Arg Val Leu Gln Val Pro Val Leu Ala His Gly Gly

180 185 190

tgc tgc aga gaa gat aaa gtg gta gct tct agg ctt act cca gag tcc 802

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

195 200 205 210

cag ggg ttg ttg aaa gtg gaa gat gtg gcc ctg acc ctc acc cct gaa 850

Gln Gly Leu Leu Lys Val Glu Asp Val Ala Leu Thr Leu Thr Pro Glu

215 220 225

tgg aca cag cag gat tca tct cag ggg aat ctc tgt aga gat gaa aag 898

Trp Thr Gln Gln Asp Ser Ser Gln Gly Asn Leu Cys Arg Asp Glu Lys

230 235 240

cag gag aac cat ggc agc ctg gtc tcc ctg ggt gat gaa aaa cag act 946

Gln Glu Asn His Gly Ser Leu Val Ser Leu Gly Asp Glu Lys Gln Thr

245 250 255

aag agc agg gac ttg cct cca gct gag gag ctt cca gaa aag gag cat 994

Lys Ser Arg Asp Leu Pro Pro Ala Glu Glu Leu Pro Glu Lys Glu His

260 265 270

ggg aag ata tcg tgc cac ctg aga gaa gac att gcc cag att cct aca 1042

Gly Lys Ile Ser Cys His Leu Arg Glu Asp Ile Ala Gln Ile Pro Thr

275 280 285 290

tgt gca gaa gct ggt gaa cag gag ggc agg cta caa aga aag cag aaa 1090

Cys Ala Glu Ala Gly Glu Gln Glu Gly Arg Leu Gln Arg Lys Gln Lys

295 300 305

aat gcc aca gga ggg agg cgg cac atc tgc cat gaa tgt gga aag agt 1138

Asn Ala Thr Gly Gly Arg Arg His Ile Cys His Glu Cys Gly Lys Ser

310 315 320

ttt gct caa agc tca ggc ctg agt aaa cac agg aga atc cac act ggt 1186

Phe Ala Gln Ser Ser Gly Leu Ser Lys His Arg Arg Ile His Thr Gly

325 330 335

gag aaa ccc tac gaa tgt gaa gag tgt ggc aaa gcc ttc att ggg agc 1234

Glu Lys Pro Tyr Glu Cys Glu Glu Cys Gly Lys Ala Phe Ile Gly Ser

340 345 350

tct gcc ctt gtc att cat cag aga gtc cac act ggt gag aag cca tat 1282

Ser Ala Leu Val Ile His Gln Arg Val His Thr Gly Glu Lys Pro Tyr

355 360 365 370

gag tgt gaa gaa tgt ggt aag gcc ttc agt cat agc tca gac ctt atc 1330

Glu Cys Glu Glu Cys Gly Lys Ala Phe Ser His Ser Ser Asp Leu Ile

375 380 385

aag cat cag aga acc cac act ggg gag aag ccc tat gag tgt gat gac 1378

Lys His Gln Arg Thr His Thr Gly Glu Lys Pro Tyr Glu Cys Asp Asp

390 395 400

tgt ggg aag acc ttc agc cag agc tgc agc ctc ctt gaa cat cac aga 1426

Cys Gly Lys Thr Phe Ser Gln Ser Cys Ser Leu Leu Glu His His Arg

405 410 415

atc cac act ggg gag aag ccg tat cag tgc agt atg tgt ggc aaa gcc 1474

Ile His Thr Gly Glu Lys Pro Tyr Gln Cys Ser Met Cys Gly Lys Ala

420 425 430

ttt agg cga agt tca cat ctc ctg aga cat cag agg atc cat act ggg 1522

Phe Arg Arg Ser Ser His Leu Leu Arg His Gln Arg Ile His Thr Gly

435 440 445 450

gat aaa aat gtt cag gaa cct gag cag gga gag gcc tgg aaa agt agg 1570

Asp Lys Asn Val Gln Glu Pro Glu Gln Gly Glu Ala Trp Lys Ser Arg

455 460 465

atg gaa agc cag ttg gaa aat gtt gaa act ccc atg tct tat aaa tgt 1618

Met Glu Ser Gln Leu Glu Asn Val Glu Thr Pro Met Ser Tyr Lys Cys

470 475 480

aat gag tgt gaa aga agt ttc act cag aat aca ggc ctc att gaa cat 1666

Asn Glu Cys Glu Arg Ser Phe Thr Gln Asn Thr Gly Leu Ile Glu His

485 490 495

caa aaa atc cac act ggt gag aaa ccc tat cag tgt aat gcg tgt gga 1714

Gln Lys Ile His Thr Gly Glu Lys Pro Tyr Gln Cys Asn Ala Cys Gly

500 505 510

aaa ggc ttc acc cga att tca tac ctt gtt caa cat cag aga agc cat 1762

Lys Gly Phe Thr Arg Ile Ser Tyr Leu Val Gln His Gln Arg Ser His

515 520 525 530

gta ggg aaa aac atc cta tca cag tga cccatgccat acatgccaga 1809

Val Gly Lys Asn Ile Leu Ser Gln

535

gttggtgctc atttgtcact gatctgaagc cactccccct ggagtctcaa ctatagaaat 1869

tgtgggctgg gctttattta ccactacaca ttatcaggtg ttagaaaatg tagactgggt 1929

tagacaaatt atcttctaag ttctagaagg ggtttgtaat caaaacatat ttgataggag 1989

gctacgggag agttgtctag aagaggtaag acctgaaact gtttgttctc tcccactaga 2049

attaaatgga tgtttagact ggctacttgc cctaaaccag cacttggtga tgtctactgg 2109

gtggatggtt gtgatttggg ggctgcttct ctgaccattc tttttgactg taatgaatcc 2169

tccacagttg gtcagattca taaagtctta tatccccctc tgtgcctttt ctgcaggtgc 2229

taataaatgt ttattgaatg t 2250

<210>4

<211>538

<212>PRT

＜213〉people

<400>4

Met Ala Arg Glu Leu Ser Glu Ser Thr Ala Leu Asp Ala Gln Ser Thr

1 5 10 15

Glu Asp Gln Met Glu Leu Leu Val Ile Lys Val Glu Glu Glu Glu Ala

20 25 30

Gly Phe Pro Ser Ser Pro Asp Leu Gly Ser Glu Gly Ser Arg Glu Arg

35 40 45

Phe Arg Gly Phe Arg Tyr Pro Glu Ala Ala Gly Pro Arg Glu Ala Leu

50 55 60

Ser Arg Leu Arg Glu Leu Cys Arg Gln Trp Leu Gln Pro Glu Met His

65 70 75 80

Ser Lys Glu Gln Ile Leu Glu Leu Leu Val Leu Glu Gln Phe Leu Thr

85 90 95

Ile Leu Pro Gly Asn Leu Gln Ser Trp Val Arg Glu Gln His Pro Glu

100 105 110

Ser Gly Glu Glu Val Val Val Leu Leu Glu Tyr Leu Glu Arg Gln Leu

115 120 125

Asp Glu Pro Ala Pro Gln Val Ser Gly Val Asp Gln Gly Gln Glu Leu

130 135 140

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

145 150 155 160

Ser Gln Phe Gln Leu Met Lys Ala Leu Leu Lys His Glu Ser Val Gly

165 170 175

Ser Gln Pro Leu Gln Asp Arg Val Leu Gln Val Pro Val Leu Ala His

180 185 190

Gly Gly Cys Cys Arg Glu Asp Lys Val Val Ala Ser Arg Leu Thr Pro

195 200 205

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

210 215 220

Pro Glu Trp Thr Gln Gln Asp Ser Ser Gln Gly Asn Leu Cys Arg Asp

225 230 235 240

Glu Lys Gln Glu Asn His Gly Ser Leu Val Ser Leu Gly Asp Glu Lys

245 250 255

Gln Thr Lys Ser Arg Asp Leu Pro Pro Ala Glu Glu Leu Pro Glu Lys

260 265 270

Glu His Gly Lys Ile Ser Cys His Leu Arg Glu Asp Ile Ala Gln Ile

275 280 285

Pro Thr Cys Ala Glu Ala Gly Glu Gln Glu Gly Arg Leu Gln Arg Lys

290 295 300

Gln Lys Asn Ala Thr Gly Gly Arg Arg His Ile Cys His Glu Cys Gly

305 310 315 320

Lys Ser Phe Ala Gln Ser Ser Gly Leu Ser Lys His Arg Arg Ile His

325 330 335

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

340 345 350

Gly Ser Ser Ala Leu Val Ile His Gln Arg Val His Thr Gly Glu Lys

355 360 365

Pro Tyr Glu Cys Glu Glu Cys Gly Lys Ala Phe Ser His Ser Ser Asp

370 375 380

Leu Ile Lys His Gln Arg Thr His Thr Gly Glu Lys Pro Tyr Glu Cys

385 390 395 400

Asp Asp Cys Gly Lys Thr Phe Ser Gln Ser Cys Ser Leu Leu Glu His

405 410 415

His Arg Ile His Thr Gly Glu Lys Pro Tyr Gln Cys Ser Met Cys Gly

420 425 430

Lys Ala Phe Arg Arg Ser Ser His Leu Leu Arg His Gln Arg Ile His

435 440 445

Thr Gly Asp Lys Asn Val Gln Glu Pro Glu Gln Gly Glu Ala Trp Lys

450 455 460

Ser Arg Met Glu Ser Gln Leu Glu Asn Val Glu Thr Pro Met Ser Tyr

465 470 475 480

Lys Cys Asn Glu Cys Glu Arg Ser Phe Thr Gln Asn Thr Gly Leu Ile

485 490 495

Glu His Gln Lys Ile His Thr Gly Glu Lys Pro Tyr Gln Cys Asn Ala

500 505 510

Cys Gly Lys Gly Phe Thr Arg Ile Ser Tyr Leu Val Gln His Gln Arg

515 520 525

Ser His Val Gly Lys Asn Ile Leu Ser Gln

530 535

<210>5

<211>2253

<212>DNA

＜213〉people

<220>

<221>CDS

<222>(743)..(1414)

<400>5

ggcgaccaga gttgctgggc agtcttgggt ttagtgccgc aatgctgcac ttctgacagg 60

ctgcttcctg gatccctgca gggacctggc cagggcattg gccccaggtc acctgtggct 120

ttggggagag cagttgcagg gctggcctgg gctgtgggag gcagctgtgc cgttttctga 180

cagccattgg gaccgcccca gtacccgaag aggtggctgc cctgagttgc cacgggaaac 240

tgcagggtgg ccctggctca gcagccaacc ttggggagac ctggaggaac agtgcagtgt 300

aaagatggcc tcgcgctgag cgggtccccg cagtcggcac tcccaggaga accagcgggc 360

tgtgcatgtg acccgccagc ctgcaccgcg ggcacgtcct tcccacccag aggaccctgc 420

actgccttca gggccacagc agtcctccgg ggcccagcct caggcaggaa acagcaaatg 480

cttttgctgc caaccaagtg tggctgtcgc tttcgatgat gccagtggcg ctggtgcgac 540

tgccgggcag catgtgtggg cggggatggc tcggccactg accgtctccc cgcagtggtg 600

gtcctgctcc atgatgtccg tgatgtgagc gtggaggagg agaaggtccg gtacttcggg 660

aaaggctaca tggtggtgct ccggcttgcg acgggcttct cccaccccct cacgcagagt 720

gcagtcatgg gccaccgcag tg atg tgg aag cca tcg cca agc tca tca cca 772

Met Trp Lys Pro Ser Pro Ser Ser Ser Pro

1 5 10

gct tcc tgg agc tgc act gcc ttg aga gcc cca cag agc tgt ctc aga 820

Ala Ser Trp Ser Cys Thr Ala Leu Arg Ala Pro Gln Ser Cys Leu Arg

15 20 25

gca gcg aca gtg agg ccg gtg acc ctg caa gcc aga gct gac agc ccc 868

Ala Ala Thr Val Arg Pro Val Thr Leu Gln Ala Arg Ala Asp Ser Pro

30 35 40

act gtg cct gag ccc gtg cac cgc cca cag gac cca tgg cac att ccc 916

Thr Val Pro Glu Pro Val His Arg Pro Gln Asp Pro Trp His Ile Pro

45 50 55

ggt gtg cct gag ccc gtg cac cgc cca cag gac ccg tgg cac att ccc 964

Gly Val Pro Glu Pro Val His Arg Pro Gln Asp Pro Trp His Ile Pro

60 65 70

ggt gtg cct gag ccc gtg cac cgc cca cag gac ccg tgg cac att ccc 1012

Gly Val Pro Glu Pro Val His Arg Pro Gln Asp Pro Trp His Ile Pro

75 80 85 90

ggt gtg cct gag ccc gtg cac cgc cca cag gac ccg tgg cac att ccc 1060

Gly Val Pro Glu Pro Val His Arg Pro Gln Asp Pro Trp His Ile Pro

95 100 105

ggt gtg cct gag ccc gtg cac cgc cca cag gac ccg tgg cct tgg ctt 1108

Gly Val Pro Glu Pro Val His Arg Pro Gln Asp Pro Trp Pro Trp Leu

110 115 120

cag ttg gtg cct cca gcc gag ttg gcc tat tgc ctg ctc atg ctg ctg 1156

Gln Leu Val Pro Pro Ala Glu Leu Ala Tyr Cys Leu Leu Met Leu Leu

125 130 135

ctt gca cac tgc atg aaa cag cag gcc aga cca gga cat cca gac ttt 1204

Leu Ala His Cys Met Lys Gln Gln Ala Arg Pro Gly His Pro Asp Phe

140 145 150

ctc cat cgt gag gcc tgg gcc tgc ctt tct gca gcc gga ggt ctc gcc 1252

Leu His Arg Glu Ala Trp Ala Cys Leu Ser Ala Ala Gly Gly Leu Ala

155 160 165 170

agc cct gga ctc ctg ctt tgg gcc aca gca aga cct cgg gcg agt gga 1300

Ser Pro Gly Leu Leu Leu Trp Ala Thr Ala Arg Pro Arg Ala Ser Gly

175 180 185

gag gcg ggg cca ggc cgg gcc ctt gtg ggt gct gat gct gca tgt tgt 1348

Glu Ala Gly Pro Gly Arg Ala Leu Val Gly Ala Asp Ala Ala Cys Cys

190 195 200

ccc cga cac agc gtc ctc tcc ctg gtg gac atc ccc agc ggt cag gtg 1396

Pro Arg His Ser Val Leu Ser Leu Val Asp Ile Pro Ser Gly Gln Val

205 210 215

ctt ccc caa ggg cag tga ggggtgaaca tccagggcct acctggctgt 1444

Leu Pro Gln Gly Gln

220

gacgctgaag ccacactgtg gaagctgccc ctccccgagg acccgcctcc cttgctgatg 1504

ccaggatctc ggcgcataga ccactctgcc ccagcggtcg tcacagaaag gtctctctgt 1564

tcctcacact cagcttcagc ataagctgtg aggccagaaa aaaggtcagc tcttctagta 1624

tcgtgcagtg cttaaaaacc gggagctcca gccgggcgca gtggttcatg ccagtaatcc 1684

cagcactttc ggaggccgag gtgggaggat tgcttgaggc caggagttca agaccagcct 1744

gggcaacaca gcaagatcct gtctttgtaa aaaaactaac caaacaggaa aaactgggag 1804

attttctgca gaaattgagt tccagcctct ctcgaacctg ggaagacctg gcaggagggg 1864

gctgggctct cggctacaga cttctcccca ccccgtagga gctgaacgcc aaccatcctg 1924

acccgccagt gctcttggtc tcctgagtgt acccaggtcc tcccaggtgc ggtgtgcacc 1984

gagcgcgcct ggcctgatgc cctggcctgt gagctgggga ctcctgggcc ctgtgagccc 2044

ctaggcggca ggcccaggaa tggctgggta ggacagggaa cacctttgcc ccacgtctgg 2104

ctgtgacctc ggtgaaagcc gacaggagag agatgggacc ctcctcctca gtagtggctg 2164

ccagtccctc gttgcaggac agggtcatca taaccataaa taacccttca cgtgtcaaaa 2224

aaaaaaaaaa aaaaaaaaaa aaaaaaaaa 2253

<210>6

<211>223

<212>PRT

＜213〉people

<400>6

Met Trp Lys Pro Ser Pro Ser Ser Ser Pro Ala Ser Trp Ser Cys Thr

1 5 10 15

Ala Leu Arg Ala Pro Gln Ser Cys Leu Arg Ala Ala Thr Val Arg Pro

20 25 30

Val Thr Leu Gln Ala Arg Ala Asp Ser Pro Thr Val Pro Glu Pro Val

35 40 45

His Arg Pro Gln Asp Pro Trp His Ile Pro Gly Val Pro Glu Pro Val

50 55 60

His Arg Pro Gln Asp Pro Trp His Ile Pro Gly Val Pro Glu Pro Val

65 70 75 80

His Arg Pro Gln Asp Pro Trp His Ile Pro Gly Val Pro Glu Pro Val

85 90 95

His Arg Pro Gln Asp Pro Trp His Ile Pro Gly Val Pro Glu Pro Val

100 105 110

His Arg Pro Gln Asp Pro Trp Pro Trp Leu Gln Leu Val Pro Pro Ala

115 120 125

Glu Leu Ala Tyr Cys Leu Leu Met Leu Leu Leu Ala His Cys Met Lys

130 135 140

Gln Gln Ala Arg Pro Gly His Pro Asp Phe Leu His Arg Glu Ala Trp

145 150 155 160

Ala Cys Leu Ser Ala Ala Gly Gly Leu Ala Ser Pro Gly Leu Leu Leu

165 170 175

Trp Ala Thr Ala Arg Pro Arg Ala Ser Gly Glu Ala Gly Pro Gly Arg

180 185 190

Ala Leu Val Gly Ala Asp Ala Ala Cys Cys Pro Arg His Ser Val Leu

195 200 205

Ser Leu Val Asp Ile Pro Ser Gly Gln Val Leu Pro Gln Gly Gln

210 215 220

<210>7

<211>2529

<212>DNA

＜213〉people

<220>

<221>CDS

<222>(203)..(1318)

<400>7

gagagagaga gagagagaga actagtctcg aggtcgacgc ggccgcgaat tcgcggccgc 60

gtcgaccgaa gacgcaggct ctatttagag ccgggtaggg gagcgcacgg ccagatacct 120

cagcgctacc tggcggaact ggatttctct cccgcctgcc ggcctgcctg ccacagccgg 180

actccgccac tccggtagcc tc atg gct gca acc tgt gag att agc aac att 232

Met Ala Ala Thr Cys Glu Ile Ser Asn Ile

1 5 10

ttt agc aac tac ttc agt gcg atg tac agc tcg gag gac tcc acc ctg 280

Phe Ser Asn Tyr Phe Ser Ala Met Tyr Ser Ser Glu Asp Ser Thr Leu

15 20 25

gcc tct gtt ccc cct gct gcc acc ttt ggg gcc gat gac ttg gta ctg 328

Ala Ser Val Pro Pro Ala Ala Thr Phe Gly Ala Asp Asp Leu Val Leu

30 35 40

acc ctg agc aac ccc cag atg tca ttg gag ggt aca gag aag gcc agc 376

Thr Leu Ser Asn Pro Gln Met Ser Leu Glu Gly Thr Glu Lys Ala Ser

45 50 55

tgg ttg ggg gaa cag ccc cag ttc tgg tcg aag acg cag gtt ctg gac 424

Trp Leu Gly Glu Gln Pro Gln Phe Trp Ser Lys Thr Gln Val Leu Asp

60 65 70

tgg atc agc tac caa gtg gag aag aac aag tac gac gca agc gcc att 472

Trp Ile Ser Tyr Gln Val Glu Lys Asn Lys Tyr Asp Ala Ser Ala Ile

75 80 85 90

gac ttc tca cga tgt gac atg gat ggc gcc acc ctc tgc aat tgt gcc 520

Asp Phe Ser Arg Cys Asp Met Asp Gly Ala Thr Leu Cys Asn Cys Ala

95 100 105

ctt gag gag ctg cgt ctg gtc ttt ggg cct ctg ggg gac caa ctc cat 568

Leu Glu Glu Leu Arg Leu Val Phe Gly Pro Leu Gly Asp Gln Leu His

110 115 120

gcc cag ctg cga gac ctc act tcc agc tct tct gat gag ctc agt tgg 616

Ala Gln Leu Arg Asp Leu Thr Ser Ser Ser Ser Asp Glu Leu Ser Trp

125 130 135

atc att gag ctg ctg gag aag gat ggc atg gcc ttc cag gag gcc cta 664

Ile Ile Glu Leu Leu Glu Lys Asp Gly Met Ala Phe Gln Glu Ala Leu

140 145 150

gac cca ggg ccc ttt gac cag ggc agc ccc ttt gcc cag gag ctg ctg 712

Asp Pro Gly Pro Phe Asp Gln Gly Ser Pro Phe Ala Gln Glu Leu Leu

155 160 165 170

gac gac ggt cag caa gcc agc ccc tac cac ccc ggc agc tgt ggc gca 760

Asp Asp Gly Gln Gln Ala Ser Pro Tyr His Pro Gly Ser Cys Gly Ala

175 180 185

gga gcc ccc tcc ccc ggc agc tct gac gtc tcc acc gca ggg act ggt 808

Gly Ala Pro Ser Pro Gly Ser Ser Asp Val Ser Thr Ala Gly Thr Gly

190 195 200

gct tct cgg agc tcc cac tcc tca gac tcc ggt gga agt gac gtg gac 856

Ala Ser Arg Ser Ser His Ser Ser Asp Ser Gly Gly Ser Asp Val Asp

205 210 215

ctg gat ccc act gat ggc aag ctc ttc ccc agc gat ggt ttt cgt gac 904

Leu Asp Pro Thr Asp Gly Lys Leu Phe Pro Ser Asp Gly Phe Arg Asp

220 225 230

tgc aag aag ggg gat ccc aag cac ggg aag cgg aaa cga ggc cgg ccc 952

Cys Lys Lys Gly Asp Pro Lys His Gly Lys Arg Lys Arg Gly Arg Pro

235 240 245 250

cga aag ctg agc aaa gag tac tgg gac tgt ctc gag ggc aag aag agc 1000

Arg Lys Leu Ser Lys Glu Tyr Trp Asp Cys Leu Glu Gly Lys Lys Ser

255 260 265

aag cac gcg ccc aga ggc acc cac ctg tgg gag ttc atc cgg gac atc 1048

Lys His Ala Pro Arg Gly Thr His Leu Trp Glu Phe Ile Arg Asp Ile

270 275 280

ctc atc cac ccg gag ctc aac gag ggc ctc atg aag tgg gag aat cgg 1096

Leu Ile His Pro Glu Leu Asn Glu Gly Leu Met Lys Trp Glu Asn Arg

285 290 295

cat gaa ggc gtc ttc aag ttc ctg cgc tcc gag gct gtg gcc caa cta 1144

His Glu Gly Val Phe Lys Phe Leu Arg Ser Glu Ala Val Ala Gln Leu

300 305 310

tgg ggc caa aag aaa aag aac agc aac atg acc tac gag aag ctg agc 1192

Trp Gly Gln Lys Lys Lys Asn Ser Asn Met Thr Tyr Glu Lys Leu Ser

315 320 325 330

cgg gcc atg agg tac tac tac aaa cgg gag atc ctg gaa cgg gtg gat 1240

Arg Ala Met Arg Tyr Tyr Tyr Lys Arg Glu Ile Leu Glu Arg Val Asp

335 340 345

ggc cgg cga ctc gtc tac aag ttt ggc aaa aac tca agc ggc tgg aag 1288

Gly Arg Arg Leu Val Tyr Lys Phe Gly Lys Asn Ser Ser Gly Trp Lys

350 355 360

gag gaa gag gtt ctc cag agt cgg aac tga gggttggaac tatacccggg 1338

Glu Glu Glu Val Leu Gln Ser Arg Asn

365 370

accaaactca cggaccactc gaggcctgca aaccttcctg ggaggacagg caggccagat 1398

ggcccctcca ctggggaatg ctcccagctg tgctgtggag agaagctgat gttttggtgt 1458

attgtcagcc atcgtccttg gactcggaga ctatggcctc gcctccccac cctcctcttg 1518

gaattacaag ccctggggtt tgaagctgac tttatagctg caagtgtatc tccttttatc 1578

tggtgcctcc tcaaacccag tctcagacac taaatgcaga caacaccttc ctcctgcaga 1638

cacttggact gagccaagga ggcttgggag gccctaggga gcaccgtgat ggagaggaca 1698

gagcaggggc tccagcactt ctttctggac tggcgttcac ctccctgctc agtgcttggg 1758

ctccacgggc aggggtcaga gcactcccta atttatgtgc tatataaata tatcagatgt 1818

acatagagat ctattttttc taaaacattc ccctccccac tcctctccca cagagtgctg 1878

gactgttcca ggccctccag tgggctgatg ctgggaccct taggatgggg ctcccagctc 1938

ctttctcctg tgaatggagg cagagacctc caataaagtg ccttctgggc tttttctaac 1998

ctttgtctta gctacctgtg tactgaaatt tgggcctttg gatcgaatat ggtcaagagg 2058

ttggagggga ggaaaatgaa ggtctaccag gatgagggtg agggcaaagg ctgacgaaga 2118

ggggagttac agatttcctg tagcaggtgt gggcttacag acacatggac tgggctggga 2178

ggcgagcaaa ggaagcagct gagactgttg gagaacgtta caagacttca tgcaagaagg 2238

acatgaactc agaacactga ggtcagaagc atctgctgtc atgacaccgc tcgagtgacc 2298

ttgaccttga ccaagtcttg tccttgttta ggactgattt ttcctattag gctagggttt 2358

ggacctgatg ttctcaagat gtctagaatt gcatggctgg ccttgtggaa tagatggttt 2418

tgcattccag ccaagtgtgc tgtaaactgt atatctgtaa tatgaatccc agcttttgag 2478

tctgacaaaa tcagagttag gatcttgtaa aggaaaaaaa aaaaaaaaaa a 2529

<210>8

<211>371

<212>PRT

＜213〉people

<400>8

Met Ala Ala Thr Cys Glu Ile Ser Asn Ile Phe Ser Asn Tyr Phe Ser

1 5 10 15

Ala Met Tyr Ser Ser Glu Asp Ser Thr Leu Ala Ser Val Pro Pro Ala

20 25 30

Ala Thr Phe Gly Ala Asp Asp Leu Val Leu Thr Leu Ser Asn Pro Gln

35 40 45

Met Ser Leu Glu Gly Thr Glu Lys Ala Ser Trp Leu Gly Glu Gln Pro

50 55 60

Gln Phe Trp Ser Lys Thr Gln Val Leu Asp Trp Ile Ser Tyr Gln Val

65 70 75 80

Glu Lys Asn Lys Tyr Asp Ala Ser Ala Ile Asp Phe Ser Arg Cys Asp

85 90 95

Met Asp Gly Ala Thr Leu Cys Asn Cys Ala Leu Glu Glu Leu Arg Leu

100 105 110

Val Phe Gly Pro Leu Gly Asp Gln Leu His Ala Gln Leu Arg Asp Leu

115 120 125

Thr Ser Ser Ser Ser Asp Glu Leu Ser Trp Ile Ile Glu Leu Leu Glu

130 135 140

Lys Asp Gly Met Ala Phe Gln Glu Ala Leu Asp Pro Gly Pro Phe Asp

145 150 155 160

Gln Gly Ser Pro Phe Ala Gln Glu Leu Leu Asp Asp Gly Gln Gln Ala

165 170 175

Ser Pro Tyr His Pro Gly Ser Cys Gly Ala Gly Ala Pro Ser Pro Gly

180 185 190

Ser Ser Asp Val Ser Thr Ala Gly Thr Gly Ala Ser Arg Ser Ser His

195 200 205

Ser Ser Asp Ser Gly Gly Ser Asp Val Asp Leu Asp Pro Thr Asp Gly

210 215 220

Lys Leu Phe Pro Ser Asp Gly Phe Arg Asp Cys Lys Lys Gly Asp Pro

225 230 235 240

Lys His Gly Lys Arg Lys Arg Gly Arg Pro Arg Lys Leu Ser Lys Glu

245 250 255

Tyr Trp Asp Cys Leu Glu Gly Lys Lys Ser Lys His Ala Pro Arg Gly

260 265 270

Thr His Leu Trp Glu Phe Ile Arg Asp Ile Leu Ile His Pro Glu Leu

275 280 285

Asn Glu Gly Leu Met Lys Trp Glu Asn Arg His Glu Gly Val Phe Lys

290 295 300

Phe Leu Arg Ser Glu Ala Val Ala Gln Leu Trp Gly Gln Lys Lys Lys

305 310 315 320

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

325 330 335

Tyr Lys Arg Glu Ile Leu Glu Arg Val Asp Gly Arg Arg Leu Val Tyr

340 345 350

Lys Phe Gly Lys Asn Ser Ser Gly Trp Lys Glu Glu Glu Val Leu Gln

355 360 365

Ser Arg Asn

370

<210>9

<211>1105

<212>DNA

＜213〉people

<220>

<221>CDS

<222>(85)..(867)

<400>9

ggggcttgtg ggtctttgag acccgaaaat tgagagcgtt ttcgcactcc agcggctgct 60

cctggcggct ctgcggccgt cacc atg cca cag aat gaa tat att gaa tta 111

Met Pro Gln Asn Glu Tyr Ile Glu Leu

1 5

cac cgt aaa cgc tat gga tac cgt ttg gat tac cat gag aaa aag aga 159

His Arg Lys Arg Tyr Gly Tyr Arg Leu Asp Tyr His Glu Lys Lys Arg

10 15 20 25

aag aag gaa agt cga gag gct cat gaa cgt tca aag aag gca aag aaa 207

Lys Lys Glu Ser Arg Glu Ala His Glu Arg Ser Lys Lys Ala Lys Lys

30 35 40

atg att ggt ctg aag gct aag ctt tac cat aaa cag cgt cat gct gag 255

Met Ile Gly Leu Lys Ala Lys Leu Tyr His Lys Gln Arg His Ala Glu

45 50 55

aaa ata caa atg aaa aag act atc aag atg cat gaa aag aga aac acc 303

Lys Ile Gln Met Lys Lys Thr Ile Lys Met His Glu Lys Arg Asn Thr

60 65 70

aaa caa aag aat gat gaa aag aca cca cag gga gca gta cct gcc tat 351

Lys Gln Lys Asn Asp Glu Lys Thr Pro Gln Gly Ala Val Pro Ala Tyr

75 80 85

ctg ctg gac aga gag gga caa tct cga gct aaa gta ctt tcc aat atg 399

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

90 95 100 105

att aaa cag aaa aga aaa gag aag gcg gga aaa tgg gaa gtc cct ctg 447

Ile Lys Gln Lys Arg Lys Glu Lys Ala Gly Lys Trp Glu Val Pro Leu

110 115 120

cct aaa gta cgt gcc cag gga gaa aca gaa gta tta aaa gtt att cga 495

Pro Lys Val Arg Ala Gln Gly Glu Thr Glu Val Leu Lys Val Ile Arg

125 130 135

aca gga aag aga aag aag aag gca tgg aag aga atg gtt act aaa gtg 543

Thr Gly Lys Arg Lys Lys Lys Ala Trp Lys Arg Met Val Thr Lys Val

140 145 150

tgc ttt gtt gga gat ggc ttt aca aga aaa cca cct aaa tat gaa aga 591

Cys Phe Val Gly Asp Gly Phe Thr Arg Lys Pro Pro Lys Tyr Glu Arg

155 160 165

ttc atc agg cca atg ggc ttg cgt ttc aag aaa gcc cat gta aca cat 639

Phe Ile Arg Pro Met Gly Leu Arg Phe Lys Lys Ala His Val Thr His

170 175 180 185

cct gaa ctg aaa gcc acc ttt tgc cta cca ata ctt ggt gta aag aag 687

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

190 195 200

aat ccc tca tcc cca ctg tat aca act ttg ggt gtt att acc aaa ggt 735

Asn Pro Ser Ser Pro Leu Tyr Thr Thr Leu Gly Val Ile Thr Lys Gly

205 210 215

act gtc att gaa gta aat gtg agc gaa ttg ggc ctt gtg aca caa gga 783

Thr Val Ile Glu Val Asn Val Ser Glu Leu Gly Leu Val Thr Gln Gly

220 225 230

ggc aaa gtt att tgg gga aaa tat gcc cag gtt acc aac aat cct gaa 831

Gly Lys Val Ile Trp Gly Lys Tyr Ala Gln Val Thr Asn Asn Pro Glu

235 240 245

aat gat gga tgt ata aat gca gtc tta ctg gtt tga cagcaatttc 877

Asn Asp Gly Cys Ile Asn Ala Val Leu Leu Val

250 255 260

atatataatt attgaggact acacaccaat tgaagaaact gccattactg tgatgtttct 937

gaatactacc aaacagccat acatgtctgc aatgaagaga tttattaaat tgtaaacatt 997

aaagtggtcc agttttataa atggtcttta ttttgaaata cgctttgacc ccatgttcat 1057

aaaactgaat gattgaaaaa aagcaaatat acaaatatcc tacttcat 1105

<210>10

<211>260

<212>PRT

＜213〉people

<400>10

Met Pro Gln Asn Glu Tyr Ile Glu Leu His Arg Lys Arg Tyr Gly Tyr

1 5 10 15

Arg Leu Asp Tyr His Glu Lys Lys Arg Lys Lys Glu Ser Arg Glu Ala

20 25 30

His Glu Arg Ser Lys Lys Ala Lys Lys Met Ile Gly Leu Lys Ala Lys

35 40 45

Leu Tyr His Lys Gln Arg His Ala Glu Lys Ile Gln Met Lys Lys Thr

50 55 60

Ile Lys Met His Glu Lys Arg Asn Thr Lys Gln Lys Asn Asp Glu Lys

65 70 75 80

Thr Pro Gln Gly Ala Val Pro Ala Tyr Leu Leu Asp Arg Glu Gly Gln

85 90 95

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

100 105 110

Lys Ala Gly Lys Trp Glu Val Pro Leu Pro Lys Val Arg Ala Gln Gly

115 120 125

Glu Thr Glu Val Leu Lys Val Ile Arg Thr Gly Lys Arg Lys Lys Lys

130 135 140

Ala Trp Lys Arg Met Val Thr Lys Val Cys Phe Val Gly Asp Gly Phe

145 150 155 160

Thr Arg Lys Pro Pro Lys Tyr Glu Arg Phe Ile Arg Pro Met Gly Leu

165 170 175

Arg Phe Lys Lys Ala His Val Thr His Pro Glu Leu Lys Ala Thr Phe

180 185 190

Cys Leu Pro Ile Leu Gly Val Lys Lys Asn Pro Ser Ser Pro Leu Tyr

195 200 205

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

210 215 220

Ser Glu Leu Gly Leu Val Thr Gln Gly Gly Lys Val Ile Trp Gly Lys

225 230 235 240

Tyr Ala Gln Val Thr Asn Asn Pro Glu Asn Asp Gly Cys Ile Asn Ala

245 250 255

Val Leu Leu Val

260

<210>11

<211>1333

<212>DNA

＜213〉people

<220>

<221>CDS

<222>(141)..(788)

<400>11

agttccaggc gacggcggac ggtggtacgg tcctggaggg cccagtgcgc ggggctagcc 60

gtggctggag agcttcgaag agccttgaaa tgtgaggagg aggaagatag ctgttgcaga 120

agtagtggcc aaggcaaaag atg gcc aag gaa aga tgc ctg aaa aag tcc ttt 173

Met Ala Lys Glu Arg Cys Leu Lys Lys Ser Phe

1 5 10

caa gat agt ctt gaa gac ata aag aag cga atg aaa gag aaa agg aat 221

Gln Asp Ser Leu Glu Asp Ile Lys Lys Arg Met Lys Glu Lys Arg Asn

15 20 25

aaa aac ttg gca gag att ggc aaa cgc agg tct ttt ata gct gca cca 269

Lys Asn Leu Ala Glu Ile Gly Lys Arg Arg Ser Phe Ile Ala Ala Pro

30 35 40

tgc caa ata atc acc aac act tct aca ctg ctg aaa aat tac caa gac 317

Cys Gln Ile Ile Thr Asn Thr Ser Thr Leu Leu Lys Asn Tyr Gln Asp

45 50 55

aac aac aaa atg tta gtt tta gct ttg gaa aat gaa aaa tcc aaa gtg 365

Asn Asn Lys Met Leu Val Leu Ala Leu Glu Asn Glu Lys Ser Lys Val

60 65 70 75

aaa gaa gcc caa gat atc atc cta cag ctg aga aaa gaa tgt tac tat 413

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

80 85 90

ctc aca tgt cag cta tat gca ttg aaa gga aaa ctt aca tca caa caa 461

Leu Thr Cys Gln Leu Tyr Ala Leu Lys Gly Lys Leu Thr Ser Gln Gln

95 100 105

aca gta gaa cct gct cag aac cag gaa ata tgt tcc tct gga atg gac 509

Thr Val Glu Pro Ala Gln Asn Gln Glu Ile Cys Ser Ser Gly Met Asp

110 115 120

ccc aat agt gat gac agc tcc aga aat tta ttt gtg aag gat tta ccg 557

Pro Asn Ser Asp Asp Ser Ser Arg Asn Leu Phe Val Lys Asp Leu Pro

125 130 135

caa att cct ctt gaa gaa act gaa ctt cca gga caa gga gaa tca ttt 605

Gln Ile Pro Leu Glu Glu Thr Glu Leu Pro Gly Gln Gly Glu Ser Phe

140 145 150 155

caa ata gaa gat cag ata cct act att cct caa gac aca ctg gga gtt 653

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

160 165 170

gat ttt gat tca gga gcc ctg gag gta tca cct gcc aaa gaa gca att 701

Asp Phe Asp Ser Gly Ala Leu Glu Val Ser Pro Ala Lys Glu Ala Ile

175 180 185

ttt att tta tat tat gtt cga gaa ttt gtt tcg aga ttc cca gac tgt 749

Phe Ile Leu Tyr Tyr Val Arg Glu Phe Val Ser Arg Phe Pro Asp Cys

190 195 200

agg aaa tgt aaa ctt gaa acc cac atc tgc ttg agg taa agtccacggg 798

Arg Lys Cys Lys Leu Glu Thr His Ile Cys Leu Arg

205 210 215

cttcacatcc ttagaaaact tttttagtcc atgcattcac taatgtatgc agacctttaa 858

tagattctag ctttatgtgt tgggggagag atggaggggt gttgacagtc tgtttcactc 918

agcctacata cataataaaa tctaagccct ttaattagag atagcaactt tcccagggtg 978

accgaggaat cagtacgtgt tgaacccctc ttttaagttt ccttttggtt gtgtcacttg 1038

gagattgcct tttctttctt gtaagctcag ctgtgtcctc aaatatattg cattttaccc 1098

agcatttcca ggtgttttca ggggtcaaag tttttaagtt atcttagtct ttgatattgt 1158

gtaagcgaaa ttctccaatc ccatctccta gatgattttt ttttaaatca caaaatgtga 1218

ttatgcttaa cagctgtttc ctgtttctct taaaataaaa tccatttcct atttctctta 1278

aaataaaatc cattattctt aaaacaaaaa aaaaaaaaaa aaaaaaaaaa aaaaa 1333

<210>12

<211>215

<212>PRT

＜213〉people

<400>12

Met Ala Lys Glu Arg Cys Leu Lys Lys Ser Phe Gln Asp Ser Leu Glu

1 5 10 15

Asp Ile Lys Lys Arg Met Lys Glu Lys Arg Asn Lys Asn Leu Ala Glu

20 25 30

Ile Gly Lys Arg Arg Ser Phe Ile Ala Ala Pro Cys Gln Ile Ile Thr

35 40 45

Asn Thr Ser Thr Leu Leu Lys Asn Tyr Gln Asp Asn Asn Lys Met Leu

50 55 60

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

65 70 75 80

Ile Ile Leu Gln Leu Arg Lys Glu Cys Tyr Tyr Leu Thr Cys Gln Leu

85 90 95

Tyr Ala Leu Lys Gly Lys Leu Thr Ser Gln Gln Thr Val Glu Pro Ala

100 105 110

Gln Asn Gln Glu Ile Cys Ser Ser Gly Met Asp Pro Asn Ser Asp Asp

115 120 125

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

130 135 140

Glu Thr Glu Leu Pro Gly Gln Gly Glu Ser Phe Gln Ile Glu Asp Gln

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Glu Thr His Ile Cys Leu Arg

210 215

<210>13

<211>2513

<212>DNA

＜213〉people

<220>

<221>CDS

<222>(94)..(2085)

<400>13

gaaaggaggg caggaagccc acggcccaca ggggtgtagc ccgagaccca cctgcagccc 60

ccagcccttg ccaggaaagc agcagccgca gcc atg gcg ggg atg aag aca gcc 114

Met Ala Gly Met Lys Thr Ala

1 5

tcc ggg gac tac atc gac tcg tca tgg gag ctg cgg gtg ttt gtg gga 162

Ser Gly Asp Tyr Ile Asp Ser Ser Trp Glu Leu Arg Val Phe Val Gly

10 15 20

gag gag gac cca gag gcc gag tcg gtc acc ctg cgg gtc act ggg gag 210

Glu Glu Asp Pro Glu Ala Glu Ser Val Thr Leu Arg Val Thr Gly Glu

25 30 35

tcg cac atc ggc ggg gtg ctc ctg aag att gtg gag cag atc aat cgc 258

Ser His Ile Gly Gly Val Leu Leu Lys Ile Val Glu Gln Ile Asn Arg

40 45 50 55

aag cag gac tgg tca gac cat gct att tgg tgg gaa cag aag agg cag 306

Lys Gln Asp Trp Ser Asp His Ala Ile Trp Trp Glu Gln Lys Arg Gln

60 65 70

tgg ctg ctg cag acc cac tgg aca ctg gac aag tac ggg atc ctg gcc 354

Trp Leu Leu Gln Thr His Trp Thr Leu Asp Lys Tyr Gly Ile Leu Ala

75 80 85

gac gca cgc ctc ttc ttt ggg ccc cag cac cgg ccc gtc atc ctt cgg 402

Asp Ala Arg Leu Phe Phe Gly Pro Gln His Arg Pro Val Ile Leu Arg

90 95 100

ttg ccc aac cgc cgc gca ctg cgc ctc cgt gcc agc ttc tcc cag ccc 450

Leu Pro Asn Arg Arg Ala Leu Arg Leu Arg Ala Ser Phe Ser Gln Pro

105 110 115

ctc ttc cag gct gtg gct gcc atc tgc cgc ctc ctc agc atc cgg cac 498

Leu Phe Gln Ala Val Ala Ala Ile Cys Arg Leu Leu Ser Ile Arg His

120 125 130 135

ccc gag gag ctg tcc ctg ctc cgg gct cct gag aag aag gag aag aag 546

Pro Glu Glu Leu Ser Leu Leu Arg Ala Pro Glu Lys Lys Glu Lys Lys

140 145 150

aag aaa gag aag gag cca gag gaa gag ctc tat gac ttg agc aag gtt 594

Lys Lys Glu Lys Glu Pro Glu Glu Glu Leu Tyr Asp Leu Ser Lys Val

155 160 165

gtc ttg gct ggg ggc gtg gca cct gca ctg ttc cgg ggg atg cca gct 642

Val Leu Ala Gly Gly Val Ala Pro Ala Leu Phe Arg Gly Met Pro Ala

170 175 180

cac ttc tcg gac agc gcc cag act gag gcc tgc tac cac atg ctg agc 690

His Phe Ser Asp Ser Ala Gln Thr Glu Ala Cys Tyr His Met Leu Ser

185 190 195

cgg ccc cag ccg cca ccc gac ccc ctc ctg ctc cag cgt ctg cca cgg 738

Arg Pro Gln Pro Pro Pro Asp Pro Leu Leu Leu Gln Arg Leu Pro Arg

200 205 210 215

ccc agc tcc ctg tca gac aag acc cag ctc cac agc agg tgg ctg gac 786

Pro Ser Ser Leu Ser Asp Lys Thr Gln Leu His Ser Arg Trp Leu Asp

220 225 230

tcg tcg cgg tgt ctc atg cag cag ggc atc aag gct ggg gac gca ctc 834

Ser Ser Arg Cys Leu Met Gln Gln Gly Ile Lys Ala Gly Asp Ala Leu

235 240 245

tgg ctg cgc ttc aag tac tac agc ttc ttc gat ttg gat ccc aag aca 882

Trp Leu Arg Phe Lys Tyr Tyr Ser Phe Phe Asp Leu Asp Pro Lys Thr

250 255 260

gac ccc gtg cgg ctg aca cag ctg tat gag cag gcc cgg tgg gac ctg 930

Asp Pro Val Arg Leu Thr Gln Leu Tyr Glu Gln Ala Arg Trp Asp Leu

265 270 275

ctg ctg gag gag att gac tgc acc gag gag gag atg atg gtg ttt gcc 978

Leu Leu Glu Glu Ile Asp Cys Thr Glu Glu Glu Met Met Val Phe Ala

280 285 290 295

gcc ctg cag tac cac atc aac aag ctg tcc cag agc ggg gag gtg ggg 1026

Ala Leu Gln Tyr His Ile Asn Lys Leu Ser Gln Ser Gly Glu Val Gly

300 305 310

gag ccg gct ggc aca gac cca ggg ctg gac gac ctg gat gtg gcc ctg 1074

Glu Pro Ala Gly Thr Asp Pro Gly Leu Asp Asp Leu Asp Val Ala Leu

315 320 325

agc aac ctg gag gtg aag ctg gag ggg tcg gcg ccc aca gat gtg ctg 1122

Ser Asn Leu Glu Val Lys Leu Glu Gly Ser Ala Pro Thr Asp Val Leu

330 335 340

gac agc ctc acc acc atc cca gag ctc aag gac cat ctc cga atc ttt 1170

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

345 350 355

cgg ccc cgg aag ctg acc ctg aag ggc tac cgc caa cac tgg gtg gtg 1218

Arg Pro Arg Lys Leu Thr Leu Lys Gly Tyr Arg Gln His Trp Val Val

360 365 370 375

ttc aag gag acc aca ctg tcc tac tac aag agc cag gac gag gcc cct 1266

Phe Lys Glu Thr Thr Leu Ser Tyr Tyr Lys Ser Gln Asp Glu Ala Pro

380 385 390

ggg gac ccc att cag cag ctc aac ctc aag ggc tgt gag gtg gtt ccc 1314

Gly Asp Pro Ile Gln Gln Leu Asn Leu Lys Gly Cys Glu Val Val Pro

395 400 405

gat gtt aac gtc tcc ggc cag aag ttc tgc att aaa ctc cta gtg ccc 1362

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

410 415 420

tcc cct gag ggc atg agt gag atc tac ctg cgg tgc cag gat gag cag 1410

Ser Pro Glu Gly Met Ser Glu Ile Tyr Leu Arg Cys Gln Asp Glu Gln

425 430 435

cag tat gcc cgc tgg atg gct ggc tgc cgc ctg gcc tcc aaa ggc cgc 1458

Gln Tyr Ala Arg Trp Met Ala Gly Cys Arg Leu Ala Ser Lys Gly Arg

440 445 450 455

acc atg gcc gac agc agc tac acc agc gag gtg cag gcc atc ctg gcc 1506

Thr Met Ala Asp Ser Ser Tyr Thr Ser Glu Val Gln Ala Ile Leu Ala

460 465 470

ttc ctc agc ctg cag cgc acg ggc agt ggg ggc ccg ggc aac cac ccc 1554

Phe Leu Ser Leu Gln Arg Thr Gly Ser Gly Gly Pro Gly Asn His Pro

475 480 485

cac ggc cct gat gcc tct gcc gag ggc ctc aac ccc tac ggc ctc gtt 1602

His Gly Pro Asp Ala Ser Ala Glu Gly Leu Asn Pro Tyr Gly Leu Val

490 495 500

gcc ccc cgt ttc cag cga aag ttc aag gcc aag cag ctc acc cca cgg 1650

Ala Pro Arg Phe Gln Arg Lys Phe Lys Ala Lys Gln Leu Thr Pro Arg

505 510 515

atc ctg gaa gcc cac cag aat gtg gcc cag ttg tcg ctg gca gag gcc 1698

Ile Leu Glu Ala His Gln Asn Val Ala Gln Leu Ser Leu Ala Glu Ala

520 525 530 535

cag ctg cgc ttc atc cag gcc tgg cag tcc ctg ccc gac ttc ggc atc 1746

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

540 545 550

tcc tat gtc atg gtc agg ttc aag ggc agc agg aaa gac gag atc ctg 1794

Ser Tyr Val Met Val Arg Phe Lys Gly Ser Arg Lys Asp Glu Ile Leu

555 560 565

ggc atc gcc aac aac cga ctg atc cgc atc gac ttg gcc gtg ggc gac 1842

Gly Ile Ala Asn Asn Arg Leu Ile Arg Ile Asp Leu Ala Val Gly Asp

570 575 580

gtg gtc aag acc tgg cgt ttc agc aac atg cgc cag tgg aat gtc aac 1890

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

585 590 595

tgg gac atc cgg cag gtg gcc atc gag ttt gat gaa cac atc aat gtg 1938

Trp Asp Ile Arg Gln Val Ala Ile Glu Phe Asp Glu His Ile Asn Val

600 605 610 615

gcc ttc agc tgc gtg tct gcc agc tgc cga att gta cac gag tat atc 1986

Ala Phe Ser Cys Val Ser Ala Ser Cys Arg Ile Val His Glu Tyr Ile

620 625 630

ggg ggc tac att ttc ctg tcg acg cgg gag cgg gcc cgt ggg gag gag 2034

Gly Gly Tyr Ile Phe Leu Ser Thr Arg Glu Arg Ala Arg Gly Glu Glu

635 640 645

ctg gat gaa gac ctc ttc ctg cag ctc acc ggg ggc cat gag gcc ttc 2082

Leu Asp Glu Asp Leu Phe Leu Gln Leu Thr Gly Gly His Glu Ala Phe

650 655 660

tga gggctgtctg attgcccctg ccctgctcac cacctgtcac agccactccc 2135

aagcccacac ccacaggggc tcactgcccc acacccgctc caggcaggca cccagctggg 2195

catttcacct gctgtcactg actttgtgca ggccaaggac ctggcagggc cagacgctgt 2255

accatcaccc aggccaggga tgggggtggg ggtccctgag ctcatgtggt gccccctttc 2315

cttgtctgag tggctgaggc tgatacccct gacctatctg cagtccccca gcacacaagg 2375

aagaccagat gtagctacag gatgatgaaa catggtttca aacgagttct ttcttgttac 2435

tttttaaaat ttctttttta taaattaata ttttattgtt ggaaaaaaaa aaaaaaaaaa 2495

aaaaaaaaaa aaaaaaac 2513

<210>14

<211>663

<212>PRT

＜213〉people

<400>14

Met Ala Gly Met Lys Thr Ala Ser Gly Asp Tyr Ile Asp Ser Ser Trp

1 5 10 15

Glu Leu Arg Val Phe Val Gly Glu Glu Asp Pro Glu Ala Glu Ser Val

20 25 30

Thr Leu Arg Val Thr Gly Glu Ser His Ile Gly Gly Val Leu Leu Lys

35 40 45

Ile Val Glu Gln Ile Asn Arg Lys Gln Asp Trp Ser Asp His Ala Ile

50 55 60

Trp Trp Glu Gln Lys Arg Gln Trp Leu Leu Gln Thr His Trp Thr Leu

65 70 75 80

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

85 90 95

His Arg Pro Val Ile Leu Arg Leu Pro Asn Arg Arg Ala Leu Arg Leu

100 105 110

Arg Ala Ser Phe Ser Gln Pro Leu Phe Gln Ala Val Ala Ala Ile Cys

115 120 125

Arg Leu Leu Ser Ile Arg His Pro Glu Glu Leu Ser Leu Leu Arg Ala

130 135 140

Pro Glu Lys Lys Glu Lys Lys Lys Lys Glu Lys Glu Pro Glu Glu Glu

145 150 155 160

Leu Tyr Asp Leu Ser Lys Val Val Leu Ala Gly Gly Val Ala Pro Ala

165 170 175

Leu Phe Arg Gly Met Pro Ala His Phe Ser Asp Ser Ala Gln Thr Glu

180 185 190

Ala Cys Tyr His Met Leu Ser Arg Pro Gln Pro Pro Pro Asp Pro Leu

195 200 205

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

210 215 220

Leu His Ser Arg Trp Leu Asp Ser Ser Arg Cys Leu Met Gln Gln Gly

225 230 235 240

Ile Lys Ala Gly Asp Ala Leu Trp Leu Arg Phe Lys Tyr Tyr Ser Phe

245 250 255

Phe Asp Leu Asp Pro Lys Thr Asp Pro Val Arg Leu Thr Gln Leu Tyr

260 265 270

Glu Gln Ala Arg Trp Asp Leu Leu Leu Glu Glu Ile Asp Cys Thr Glu

275 280 285

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

290 295 300

Ser Gln Ser Gly Glu Val Gly Glu Pro Ala Gly Thr Asp Pro Gly Leu

305 310 315 320

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

325 330 335

Ser Ala Pro Thr Asp Val Leu Asp Ser Leu Thr Thr Ile Pro Glu Leu

340 345 350

Lys Asp His Leu Arg Ile Phe Arg Pro Arg Lys Leu Thr Leu Lys Gly

355 360 365

Tyr Arg Gln His Trp Val Val Phe Lys Glu Thr Thr Leu Ser Tyr Tyr

370 375 380

Lys Ser Gln Asp Glu Ala Pro Gly Asp Pro Ile Gln Gln Leu Asn Leu

385 390 395 400

Lys Gly Cys Glu Val Val Pro Asp Val Asn Val Ser Gly Gln Lys Phe

405 410 415

Cys Ile Lys Leu Leu Val Pro Ser Pro Glu Gly Met Ser Glu Ile Tyr

420 425 430

Leu Arg Cys Gln Asp Glu Gln Gln Tyr Ala Arg Trp Met Ala Gly Cys

435 440 445

Arg Leu Ala Ser Lys Gly Arg Thr Met Ala Asp Ser Ser Tyr Thr Ser

450 455 460

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

465 470 475 480

Gly Gly Pro Gly Asn His Pro His Gly Pro Asp Ala Ser Ala Glu Gly

485 490 495

Leu Asn Pro Tyr Gly Leu Val Ala Pro Arg Phe Gln Arg Lys Phe Lys

500 505 510

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

515 520 525

Gln Leu Ser Leu Ala Glu Ala Gln Leu Arg Phe Ile Gln Ala Trp Gln

530 535 540

Ser Leu Pro Asp Phe Gly Ile Ser Tyr Val Met Val Arg Phe Lys Gly

545 550 555 560

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

565 570 575

Ile Asp Leu Ala Val Gly Asp Val Val Lys Thr Trp Arg Phe Ser Asn

580 585 590

Met Arg Gln Trp Asn Val Asn Trp Asp Ile Arg Gln Val Ala Ile Glu

595 600 605

Phe Asp Glu His Ile Asn Val Ala Phe Ser Cys Val Ser Ala Ser Cys

610 615 620

Arg lle Val His Glu Tyr Ile Gly Gly Tyr Ile Phe Leu Ser Thr Arg

625 630 635 640

Glu Arg Ala Arg Gly Glu Glu Leu Asp Glu Asp Leu Phe Leu Gln Leu

645 650 655

Thr Gly Gly His Glu Ala Phe

660

Claims (16)

1. isolating polynucleotide is characterized in that, described polynucleotide contain the nucleotide sequence that coding has the polypeptide that promotes the cell proliferation function, and this nucleotide sequence is selected from:

(a) polynucleotide of polypeptide that contain the aminoacid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14 with coding have the polynucleotide of at least 70% similarity;

(b) coding contains the polynucleotide of polypeptide that aminoacid sequence with SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14 has the aminoacid sequence of at least 70% similarity;

(c) with (a) or polynucleotide complementary polynucleotide (b).

2. polynucleotide as claimed in claim 1, it is characterized in that the polypeptide of described polynucleotide encoding has the aminoacid sequence of the group of being selected from down: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:12, SEQ ID NO:14.

3. polynucleotide as claimed in claim 1 is characterized in that, the sequence of described polynucleotide is shown at least 85% similarity with the nucleotides sequence that is selected from down group:

(a) coding region sequence or the full length sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ IDNO:11, SEQ ID NO:13;

(b) at least one sequence of the sequence of in genetic code degeneracy scope, mentioning in corresponding to (a);

(c) with (a) or at least one sequence of the sequence complementary sequence hybridization of mentioning (b).

4. polynucleotide as claimed in claim 3, it is characterized in that the sequence of described polynucleotide is selected from coding region sequence or the full length sequence of SEQ ID NO:1, SEQ IDNO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13.

5. the polypeptide of the described polynucleotide encoding of claim 1, it is characterized in that described polypeptide comprises the polypeptide that is selected from down the aminoacid sequence in the group: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQID NO:12, SEQ ID NO:14; Or the polypeptide that has at least 90% similarity with above arbitrary aminoacid sequence; Or its conservative property variation polypeptide or its active fragments or its reactive derivative.

6. the described polypeptide of claim 5, it is characterized in that described polypeptide has the aminoacid sequence of the group of being selected from down: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14.

7. a carrier is characterized in that, described carrier contains the described polynucleotide of claim 1.

8. a genetically engineered host cell is characterized in that, described host cell is selected from:

(a) host cell that transforms or transduce with the described carrier of claim 7;

(b) host cell that transforms or transduce with the described polynucleotide of claim 1.

9. an antibody is characterized in that, described antibody be can with the described polypeptid specificity bonded of claim 5 antibody.

10. a nucleic acid fragment is characterized in that, described nucleic acid fragment contains 8-100 successive Nucleotide in the described arbitrary polynucleotide of claim 1.

11. the application of the described polynucleotide of claim 1 in promoting cell proliferation is characterized in that described polynucleotide heterogenous expression in host cell can promote cell proliferation.

12. the described application of claim 11 is characterized in that described host cell is selected from the described host cell of claim 8.

13. described polynucleotide of claim 1 or the described polypeptide of claim 5 prevent and/or treat purposes in the medicine with the body cell multiplication diseases associated in preparation.

14. purposes according to claim 13, wherein said disease are selected from autoimmune disease, tumour or disease and post-traumatic injury repairing.

15. contain the pharmaceutical composition of described polypeptide of claim 5 and pharmaceutically acceptable carrier.

16. the external detection method of autoimmune disease or tumour is characterized in that, utilizes described antibody of claim 9 or claim 10 described nucleic acid fragments to detect the existence or the level of the polypeptide in host's sample.

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