CN1952132A - Polynucleotide having influence on ELK1 activity 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 human protein with the function of influencing the activity of ELK1. The invention also discloses the application of the influence of such novel polynucleotide exogenous expressing in host cells on ELK1 activity. The invention also discloses the use of the said polypeptide, antibodies in the drug preparation for the prevention and treatment of nervous system diseases and cancer.

Description

Influence the active polynucleotide of ELK1 and coded polypeptide and purposes

Technical field

The invention discloses the new coding of a class and have proteic polynucleotide of the people who influences the active function of ELK1 and encoded polypeptides thereof, the antibody of polypeptide.The present invention also disclose the new polynucleotide of this class in host cell heterogenous expression to the application of the active influence of ELK1.The invention also discloses described polypeptide, the purposes of antibody on the medicine of preparation prevention and treatment nervous system disorders and tumour.

Background technology

ELK1 (member of ETS oncogene family), the assignment of genes gene mapping is in Xp11.2, and the paper that Rao in 1989 etc. deliver is pointed out, two new ETS oncogene have been found, ELK1 by name and ELK2, at testis and two organs of lung, ELK and similar sequences thereof all show functional transcription.The protein product of this genes encoding is ELK1, is a member of ETS oncogene family (transcription factor), belongs to TCF (the ternary complex factor) subfamily.The protein product of TCF subfamily genes encoding forms one three mixture and plays a role by in conjunction with the serum response element (SRE) and the serum response factor (SRF) that are positioned at the upstream promoter district of c-fos proto-oncogene.The albumen of this genes encoding is a molecule of appraising and deciding of ras-raf-MAPK signal transduction cascade.

The ETS DNA-binding domain is positioned at the N end, and The B-box region is the protein-protein interaction position that Elk1 is incorporated into SRF, has promoted the formation of three mixtures.The transcriptional activation domain (TAD) comprises a plurality of serine/threonine residues, is the mapk kinase binding site of phosphorylation; The D-domain and the FxF motif are the docking sites of mapk kinase; ETS DNA-binding domain and the R-motif have so that activity of gene expression.Above structural domain is guarded in vertebrates, and it has important function the conservative explanation in the evolution.

The biological function of Elk1 is very complicated, can be divided into the function of two classes: Elk1 and the function that the TCF mixture forms substantially.Studies show that: the Elk1 gene may play a role in neural system.At first, Elk1 expresses in the multiple neurocyte of the brain of rat, and is that phosphorylation takes place in glutamate receptor activation; In addition, the Elk1 that N end disappearance in the neurocyte atomization, occurred.But, the effect of Elk1 in clone is the adjusting realization of early protein such as c-fos that opposes after activating by Elk1.

After the Elk1 activation, serum response element (SRE) and serum response factor (SRF) by in conjunction with the upstream promoter district that is positioned at upright early protein gene form one three mixture, have mediated the rapid reaction of cell to stimulating.What discovery had regulating effect the earliest is the c-fos proto-oncogene, at egr-1 and jun-B etc. effect was arranged also afterwards, but now and do not know whole albumen of its regulation and control, also do not know whether to exist the mode of action of the non-dependence of SRF, and whether they can be in the different sites performance functions of promoter region.In fact, Elk1 can form the promotor coupled complex with the form and the Pax-5 of the non-dependence of The B-box region, and that play a role this moment is The ETS DNA-binding domain and Pax-5.

Classical theory is thought: the main effect of Elk1 is to form Elk-1-SRF-SRE three mixtures, and this mixture is the decision cell proliferation or the molecular switch of apoptosis; Elk1 is a molecule of appraising and deciding of ras-raf-MAPK signal transduction cascade.Therefore, though we can't determine the stagnation point of its adjusting, we can determine that it is in cell proliferation and regulation of apoptosis, neural system, tumour

Have effect:

1.Elk1 and tumour

Elk1 to the possible action mechanisms of tumour may for: Elk1 is a molecule of appraising and deciding of ras-raf-MAPK signal transduction cascade, after it is activated phosphorylation by the MAPK path, form Elk-1-SRF-SRE three mixtures, increased the expression of upright early genes such as c-fos proto-oncogene, the molecular switch that has determined cell proliferation/apoptosis is to suppressing apoptosis and promoting the propagation aspect to transform, the malignant proliferation that causes cell, and and then cause the generation of tumour.Experimental evidence comprises: in Helicobacter inductive stomach small intestine sample worsens and villin causes Elk1 and the activation height correlation of SRF; In prostate cancer cell, the activation of Elk1 has caused the significance propagation of cell etc.

2.Elk1 relation with nerve growth, Regular Insulin

Mechanism about this respect also is not very clear, but more and more evidences shows that there is contact in they: as there are some researches show that in EGF inductive pheochromocytoma PC12 and insulinoma INS-1 propagation, Elk1 plays the vital role that regulatory gene is transcribed; Elk1 expresses in the multiple neurocyte of the brain of rat, and is that phosphorylation takes place in glutamate receptor activation; In addition, the Elk1 that N end disappearance in the neurocyte atomization, occurred.It is on the knees of the gods whether but this regulating and controlling effect needs to form Elk-1-SRF-SRE three mixtures.

3. the effect of bringing into play as Elk-1-SRF-SRE three mixtures

This mixture is in the muscle hyperplasia and reinvent, play a role in the aging.See patent for details and " influence the active polynucleotide of SRE and coded polypeptide and purposes " (Gao Xia etc., 200510102772.7).

Since the generation of a lot of diseases is relevant unusually with Elk1 and regulation and control thereof with development, it is logical so some disease being carried out therapeutic intervention.Relevant experimentation on animals also shows, to the sudden change of the sealing of Elk1 or reactive site in the change of aspects such as tumour, nervous system abnormality, but up to the present, do not see the report that relevant this gene or its expressing protein are used for the treatment of as yet, or the example that regulation and control intervention of this path is used for the treatment of.Therefore, Elk1 signal transduction and Regulation Mechanism research thereof are for a lot of 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 ELK1IF1 of the people who influences the ELK1 active function, 2,3,4,5,6,7,8.

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 new polynucleotide of this class heterogenous expression in host cell influences the active application of ELK1.

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 influence the active function of ELK1, and this nucleotide sequence is selected from: the polynucleotide that at least 70% similarity (a) is arranged with the polynucleotide of the polypeptide of the aminoacid sequence contain SEQ ID NO:2, SEQ ID NO:4, SEQID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16 of encoding; (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, SEQ ID NO:16 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, SEQ ID NO:16.

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, SEQ ID NO:15; (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, SEQ ID NO:15.

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, SEQ ID NO:16; 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, SEQ ID NO:16.

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 heterogenous expression in host cell to influence the active application of ELK1.Preferably, this application comprises that the polynucleotide external source expression inhibiting ELK1 activatory in host cell with the coding region that is selected among SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, the SEQ ID NO:9 or full length sequence is used and polynucleotide external source expressing promoting in host cell with the coding region that is selected among SEQ ID NO:11, SEQ ID NO:13, the SEQ ID NO:15 or full length sequence is advanced the ELK1 activatory and used.

In a sixth aspect of the present invention, provide above-mentioned polynucleotide and polypeptide to prevent and/or treat purposes in the medicine of neural disease or tumour in preparation.

Aspect the present invention ground the 7th, a kind of pharmaceutical composition is provided, it contains polypeptide and the pharmaceutically acceptable carrier that influences the ELK1 active 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 nervous system disorders 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 of cell death inducing 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 the ELK1IF1 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 ELK1IF1 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 SEQID NO:1 has difference.Have the polypeptide that influences the ELK1 active 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 influences the ELK1 active function.Term " fragment ", " derivative " and " analogue " be meant keep basically with of the present invention natural have influence identical biological function of the people of ELK1 active function protein polypeptide or active polypeptide.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 the protein polypeptide that having of reorganization expressed or produced to polynucleotide sequence of the present invention influences the ELK1 active function (Science, 1984 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, coding has the polynucleotide sequence that influences the people of ELK1 active function protein polypeptide and 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, et al.MolecularCloning, a Laboratory Manual, Cold Spring Harbor Laboratory.New York, 1989) such as extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant DNA technologies of body.

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 influences the ELK1 active 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; 293T, 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 influence the ELK1 active 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 nervous system disorders and tumour etc.; Also can be used for screening and promote or antagonism has proteic antibody, polypeptide or other part that influence the active function of ELK1, for example, screen the antibody that can be used for promoting or suppressing 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 the proteic polynucleotide of the people who influences the ELK1 active function and also can be used for multiple therapeutic purpose.Can be used on to treat in the gene therapy technology and influence the people of the active function of ELK1 protein abnormal expression or the active disease that causes unusually owing to having.The gene therapy vector (as virus vector) of reorganization can be designed to express people's albumen that having of variation influences the active function of ELK1, suppresses the endogenic proteic activity of the people who influences the ELK1 active function that has.The having of reorganization influences the people of ELK1 active function protein gene and 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 with cell death inducing function in the living specimen.Can also be used for clinical diagnosis, treatment, therapeutic evaluation of the disease relevant etc. clinically with people's albumen with cell death inducing 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 to influence the relevant disease of the people of ELK1 active function albumen with having.The antibody that gives suitable dosage can stimulate or block and has proteic generation of the people who influences the ELK1 active function or activity.

The invention still further relates to quantitatively and detection and localization has the diagnostic testing process of the protein level that influences the ELK1 active 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 influences the ELK1 active function, can have the importance of albumen in various diseases that influences the ELK1 active function with laying down a definition and be used to diagnose to have the disease that the albumen that influences the ELK1 active function works.

Have the proteic polynucleotide that influence the ELK1 active function and can be used for having the diagnosis and the treatment of the protein related diseases that influences the ELK1 active function.Aspect diagnosis, have the proteic polynucleotide that influence the ELK1 active function can be used for detecting have influence the ELK1 active function proteic expression whether, or under morbid state, have the abnormal exprssion that influences the ELK1 active function.As have the proteic dna sequence dna that influences the ELK1 active function and can be used for that the hybridization of biopsy specimen is had the proteic abnormal expression that influences the ELK1 active 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.The special primer of the albumen of the influential ELK1 active function of apparatus carries out RNA-polymerase chain reaction (RT-PCR) amplification in vitro and also can detect and have the proteic transcription product that influences the ELK1 active function.

The sudden change that detection has a protein gene that influences the ELK1 active function also can be used for diagnosing and has the relevant disease of albumen that influences the ELK1 active function.Mutant form with the protein gene that influences the ELK1 active function comprises that to influence point mutation that the proteic dna sequence dna of ELK1 active function compares, transposition, disappearance, reorganization and other any unusual etc. with having of normal wild type.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 ELK1IF1,2,3,4,5,6,7,8 healthy tissues that all are used in experiment, fetal tissue, the tumor tissues expression is arranged all, illustrate that it is the important ELK1 factor of influence 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 two luciferase reporter gene methods to detect the influence of ELK1IF1,2,3,4,5,6,7,8 gene pairs ELK1 then.What this method adopted is signal transduction pathway cis report system in the body, wherein used ELK1-luc reporter plasmid is loaded with firefly luciferase gene, this expression of gene is regulated and control by the synthetic promotor, wherein comprise basic promoter element (TATA box), and the binding site of transcription factor ELK1, the structure iron of ELK1-luc reporter plasmid is as shown in Figure 2.When intracellular ELK1 is activated by certain signal transduction pathway, ELK1 just can be incorporated into the enhancer element of reporter plasmid ELK1-luc, thereby start transcribing of reporter gene firefly luciferase gene, further translate into luciferase in the born of the same parents, cause luciferase quantity increase in the cell, increased activity; On the contrary, when intracellular ELK1 activation is suppressed, the expression amount of luciferase and active just low.So,, can reflect just whether the interior ELK1 of goal gene pair cell of different stimulated thing and cotransfection has the activation of promotion or suppress the activatory function by detecting the activity of luciferase.PRL-SV40 reporter plasmid carrier as shown in Figure 2, contain the jellyfish luciferases gene, this expression of gene is regulated and control by simian virus 40 (SV40) enhancers/promoters, can produce the expression of the jellyfish luciferases of stronger basic horizontal behind the transfection mammalian cell, and the adjusting whether activity of expressing is not activated by ELK1 is so be used as the internal reference reporter gene in experiment.Experiment shows that polypeptide of the present invention has remarkable, the stable active effect of ELK1 that influences.

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, human new functional gene ELK1IF1,2,3,4,5,6,7,8 cDNA sequence and coded polypeptide thereof are provided;

3, find that first human new functional gene ELK1IF1,2,3,4,5,6,7,8 has the active effect of the ELK1 of influence, and efficient, stable;

4, ELK1IF1,2,3,4,5,6,7,8 expresses at the most normal cells of body, illustrates that it is self important ELK1 regulatory molecule.

5, based on 4 above-mentioned advantages, the present invention be further research ELK1IF1,2,3,4,5,6,7,8 and ELK1 between regulation relationship, and the novel drugs of exploitation treatment and propagation and muscle differentiation phase related disorders or tumour, 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-ELK1IFx one of (x be selected from 1,2,3,4,5,6,7,8).

The structure iron (2A) of Fig. 2, pELK1-luc luciferase gene reporter plasmid and pRL-TK reporter plasmid structure iron (2B).

Fig. 3, ELK1IF1,2,3,4,5 heterogenous expressions suppress the activation of PMA to ELK1

Fig. 4, ELK1IF6,7,8 heterogenous expressions promote the ELK1 activation

Embodiment

Below in conjunction with specific embodiments and the drawings, further set forth the present invention.These embodiment and accompanying drawing 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 by the BLASTn method and proofread and correct, the sequence that finally obtains is set at down the group sequence: SEQID 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, SEQ ID NO:15.According to this type of sequences Design gene ELK1IF1,2,3,4,5,6,7,8 special primer:

The gene title	Upstream primer (5 '-3 ')	Downstream primer (5 '-3 ')
			ELK1IF1 ELK1IF2 ELK1IF3 ELK1IF4 ELK1IF5 ELK1IF6 ELK1IF7 ELK1IF8	ccatgctgcatccagagacctc tcttcgtgcttcagatttgtttacc gcagagggcagtagagatggc ccttacactttgcttcaggctcc cgtagttgaaatgaggaagaatgagag tggaatggaagagccaacagc gcaagatggtgtcctggatgatc tagtggcctatgtcccttgctc	tcaggaccctgggcccc gctccatttgctattcaattcgtc aatgagatggctgagcagatgtg tctgttatgttctaaagagtttgtgtttcc ctggacagatgaataagtgaaaggc gagtaagttgttgggagtttagaccg gaccctagctgtccacgtctgag ttcttcccaagtttctcaatgtctg

(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, all there are gene ELK1IF1,2,3,4,5,6,7,8 cDNA in the cell of these tissues, illustrate that ELK1IF1,2,3,4,5,6,7,8 has produced gene ELK1IF1,2,3,4,5,6,7,8 transcription product 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, has selected and has inserted segmental positive colony, select correct forward by order-checking (ABI PRISM 3700 DNA analysis instrument) and insert clone, called after pcDT-ELK1IF1,2,3,4,5,6,7,8 separately.

Collect nutrient solution simultaneously, analyze protein precipitation, obtain ELK1IF1,2,3,4,5,6,7,8 polypeptide with SDS-PAGE.

ELK1IF1,2,3,4,5,6,7,8 analysis of protein results show: ELK1IF1,2,3,4,5,6,7, following group of sequence of 8 protein sequences: 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, SEQ ID NO:14, the SEQ ID NO:16.

Embodiment 3, two luciferase reporter gene method are measured goal gene the PMA+ ionomycin are induced ELK1 activatory restraining effect.

With goal gene and reporter gene pELK1-luc, pRL-TK cotransfection human embryo kidney 293T cell is measured the activity of ELK1 by detecting two kinds of uciferase activities respectively.The method of cotransfection is the non-fat infection protocol of positively charged ion, adopts prestige lattice Lars biotechnologys (Beijing) company limited, is undertaken by product description is described.

The transfection operation steps is as follows:

(1) cell cultures: with 293T cell (ATCC Number:CRL-11268) (2.0 * 104) 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) on, inoculum density 1.2 ten thousand/hole, culture volume 100ul/ hole, at 5%CO2, cultivated 18-24 hour in 37 ℃ the incubator.

(2) preparation transfection composite:

Get 2.5 μ l physiological saline dilution 40ng reporter gene pELK1-luc, 4ng pRL-TK and 50ng purpose plasmid slowly mix; With the vigofect (being generally 0.04 μ l/ hole) of 2.5 μ l physiological saline dilution appropriate amount, slowly mix equally, at room temperature placed 5 minutes, slowly mix with the DNA of dilution, room temperature was placed 15 minutes, to form transfection composite.

(3) transfection: transfection composite is slowly splashed into Tissue Culture Plate (5 μ l/ hole), slightly shake up.5%CO ₂, cultivated 18-24 hour in 37 ℃ the incubator.

With PMA (TETRADECONIC ACID Buddhist ripple ester, Calbiochem, 524400, final concentration 50ng/ml)+ionomycin (Calbiochem, 407950, final concentration 1 μ M) irritation cell, 37 ℃ of incubators are cultivated after transfection 18-24 hour.Discard nutrient solution after 6-8 hour, (Promega E1960), puts into-80 ℃ of refrigerators to add Passive Lysis Buffer.During detection, take out cell plate, thaw naturally under the room temperature, make the complete cracking of cell, 10 μ l cell pyrolysis liquids move to the white fluorescent plate, add the two reporting systems of Dual-luciferase and detect substrate, (Genios Pro Tecan) detects uciferase activity with the microwell plate microplate reader.

Intracellular uciferase activity ratio (Photinus pyralis LUC fluorescence intensity/jellyfish luciferases fluorescence intensity is represented) is 1 when setting transfection pcDB empty carrier, the stimulation of PMA+ ionomycin, intracellular uciferase activity ratio is represented with relative value as standard under other conditions.The result is with reference to Fig. 3, use plasmid to be checked and pELK1-luc cotransfection 293T cell respectively, the activity of luciferase is reduced significantly in the 293T cell that stimulates with the PMA+ ionomycin, shows that the expression product of ELK1IF1,2,3,4,5 in the 293T cell induce the ELK1 activation that strong restraining effect is arranged to the PMA+ ionomycin.

Can determine that according to above experimental result when the rise of ELK1 expression level caused increase corresponding with the transcriptional expression of some disease related gene, ELK1IF1,2,3,4,5 pairs of ELK1 activatory restraining effect can stop the generation and the development of this disease.Therefore, the present invention can be used to prepare prevention and treatment because ELK1 crosses the nervous system disorders that expression causes or the medicine of tumour.

Embodiment 4, two luciferase reporter gene method are measured goal gene to ELK1 activatory promoter action.

Except that stimulating without stimulator, all the other experimentations and step are with embodiment 3.

The result is with reference to Fig. 4, use plasmid to be checked and pELK1-luc cotransfection 293T cell respectively, uciferase activity ratio is standard 100% during with transfection empty carrier pcDB, plasmid to be checked can obviously promote the activation of ELK1, the pELK1-luc uciferase activity obviously raises, and shows that activation has strong promoter action to the expression product of ELK1IF6,7,8 in the 293T cell to ELK1.

Can determine that according to above experimental result when the downward modulation of ELK1 expression level caused minimizing corresponding with the transcriptional expression of some disease related gene, ELK1IF6,7,8 pairs of ELK1 activatory promoter actions can stop the generation and the development of this disease.Therefore, the present invention can be used to prepare prevention and treat because ELK1 expresses the medicine of less nervous system disorders that causes or tumour.

Embodiment 5, Antibody Preparation

Antigen is selected ELK1IF1,2,3,4,5,6,7,8 albumen total lengths or the partial peptide section of prokaryotic cell prokaryocyte or eukaryotic cell expression for use, also can synthesize polypeptide as antigen.

Polyclonal Antibody Preparation: immune animal is selected bull new zealand rabbit or BALb/c mouse for use, after initial immunity uses 200ug (new zealand rabbit) or 20ug (BALb/c mouse) antigen and equal-volume Freund's complete adjuvant (FCA) fully emulsified, the subcutaneous multi-point injection in the back.Behind the initial immunity 21,42,63 days, with Freund's incomplete adjuvant (FIA) emulsive antigen protein fully, each booster immunization 1 time, consumption was the same.Each immunity back 7 ~ 10 days, the ELISA method detects serum titer, reaches 1 * 10 ^-4The time, bloodletting separation of serum .Western blot identifies antibodies specific.

Monoclonal Antibody: immune BALb/c mouse is the same, gets spleen and makes the B cell suspension, with the myeloma cell SP2/0 fusion of logarithmic phase, by HAT (H: xanthoglobulin; A: aminopterin-induced syndrome; T: thymidine) selectivity is cultivated, and obtains hybrid cell line, detects antibody titer by the ELISA method again, filters out specific hybridoma cell line, and obtains monoclonal antibody.

Sequence table

＜110〉Sinogenomax Co., Ltd.

＜120〉influence the active polynucleotide of ELK1 and coded polypeptide and purposes

<130>

<160>16

<170>PatentIn version 3.3

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<211>1989

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＜213〉people

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<222>(258)..(1598)

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ggtccggatt ctccgatgcg aggtcctcct cccagggccc ggggctgggg agcggaagcc 60

atcagtgggc tcggaggaca ctgtcccagc caggacacgg ccatcggtca ctaatctgca 120

gcaccggaat gtccccaggc caatcagcgg gcccagcctg actcctggag attgtgaata 180

gctccatcca gcctgagaaa caagccgggt ggctgagcca ggctgtgcac ggagcgcctg 240

acgggcccaa cagaccc atg ctg cat cca gag acc tcc cct ggc cgg ggg 290

Met Leu His Pro Glu Thr Ser Pro Gly Arg Gly

1 5 10

cat ctc ctg gct gtg ctc ctg gcc ctc ctt ggc acc gcc tgg gca gag 338

His Leu Leu Ala Val Leu Leu Ala Leu Leu Gly Thr Ala Trp Ala Glu

15 20 25

gtg tgg cca ccc cag ctg cag gag cag gct ccg atg gcc gga gcc ctg 386

Val Trp Pro Pro Gln Leu Gln Glu Gln Ala Pro Met Ala Gly Ala Leu

30 35 40

aac agg aag gag agt ttc ttg ctc ctc tcc ctg cac aac cgc ctg cgc 434

Asn Arg Lys Glu Ser Phe Leu Leu Leu Ser Leu His Asn Arg Leu Arg

45 50 55

agc tgg gtc cag ccc cct gcg gct gac atg cgg agg ctg gac tgg agt 482

Ser Trp Val Gln Pro Pro Ala Ala Asp Met Arg Arg Leu Asp Trp Ser

60 65 70 75

gac agc ctg gcc cag ctg gct caa gcc agg gca gcc ctc tgt gga acc 530

Asp Ser Leu Ala Gln Leu Ala Gln Ala Arg Ala Ala Leu Cys Gly Thr

80 85 90

cca acc ccg agc ctg gcg tcc ggc ctg tgg cgc acc ctg caa gtg ggc 578

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

95 100 105

tgg aac atg cag ctg cta ccc gcg ggc ttg gcg tcc ttt gtc gaa gtg 626

Trp Asn Met Gln Leu Leu Pro Ala Gly Leu Ala Ser Phe Val Glu Val

110 115 120

gtc agc cta tgg ttt gca gag ggg cag cgg tac agc cac gcg gca gga 674

Val Ser Leu Trp Phe Ala Glu Gly Gln Arg Tyr Ser His Ala Ala Gly

125 130 135

gag tgt gct cgc aac gcc acc tgc acc cac tac acg cag ctc gtg tgg 722

Glu Cys Ala Arg Asn Ala Thr Cys Thr His Tyr Thr Gln Leu Val Trp

140 145 150 155

gcc acc tca agc cag ctg ggc tgt ggg cgg cac ctg tgc tct gca ggc 770

Ala Thr Ser Ser Gln Leu Gly Cys Gly Arg His Leu Cys Ser Ala Gly

160 165 170

cag gca gcg ata gaa gcc ttt gtc tgt gcc tac tcc ccc aga ggc aac 818

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

175 180 185

tgg gag gtc aac ggg aag aca atc gtc ccc tat aag aag ggt gcc tgg 866

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

190 195 200

tgt tcg ctc tgc aca gcc agt gtc tca ggc tgc ttc aaa gcc tgg gac 914

Cys Ser Leu Cys Thr Ala Ser Val Ser Gly Cys Phe Lys Ala Trp Asp

205 210 215

cat gca ggg ggg ctc tgt gag gtc ccc agg aat cct tgt cgc atg agc 962

His Ala Gly Gly Leu Cys Glu Val Pro Arg Asn Pro Cys Arg Met Ser

220 225 230 235

tgc cag aac cat gga cgt ctc aac atc agc acc tgc cac tgc cac tgt 1010

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

240 245 250

ccc cct ggc tac acg ggc aga tac tgc caa gtg agg tgc agc ctg cag 1058

Pro Pro Gly Tyr Thr Gly Arg Tyr Cys Gln Val Arg Cys Ser Leu Gln

255 260 265

tgt gtg cac ggc cgg ttc cgg gag gag gag tgc tcg tgc gtc tgt gac 1106

Cys Val His Gly Arg Phe Arg Glu Glu Glu Cys Ser Cys Val Cys Asp

270 275 280

atc ggc tac ggg gga gcc cag tgt gcc acc aag gtg cat ttt ccc ttc 1154

Ile Gly Tyr Gly Gly Ala Gln Cys Ala Thr Lys Val His Phe Pro Phe

285 290 295

cac acc tgt gac ctg agg atc gac gga gac tgc ttc atg gtg tct tca 1202

His Thr Cys Asp Leu Arg Ile Asp Gly Asp Cys Phe Met Val Ser Ser

300 305 310 315

gag gca gac acc tat tac aga gcc agg atg aaa tgt cag agg aaa ggc 1250

Glu Ala Asp Thr Tyr Tyr Arg Ala Arg Met Lys Cys Gln Arg Lys Gly

320 325 330

ggg gtg ctg gcc cag atc aag agc cag aaa gtg cag gac atc ctc gcc 1298

Gly Val Leu Ala Gln Ile Lys Ser Gln Lys Val Gln Asp Ile Leu Ala

335 340 345

ttc tat ctg ggc cgc ctg gag acc acc aac gag gtg att gac agt gac 1346

Phe Tyr Leu Gly Arg Leu Glu Thr Thr Asn Glu Val Ile Asp Ser Asp

350 355 360

ttc gag acc agg aac ttc tgg atc ggg ctc acc tac aag acc gcc aag 1394

Phe Glu Thr Arg Asn Phe Trp Ile Gly Leu Thr Tyr Lys Thr Ala Lys

365 370 375

gac tcc ttc cgc tgg gcc aca ggg gag cac cag gcc ttc acc agt ttt 1442

Asp Ser Phe Arg Trp Ala Thr Gly Glu His Gln Ala Phe Thr Ser Phe

380 385 390 395

gcc ttt ggg cag cct gac aac cac ggg ttt ggc aac tgc gtg gag ctg 1490

Ala Phe Gly Gln Pro Asp Asn His Gly Phe Gly Asn Cys Val Glu Leu

400 405 410

cag gct tca gct gcc ttc aac tgg aac gac cag cgc tgc aaa acc cga 1538

Gln Ala Ser Ala Ala Phe Asn Trp Asn Asp Gln Arg Cys Lys Thr Arg

415 420 425

aac cgt tac atc tgc cag ttt gcc cag gag cac atc tcc cgg tgg ggc 1586

Asn Arg Tyr Ile Cys Gln Phe Ala Gln Glu His Ile Ser Arg Trp Gly

430 435 440

cca ggg tcc tga ggcctgacca catggctccc tcgcctgccc tgggagcacc 1638

Pro Gly Ser

445

ggctctgctt acctgtctgc ccacctgtct ggaacaaggg ccaggttaag accacatgcc 1698

tcatgtccaa agaggtctca gaccttgcac aatgccagaa gttgggcaga gagaggcagg 1758

gaggccagtg agggccaggg agtgagtgtt agaagaagct ggggcccttc gcctgctttt 1818

gattgggaag atgggcttca attagatggc gaaggagagg acaccgccag tggtccaaaa 1878

aggctgctct cttccacctg gcccagaccc tgtggggcag cggagcttcc ctgtggcatg 1938

aaccccacgg ggtattaaat tatgaatcag ctgaaaaaaa aaaaaaaaaa a 1989

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Met Leu His Pro Glu Thr Ser Pro Gly Arg Gly His Leu Leu Ala Val

1 5 10 15

Leu Leu Ala Leu Leu Gly Thr Ala Trp Ala Glu Val Trp Pro Pro Gln

20 25 30

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

35 40 45

Phe Leu Leu Leu Ser Leu His Asn Arg Leu Arg Ser Trp Val Gln Pro

50 55 60

Pro Ala Ala Asp Met Arg Arg Leu Asp Trp Ser Asp Ser Leu Ala Gln

65 70 75 80

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

85 90 95

Ala Ser Gly Leu Trp Arg Thr Leu Gln Val Gly Trp Asn Met Gln Leu

100 105 110

Leu Pro Ala Gly Leu Ala Ser Phe Val Glu Val Val Ser Leu Trp Phe

115 120 125

Ala Glu Gly Gln Arg Tyr Ser His Ala Ala Gly Glu Cys Ala Arg Asn

130 135 140

Ala Thr Cys Thr His Tyr Thr Gln Leu Val Trp Ala Thr Ser Ser Gln

145 150 155 160

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

165 170 175

Ala Phe Val Cys Ala Tyr Ser Pro Arg Gly Asn Trp Glu Val Asn Gly

180 185 190

Lys Thr Ile Val Pro Tyr Lys Lys Gly Ala Trp Cys Ser Leu Cys Thr

195 200 205

Ala Ser Val Ser Gly Cys Phe Lys Ala Trp Asp His Ala Gly Gly Leu

210 215 220

Cys Glu Val Pro Arg Asn Pro Cys Arg Met Ser Cys Gln Asn His Gly

225 230 235 240

Arg Leu Asn Ile Ser Thr Cys His Cys His Cys Pro Pro Gly Tyr Thr

245 250 255

Gly Arg Tyr Cys Gln Val Arg Cys Ser Leu Gln Cys Val His Gly Arg

260 265 270

Phe Arg Glu Glu Glu Cys Ser Cys Val Cys Asp Ile Gly Tyr Gly Gly

275 280 285

Ala Gln Cys Ala Thr Lys Val His Phe Pro Phe His Thr Cys Asp Leu

290 295 300

Arg Ile Asp Gly Asp Cys Phe Met Val Ser Ser Glu Ala Asp Thr Tyr

305 310 315 320

Tyr Arg Ala Arg Met Lys Cys Gln Arg Lys Gly Gly Val Leu Ala Gln

325 330 335

Ile Lys Ser Gln Lys Val Gln Asp Ile Leu Ala Phe Tyr Leu Gly Arg

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

Asp Asn His Gly Phe Gly Asn Cys Val Glu Leu Gln Ala Ser Ala Ala

405 410 415

Phe Asn Trp Asn Asp Gln Arg Cys Lys Thr Arg Asn Arg Tyr Ile Cys

420 425 430

Gln Phe Ala Gln Glu His Ile Ser Arg Trp Gly Pro Gly Ser

435 440 445

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＜213〉people

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ctagacgcgc ggcgcattca ggttctcctg gaccgtagtg gtagtcgctg gcccgagtta 60

gaggccagct gagaagtctt cgtgcttcag atttgtttac ctacaaa atg ctt cgg 116

Met Leu Arg

1

tat cca tat ttt tgt aga atg tat aaa gaa tgc ctt tca tgt tgg ttg 164

Tyr Pro Tyr Phe Cys Arg Met Tyr Lys Glu Cys Leu Ser Cys Trp Leu

5 10 15

gaa tct ggc ata cct aat tta ggt gtc tgg cca aac aga ata cat act 212

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

20 25 30 35

aca gca gaa aaa tat aga gaa tat gaa gcc cgg gag caa aca gat caa 260

Thr Ala Glu Lys Tyr Arg Glu Tyr Glu Ala Arg Glu Gln Thr Asp Gln

40 45 50

act caa gcc cag gag tta cac aga tct caa gat aga gat ttt gaa act 308

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

55 60 65

atg gct aaa tta cat att cca gta atg gtg gat gaa gtt gtt cat tgt 356

Met Ala Lys Leu His Ile Pro Val Met Val Asp Glu Val Val His Cys

70 75 80

ttg tca cca caa aaa gga cag att ttt cta gat atg aca ttt ggt tcg 404

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

85 90 95

gga ggg cac aca aaa gcc att ctg cag aag gag tca gat att gtt ctc 452

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

100 105 110 115

tat gcc ttg gac aga gac cca aca gct tat gca tta gct gaa cat ctt 500

Tyr Ala Leu Asp Arg Asp Pro Thr Ala Tyr Ala Leu Ala Glu His Leu

120 125 130

tca gag ttg tat cct aaa caa atc cga gct atg ctg ggc cag ttc agc 548

Ser Glu Leu Tyr Pro Lys Gln Ile Arg Ala Met Leu Gly Gln Phe Ser

135 140 145

cag gca gaa gcc tta tta atg aaa gct gga gtg cag cca gga act ttt 596

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

150 155 160

gat gga gtt ctt atg gat ctt ggg tgt tcc tcc atg caa ctt gat act 644

Asp Gly Val Leu Met Asp Leu Gly Cys Ser Ser Met Gln Leu Asp Thr

165 170 175

cct gaa aga ggt ttt tcc ctt cgg aaa gat ggc cct ttg gac atg aga 692

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

180 185 190 195

atg gat ggt ggc agg tac cct gac atg ccc act gct gct gat gtt gtg 740

Met Asp Gly Gly Arg Tyr Pro Asp Met Pro Thr Ala Ala Asp Val Val

200 205 210

aat gct tta gat caa cag gca ctt gca tct atc cta aga aca tac ggg 788

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

215 220 225

gag gag aag cat gcc aag aaa atc gct tca gca att gtt cag gca cgc 836

Glu Glu Lys His Ala Lys Lys Ile Ala Ser Ala Ile Val Gln Ala Arg

230 235 240

agc atc tac ccc atc acc aga acc cag cag ctt gcc agc atc gtt gca 884

Ser Ile Tyr Pro Ile Thr Arg Thr Gln Gln Leu Ala Ser Ile Val Ala

245 250 255

gaa tac tct gaa aca tat ttc cca gtg gcc caa act ctt tat tag 929

Glu Tyr Ser Glu Thr Tyr Phe Pro Val Ala Gln Thr Leu Tyr

260 265 270

gaaaatggac gaattgaata gcaaatggag catttcctcc ctctgctatt tatacacgga 989

aagacttact acagcgatct acccatattg ccaccaagac tttccaggct cttcgcatat 1049

ttgtgaacaa tgagctcaat gaactctaca cgggactgaa gacagctcag aagtttctga 1109

gacctggtgg tcgtcttgtt gccctctcct tccattcact agaggatcgc atcgtcaaaa 1169

gatttttgct tggaataagc atgacagaaa gatttaacct aagtgttaga caacaagtga 1229

tgaaaacatc tcaattgggt tcagatcacg aaaacacgga agaagtctct atgagaagag 1289

ctcctttaat gtgggaattg atacacaaga aggtacttag tccacaagat caggatgtac 1349

aagataaccc cagagggcgc tcagccaagc ttagagcagc tatcaaatta taagttatca 1409

tcatcttatt cttcaaattt ttttctcaca atttctctaa tctttactca tgttatgtcc 1469

ctgaatgtct tggtataggt ttaagtgtgg gacagtctga aaattgatag catttagcat 1529

ttcttttttc tcaaaaagaa actgtaggaa atacatgaca gagaaagtta cactcaggga 1589

gcagcagcac ctccagactg gaaaaatgtg ttaatctttg catcatattg gactcttgaa 1649

ggcaatcctt cctctggcca ggaaattttt taaaaaataa tactgtgttg tgtttatcta 1709

aatacgtaaa ctcaagctac caaagagaaa gatgttgtaa tcatatctgc atgtcctaaa 1769

ttttgaattt agatattcca atttgcatca gtctttctgt ggctcaaata atgatgatta 1829

tggaatgaat tttaatgtcc ctacttgtga ataattagct ttctcaaatg taggcttttt 1889

acaaatttta aattttaaaa tattagttta aatgtgtgtt atactgataa aatttcatct 1949

ttcaaattat agtctattat tttaaaggga tttttcagta tgatatgggc cattttgttc 2009

atctatcgca aagtaaaaat gtaaaatcct tacagagaat tgtttcacaa aacttatatt 2069

tcatgtcaat tgtatttatt ttaataatag ctcacaatgc ctttagtaag taataaagtc 2129

tcttattaga atcttgtatt ttttaattga gctaatcaaa ataattcagc caagtctatt 2189

tgaaatagaa aactgtctat ttaatatagt aaaatcaatg ctcccttaat gttgttacaa 2249

agatatggta actgtaatat gggtaaaagt ttattcaaga aaagagtact tggtagaaga 2309

ttctttaaca aattgaggag attgattcat aattcacatg tcaatttttt atagtaatat 2369

gtacttctaa tttattattt atacttaaat tgtgacctga aagatgagtt ggagataatt 2429

agtcaaaggg cacatggagt atggaagggg gcatgttgat taaccataca tagaaataaa 2489

tattctctta gttatccttg aaatcatatt tatactatta atttgctcag tgcacttttt 2549

tcaaatacag aaaaaagatt ctccattata gttacaaatt ttaggttggt gaatccaagt 2609

gaatgaaata tcagcataac tatgtgggca aaatagatag aattaaatgc atgaattttc 2669

ctcagactta ttttatctgc ctctgtaata tttaacttta gtacccaggc ttgttcacta 2729

cttctgcttt acaggcttta tttaaatctg aacaatccta cagggaaatc agaattagga 2789

aaaaaatctg gagaacaata tggttgaaat agaaacaaga gaatgcacct aggttaattc 2849

cctgaatcct acttgaacat tgtataaatt tctctttgca tataatacat atttgtgaat 2909

gagacatatt cccaaaaaat tcttatctct gtatgtgatt ggaaaagaaa agatcacatt 2969

tgtatattca acaatctttc acctatttca taagtcattt tttcaccctg tatagtatgg 3029

gaattatttt ttatgttaaa tagaaactga atgtactggg ttgaatggtg tcctctccaa 3089

aattcatgta cttcctggag cctcagtatg tgaccttatt tggaaatact gtggttgtgg 3149

ttgtaagtag ctaagatgag gtcatactgg agcagggcag gcccttaatc caatatgact 3209

ggtgttcctt ataagagaag atagggcggg catggtggct cacgcctgta atcccagcac 3269

tgtgggaggc caagccaggc aaatcgcttg aggctgagga gttcaagacc agcctggcca 3329

acatggcgaa aacccatctc tactaaaaat aaaattagcc atgcgtggtg cttgtaatgt 3389

cagctaccca ggagtctgag gcacaagaat cactcgaacc tgggaggtgg aggttgcgag 3449

atcacaccac tgcactccag tgtgagcaac agaacgagac tctgtctcaa aaaaaaaaaa 3509

aaaaaaggga agaaagagaa gatagagaca caggggagaa tgccacatga agctggaggc 3569

agagattgga gtgatacatt gtgggaaccc tcagaagata ggaaaaaagc atggaataga 3629

ttctccctca gagttccagt agttgccaac cctgctaaca tggttttgca catctagcat 3689

ccagaacagt gagaatgtat ttctgctgtt ttaaggtacc cagttcatgg taatttgtta 3749

cagcaatcct aggaagctaa tactctgaca gaaaaaaatc ttggataata tgcatgagaa 3809

aaatccttca ataaacacaa aattggtttc aaaatgtttt catgtaaaaa taaattaact 3869

tttctgtaat tagattgtgt tttatagtaa ttcttgcctg cttcttagtc tttgttgtag 3929

gatgcataat aaaatacatg aatgagtttt 3959

<210>4

<211>273

<212>PRT

＜213〉people

<400> 4

Met Leu Arg Tyr Pro Tyr Phe Cys Arg Met Tyr Lys Glu Cys Leu Ser

1 5 10 15

Cys Trp Leu Glu Ser Gly Ile Pro Asn Leu Gly Val Trp Pro Asn Arg

20 25 30

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

35 40 45

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

50 55 60

Phe Glu Thr Met Ala Lys Leu His Ile Pro Val Met Val Asp Glu Val

65 70 75 80

Val His Cys Leu Ser Pro Gln Lys Gly Gln Ile Phe Leu Asp Met Thr

85 90 95

Phe Gly Ser Gly Gly His Thr Lys Ala Ile Leu Gln Lys Glu Ser Asp

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

Gly Thr Phe Asp Gly Val Leu Met Asp Leu Gly Cys Ser Ser Met Gln

165 170 175

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

180 185 190

Asp Met Arg Met Asp Gly Gly Arg Tyr Pro Asp Met Pro Thr Ala Ala

195 200 205

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

210 215 220

Thr Tyr Gly Glu Glu Lys His Ala Lys Lys Ile Ala Ser Ala Ile Val

225 230 235 240

Gln Ala Arg Ser Ile Tyr Pro Ile Thr Arg Thr Gln Gln Leu Ala Ser

245 250 255

Ile Val Ala Glu Tyr Ser Glu Thr Tyr Phe Pro Val Ala Gln Thr Leu

260 265 270

Tyr

<210>5

<211>1680

<212>DNA

＜213〉people

<220>

<221>CDS

<222>(224)..(781)

<400>5

agagagctgt ttactaggca cgactgcgaa ggcaaggggg caccagctca ggactgcatc 60

tgcctgccat ttcccttcca ctcctccttt ctggagtctg acattagaaa gccagcgaga 120

aggaagattc aaacaaccaa ccctgatttc ctgcttctcc ttttcatgag tgttcctgtg 180

gtctctgcac ctcctttctg tcccccggca gagggcagta gag atg gcc ggc cca 235

Met Ala Gly Pro

1

agg tct cgg tgg cgc gac cag ctg ctg ttc atg agc atc ata gtc ctc 283

Arg Ser Arg Trp Arg Asp Gln Leu Leu Phe Met Ser Ile Ile Val Leu

5 10 15 20

gtg att gtg gtc atc tgc ctg atg tta tac gct ctt ctc tgg gag gct 331

Val Ile Val Val Ile Cys Leu Met Leu Tyr Ala Leu Leu Trp Glu Ala

25 30 35

ggc aac ctc act gac ctg ccc aac ctg aga atc ggc ttc tat aac ttc 379

Gly Asn Leu Thr Asp Leu Pro Asn Leu Arg Ile Gly Phe Tyr Asn Phe

40 45 50

tgc ctg tgg aat gag gac acc agc acc cta cag tgt cac cag ttc cct 427

Cys Leu Trp Asn Glu Asp Thr Ser Thr Leu Gln Cys His Gln Phe Pro

55 60 65

gag ctg gaa gcc ctg ggg gtg cct cgg gtt ggc ctg ggc ctg gcc agg 475

Glu Leu Glu Ala Leu Gly Val Pro Arg Val Gly Leu Gly Leu Ala Arg

70 75 80

ctt ggc gtg tac ggg tcc ctg gtc ctc acc ctc ttt gcc ccc cag cct 523

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

85 90 95 100

ctc ctc cta gcc cag tgc aac agt gat gag aga gtg tgg cgg ctg gca 571

Leu Leu Leu Ala Gln Cys Asn Ser Asp Glu Arg Val Trp Arg Leu Ala

105 110 115

gtg ggc ttc ctg gct gtg tcc tct gtg ctg ctg gca ggc ggc ctg ggc 619

Val Gly Phe Leu Ala Val Ser Ser Val Leu Leu Ala Gly Gly Leu Gly

120 125 130

ctc ttc ctc tcc tat gtg tgg aag tgg gtc agg ctc tcc ctc ccg ggg 667

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

135 140 145

cct ggg ttt cta gct ctg ggc agc gcc cag gcc tta ctc atc ctc ttg 715

Pro Gly Phe Leu Ala Leu Gly Ser Ala Gln Ala Leu Leu Ile Leu Leu

150 155 160

ctt ata gcc atg gct gtg ttc cct ctg agg gct gag agg gct gag agc 763

Leu Ile Ala Met Ala Val Phe Pro Leu Arg Ala Glu Arg Ala Glu Ser

165 170 175 180

aag ctt gag agc tgc taa aggcttacgt gattgcaagg gttcagttcc 811

Lys Leu Glu Ser Cys

185

aaccatggtc agaggtggca catctgctca gccatctcat tttacagcta acgctgatct 871

ccagctccag cgatggaacc cactacagag gaggtggggc ccctgtgtca aagaggccga 931

ggggcagcaa gggcagccag ggcacctgtg acttcttagt acaagattgt ctgtccttca 991

ggacttccaa ggctcccaaa gactccctaa accatgcagc tcattgtcac accaattcct 1051

gctttaatta atggatctga gcaaatcttc ctctagcttc aggagggtgg ggagggagtg 1111

attgctgtca tggggccaga cttccaggct gatttgccaa atgccaaaat gaaacctagc 1171

aaagaactta cggcaacaaa cgaggacatt aaaagagcga gcacctcagt gtctctgggg 1231

acatggttaa ggagcttcca ctcagcccac catagtgagt gggccgccat aagccatcac 1291

tggaactcca accccagagg tccaggagtg atctctgagt gactcaacaa agacaggaca 1351

catggggtac aaagacaagg cttgactgct tcaaagcttc cctggacctg aagccagaca 1411

gggcagaggc gtccgctgac aaatcactcc catgatgaga ccctggagga ctccaaatcc 1471

tcgctgtgaa caggactgga cggttgcgca caaacaaacg ctgccaccct ccacttccca 1531

acccagaact tggaaagaca ttagcacaac ttacgcattg gggaattgtg tgtattttct 1591

agcacttgtg tattggaaaa cctgtatggc agtgatttat tcatatattc ctgtccaaag 1651

ccacactgaa aacagaggca gagacatgt 1680

<210>6

<211>185

<212>PRT

＜213〉people

<400> 6

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

1 5 10 15

Ile Ile Val Leu Val Ile Val Val Ile Cys Leu Met Leu Tyr Ala Leu

20 25 30

Leu Trp Glu Ala Gly Asn Leu Thr Asp Leu Pro Asn Leu Arg Ile Gly

35 40 45

Phe Tyr Asn Phe Cys Leu Trp Asn Glu Asp Thr Ser Thr Leu Gln Cys

50 55 60

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

65 70 75 80

Gly Leu Ala Arg Leu Gly Val Tyr Gly Ser Leu Val Leu Thr Leu Phe

85 90 95

Ala Pro Gln Pro Leu Leu Leu Ala Gln Cys Asn Ser Asp Glu Arg Val

100 105 110

Trp Arg Leu Ala Val Gly Phe Leu Ala Val Ser Ser Val Leu Leu Ala

115 120 125

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

130 135 140

Ser Leu Pro Gly Pro Gly Phe Leu Ala Leu Gly Ser Ala Gln Ala Leu

145 150 155 160

Leu Ile Leu Leu Leu Ile Ala Met Ala Val Phe Pro Leu Arg Ala Glu

165 170 175

Arg Ala Glu Ser Lys Leu Glu Ser Cys

180 185

<210>7

<211>1195

<212>DNA

＜213〉people

<220>

<221>CDS

<222>(137)..(703)

<400>7

ggcacgaggc gccagtcccc taaccctgag gctgccgcgc ggcggtcact gcgccggggt 60

agtgggcccc agtgttgcgc tctctggccg ttccttacac tttgcttcag gctccagtgc 120

aggggcgtag tgggat atg gcc aac tcg ggc tgc aag gac gtc acg ggt cca 172

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

1 5 10

gat gag gag agt ttt ctg tac ttt gcc tac ggc agc aac ctg ctg aca 220

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

15 20 25

gag agg atc cac ctc cga aac ccc tcg gcg gcg ttc ttc tgt gtg gcc 268

Glu Arg Ile His Leu Arg Asn Pro Ser Ala Ala Phe Phe Cys Val Ala

30 35 40

cgc ctg cag gat ttt aag ctt gac ttt ggc aat tcc caa ggc aaa aca 316

Arg Leu Gln Asp Phe Lys Leu Asp Phe Gly Asn Ser Gln Gly Lys Thr

45 50 55 60

agt caa act tgg cat gga ggg ata gcc acc att ttt cag agt cct ggc 364

Ser Gln Thr Trp His Gly Gly Ile Ala Thr Ile Phe Gln Ser Pro Gly

65 70 75

gat gaa gtg tgg gga gta gta tgg aaa atg aac aaa agc aat tta aat 412

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

80 85 90

tct ctg gat gag caa gaa ggg gtt aaa agt gga atg tat gtt gta ata 460

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

95 100 105

gaa gtt aaa gtt gca act caa gaa gga aaa gaa ata acc tgt cga agt 508

Glu Val Lys Val Ala Thr Gln Glu Gly Lys Glu Ile Thr Cys Arg Ser

110 115 120

tat ctg atg aca aat tac gaa agt gct ccc cca tcc cca cag tat aaa 556

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

125 130 135 140

aag att att tgc atg ggt gca aaa gaa aat ggt ttg ccg ctg gag tat 604

Lys Ile Ile Cys Met Gly Ala Lys Glu Asn Gly Leu Pro Leu Glu Tyr

145 150 155

caa gag aag tta aaa gca ata gaa cca aat gac tat aca gga aag gtc 652

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

160 165 170

tca gaa gaa att gaa gac atc atc aaa aag ggg gaa aca caa act ctt 700

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

175 180 185

tag aacataacag aatatatcta agggtattct atgtgctaat ataaaatatt 753

tttaacactt gagaacaggg atctggggga tctccacgtt tgatccgttt tcagcagtgc 813

tctgaaggag tatcttactt gggtgattcc ttgtttttag actataaaaa gaaactggga 873

taggagttag acaatttaaa aggggtgtat gagggcctga aatatgtgac aaatgaatgt 933

gagtacccct tctgtgaaca ctgaaagcta ttctcttgaa ttgatcttaa gtgtctcctt 993

gctctggtaa aagatagatt tgtagctcac ttgatgatgg tgctggtgaa ttgctctgct 1053

ctgtctgaga tttttaaaaa tcagcttaat gagagtaatc tgcagacaat tgataataac 1113

attttgaaaa ttggaaagat ggtatactgt ttttagagga ataaacgtat ttgtggttta 1173

aaaaaaaaaa aaaaaaaaaa aa 1195

<210>8

<211>188

<212>PRT

＜213〉people

<400>8

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

1 5 10 15

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

20 25 30

Leu Arg Asn Pro Ser Ala Ala Phe Phe Cys Val Ala Arg Leu Gln Asp

35 40 45

Phe Lys Leu Asp Phe Gly Asn Ser Gln Gly Lys Thr Ser Gln Thr Trp

50 55 60

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

65 70 75 80

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

85 90 95

Gln Glu Gly Val Lys Ser Gly Met Tyr Val Val Ile Glu Val Lys Val

100 105 110

Ala Thr Gln Glu Gly Lys Glu Ile Thr Cys Arg Ser Tyr Leu Met Thr

115 120 125

Asn Tyr Glu Ser Ala Pro Pro Ser Pro Gln Tyr Lys Lys Ile Ile Cys

130 135 140

Met Gly Ala Lys Glu Asn Gly Leu Pro Leu Glu Tyr Gln Glu Lys Leu

145 150 155 160

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

165 170 175

Glu Asp Ile Ile Lys Lys Gly Glu Thr Gln Thr Leu

180 185

<210>9

<211>1817

<212>DNA

＜213〉people

<220>

<221>CDS

<222>(292)..(771)

<400>9

aaaaatcaga ataagaagta cctgacatac tttctacatc tgtagttgcg gaagacattt 60

taataggtct tctcatagcc tttctttgct aaggacattg tgactctcca gagagcaaca 120

gtgatggctc tagaatgtct aggaaaaaga agggcttaat gtcaggagtc tgcttggggc 180

acacaacact agaagatgtc cttctgcaca ttgtttcata tcgagtatgg aacccttcag 240

atcaaagctt accaataaat tcagtatgta gaacagatta acgtagttga a atg agg 297

Met Arg

1

aag aat gag agt tat ctc aac cag cca gca ccc cct atc ccc att ccc 345

Lys Asn Glu Ser Tyr Leu Asn Gln Pro Ala Pro Pro Ile Pro Ile Pro

5 10 15

aca ctt tcc ctc atg gga ggc tgt cgg gag cac ttc gaa aac cac tgg 393

Thr Leu Ser Leu Met Gly Gly Cys Arg Glu His Phe Glu Asn His Trp

20 25 30

aaa ggc cgg gca cgg tgg ctc atg cct gta atc cca gca ctt tgg gag 441

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

35 40 45 50

gcc aag gca ggc aga tca cct gag gtc agg agt tcg aaa cca gcc tgg 489

Ala Lys Ala Gly Arg Ser Pro Glu Val Arg Ser Ser Lys Pro Ala Trp

55 60 65

cca aca tgg cga aac ccc atc ttt act aaa aat acg aaa att agc cag 537

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

70 75 80

gta tta gaa tta ttt ctg aat tat cag tct ctc att tgt gct ttg gag 585

Val Leu Glu Leu Phe Leu Asn Tyr Gln Ser Leu Ile Cys Ala Leu Glu

85 90 95

aag cag aaa agg caa aag ggg tct ttg gcc atc ttc tgc tgg agc ttc 633

Lys Gln Lys Arg Gln Lys Gly Ser Leu Ala Ile Phe Cys Trp Ser Phe

100 105 110

cag gga gga tgt gtc tcc aag aga cca gat gta ccg agt ttg aaa tcc 681

Gln Gly Gly Cys Val Ser Lys Arg Pro Asp Val Pro Ser Leu Lys Ser

115 120 125 130

cag aag ccc aag agg aaa aga atc aca ggg agg aaa aga ctg tcc aaa 729

Gln Lys Pro Lys Arg Lys Arg Ile Thr Gly Arg Lys Arg Leu Ser Lys

135 140 145

ggc ttc tgg agt ctt ctg ttc tct aac ctt gga agg ttt tga 771

Gly Phe Trp Ser Leu Leu Phe Ser Asn Leu Gly Arg Phe

150 155

acaatatttc tcagaggata gcctttcact tattcatctg tccagcatga ctcatccccg 831

ggagtgttga gtaagtgaaa ttttgctgta ttcatgtttt tgtgacttat aaaataggat 891

gataaggaga gaacatgaac tctggagtca gacctgttac ctcggacatg atactcttag 951

ctttgtcatt tagtatttga gtaattttgg gcaagctaac atctctggtc gttctcatct 1011

gtaaaatgag aataaatgaa acccactaac cagaattggt atgaaaatga aatgtggcag 1071

aaaaaaaatg aaagtgaata gtatcaccac tgacacacaa gcactaaagg cccttcctgt 1131

ctccatcagg tatggatttg gggcaacatt tggccaggtc ttgtttatct ttctgttcat 1191

ctattctgtc taattcagtg ctttgtttac aacgaatgtc ttacaaatgc tgactgaaca 1251

ctagcatacc tgcatgaaca acaggtaaat aaattttaga tgtgttttaa tgtttattaa 1311

tctatcctgt cagagaagaa ctgccagtta tagataaata tgatgccagg tcagggctga 1371

agagttgggc aggttgttat ctgcatgggg tcactaggtt ccagtggaga ggtgggggct 1431

aagctctcac ccgctctgca gccacctggc acccggtttc agtttcctga aagggagcct 1491

tctacttgct gacgactgcc tcatctcttc tgaggtttcc tctgataaac aattttctct 1551

gctttttttt tttaattatg aaatacttaa aatgtaaagg agataatgtg acacacattt 1611

acccataatt gagattgcca taattgctat aactttttca aaattttgac ttaatttcag 1671

acttttagaa aaattggcca ggtgtgatgg ctcatgcctg taatcccagc actttgggag 1731

gccaaggtgg gtaaattact tgaacccagc agttcgagac ttgcctgggc ggcatagtga 1791

gaccttgtct ctactgaaaa caaact 1817

<210>10

<211>159

<212>PRT

＜213〉people

<400>10

Met Arg Lys Asn Glu Ser Tyr Leu Asn Gln Pro Ala Pro Pro Ile Pro

1 5 10 15

Ile Pro Thr Leu Ser Leu Met Gly Gly Cys Arg Glu His Phe Glu Asn

20 25 30

His Trp Lys Gly Arg Ala Arg Trp Leu Met Pro Val Ile Pro Ala Leu

35 40 45

Trp Glu Ala Lys Ala Gly Arg Ser Pro Glu Val Arg Ser Ser Lys Pro

50 55 60

Ala Trp Pro Thr Trp Arg Asn Pro Ile Phe Thr Lys Asn Thr Lys Ile

65 70 75 80

Ser Gln Val Leu Glu Leu Phe Leu Asn Tyr Gln Ser Leu Ile Cys Ala

85 90 95

Leu Glu Lys Gln Lys Arg Gln Lys Gly Ser Leu Ala Ile Phe Cys Trp

100 105 110

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

115 120 125

Lys Ser Gln Lys Pro Lys Arg Lys Arg Ile Thr Gly Arg Lys Arg Leu

130 135 140

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

145 150 155

<210>11

<211>1615

<212>DNA

＜213〉people

<220>

<221>CDS

<222>(193)..(1086)

<400>11

ggagcagaac cgctccccgc cggctgtccg caacctccgc cagcgtcggt ccctgggcag 60

gtgtgggtgg aatgcatcct tgggacatct ttgcatagcc ccatctagga agaagttctg 120

tgatgtgtga actgtgagtt tactcaaaca agtccaactc tttataagac ataggtagac 180

gtcaggtctg ga atg gaa gag cca aca gca gta gaa ggc cag gtc cag ctt 231

Met Glu Glu Pro Thr Ala Val Glu Gly Gln Val Gln Leu

1 5 10

cca agc ccc cac cag ggc tct ctc agg aag gct gtg gct gct gcc ctg 279

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

15 20 25

gcg ctg gat ggg gaa tcc aca atg ggg cac agg aaa aag aag agg aaa 327

Ala Leu Asp Gly Glu Ser Thr Met Gly His Arg Lys Lys Lys Arg Lys

30 35 40 45

gag tca cgc cca gaa tcc atc atc atc tac cgc tca gac aat gag aaa 375

Glu Ser Arg Pro Glu Ser Ile Ile Ile Tyr Arg Ser Asp Asn Glu Lys

50 55 60

aca gat gag gag ccc gga gaa tca gaa ggt gga gat cag cct aaa gag 423

Thr Asp Glu Glu Pro Gly Glu Ser Glu Gly Gly Asp Gln Pro Lys Glu

65 70 75

gag gag gga gat gat ttc cta gac tat cct gtg gat gat gat atg tgg 471

Glu Glu Gly Asp Asp Phe Leu Asp Tyr Pro Val Asp Asp Asp Met Trp

80 85 90

aac ctg cct ctg gac agc cgc tac gtc acc tta act ggg acc atc aca 519

Asn Leu Pro Leu Asp Ser Arg Tyr Val Thr Leu Thr Gly Thr Ile Thr

95 100 105

cga ggg aag aaa aag ggt cag atg gtg gac atc cat gtc aca ttg aca 567

Arg Gly Lys Lys Lys Gly Gln Met Val Asp Ile His Val Thr Leu Thr

110 115 120 125

gag aaa gag ctg cag gaa ctg acc aaa cct aaa gag tca tca agg gaa 615

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

130 135 140

acg acg cct gaa gga aga atg gcc tgc cag atg gga gct gac cgt ggg 663

Thr Thr Pro Glu Gly Arg Met Ala Cys Gln Met Gly Ala Asp Arg Gly

145 150 155

ccc cat gtg gtc ctc tgg acg ctg atc tgc ctg cct gtg gtt ttc atc 711

Pro His Val Val Leu Trp Thr Leu Ile Cys Leu Pro Val Val Phe Ile

160 165 170

ctt tct ttt gtt gtc tct ttc tac tac ggc act atc acc tgg tac aac 759

Leu Ser Phe Val Val Ser Phe Tyr Tyr Gly Thr Ile Thr Trp Tyr Asn

175 180 185

atc ttc ctc gtg tat aat gag gaa agg acc ttc tgg cac aag atc tcg 807

Ile Phe Leu Val Tyr Asn Glu Glu Arg Thr Phe Trp His Lys Ile Ser

190 195 200 205

tat tgc cct tgc ctc gtt ctc ttc tat cca gtg ctc atc atg gcc atg 855

Tyr Cys Pro Cys Leu Val Leu Phe Tyr Pro Val Leu Ile Met Ala Met

210 215 220

gct tct tcc ctc ggc ctc tac gct gct gtg gtc cag ctc tcg tgg tcc 903

Ala Ser Ser Leu Gly Leu Tyr Ala Ala Val Val Gln Leu Ser Trp Ser

225 230 235

tgg gaa gca tgg tgg caa gct gcc cgg gac atg gag aaa ggc ttc tgt 951

Trp Glu Ala Trp Trp Gln Ala Ala Arg Asp Met Glu Lys Gly Phe Cys

240 245 250

ggc tgg ctc tgc agc aag ctg ggt ctg gag gac tgt tct ccc tac agc 999

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

255 260 265

att gtg gag ttg ctt gaa tcc gac aat atc tca agc act ctc tcc aac 1047

Ile Val Glu Leu Leu Glu Ser Asp Asn Ile Ser Ser Thr Leu Ser Asn

270 275 280 285

aag gac ccc atc caa gaa gta gaa acc tcc acg gtc taa actcccaaca 1096

Lys Asp Pro Ile Gln Glu Val Glu Thr Ser Thr Val

290 295

acttactccc tcctctggcc ccagtagcct atatatcatc ttaaaattcc agcagattat 1156

ttctttaaat taccccctac tctccgcagt tcttctggga aatcagagtc catactgatc 1216

agttttacca tcttgagggt tccaggaggg catggagcag acaagcaatt gtgccaaagc 1276

agttcaccca atggacaaac tctttttgat tccctgccct aaaatcacca tttatttagg 1336

acaatggaac tctgctgtgt gtcgttttgg gagcctggaa gtgttactgg tgcctggaac 1396

tgaggggagt atgtgactaa atgtgtcagg gagaataaag aacctcgggg taaccaaatc 1456

caccaagata atagacaggg atggagtgag acatttagga agctggacta ccacagtgta 1516

gcagaaggta aagatttgtg tgtatcattt agatttagat ttagctgcat agaattaaaa 1576

ccctaaaata tcagtggctt aaacaaaaaa aaaaaaaaa 1615

<210>12

<211>297

<212>PRT

＜213〉people

<400>12

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

1 5 10 15

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

20 25 30

Gly Glu Ser Thr Met Gly His Arg Lys Lys Lys Arg Lys Glu Ser Arg

35 40 45

Pro Glu Ser Ile Ile Ile Tyr Arg Ser Asp Asn Glu Lys Thr Asp Glu

50 55 60

Glu Pro Gly Glu Ser Glu Gly Gly Asp Gln Pro Lys Glu Glu Glu Gly

65 70 75 80

Asp Asp Phe Leu Asp Tyr Pro Val Asp Asp Asp Met Trp Asn Leu Pro

85 90 95

Leu Asp Ser Arg Tyr Val Thr Leu Thr Gly Thr Ile Thr Arg Gly Lys

100 105 110

Lys Lys Gly Gln Met Val Asp Ile His Val Thr Leu Thr Glu Lys Glu

115 120 125

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

130 135 140

Glu Gly Arg Met Ala Cys Gln Met Gly Ala Asp Arg Gly Pro His Val

145 150 155 160

Val Leu Trp Thr Leu Ile Cys Leu Pro Val Val Phe Ile Leu Ser Phe

165 170 175

Val Val Ser Phe Tyr Tyr Gly Thr Ile Thr Trp Tyr Asn Ile Phe Leu

180 185 190

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

195 200 205

Cys Leu Val Leu Phe Tyr Pro Val Leu Ile Met Ala Met Ala Ser Ser

210 215 220

Leu Gly Leu Tyr Ala Ala Val Val Gln Leu Ser Trp Ser Trp Glu Ala

225 230 235 240

Trp Trp Gln Ala Ala Arg Asp Met Glu Lys Gly Phe Cys Gly Trp Leu

245 250 255

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

260 265 270

Leu Leu Glu Ser Asp Asn Ile Ser Ser Thr Leu Ser Asn Lys Asp Pro

275 280 285

Ile Gln Glu Val Glu Thr Ser Thr Val

290 295

<210>13

<211>1731

<212>DNA

＜213〉people

<220>

<221>CDS

<222>(454)..(1227)

<400>13

gcaagccgga gccctggggt tgggcagcac tcggttccgt gcaactttca agtgagttgc 60

gaactccgcc ctgtaggccg gtgctggtgg cccggcgcgc tggaaccgcg gcgacccgct 120

ccagcgcggg accagcagca agggccgagc gccaggttct ccgcggcaga aagggcgggt 180

gggagctgta actgccccgg ccgcggggcg cgcccgctcc caagtcggct tcctccccgc 240

cggggccgct ttgcctcggg tctccccatt ctccaggtcc cctgaactgc acagtcggag 300

gccgtgggcg gcgggctctg cctccgccga gggacagccg gatcgcccct ctgcttcccg 360

caactgccct gatcaccccc cgtcccagcc cttgagtgaa cgtccttctg agcggcttcc 420

tggggtcctc cccacgtccc aaaggccggc aag atg gtg tcc tgg atg atc tgt 474

Met Val Ser Trp Met Ile Cys

1 5

cgc ctg gtg gtg ctg gtg ttt ggg atg ctg tgt cca gct tat gct tcc 522

Arg Leu Val Val Leu Val Phe Gly Met Leu Cys Pro Ala Tyr Ala Ser

10 15 20

tat aag gct gtg aag acc aag aac att cgt gaa tat gtg cgg tgg atg 570

Tyr Lys Ala Val Lys Thr Lys Asn Ile Arg Glu Tyr Val Arg Trp Met

25 30 35

atg tac tgg att gtt ttt gca ctc ttc atg gca gca gag atc gtt aca 618

Met Tyr Trp Ile Val Phe Ala Leu Phe Met Ala Ala Glu Ile Val Thr

40 45 50 55

gac att ttt atc tcc tgg ttc cct ttc tac tat gag atc aag atg gcc 666

Asp Ile Phe Ile Ser Trp Phe Pro Phe Tyr Tyr Glu Ile Lys Met Ala

60 65 70

ttc gtg ctg tgg ctg ctc tca ccc tac acc aag ggc gcc agc ctg ctt 714

Phe Val Leu Trp Leu Leu Ser Pro Tyr Thr Lys Gly Ala Ser Leu Leu

75 80 85

tac cgc aag ttt gtc cac ccg tcc ctg tcc cgc cat gag aag gag atc 762

Tyr Arg Lys Phe Val His Pro Ser Leu Ser Arg His Glu Lys Glu Ile

90 95 100

gac gcg tac atc gtg cag gcc aag gag cgc agc tac gag acc gtg ctc 810

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

105 110 115

agc ttc ggg aag cgg ggc ctc aac att gcc gcc tcc gct gct gtg cag 858

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

120 125 130 135

gct gcc acc aag agt cag ggg gcg ctg gcc ggc agg ctg cgg agc ttc 906

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

140 145 150

tcc atg cag gac ctg cgc tcc atc tct gac gca cct gcc cct gcc tac 954

Ser Met Gln Asp Leu Arg Ser Ile Ser Asp Ala Pro Ala Pro Ala Tyr

155 160 165

cat gac ccc ctc tac ctg gag gac cag gtg tcc cac cag agg cca ccc 1002

His Asp Pro Leu Tyr Leu Glu Asp Gln Val Ser His Gln Arg Pro Pro

170 175 180

att ggg tac cgg gcc ggg ggc ctg cag gac agc gac acc gag gat gag 1050

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

185 190 195

tgt tgg tca gat act gag gca gtc ccc cgg gcg cca gcc cgg ccc cga 1098

Cys Trp Ser Asp Thr Glu Ala Val Pro Arg Ala Pro Ala Arg Pro Arg

200 205 210 215

gag aag ccc cta atc cgc agc cag agc ctg cgt gtg gtc aag agg aag 1146

Glu Lys Pro Leu Ile Arg Ser Gln Ser Leu Arg Val Val Lys Arg Lys

220 225 230

cca ccg gtg cgg gag ggc acc tcg cgc tcc ctg aag gtt cgg acg agg 1194

Pro Pro Val Arg Glu Gly Thr Ser Arg Ser Leu Lys Val Arg Thr Arg

235 240 245

aaa aag act gtg ccc tca gac gtg gac agc tag ggtctgctgc atctgccccc 1247

Lys Lys Thr Val Pro Ser Asp Val Asp Ser

250 255

ttcttacctc gtgccctgca gggctccagg gctatttgga gggaccttgg gctgcacatc 1307

tggcctgcct gcaccagctg cctgggcccc accctcctga ctcctgctga tggttaaggg 1367

ccgggagcag atgctgccaa ggccacatgc agggatgcac ccacaatgta ccaaagcagg 1427

ctgggcccag ggttctattt attgccttgc tctgccctct cccttccccg gttgtgggac 1487

aagagccctc cctgaacccc tgcaaccctc cctgaacccc tgcaaatgaa accaaacgtc 1547

cacctgggtg tgttcattcc ttcctgtcct tcaaagtact tgatagcctt tcataaggcc 1607

tggcacatgt gtcctggttg tgtgtgtgtg tgttggtgag tgaggtcagg tttgcgagtg 1667

ttttgataaa taaatacata aaggggcaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1727

aaaa 1731

<210>14

<211>257

<212>PRT

＜213〉people

<400>14

Met Val Ser Trp Met Ile Cys Arg Leu Val Val Leu Val Phe Gly Met

1 5 10 15

Leu Cys Pro Ala Tyr Ala Ser Tyr Lys Ala Val Lys Thr Lys Asn Ile

20 25 30

Arg Glu Tyr Val Arg Trp Met Met Tyr Trp Ile Val Phe Ala Leu Phe

35 40 45

Met Ala Ala Glu Ile Val Thr Asp Ile Phe Ile Ser Trp Phe Pro Phe

50 55 60

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

65 70 75 80

Thr Lys Gly Ala Ser Leu Leu Tyr Arg Lys Phe Val His Pro Ser Leu

85 90 95

Ser Arg His Glu Lys Glu Ile Asp Ala Tyr Ile Val Gln Ala Lys Glu

100 105 110

Arg Ser Tyr Glu Thr Val Leu Ser Phe Gly Lys Arg Gly Leu Asn Ile

115 120 125

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

130 135 140

Ala Gly Arg Leu Arg Ser Phe Ser Met Gln Asp Leu Arg Ser Ile Ser

145 150 155 160

Asp Ala Pro Ala Pro Ala Tyr His Asp Pro Leu Tyr Leu Glu Asp Gln

165 170 175

Val Ser His Gln Arg Pro Pro Ile Gly Tyr Arg Ala Gly Gly Leu Gln

180 185 190

Asp Ser Asp Thr Glu Asp Glu Cys Trp Ser Asp Thr Glu Ala Val Pro

195 200 205

Arg Ala Pro Ala Arg Pro Arg Glu Lys Pro Leu Ile Arg Ser Gln Ser

210 215 220

Leu Arg Val Val Lys Arg Lys Pro Pro Val Arg Glu Gly Thr Ser Arg

225 230 235 240

Ser Leu Lys Val Arg Thr Arg Lys Lys Thr Val Pro Ser Asp Val Asp

245 250 255

Ser

<210>15

<211>573

<212>DNA

＜213〉people

<220>

<221>CDS

<222>(25)..(789)

<400>15

ggcacgaggc tgccctagtg gcct atg tcc ctt gct cgg ggc cat gga gac 51

Met Ser Leu Ala Arg Gly His Gly Asp

1 5

act gcg gcc agt acg gcg gcg cct ctg tct gaa gaa ggg gaa gtg acc 99

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

10 15 20 25

tcc ggc ctc cag gct ctg gcc gtg gag gat acc gga ggc ccc tct gcc 147

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

30 35 40

tcg gcc ggt aag gcc gag gac gag ggg gaa gga ggc cga gag gag acc 195

Ser Ala Gly Lys Ala Glu Asp Glu Gly Glu Gly Gly Arg Glu Glu Thr

45 50 55

gag cgt gag ggg tcc ggg ggc gag gag gcg cag gga gaa gtc ccc agc 243

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

60 65 70

gct ggg gga gaa gag cct gcc gag gag gac tcc gag gac tgg tgc gtg 291

Ala Gly Gly Glu Glu Pro Ala Glu Glu Asp Ser Glu Asp Trp Cys Val

75 80 85

ccc tgc agc gac gag gag gtg gag ctg cct gcg gat ggg cag ccc tgg 339

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

90 95 100 105

atg ccc ccg ccc tcc gaa atc cag cgg ctc tat gaa ctg ctg gct gcc 387

Met Pro Pro Pro Ser Glu Ile Gln Arg Leu Tyr Glu Leu Leu Ala Ala

110 115 120

cac ggt act ctg gag ctg caa gcc gag atc ctg ccc cgc cgg cct ccc 435

His Gly Thr Leu Glu Leu Gln Ala Glu Ile Leu Pro Arg Arg Pro Pro

125 130 135

acg ccg gag gcc cag agc gaa gag gag aga tcc gat gag gag ccg gag 483

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

140 145 150

gcc aaa gaa gag gaa gag gaa aaa cca cac atg ccc acg gaa ttt gat 531

Ala Lys Glu Glu Glu Glu Glu Lys Pro His Met Pro Thr Glu Phe Asp

155 160 165

ttt gat gat gag cca gtg aca cca aag gac tcc ctg att gac cgg aga 579

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

170 175 180 185

cgc acc cca gga agc tca gcc cgg agc cag aaa cgg gag gcc cgc ctg 627

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

190 195 200

gac aag gtg ctg tcg gac atg aag aga cac aag aag ctg gag gag cag 675

Asp Lys Val Leu Ser Asp Met Lys Arg His Lys Lys Leu Glu Glu Gln

205 210 215

atc ctt cgt acc ggg agg gac ctc ttc agc ctg gac tcg gag gac ccc 723

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

220 225 230

agc ccc gcc agc ccc cca ctc cga tcc tcc ggg agt agt ctc ttc cct 771

Ser Pro Ala Ser Pro Pro Leu Arg Ser Ser Gly Ser Ser Leu Phe Pro

235 240 245

cgg cag cgg aaa tac tga ttcccactgc tcctgcctct agggtgcagt 819

Arg Gln Arg Lys Tyr

250

gtccgtacct gctggagcct gggccctcct tccccagccc agacattgag aaacttggga 879

agaagagaga aacctcaagc tcccaaacag cacgttgcgg gaaagaggaa gagagagtgt 939

gagtgtgtgt gtgtgttttt tctattgaac acctgtagag tgtgtgtgtg tgttttctat 999

tgaacaccta tagagagagt gtgtgtgttt tctattgaac atctatatag agagagtgtg 1059

tgagtgtgtg ttttctattg aacacctatt cagagacctg gactgaattt tctgagtctg 1119

aaataaaaga tgcagagcta tcatctctta aaaaaaaaaa aaaaaaaaaa aaaa 1173

<210>16

<211>254

<212>PRT

＜213〉people

<400>16

Met Ser Leu Ala Arg Gly His Gly Asp Thr Ala Ala Ser Thr Ala Ala

1 5 10 15

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

20 25 30

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

35 40 45

Glu Gly Glu Gly Gly Arg Glu Glu Thr Glu Arg Glu Gly Ser Gly Gly

50 55 60

Glu Glu Ala Gln Gly Glu Val Pro Ser Ala Gly Gly Glu Glu Pro Ala

65 70 75 80

Glu Glu Asp Ser Glu Asp Trp Cys Val Pro Cys Ser Asp Glu Glu Val

85 90 95

Glu Leu Pro Ala Asp Gly Gln Pro Trp Met Pro Pro Pro Ser Glu Ile

100 105 110

Gln Arg Leu Tyr Glu Leu Leu Ala Ala His Gly Thr Leu Glu Leu Gln

115 120 125

Ala Glu Ile Leu Pro Arg Arg Pro Pro Thr Pro Glu Ala Gln Ser Glu

130 135 140

Glu Glu Arg Ser Asp Glu Glu Pro Glu Ala Lys Glu Glu Glu Glu Glu

145 150 155 160

Lys Pro His Met Pro Thr Glu Phe Asp Phe Asp Asp Glu Pro Val Thr

165 170 175

Pro Lys Asp Ser Leu Ile Asp Arg Arg Arg Thr Pro Gly Ser Ser Ala

180 185 190

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

195 200 205

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

210 215 220

Leu Phe Ser Leu Asp Ser Glu Asp Pro Ser Pro Ala Ser Pro Pro Leu

225 230 235 240

Arg Ser Ser Gly Ser Ser Leu Phe Pro Arg Gln Arg Lys Tyr

245 250

Claims (16)

1. isolating polynucleotide is characterized in that, described polynucleotide contain the nucleotide sequence that coding has the polypeptide that influences the active function of ELK1, 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, SEQ ID NO:16 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, SEQ ID NO:16 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, SEQ ID NO:16.

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, SEQ ID NO:15;

(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, SEQ IDNO:15.

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, SEQ ID NO:16; 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, SEQ ID NO:16.

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 isolating polynucleotide in influencing the ELK1 activity is characterized in that described polynucleotide are selected from the described polynucleotide of claim 1, these polynucleotide heterogenous expression in host cell can influence the ELK1 activity.

12. the described application of claim 11, 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 ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, these polynucleotide heterogenous expression in host cell can suppress the activation of ELK1.

13. the described application of claim 11, 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:11, SEQ ID NO:13, SEQ ID NO:15, these polynucleotide heterogenous expression in host cell can promote the activation of ELK1.

14. described polynucleotide of claim 1 or the described polypeptide of claim 5 prevent and/or treat purposes in the medicine of nervous system disorders or tumour in preparation.

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

16. the external detection method of nervous system disorders 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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