CN1438320A - Diagnosis, treatment method for tumor using human CYLD gene and medicine development method - Google Patents

Abstract

The invention discloses a method for diagnosing tumour, especially trichoepithelioma; it includes checking if individual CYLD gene, transcription and/or albumen have aberrance or not comparing with the normal, having the aberrance testifies that the possibility in suffing tumour of the individual is bigger than normal public. The invention also discloses correspondent checking reagent box, the method and remedy for curing tumour.

Description

Utilize human's CYLD gene to tumour diagnose, the method for treatment and drug development

Technical field

The present invention relates to biotechnology and medical field.More specifically, the present invention relates to utilize the especially method of trichoepithelioma of diagnosis of human's CYLD gene and coded product thereof and treatment tumour, and contain CYLD gene and/or proteic pharmaceutical composition.

Background technology

Trichoepithelioma is a kind of dermatoma.It is fallen ill at preadolescence usually, good sending out, especially nasolabial groove and upper lip in face, and the back, scalp, neck, trunk, four limbs also can be got involved.Its skin decreases and is semisphere or taper shape, and quality is solid, and is translucent, and yellow or incarnadine, bigger sometimes skin decrease and show visible telangiectasis.Trichoepithelioma not only influences beauty treatment, because it makes the back, and scalp, neck, trunk, four limbs are got involved, so severe patient also can be met difficulty in life.This disease is an autosome dominant disease, and the mechanism of its morbidity is not clear at present.Still there is not at present satisfied methods of treatment for this disease.

Yet, up to now, still not fully do not disclose the definite reason of trichoepithelioma, also nobody discloses trichoepithelioma and there is direct dependency in certain albumen.In addition, this area also lacks the effective ways of early diagnosis trichoepithelioma disease and the effective means of non-operative treatment trichoepithelioma.

CYLD is a kind of known protein, and its essential information is as follows:

English: Gene from:Homo sapiens chromosome 16 working draft sequencesegment.

NCBI：Contig?NT_030834

GENE：Gene?from：Homo?sapiens?chromosome?16?working?draft?sequencesegment.gi|17488444：243550-308161

The dna sequence dna of CYLD is shown in SEQ ID NO:1, and ORF is positioned at the 392-3262 position, 956 amino acid whose protein of the total length of encoding (SEQ ID NO:2).Other information of CYLD can be from Http: //www.ncbi.nlm.nih.gov obtains.

Once the be in the news sudden change of its exon of CYLD gene has caused generation cylindromatous, but does not still have the relation of any article report CYLD gene and trichoepithelioma in the world.The hereditary mechanistic information of trichoepithelioma can obtain from the OMIM of NCBI.

Therefore, this area presses for the effective ways of new diagnosis of exploitation and treatment trichoepithelioma, and relevant medicine, diagnostic techniques and reagent.

Summary of the invention

One object of the present invention just provides a kind of new diagnosis (especially early diagnosis) tumour especially method and the detection kit of trichoepithelioma.

Another object of the present invention provides the especially method of trichoepithelioma of a kind of new treatment tumour.

A further object of the present invention provides a kind of especially pharmaceutical composition of trichoepithelioma of tumour for the treatment of.

In a first aspect of the present invention, a kind of method that the tumor susceptibility of individuality is diagnosed is provided, it comprises step:

Detect this individual CYLD gene, transcript and/or albumen, and compare with normal CYLD gene, transcript and/or albumen,

The possibility that there are differences with regard to showing this individuality trouble tumour is higher than normal population.

Preferably, detected is gene or the transcript of CYLD, and with normal CYLD nucleotide sequence comparing difference.More preferably, described tumour is a trichoepithelioma, and described difference is selected from: the 1853rd A disappearance among the SEQ ID NO:1.

In a second aspect of the present invention, a kind of method for the treatment of tumour is provided, it comprises step: the normal CYLD albumen of using safe and effective amount to the patient of the described treatment of needs.Preferably, CYLD albumen is locally applied to affected area.

In a third aspect of the present invention, provide a kind of CYLD albumen aspect pharmaceutical compositions purposes and corresponding pharmaceutical compositions, it contains the people CYLD albumen and the pharmaceutically acceptable carrier of safe and effective amount.Preferably, it is an ointment.

In a fourth aspect of the present invention, a kind of test kit that detects tumour is provided, it comprises the primer of specific amplification CYLD gene or transcript.Preferably, it also contain with mutable site bonded probe and/or identification the mutational site enzyme.

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

Description of drawings

Fig. 1 has shown the pedigree chart of the trichoepithelioma family of an autosomal dominant inheritance.

Fig. 2 is typical patient's a facial photo.

Fig. 3 is patient's a biopsy pathology photo.

Fig. 4 has shown the sequence variation in the CYLD gene, and wherein the 1853rd of SEQ ID NO:1 the disappearance of base has taken place, thereby has caused phase shift mutation, and its encoded protein is shortened.

Embodiment

The inventor is through extensive and deep research, and find first and proved that CYLD and trichoepithelioma are closely related, and found its new function: the change of CYLD will directly cause trichoepithelioma.Further research hints that also CYLD is a cancer suppressor gene, and its sudden change can cause the generation of kinds of tumors.Finished the present invention on this basis.

By the trichoepithelioma family of an autosomal dominant inheritance being carried out genetic linkage analysis, candidate gene screening and large scale sequencing, confirmed that all patients in this family are in 1853 disappearances that base takes place of No. 10 exon of CYLD gene, thereby caused phase shift mutation, its amino acid coding is shortened, protein translation is unusual, has finally caused the generation of trichoepithelioma.The disappearance of base A if taken place at 1853 places then the total length of only encoding is 496 amino acid whose albumen (SEQ ID NO:5) and the improper little peptide of part in normal CYLD coding 956 amino acid whose albumen of total length (SEQ ID NO:2).

The sudden change of CYLD has caused the human individual to show tumour, especially trichoepithelioma.We studies show that, the existence of normal CYLD is the key of skin normal physiological state, and the sudden change of CYLD simultaneously also can cause other many tumours.

In view of the variation of human CYLD is to cause one of immediate cause of tumours such as trichoepithelioma.Therefore, can be used for diagnosing and treating human tumour according to the medicine and the diagnoses and treatment technology of this gene and expression product thereof design, comprising but be not limited to: trichoepithelioma, eccrine spiradenoma, melanoma, cylindroma, cancer of the stomach, liver cancer etc.

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

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

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

The CYLD encoding sequence is inserted suitable expression vector, change host cell again over to, just can isolate CYLD albumen.

Based on new risk of the present invention, CYLD albumen or polypeptide have many-sided new purposes.These purposes include, but is not limited to: the direct disease (as tumours such as trichoepitheliomas) due to the low or forfeiture and be used to screen the material that promotes the CYLD protein function as pharmacological agent CYLD protein function, and as antibody, polypeptide or other part.The peptide molecule that can stimulate people CYLD protein function that can be used for seeking therapeutic value with the recombinant human CYLD protein screening peptide library of expressing.

On the other hand, the present invention also comprises people CYLD DNA or the polypeptide of its fragment coding has specific polyclonal antibody and monoclonal antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into human's CYLD gene product or fragment.Preferably, refer to that those can combine with human's CYLD gene product or fragment but nonrecognition and be incorporated into the antibody of other irrelevant antigen molecule.Among the present invention antibody comprise those can in conjunction with and suppress the proteic molecule of people CYLD, comprise that also those do not influence the antibody of people CYLD protein function.

The present invention not only comprises complete mono-clonal or polyclonal antibody, but also comprises having immunocompetent antibody fragment, as Fab ' or (Fab) ₂Fragment; Heavy chain of antibody; Light chain of antibody; Genetically engineered strand Fv molecule; Or chimeric antibody.

Antibody of the present invention can be prepared by the known various technology of those skilled in that art.For example, the human's CYLD gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing human CYLD albumen or its have antigenic segmental cell and can be used to immune animal and produce antibody.Multiple adjuvant can be used for the enhancing immunity reaction, includes but not limited to freund's adjuvant etc.

Antibody of the present invention also can be monoclonal antibody.This type of monoclonal antibody can utilize hybridoma technology to prepare.Antibody of the present invention comprises the antibody that can block people CYLD protein function and the antibody that does not influence people CYLD protein function.Each antibody-like of the present invention can utilize the fragment or the functional zone of human's CYLD gene product, obtains by the routine immunization technology.These fragments or functional zone can utilize recombinant methods or utilize Peptide synthesizer synthetic.Can come immune animal and produce with the gene product of producing in the prokaryotic cell prokaryocyte (for example E.Coli) with the unmodified form bonded antibody of human's CYLD gene product; With posttranslational modification form bonded antibody (as the albumen or the polypeptide of glycosylation or phosphorylation), can come immune animal and obtain with the gene product that produces in the eukaryotic cell (for example yeast or insect cell).

The proteic antibody of anti-people CYLD can be used in the immunohistochemistry technology, detects the people CYLD albumen in the biopsy specimen.A kind of preferred anti-CYLD antibody is that the antibody of the normal CYLD of nonrecognition but identification sudden change CYLD is (because the coded albumen of the CYLD of the sudden change albumen more coded than normal CYLD is much smaller, so can design the antibody of only discerning unusual small protein), perhaps discern normal CYLD but the antibody of nonrecognition sudden change CYLD.Utilize this antibody to the proteic specificity difference of normal and unusual CYLD, the trichoepithelioma susceptibility that can carry out protein level easily detects.

Utilize CYLD albumen of the present invention,, can filter out with CYLD albumen interactional material takes place, as inhibitor, agonist or antagonist etc. by various conventional screening methods.

CYLD albumen of the present invention and antibody thereof, inhibitor, agonist, antagonist etc. when using (administration) in treatment, can provide different effects.Usually, can these materials are formulated in nontoxic, inert and the pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5-8 usually, and preferably pH is about 6-8, although the pH value can be with being changed to some extent by preparation Substance Properties and illness to be treated.The pharmaceutical composition for preparing can carry out administration by conventional route, comprising (but being not limited to): intramuscular, intravenously, subcutaneous or topical (comprising affected area).Be preferably topical.

Normal CYLD polypeptide can be directly used in disease treatment, for example, is used for the treatment of trichoepithelioma aspect.When using CYLD albumen of the present invention, also can use the medicament of other treatment tumour simultaneously.

The present invention also provides a kind of pharmaceutical composition, and it contains CYLD albumen of the present invention and the pharmaceutically acceptable carrier or the vehicle of safe and effective amount.This class carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Pharmaceutical preparation should be complementary with administering mode.Pharmaceutical composition of the present invention can be made into the injection form, for example is prepared by ordinary method with the physiological saline or the aqueous solution that contains glucose and other assistant agents.Pharmaceutical composition such as ointment, tablet and capsule can be prepared by ordinary method.Pharmaceutical composition such as ointment, injection, solution, tablet and capsule should be made under aseptic condition.The dosage of activeconstituents is the treatment significant quantity, for example every day about 0.1 microgram/kg body weight-Yue 10 mg/kg body weight.In addition, polypeptide of the present invention also can use with the other treatment agent.

When making pharmaceutical composition, be that the CYLD albumen of safe and effective amount or its antagonist, agonist are applied to Mammals, wherein this safe and effective amount is usually at least about 0.1 microgram/kg body weight, and in most of the cases be no more than about 10 mg/kg body weight, preferably this dosage is about 0.1 microgram/kg body weight-Yue 100 micrograms/kg body weight.Certainly, concrete dosage also should be considered factors such as route of administration, patient health situation, and these all are within the skilled practitioners skill.

The proteic polynucleotide of people CYLD also can be used for multiple therapeutic purpose.Gene therapy technology can be used for treating because cell proliferation, growth or the metabolic disturbance due to the proteic expression of CYLD of the proteic nothing expression of CYLD or unusual/non-activity.The method that structure carries the recombinant viral vector of CYLD gene is found in existing document (Sambrook, et al.).The human's CYLD gene of recombinating in addition can be packaged in the liposome, and then is transferred in the cell.

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

The invention still further relates to the diagnostic testing process of quantitative and detection and localization people CYLD protein level.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.

Whether having the proteic method of CYLD in a kind of detection test sample is to utilize the proteic specific antibody of CYLD to detect, and it comprises: sample is contacted with the CYLD protein specific antibody; Observe whether form antibody complex, formed antibody complex and just represented to exist in the sample CYLD albumen.

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

Whether unusual the present invention also provide a kind of human's CYLD gene that detects to express method, and it comprises step: (a) determine the 1853rd Nucleotide in sequence shown in the SEQ of the human's CYLD gene ID NO:1; (b) whether detection exists base A disappearance in described position.

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

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

Embodiment 1

Determining of autosomal dominant trichoepithelioma family

1.1 object

The big family of trichoepithelioma, totally 4 generations, 53 people.Wherein the trichoepithelioma patient has 20 people (Fig. 1).

Embodiment 2

Determine that the CYLD sudden change is the immediate cause that causes trichoepithelioma

2.1 genetic linkage analysis

Utilize microsatellite marker to carry out the linkage analysis of this family.Utilize 384 pairs of micro-satellite primers (RESEARCH GENETICS) that whole genome is scanned altogether, with this family trichoepithelioma assignment of genes gene mapping on people's No. 16 karyomit(e).Further synthetic primer navigates to Disease-causing gene in the genetic distance of 14 between microsatellite marker d16s753 and the d16s3253 (cM) centimorgan, as shown in Figure 1.Nearly 1,000 4 hundred ten thousand base pairs of this physical distance, more than 100 gene.

Mark				The Lod value at θ place=						??0.4	Maximum value
														Position (cm)	??n	??het	??0.0	??0.01	??0.05	??0.1	??0.2	??0.3		??Lod	???θ
??D16s3093	??52.26	??6	??0.7315	??-inf.	??-5.24	??-1.95	??-0.72	??0.18	??0.36	??0.22	??0.36	??0.3
													??D16s753	??57.79	??6	??/	??-inf.	??1.27	??1.77	??1.80	??1.51	??1.01	??0.45	??1.80	??0.1
??D16s411	??59.68	??7	??0.7943	??6.32	??6.22	??5.80	??5.25	??4.05	??2.71	??1.22	??6.32	??0.0
													??D16s3396	??63.78	??5	??/	??2.96	??2.91	??2.70	??2.43	??1.83	??1.16	??0.46	??2.96	??0.0
??D16s757	??65.10	??4	??/	??6.29	??6.19	??5.77	??5.22	??4.02	??2.68	??1.20	??6.29	??0.0
													??D16s770	??65.77	??4	??/	??3.26	??3.20	??2.96	??2.64	??1.96	??1.21	??0.43	??3.26	??0.0
??D16s2623	??66.10	??5	??/	??6.15	??6.05	??5.63	??5.09	??3.92	??2.60	??1.16	??6.15	??0.0
													??D16s3253	??71.77	??6	??/	??-inf.	??0.49	??1.05	??1.17	??1.05	??0.77	??0.42	??1.17	??0.1
??D16s2260	??81.15	??5	??/	??-inf.	??-5.50	??-2.26	??-1.06	??-0.16	??0.09	??0.06	??0.09	??0.3

2.2 candidate gene

With DNA extraction agent box (QIAGEN company) from family's blood sample the extracting genomic dna as template.

To candidate gene, design PCR primer, pcr amplification goes out candidate gene, checks order then.Detect through order-checking, find that the trichoepithelioma of CYLD and this family is closely related candidate gene.

CYLD has been designed 34 pairs of primers to check order.Wherein, when the PCR product that goes out with following a pair of primer amplification checks order, the normal people that the middle CYLD that finds each patient of this trichoepithelioma family contrasts this family finds 1853 the disappearances that base take place of this gene at No. 10 exon, be the 1853rd disappearance that base A has taken place of SEQ ID NO:1, thereby caused phase shift mutation, made its encoded protein shorten to 496 amino acid.This sudden change has finally caused the generation (see figure 4) of trichoepithelioma.

Title	Sequence (5 ú 3i)	Numbering
			The CYLD_15 forward	tctgcagtga?tagcttttct?gaca	SEQ?ID?NO：3
CYLD_15 is reverse	cagtctcacc?aagatgccca?atac	SEQ?ID?NO：4

In addition, in order to verify the specificity of this site to the generation of trichoepithelioma, casual inspection 100 situations that do not have the people of close source relation in this site, do not find any sudden change.This illustrates that this site mutation is not due to the polymorphism among the crowd, but the specific mutant site of trichoepithelioma.Therefore shown that CYLD is closely related with the generation of the mankind's trichoepithelioma, this gene and coded product thereof can play an important role to the mankind's trichoepithelioma.

Embodiment 3

The preparation of trichoepithelioma detection kit

Prepare a kit; It contains: title sequence (5 ú 3i) numbering concentration forward primer tctgcagtga tagcttttct gaca SEQ ID NO:3 dry powder 2OD reverse primer cagtctcacc aagatgccca atac SEQ ID NO:4 dry powder 2ODPCR reactant liquor contains Taq enzyme dNTP magnesium ion PCR reaction buffer PCR product purification box and contains the in a small amount solution of PCR product purification, and DNA adsorption column sequencing reaction liquid contains Big Dye

Extract patients'blood 3ml to be detected, use ordinary method (or using specific test kit) from blood, to extract DNA.PCR primer in the trichoepithelioma detection kit is diluted to 1 μ mol/ μ l, is that template is carried out the PCR reaction with the primer that is provided with the DNA that is extracted.Use the PRC product purification box that detection kit provided that the PCR product is carried out purifying.The product of purifying is carried out directly checking order behind the sequencing reaction.Whether the resulting sequence of observation order-checking has phase shift mutation.

Embodiment 4

Preparation of drug combination

Normal protein that CYLD is coded and common ointment material are made the ointment that contains the coded normal protein of CYLD by specific mixed.During use, ointment is imposed on the affected part, CYLD coded normal protein in affected part is replenished, and makes patient owing to the dermatosis that the coded normal protein of shortage CYLD causes is alleviated, until last disappearance.Or the normal protein that CYLD is coded makes injection, during use subcutaneous injection carried out in the affected part, directly replenishes the CYLD proteins encoded of affected area, the physiological situation of patient's skin improved, thereby reach the purpose of treatment.

All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

＜110〉＜120〉CYLD、＜130〉020614＜160〉5＜170〉PatentIn version 3.0＜210〉1＜211〉5371＜212〉DNA＜213〉 ( Homo sapiens )＜220〉＜221〉CDS＜222〉 ( 392 ) .. ( 3262 )＜400〉1gggggcgggc ccaggtagca ggtttggctg cgcgggggcc gcgcgtcgga gtttccccct 60ttctagggtg aggatggttc tacacagcca cccggagttc cttagttgaa aggtgcgccc 120tgctgtgaca gaatgtggta attgtaatct ttaacatttt catgtaaaac atatttcctg 180atcatctttc cattgtcttc atggaaaatt gataaatatt tgtgccttcc aactctcgtc 240ttggttgaat gacttcatct taatacaaca tggacaccac gttgctgaaa acatgctttg 300ggactgccac tgaatttatc ttttgcggtt ttatgacaaa gttattagta gtttcccttt 360tttgaattag tattttgaag ttaatatcac a atg agt tca ggc tta tgg agc 412

Met?Ser?Ser?Gly?Leu?Trp?Ser

1???????????????5caa?gaa?aaa?gtc?act?tca?ccc?tac?tgg?gaa?gag?cgg?att?ttt?tac?ttg??????460Gln?Glu?Lys?Val?Thr?Ser?Pro?Tyr?Trp?Glu?Glu?Arg?Ile?Phe?Tyr?Leu

10??????????????????15?????????????????20ctt?ctt?caa?gaa?tgc?agc?gtt?aca?gac?aaa?caa?aca?caa?aag?ctc?ctt??????508Leu?Leu?Gln?Glu?Cys?Ser?Val?Thr?Asp?Lys?Gln?Thr?Gln?Lys?Leu?Leu

25?????????????????30??????????????????35aaa?gta?ccg?aag?gga?agt?ata?gga?cag?tat?att?caa?gat?cgt?tct?gtg??????556Lys?Val?Pro?Lys?Gly?Ser?Ile?Gly?Gln?Tyr?Ile?Gln?Asp?Arg?Ser?Val40??????????????????45?????????????????50??????????????????55ggg?cat?tca?agg?att?cct?tct?gca?aaa?ggc?aag?aaa?aat?cag?att?gga??????604Gly?His?Ser?Arg?Ile?Pro?Ser?Ala?Lys?Gly?Lys?Lys?Asn?Gln?Ile?Gly

60??????????????????65??????????????????70tta?aaa?att?cta?gag?caa?cct?cat?gca?gtt?ctc?ttt?gtt?gat?gaa?aag??????652Leu?Lys?Ile?Leu?Glu?Gln?Pro?His?Ala?Val?Leu?Phe?Val?Asp?Glu?Lys

75??????????????????80??????????????????85gat?gtt?gta?gag?ata?aat?gaa?aag?ttc?aca?gag?tta?ctt?ttg?gca?att??????700Asp?Val?Val?Glu?Ile?Asn?Glu?Lys?Phe?Thr?Glu?Leu?Leu?Leu?Ala?Ile

90??????????????????95??????????????????100acc?aat?tgt?gag?gag?agg?ttc?agc?ctg?ttt?aaa?aac?aga?aac?aga?cta??????748Thr?Asn?Cys?Glu?Glu?Arg?Phe?Ser?Leu?Phe?Lys?Asn?Arg?Asn?Arg?Leu

105?????????????????110?????????????????115agt?aaa?ggc?ctc?caa?ata?gac?gtg?ggc?tgt?cct?gtg?aaa?gta?cag?ctg??????796Ser?Lys?Gly?Leu?Gln?Ile?Asp?Val?Gly?Cys?Pro?Val?Lys?Val?Gln?Leu120?????????????????125?????????????????130?????????????????135aga?tct?ggg?gaa?gaa?aaa?ttt?cct?gga?gtt?gta?cgc?ttc?aga?gga?ccc??????844Arg?Ser?Gly?Glu?Glu?Lys?Phe?Pro?Gly?Val?Val?Arg?Phe?Arg?Gly?Pro

140?????????????????145?????????????????150ctg?tta?gca?gag?agg?aca?gtc?tcc?gga?ata?ttc?ttt?gga?gtt?gaa?ttg??????892Leu?Leu?Ala?Glu?Arg?Thr?Val?Ser?Gly?Ile?Phe?Phe?Gly?Val?Glu?Leu

155?????????????????160?????????????????165ctg?gaa?gaa?ggt?cgt?ggt?caa?ggt?ttc?act?gac?ggg?gtg?tac?caa?ggg??????940Leu?Glu?Glu?Gly?Arg?Gly?Gln?Gly?Phe?Thr?Asp?Gly?Val?Tyr?Gln?Gly

170?????????????????175?????????????????180aaa?cag?ctt?ttt?cag?tgt?gat?gaa?gat?tgt?ggc?gtg?ttt?gtt?gca?ttg??????988Lys?Gln?Leu?Phe?Gln?Cys?Asp?Glu?Asp?Cys?Gly?Val?Phe?Val?Ala?Leu

185?????????????????190?????????????????195gac?aag?cta?gaa?ctc?ata?gaa?gat?gat?gac?act?gca?ttg?gaa?agt?gat?????1036Asp?Lys?Leu?Glu?Leu?Ile?Glu?Asp?Asp?Asp?Thr?Ala?Leu?Glu?Ser?Asp200?????????????????205?????????????????210?????????????????215tac?gca?ggt?cct?ggg?gac?aca?atg?cag?gtc?gaa?ctt?cct?cct?ttg?gaa?????1084Tyr?Ala?Gly?Pro?Gly?Asp?Thr?Met?Gln?Val?Glu?Leu?Pro?Pro?Leu?Glu

220?????????????????225?????????????????230ata?aac?tcc?aga?gtt?tct?ttg?aag?gtt?gga?gaa?aca?ata?gaa?tct?gga?????1132Ile?Asn?Ser?Arg?Val?Ser?Leu?Lys?Val?Gly?Glu?Thr?Ile?Glu?Ser?Gly

235?????????????????240?????????????????245aca?gtt?ata?ttc?tgt?gat?gtt?ttg?cca?gga?aaa?gaa?agc?tta?gga?tat?????1180Thr?Val?Ile?Phe?Cys?Asp?Val?Leu?Pro?Gly?Lys?Glu?Ser?Leu?Gly?Tyr

250?????????????????255?????????????????260ttt?gtt?ggt?gtg?gac?atg?gat?aac?cct?att?ggc?aac?tgg?gat?gga?aga?????1228Phe?Val?Gly?Val?Asp?Met?Asp?Asn?Pro?Ile?Gly?Asn?Trp?Asp?Gly?Arg

265?????????????????270?????????????????275ttt?gat?gga?gtg?cag?ctt?tgt?agt?ttt?gcg?tgt?gtt?gaa?agt?aca?att?????1276Phe?Asp?Gly?Val?Gln?Leu?Cys?Ser?Phe?Ala?Cys?Val?Glu?Ser?Thr?Ile280?????????????????285?????????????????290?????????????????295cta?ttg?cac?atc?aat?gat?atc?atc?cca?gct?tta?tca?gag?agt?gtg?acg?????1324Leu?Leu?His?Ile?Asn?Asp?Ile?Ile?Pro?Ala?Leu?Ser?Glu?Ser?Val?Thr

300?????????????????305?????????????????310cag?gaa?agg?agg?cct?ccc?aaa?ctt?gcc?ttt?atg?tca?aga?ggt?gtt?ggg?????1372Gln?Glu?Arg?Arg?Pro?Pro?Lys?Leu?Ala?Phe?Met?Ser?Arg?Gly?Val?Gly

315?????????????????320?????????????????325gac?aaa?ggt?tca?tcc?agt?cat?aat?aaa?cca?aag?gct?aca?gga?tct?acc?????1420Asp?Lys?Gly?Ser?Ser?Ser?His?Asn?Lys?Pro?Lys?Ala?Thr?Gly?Ser?Thr

330?????????????????335?????????????????340tca?gac?cct?gga?aat?aga?aac?aga?tct?gaa?tta?ttt?tat?acc?tta?aat?????1468Ser?Asp?Pro?Gly?Asn?Arg?Asn?Arg?Ser?Glu?Leu?Phe?Tyr?Thr?Leu?Asn

345?????????????????350?????????????????355ggg?tct?tct?gtt?gac?tca?caa?cca?caa?tcc?aaa?tca?aaa?aat?aca?tgg?????1516Gly?Ser?Ser?Val?Asp?Ser?Gln?Pro?Gln?Ser?Lys?Ser?Lys?Asn?Thr?Trp360?????????????????365?????????????????370?????????????????375tac?att?gat?gaa?gtt?gca?gaa?gac?cct?gca?aaa?tct?ctt?aca?gag?ata?????1564Tyr?Ile?Asp?Glu?Val?Ala?Glu?Asp?Pro?Ala?Lys?Ser?Leu?Thr?Glu?Ile

380?????????????????385?????????????????390tct?aca?gac?ttt?gac?cgt?tct?tca?cca?cca?ctc?cag?cct?cct?cct?gtg?????1612Ser?Thr?Asp?Phe?Asp?Arg?Ser?Ser?Pro?Pro?Leu?Gln?Pro?Pro?Pro?Val

395?????????????????400?????????????????405aac?tca?ctg?acc?acc?gag?aac?aga?ttc?cac?tct?tta?cca?ttc?agt?ctc?????1660Asn?Ser?Leu?Thr?Thr?Glu?Asn?Arg?Phe?His?Ser?Leu?Pro?Phe?Ser?Leu

410?????????????????415?????????????????420acc?aag?atg?ccc?aat?acc?aat?gga?agt?att?ggc?cac?agt?cca?ctt?tct?????1708Thr?Lys?Met?Pro?Asn?Thr?Asn?Gly?Ser?Ile?Gly?His?Ser?Pro?Leu?Ser

425?????????????????430?????????????????435ctg?tca?gcc?cag?tct?gta?atg?gaa?gag?cta?aac?act?gca?ccc?gtc?caa?????1756Leu?Ser?Ala?Gln?Ser?Val?Met?Glu?Glu?Leu?Asn?Thr?Ala?Pro?Val?Gln440?????????????????445?????????????????450?????????????????455gag?agt?cca?ccc?ttg?gcc?atg?cct?cct?ggg?aac?tca?cat?ggt?cta?gaa?????1804Glu?Ser?Pro?Pro?Leu?Ala?Met?Pro?Pro?Gly?Asn?Ser?His?Gly?Leu?Glu

460?????????????????465?????????????????470gtg?ggc?tca?ttg?gct?gaa?gtt?aag?gag?aac?cct?cct?ttc?tat?ggg?gta?????1852Val?Gly?Ser?Leu?Ala?Glu?Val?Lys?Glu?Asn?Pro?Pro?Phe?Tyr?Gly?Val

475?????????????????480?????????????????485atc?cgt?tgg?atc?ggt?cag?cca?cca?gga?ctg?aat?gaa?gtg?ctc?gct?gga?????1900Ile?Arg?Trp?Ile?Gly?Gln?Pro?Pro?Gly?Leu?Asn?Glu?Val?Leu?Ala?Gly

490?????????????????495?????????????????500ctg?gaa?ctg?gaa?gat?gag?tgt?gca?ggc?tgt?acg?gat?gga?acc?ttc?aga?????1948Leu?Glu?Leu?Glu?Asp?Glu?Cys?Ala?Gly?Cys?Thr?Asp?Gly?Thr?Phe?Arg

505?????????????????510?????????????????515ggc?act?cgg?tat?ttc?acc?tgt?gcc?ctg?aag?aag?gcg?ctg?ttt?gtg?aaa?????1996Gly?Thr?Arg?Tyr?Phe?Thr?Cys?Ala?Leu?Lys?Lys?Ala?Leu?Phe?Val?Lys520?????????????????525?????????????????530?????????????????535ctg?aag?agc?tgc?agg?cct?gac?tct?agg?ttt?gca?tca?ttg?cag?ccg?gtt?????2044Leu?Lys?Ser?Cys?Arg?Pro?Asp?Ser?Arg?Phe?Ala?Ser?Leu?Gln?Pro?Val

540?????????????????545?????????????????550tcc?aat?cag?att?gag?cgc?tgt?aac?tct?tta?gca?ttt?gga?ggc?tac?tta?????2092Ser?Asn?Gln?Ile?Glu?Arg?Cys?Asn?Ser?Leu?Ala?Phe?Gly?Gly?Tyr?Leu

555?????????????????560?????????????????565agt?gaa?gta?gta?gaa?gaa?aat?act?cca?cca?aaa?atg?gaa?aaa?gaa?ggc?????2140Ser?Glu?Val?Val?Glu?Glu?Asn?Thr?Pro?Pro?Lys?Met?Glu?Lys?Glu?Gly

570?????????????????575?????????????????580ttg?gag?ata?atg?att?ggg?aag?aag?aaa?ggc?atc?cag?ggt?cat?tac?aat?????2188Leu?Glu?Ile?Met?Ile?Gly?Lys?Lys?Lys?Gly?Ile?Gln?Gly?His?Tyr?Asn

585?????????????????590?????????????????595tct?tgt?tac?tta?gac?tca?acc?tta?ttc?tgc?tta?ttt?gct?ttt?agt?tct?????2236Ser?Cys?Tyr?Leu?Asp?Ser?Thr?Leu?Phe?Cys?Leu?Phe?Ala?Phe?Ser?Ser600?????????????????605?????????????????610?????????????????615gtt?ctg?gac?act?gtg?tta?ctt?aga?ccc?aaa?gaa?aag?aac?gat?gta?gaa?????2284Val?Leu?Asp?Thr?Val?Leu?Leu?Arg?Pro?Lys?Glu?Lys?Asn?Asp?Val?Glu

620?????????????????625?????????????????630tat?tat?agt?gaa?acc?caa?gag?cta?ctg?agg?aca?gaa?att?gtt?aat?cct?????2332Tyr?Tyr?Ser?Glu?Thr?Gln?Glu?Leu?Leu?Arg?Thr?Glu?Ile?Val?Asn?Pro

635?????????????????640?????????????????645ctg?aga?ata?tat?gga?tat?gtg?tgt?gcc?aca?aaa?att?atg?aaa?ctg?agg?????2380Leu?Arg?Ile?Tyr?Gly?Tyr?Val?Cys?Ala?Thr?Lys?Ile?Met?Lys?Leu?Arg

650?????????????????655?????????????????660aaa?ata?ctt?gaa?aag?gtg?gag?gct?gca?tca?gga?ttt?acc?tct?gaa?gaa?????2428Lys?Ile?Leu?Glu?Lys?Val?Glu?Ala?Ala?Ser?Gly?Phe?Thr?Ser?Glu?Glu

665?????????????????670?????????????????675aaa?gat?cct?gag?gaa?ttc?ttg?aat?att?ctg?ttt?cat?cat?att?tta?agg?????2476Lys?Asp?Pro?Glu?Glu?Phe?Leu?Asn?Ile?Leu?Phe?His?His?Ile?Leu?Arg680?????????????????685?????????????????690?????????????????695gta?gaa?cct?ttg?cta?aaa?ata?aga?tca?gca?ggt?caa?aag?gta?caa?gat?????2524Val?Glu?Pro?Leu?Leu?Lys?Ile?Arg?Ser?Ala?Gly?Gln?Lys?Val?Gln?Asp

700?????????????????705?????????????????710tgt?tac?ttc?tat?caa?att?ttt?atg?gaa?aaa?aat?gag?aaa?gtt?ggc?gtt?????2572Cys?Tyr?Phe?Tyr?Gln?Ile?Phe?Met?Glu?Lys?Asn?Glu?Lys?Val?Gly?Val

715?????????????????720?????????????????725ccc?aca?att?cag?cag?ttg?tta?gaa?tgg?tct?ttt?atc?aac?agt?aac?ctg?????2620Pro?Thr?Ile?Gln?Gln?Leu?Leu?Glu?Trp?Ser?Phe?Ile?Asn?Ser?Asn?Leu

730?????????????????735?????????????????740aaa?ttt?gca?gag?gca?cca?tca?tgt?ctg?att?att?cag?atg?cct?cga?ttt?????2668Lys?Phe?Ala?Glu?Ala?Pro?Ser?Cys?Leu?Ile?Ile?Gln?Met?Pro?Arg?Phe

745?????????????????750?????????????????755gga?aaa?gac?ttt?aaa?cta?ttt?aaa?aaa?att?ttt?cct?tct?ctg?gaa?tta?????2716Gly?Lys?Asp?Phe?Lys?Leu?Phe?Lys?Lys?Ile?Phe?Pro?Ser?Leu?Glu?Leu760?????????????????765?????????????????770?????????????????775aat?ata?aca?gat?tta?ctt?gaa?gac?act?ccc?aga?cag?tgc?cgg?ata?tgt?????2764Asn?Ile?Thr?Asp?Leu?Leu?Glu?Asp?Thr?Pro?Arg?Gln?Cys?Arg?Ile?Cys

780?????????????????785?????????????????790gga?ggg?ctt?gca?atg?tat?gag?tgt?aga?gaa?tgc?tac?gac?gat?ccg?gac?????2812Gly?Gly?Leu?Ala?Met?Tyr?Glu?Cys?Arg?Glu?Cys?Tyr?Asp?Asp?Pro?Asp

795?????????????????800?????????????????805atc?tca?gct?gga?aaa?atc?aag?cag?ttt?tgt?aaa?acc?tgc?aac?act?caa?????2860Ile?Ser?Ala?Gly?Lys?Ile?Lys?Gln?Phe?Cys?Lys?Thr?Cys?Asn?Thr?Gln

810?????????????????815?????????????????820gtc?cac?ctt?cat?ccg?aag?agg?ctg?aat?cat?aaa?tat?aac?cca?gtg?tca?????2908Val?His?Leu?His?Pro?Lys?Arg?Leu?Asn?His?Lys?Tyr?Asn?Pro?Val?Ser

825?????????????????830?????????????????835ctt?ccc?aaa?gac?tta?ccc?gac?tgg?gac?tgg?aga?cac?ggc?tgc?atc?cct?????2956Leu?Pro?Lys?Asp?Leu?Pro?Asp?Trp?Asp?Trp?Arg?His?Gly?Cys?Ile?Pro840?????????????????845?????????????????850?????????????????855tgc?cag?aat?atg?gag?tta?ttt?gct?gtt?ctc?tgc?ata?gaa?aca?agc?cac?????3004Cys?Gln?Asn?Met?Glu?Leu?Phe?Ala?Val?Leu?Cys?Ile?Glu?Thr?Ser?His

860?????????????????865?????????????????870tat?gtt?gct?ttt?gtg?aag?tat?ggg?aag?gac?gat?tct?gcc?tgg?ctc?ttc?????3052Tyr?Val?Ala?Phe?Val?Lys?Tyr?Gly?Lys?Asp?Asp?Ser?Ala?Trp?Leu?Phe

875?????????????????880?????????????????885ttt?gac?agc?atg?gcc?gat?cgg?gat?ggt?ggt?cag?aat?ggc?ttc?aac?att?????3100Phe?Asp?Ser?Met?Ala?Asp?Arg?Asp?Gly?Gly?Gln?Asn?Gly?Phe?Asn?Ile

890?????????????????895?????????????????900cct?caa?gtc?acc?cca?tgc?cca?gaa?gta?gga?gag?tac?ttg?aag?atg?tct?????3148Pro?Gln?Val?Thr?Pro?Cys?Pro?Glu?Val?Gly?Glu?Tyr?Leu?Lys?Met?Ser

905?????????????????910?????????????????915ctg?gaa?gac?ctg?cat?tcc?ttg?gac?tcc?agg?aga?atc?caa?ggc?tgt?gca?????3196Leu?Glu?Asp?Leu?His?Ser?Leu?Asp?Ser?Arg?Arg?Ile?Gln?Gly?Cys?Ala920?????????????????925?????????????????930?????????????????935cga?aga?ctg?ctt?tgt?gat?gca?tat?atg?tgc?atg?tac?cag?agt?cca?aca?????3244Arg?Arg?Leu?Leu?Cys?Asp?Ala?Tyr?Met?Cys?Met?Tyr?Gln?Ser?Pro?Thr

940?????????????????945?????????????????950atg?agt?ttg?tac?aaa?taa?ctggggtcat?cgggaaaggc?aaagaaactg????????????3292Met?Ser?Leu?Tyr?Lys

955aaggcagagt cctaacgttg catcttattc gagctggcag ttctgttcac gtccattgcc 3352ggcaatggat gtctttgtgg tgatgatcct tcagaaaagg atgcctctgt ttaaaaacaa 3412attgcttttg tgtccctgaa gtatttaata agaagcattt tgcactctag aaagtatgtt 3472tgtgttggtt ttttaagaag tctaaatgaa gttattaata cctgaagctt taagttaagt 3532gcattgatca tatgatattt ttggaagcat acaattttaa ttgtggaagt ttaaagcctc 3592ttttagtcca ttgagaatgt aaataaatgt gtcttcttta tggaccaagg atatgaaatc 3652atttttcttt tgtagctaac ggttgccttg aggaagaaat aatttggttt tattaagagt 3712ctactctcaa tccagttatt agagatgtac tgagtttgat ttgttaatcc tttctatata 3772ctgctgatct tgcatgtcta caatctgctc agtttttctg tgtttctgca atagtggtca 3832gaaaaatact taaattccct taatggtgtt gttttctatt tgttctggtt ttgagataaa 3892tgagtgattc tgtccccaaa tgtccatttt tgaagtgatt ttcctggagg attagggtat 3952ttagcagttg aagctcttca ttcatagtag ttactgtcag ctaacaggtt ttttaaggct 4012tttaactatt aatattttat ggaatggggc aaagtaaatt gatgaaagaa ttggagtgat 4072aatagtcctt tacaaacata cagtccataa gaaaatgaat ttggcatata gaattattac 4132aatttcctgg gagagatgga tatttaaacc tctattattt tagacaagac tgtctagaac 4192ttaagtttga tctgtcagcc agtactccca ttaaattcag tgtagtttca cttgatagaa 4252tcagatatgt tatcgaaatg ttagcagcag cttcatcctc cttctgatta aagtaagtag 4312aaatgggatg ttttgtttaa taacagccat agtgtgtgtt tagaccacag cggatgttgt 4372agaccaggac catagatgat acatgtcagt gctgtggaat gtgcattctc tgagtgttgt 4432tttgtggtat cattgtcttt cctgaatgac tttctaactg tgcagaaagg cagaaaagtc 4492atcatatgta tatgtcatat gactttataa aatatttaat gtgacaaaaa gtggaaagaa 4552tctttacaaa ccctgcaatt acttttttaa aggcactttt actctttggt tttatcattc 4612cattttgcta atatttacta gctttataaa ttacagtaag gtacaaaaac tcatcttgta 4672atattttcat ttttgaagtg aaaaagtaca tatattttgc acaaggtttt atactgctaa 4732gtgcttggtt ggggtggtga gatgatgatt agatcagggg tgaggctgag agactctggg 4792tttagggcta gccctgcctc catctccctt gggtaaaatg aagggtgtgg ggtaaaagat 4852gcataaggcc ttttctagct ctgacagcct agaagtccaa tcaccctgta ataaatatgt 4912gttgaatgaa gaaatgggtg aatgagcttg tcaatgtgat tttaaaaaat tgactacctg 4972gaggaatgat taggaatcta aatgaagcca gccctcggta tctgcaggtt tctcatccat 5032ggattcaacc aactgcaaat ggaaaatacg atttttttta aaaaaaggat ggttacatcc 5092gtattgaaca tgtacagact tttttcttgt cattattctc tgaacaatac aagaactctt 5152tatgtagcat ttacatttat taggtattat aagtaatcta gagattattt aattaaaata 5212tacaggagga tgtgtgttta tatgccagaa attctgtacc attttgtatc agggaattga 5272gcatcttcag atgttggtat ctgcagggat cctggaacca aacccctgca gatactaagg 5332gctgacgatc taggtaagac tggatttaac agttggaaa 5371＜210〉2＜211〉956＜212〉PRT＜213〉 ( Homo sapiens )＜400〉2Met Ser Ser Gly Leu Trp Ser Gln Glu Lys Val Thr Ser Pro Tyr Trp1 5 10 15Glu Glu Arg Ile Phe Tyr Leu Leu Leu Gln Glu Cys Ser Val Thr Asp

20??????????????????25??????????????????30Lys?Gln?Thr?Gln?Lys?Leu?Leu?Lys?Val?Pro?Lys?Gly?Ser?Ile?Gly?Gln

35??????????????????40??????????????????45Tyr?Ile?Gln?Asp?Arg?Ser?Val?Gly?His?Ser?Arg?Ile?Pro?Ser?Ala?Lys

50??????????????????55??????????????????60Gly?Lys?Lys?Asn?Gln?Ile?Gly?Leu?Lys?Ile?Leu?Glu?Gln?Pro?His?Ala65??????????????????70??????????????????75??????????????????80Val?Leu?Phe?Val?Asp?Glu?Lys?Asp?Val?Val?Glu?Ile?Asn?Glu?Lys?Phe

85??????????????????90??????????????????95Thr?Glu?Leu?Leu?Leu?Ala?Ile?Thr?Asn?Cys?Glu?Glu?Arg?Phe?Ser?Leu

100?????????????????105?????????????????110Phe?Lys?Asn?Arg?Asn?Arg?Leu?Ser?Lys?Gly?Leu?Gln?Ile?Asp?Val?Gly

115?????????????????120?????????????????125Cys?Pro?Val?Lys?Val?Gln?Leu?Arg?Ser?Gly?Glu?Glu?Lys?Phe?Pro?Gly

130?????????????????135?????????????????140Val?Val?Arg?Phe?Arg?Gly?Pro?Leu?Leu?Ala?Glu?Arg?Thr?Val?Ser?Gly145?????????????????150?????????????????155?????????????????160Ile?Phe?Phe?Gly?Val?Glu?Leu?Leu?Glu?Glu?Gly?Arg?Gly?Gln?Gly?Phe

165?????????????????170?????????????????175Thr?Asp?Gly?Val?Tyr?Gln?Gly?Lys?Gln?Leu?Phe?Gln?Cys?Asp?Glu?Asp

180?????????????????185?????????????????190Cys?Gly?Val?Phe?Val?Ala?Leu?Asp?Lys?Leu?Glu?Leu?Ile?Glu?Asp?Asp

195?????????????????200?????????????????205Asp?Thr?Ala?Leu?Glu?Ser?Asp?Tyr?Ala?Gly?Pro?Gly?Asp?Thr?Met?Gln

210?????????????????215?????????????????220Val?Glu?Leu?Pro?Pro?Leu?Glu?Ile?Asn?Ser?Arg?Val?Ser?Leu?Lys?Val225?????????????????230?????????????????235?????????????????240Gly?Glu?Thr?Ile?Glu?Ser?Gly?Thr?Val?Ile?Phe?Cys?Asp?Val?Leu?Pro

245?????????????????250?????????????????255Gly?Lys?Glu?Ser?Leu?Gly?Tyr?Phe?Val?Gly?Val?Asp?Met?Asp?Asn?Pro

260?????????????????265?????????????????270Ile?Gly?Asn?Trp?Asp?Gly?Arg?Phe?Asp?Gly?Val?Gln?Leu?Cys?Ser?Phe

275?????????????????280?????????????????285Ala?Cys?Val?Glu?Ser?Thr?Ile?Leu?Leu?His?Ile?Asn?Asp?Ile?Ile?Pro

290?????????????????295?????????????????300Ala?Leu?Ser?Glu?Ser?Val?Thr?Gln?Glu?Arg?Arg?Pro?Pro?Lys?Leu?Ala305?????????????????310?????????????????315?????????????????320Phe?Met?Ser?Arg?Gly?Val?Gly?Asp?Lys?Gly?Ser?Ser?Ser?His?Asn?Lys

325?????????????????330?????????????????335Pro?Lys?Ala?Thr?Gly?Ser?Thr?Ser?Asp?Pro?Gly?Asn?Arg?Asn?Arg?Ser

340?????????????????345?????????????????350Glu?Leu?Phe?Tyr?Thr?Leu?Asn?Gly?Ser?Ser?Val?Asp?Ser?Gln?Pro?Gln

355?????????????????360?????????????????365Ser?Lys?Ser?Lys?Asn?Thr?Trp?Tyr?Ile?Asp?Glu?Val?Ala?Glu?Asp?Pro

370?????????????????375?????????????????380Ala?Lys?Ser?Leu?Thr?Glu?Ile?Ser?Thr?Asp?Phe?Asp?Arg?Ser?Ser?Pro385?????????????????390?????????????????395?????????????????400Pro?Leu?Gln?Pro?Pro?Pro?Val?Asn?Ser?Leu?Thr?Thr?Glu?Asn?Arg?Phe

405?????????????????410?????????????????415His?Ser?Leu?Pro?Phe?Ser?Leu?Thr?Lys?Met?Pro?Asn?Thr?Asn?Gly?Ser

420?????????????????425?????????????????430Ile?Gly?His?Ser?Pro?Leu?Ser?Leu?Ser?Ala?Gln?Ser?Val?Met?Glu?Glu

435?????????????????440?????????????????445Leu?Asn?Thr?Ala?Pro?Val?Gln?Glu?Ser?Pro?Pro?Leu?Ala?Met?Pro?Pro

450?????????????????455?????????????????460Gly?Asn?Ser?His?Gly?Leu?Glu?Val?Gly?Ser?Leu?Ala?Glu?Val?Lys?Glu465?????????????????470?????????????????475?????????????????480Asn?Pro?Pro?Phe?Tyr?Gly?Val?Ile?Arg?Trp?Ile?Gly?Gln?Pro?Pro?Gly

485?????????????????490?????????????????495Leu?Asn?Glu?Val?Leu?Ala?Gly?Leu?Glu?Leu?Glu?Asp?Glu?Cys?Ala?Gly

500?????????????????505?????????????????510Cys?Thr?Asp?Gly?Thr?Phe?Arg?Gly?Thr?Arg?Tyr?Phe?Thr?Cys?Ala?Leu

515?????????????????520?????????????????525Lys?Lys?Ala?Leu?Phe?Val?Lys?Leu?Lys?Ser?Cys?Arg?Pro?Asp?Ser?Arg

530?????????????????535?????????????????540Phe?Ala?Ser?Leu?Gln?Pro?Val?Ser?Asn?Gln?Ile?Glu?Arg?Cys?Asn?Ser545?????????????????550?????????????????555?????????????????560Leu?Ala?Phe?Gly?Gly?Tyr?Leu?Ser?Glu?Val?Val?Glu?Glu?Asn?Thr?Pro

565?????????????????570?????????????????575Pro?Lys?Met?Glu?Lys?Glu?Gly?Leu?Glu?Ile?Met?Ile?Gly?Lys?Lys?Lys

580?????????????????585?????????????????590Gly?Ile?Gln?Gly?His?Tyr?Asn?Ser?Cys?Tyr?Leu?Asp?Ser?Thr?Leu?Phe

595?????????????????600?????????????????605Cys?Leu?Phe?Ala?Phe?Ser?Ser?Val?Leu?Asp?Thr?Val?Leu?Leu?Arg?Pro

610?????????????????615?????????????????620Lys?Glu?Lys?Asn?Asp?Val?Glu?Tyr?Tyr?Ser?Glu?Thr?Gln?Glu?Leu?Leu625?????????????????630?????????????????635?????????????????640Arg?Thr?Glu?Ile?Val?Asn?Pro?Leu?Arg?Ile?Tyr?Gly?Tyr?Val?Cys?Ala

645?????????????????650?????????????????655Thr?Lys?Ile?Met?Lys?Leu?Arg?Lys?Ile?Leu?Glu?Lys?Val?Glu?Ala?Ala

660?????????????????665?????????????????670Ser?Gly?Phe?Thr?Ser?Glu?Glu?Lys?Asp?Pro?Glu?Glu?Phe?Leu?Asn?Ile

675?????????????????680?????????????????685Leu?Phe?His?His?Ile?Leu?Arg?Val?Glu?Pro?Leu?Leu?Lys?Ile?Arg?Ser

690?????????????????695?????????????????700Ala?Gly?Gln?Lys?Val?Gln?Asp?Cys?Tyr?Phe?Tyr?Gln?Ile?Phe?Met?Glu705?????????????????710?????????????????715?????????????????720Lys?Asn?Glu?Lys?Val?Gly?Val?Pro?Thr?Ile?Gln?Gln?Leu?Leu?Glu?Trp

725?????????????????730?????????????????735Ser?Phe?Ile?Asn?Ser?Asn?Leu?Lys?Phe?Ala?Glu?Ala?Pro?Ser?Cys?Leu

740?????????????????745?????????????????750Ile?Ile?Gln?Met?Pro?Arg?Phe?Gly?Lys?Asp?Phe?Lys?Leu?Phe?Lys?Lys

755?????????????????760?????????????????765Ile?Phe?Pro?Ser?Leu?Glu?Leu?Asn?Ile?Thr?Asp?Leu?Leu?Glu?Asp?Thr

770?????????????????775?????????????????780Pro?Arg?Gln?Cys?Arg?Ile?Cys?Gly?Gly?Leu?Ala?Met?Tyr?Glu?Cys?Arg785?????????????????790?????????????????795?????????????????800Glu?Cys?Tyr?Asp?Asp?Pro?Asp?Ile?Ser?Ala?Gly?Lys?Ile?Lys?Gln?Phe

805?????????????????810?????????????????815Cys?Lys?Thr?Cys?Asn?Thr?Gln?Val?His?Leu?His?Pro?Lys?Arg?Leu?Asn

820????????????????825?????????????????830His?Lys?Tyr?Asn?Pro?ValSer?Leu?Pro?Lys?Asp?Leu?Pro?Asp?Trp?Asp

835????????????????840?????????????????845Trp?Arg?His?Gly?Cys?Ile?Pro?Cys?Gln?Asn?Met?Glu?Leu?Phe?Ala?Val

850?????????????????855?????????????????860Leu?Cys?Ile?Glu?Thr?Ser?His?Tyr?Val?Ala?Phe?Val?Lys?Tyr?Gly?Lys865?????????????????870?????????????????875?????????????????880Asp?Asp?Ser?Ala?Trp?Leu?Phe?Phe?Asp?Ser?Met?Ala?Asp?Arg?Asp?Gly

885?????????????????890?????????????????895Gly?Gln?Asn?Gly?Phe?Asn?Ile?Pro?Gln?Val?Thr?Pro?Cys?Pro?Glu?Val

900?????????????????905?????????????????910Gly?Glu?Tyr?Leu?Lys?Met?Ser?Leu?Glu?Asp?Leu?His?Ser?Leu?Asp?Ser

915?????????????????920?????????????????925Arg?Arg?Ile?Gln?Gly?Cys?Ala?Arg?Arg?Leu?Leu?Cys?Asp?Ala?Tyr?Met

930 935 940Cys Met Tyr Gln Ser Pro Thr Met Ser Leu Tyr Lys945 950 955＜210〉3＜211〉24＜212〉DNA＜213〉artificial sequence＜220〉＜221〉misc_feature＜223〉primer＜400〉3tctgcagtga tagcttttct gaca＜210〉4＜211〉24＜212〉DNA＜213〉artificial sequence＜220〉＜221〉misc_feature＜223〉primer＜400〉4cagtctcacc aagatgccca atac 24＜210〉5＜211〉496＜212〉PRT＜213〉homo sapiens (Homo sapiens)＜400〉5Met Ser Ser Gly Leu Trp Ser Gln Glu Lys Val Thr Ser Pro Tyr Trp1,5 10 15Glu Glu Arg Ile Phe Tyr Leu Leu Leu Gln Glu Cys Ser Val Thr Asp

20??????????????????25??????????????????30Lys?Gln?Thr?Gln?Lys?Leu?Leu?Lys?Val?Pro?Lys?Gly?Ser?Ile?Gly?Gln

35??????????????????40??????????????????45Tyr?Ile?Gln?Asp?Arg?Ser?Val?Gly?His?Ser?Arg?Ile?Pro?Ser?Ala?Lys

50??????????????????55??????????????????60Gly?Lys?Lys?Asn?Gln?Ile?Gly?Leu?Lys?Ile?Leu?Glu?Gln?Pro?His?Ala65??????????????????70??????????????????75??????????????????80Val?Leu?Phe?Val?Asp?Glu?Lys?Asp?Val?Val?Glu?Ile?Asn?Glu?Lys?Phe

85??????????????90??????????????????????95Thr?Glu?Leu?Leu?Leu?Ala?Ile?Thr?Asn?Cys?Glu?Glu?Arg?Phe?Ser?Leu

100?????????????????105?????????????????110Phe?Lys?Asn?Arg?Asn?Arg?Leu?Ser?Lys?Gly?Leu?Gln?Ile?Asp?Val?Gly

115?????????????????120?????????????????125Cys?Pro?Val?Lys?Val?Gln?Leu?Arg?Ser?Gly?Glu?Glu?Lys?Phe?Pro?Gly

130?????????????????135?????????????????140Val?Val?Arg?Phe?Arg?Gly?Pro?Leu?Leu?Ala?Glu?Arg?Thr?Val?Ser?Gly145?????????????????150?????????????????155?????????????????160Ile?Phe?Phe?Gly?Val?Glu?Leu?Leu?Glu?Glu?Gly?Arg?Gly?Gln?Gly?Phe

165?????????????????170?????????????????175Thr?Asp?Gly?Val?Tyr?Gln?Gly?Lys?Gln?Leu?Phe?Gln?Cys?Asp?Glu?Asp

180?????????????????185?????????????????190Cys?Gly?Val?Phe?Val?Ala?Leu?Asp?Lys?Leu?Glu?Leu?Ile?Glu?Asp?Asp

195?????????????????200?????????????????205Asp?Thr?Ala?Leu?Glu?Ser?Asp?Tyr?Ala?Gly?Pro?Gly?Asp?Thr?Met?Gln

210?????????????????215?????????????????220Val?Glu?Leu?Pro?Pro?Leu?Glu?Ile?Asn?Ser?Arg?Val?Ser?Leu?Lys?Val225?????????????????230?????????????????235?????????????????240Gly?Glu?Thr?Ile?Glu?Ser?Gly?Thr?Val?Ile?Phe?Cys?Asp?Val?Leu?Pro

245?????????????????250?????????????????255Gly?Lys?Glu?Ser?Leu?Gly?Tyr?Phe?Val?Gly?Val?Asp?Met?Asp?Asn?Pro

260?????????????????265?????????????????270Ile?Gly?Asn?Trp?Asp?Gly?Arg?Phe?Asp?Gly?Val?Gln?Leu?Cys?Ser?Phe

275?????????????????280?????????????????285Ala?Cys?Val?Glu?Ser?Thr?Ile?Leu?Leu?His?Ile?Asn?Asp?Ile?Ile?Pro

290?????????????????295?????????????????300Ala?Leu?Ser?Glu?Ser?Val?Thr?Gln?Glu?Arg?Arg?Pro?Pro?Lys?Leu?Ala305?????????????????310?????????????????315?????????????????320Phe?Met?Ser?Arg?Gly?Val?Gly?Asp?Lys?Gly?Ser?Ser?Ser?His?Asn?Lys

325?????????????????330?????????????????335Pro?Lys?Ala?Thr?Gly?Ser?Thr?Ser?Asp?Pro?Gly?Asn?Arg?Asn?Arg?Ser

340?????????????????345?????????????????350Glu?Leu?Phe?Tyr?Thr?Leu?Asn?Gly?Ser?Ser?Val?Asp?Ser?Gln?Pro?Gln

355?????????????????360?????????????????365Ser?Lys?Ser?Lys?Asn?Thr?Trp?Tyr?Ile?Asp?Glu?Val?Ala?Glu?Asp?Pro

370?????????????????375?????????????????380Ala?Lys?Ser?Leu?Thr?Glu?Ile?Ser?Thr?Asp?Phe?Asp?Arg?Ser?Ser?Pro385?????????????????390?????????????????395?????????????????400Pro?Leu?Gln?Pro?Pro?Pro?Val?Asn?Ser?Leu?Thr?Thr?Glu?Asn?Arg?Phe

405?????????????????410?????????????????415His?Ser?Leu?Pro?Phe?Ser?Leu?Thr?Lys?Met?Pro?Asn?Thr?Asn?Gly?Ser

420?????????????????425?????????????????430Ile?Gly?His?Ser?Pro?Leu?Ser?Leu?Ser?Ala?Gln?Ser?Val?Met?Glu?Glu

435?????????????????440?????????????????445Leu?Asn?Thr?Ala?Pro?Val?Gln?Glu?Ser?Pro?Pro?Leu?Ala?Met?Pro?Pro

450?????????????????455?????????????????460Gly?Asn?Ser?His?Gly?Leu?Glu?Val?Gly?Ser?Leu?Ala?Glu?Val?Lys?Glu465?????????????????470?????????????????475?????????????????480Asn?Pro?Pro?Phe?Tyr?Gly?Val?Ser?Val?Gly?Ser?Val?Ser?His?Gln?Asp

485?????????????????490?????????????????495

Claims (10)

1. method that the tumor susceptibility of individuality is diagnosed is characterized in that it comprises step:

Detect this individual CYLD gene, transcript and/or albumen, and compare with normal CYLD gene, transcript and/or albumen,

The possibility that there are differences with regard to showing this individuality trouble tumour is higher than normal population.

2. the method for claim 1 is characterized in that, detection be gene or the transcript of CYLD, and with normal CYLD nucleotide sequence comparing difference.

3. the method for claim 1 is characterized in that, described tumour is a trichoepithelioma, and described difference is selected from: the 1853rd A disappearance among the SEQ ID NO:1.

4. a method for the treatment of tumour is characterized in that, comprises step: the normal CYLD albumen of using safe and effective amount to the patient of the described treatment of needs.

5. method as claimed in claim 4 is characterized in that, described CYLD albumen is locally applied to affected area.

6. the proteic purposes of CYLD is characterized in that, is used to prepare the pharmaceutical composition for the treatment of tumour.

7. purposes as claimed in claim 6 is characterized in that, described pharmaceutical composition is ointment or injection.

8. a test kit that detects tumour is characterized in that, it comprises the primer of specific amplification CYLD gene or transcript.

9. test kit as claimed in claim 8 is characterized in that, it also contains the reagent that is selected from down group:

(a) with mutable site bonded probe;

(b) restriction enzyme in identification mutational site.

10. test kit as claimed in claim 9 is characterized in that, described sudden change is selected from: the 1853rd A disappearance among the SEQ ID NO:1.

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