CN107201411A - MYLK genes as diagnosis of endometrial carcinoma mark - Google Patents

Abstract

The invention discloses a kind of diagnosis marker of carcinoma of endometrium, the diagnosis marker is MYLK genes or MYLK albumen, and the purpose of diagnosis is realized by detecting the expression of MYLK genes.Research of the present invention has shown that compared with normal endometrial tissue the mRNA expressions of MYLK genes are remarkably decreased in endometrial.According to the correlation existed between MYLK genes and carcinoma of endometrium, the kit of diagnosis of endometrial carcinoma can be prepared, the kit can clinically extensive use.

Description

MYLK genes as diagnosis of endometrial carcinoma mark

Technical field

The invention belongs to diagnostic field, it is related to a kind of diagnostic tool of carcinoma of endometrium, further relates to MYLK genes in preparation Application in carcinoma of endometrium diagnostic tool.

Background technology

Carcinoma of endometrium (endometrial carcinoma or carcinoma of endometrium, EC) is that occur It is most common with the gland cancer from endometrial gland in one group of epithelial malignancy of endometrium, it is female genital tract One of three big malignant tumours, account for women whole body malignant tumour 7%, its incidence of disease is variant in worldwide different regions, is The most common gynecologic malignant tumor of western industrialization country, North America, Europe incidence of disease highest, Asia Japan, India and in The area such as South America incidence of disease is relatively low.China still lacks the more detailed epidemiology survey data of carcinoma of endometrium, estimation and day This incidence is similar.North America and countries in Europe are higher than developing country, and the former is 10 times of the latter, and its incidence of disease is located at It is the 4th common cancer of women whole body after breast cancer, colorectal cancer, lung cancer, occupies female genital tract malignant tumour One.

Although carcinoma of endometrium symptom occurs, relatively early, diagnosis is relative to be easier to.But on son in actual clinical work The diagnosis and treatment of endometrial carcinoma still suffer from some disputes.The grade malignancy and extent of disease of tumour, including Surgical staging, tissue Learn transfer etc. outside type, tumor grade, Myometrial invasion, Lymph Node Metastasis and uterus relevant with the prognosis of carcinoma of endometrium.Cause This, early diagnosis carcinoma of endometrium is extremely important.At present, the diagnosis Main Basiss medical history of carcinoma of endometrium and clinical manifestation, B The imageological examinations such as super, CT and MRI, diagnostic curettage, hysteroscope, the auxiliary examination such as tumor markers CA-125.Wherein CA- 125 are also used as the index of observation of curative effect.But CA-125 inorganizable Sensitivity and Specificity, it is swollen in ovarian epithelial The rise of CA-125 levels is can detect in the diseases such as knurl, carcinoma of endometrium, endometriosis, digestive system tumor.

In recent years, gene diagnosis becomes the new development trend of in-vitro diagnosis industry.Gene diagnosis, also referred to as DNA are examined The diagnosis of disconnected or molecule, is applied molecular biology method, the change of inhereditary material structure or expression in detection patient's body, after And make the technology of diagnosis, it is the prevention of disease, prediction, diagnosis, treatment and prognosis of disease more accurate information is provided.Mirror The disadvantages described above existed in current carcinoma of endometrium diagnostic method, is badly in need of developing a kind of cheap, easy to operate, diagnoses sensitive Degree and the method for the high gene diagnosis available for diagnosis of endometrial carcinoma of specificity.

The content of the invention

Present invention firstly discovers that the content in MYLK gene endometrial tissues is lower than normal endometrial tissue very It is many, the differential expressions of MYLK genes can as diagnosis of endometrial carcinoma a kind of method, diagnosis endometrium can be developed accordingly The instrument of cancer.

Specifically, the invention provides the product of detection MYLK gene expressions in the instrument of diagnosis of endometrial carcinoma is prepared Application.

Further, detection product mentioned above includes：Pass through RT-PCR, real-time quantitative PCR, immune detection, original position The expression of hybridization, chip or high-flux sequence detection of platform MYLK genes is with the product of diagnosis of endometrial carcinoma.

Further, the product of the use RT-PCR diagnosis of endometrial carcinoma at least includes a pair of specific amplified MYLK genes Primer；The product of the use real-time quantitative PCR diagnosis of endometrial carcinoma at least includes the primer of a pair of specific amplified MYLK genes； The product of the use immune detection diagnosis of endometrial carcinoma includes：The antibody combined with MYLK protein-specifics；It is described to use in situ The product of hybridization diagnosis of endometrial carcinoma includes：With the probe of the nucleic acid array hybridizing of MYLK genes；It is described to diagnose son with chip The product of endometrial carcinoma includes：Protein chip and genetic chip；Wherein, protein chip includes what is combined with MYLK protein-specifics Antibody, genetic chip includes the probe with the nucleic acid array hybridizing of MYLK genes.

In specific embodiments of the present invention, the product with real-time quantitative PCR diagnosis of endometrial carcinoma is at least wrapped The sequence of primer of a pair of specific amplified MYLK genes is included as shown in SEQ ID NO.3 and SEQ ID NO.4.

Preferably, the diagnostic tool includes chip, kit, test paper or high-flux sequence platform.Wherein, high pass is measured Sequence platform is a kind of special diagnostic tool, and the product of detection MYLK gene expressions can apply to the platform and realize to MYLK bases The detection of the expression of cause.With the development of high throughput sequencing technologies, the structure of the gene expression profile of a people will be turned into Very easily work.By contrasting the gene expression profile of Disease and normal population, the different of which gene is easily analyzed It is often related to disease.Therefore, know that the exception of MYLK genes is related to carcinoma of endometrium in high-flux sequence and fall within MYLK The purposes of gene, equally within protection scope of the present invention.

Present invention also offers a kind of instrument of diagnosis of endometrial carcinoma, the product include chip, kit, test paper, Or high-flux sequence platform.

Wherein, the chip includes genetic chip, protein-chip；The genetic chip includes solid phase carrier and fixation In the oligonucleotide probe of solid phase carrier, the oligonucleotide probe includes being used to detect being directed to for MYLK gene transcription levels The oligonucleotide probe of MYLK genes；The protein-chip includes solid phase carrier and is fixed on the MYLK albumen of solid phase carrier Specific antibody；The genetic chip can be used for multiple genes of the detection including MYLK genes (for example, and endometrium The related multiple genes of cancer) expression.The protein-chip can be used for multiple eggs of the detection including MYLK albumen The expression of white matter (such as multiple protein related to carcinoma of endometrium).By by multiple with carcinoma of endometrium mark Thing is detected simultaneously, is greatly improved the accuracy rate of carcinoma of endometrium diagnosis.

Wherein, the kit includes gene detecting kit and protein immunization detection kit；The genetic test examination Agent box includes the reagent for being used to detect MYLK gene transcription levels；The protein immunization detection kit includes the spy of MYLK albumen Heterogenetic antibody.Further, the reagent is including the use of RT-PCR, real-time quantitative PCR, immune detection, in situ hybridization or chip side Reagent needed for during method detection MYLK gene expression doses.Preference, the reagent includes the primer for MYLK genes And/or probe.Easily designed according to the nucleotide sequence information of MYLK genes and can be used for detecting MYLK gene expression doses Primer and probe.

The high-flux sequence platform includes the reagent of detection MYLK gene expression doses.

The test paper includes test paper carrier and the oligonucleotides being fixed on test paper carrier, and the oligonucleotides can be detected The transcriptional level of MYLK genes.

Probe with the nucleic acid array hybridizing of MYLK genes can be that DNA, RNA, DNA-RNA chimera, PNA or other spread out It is biological.The length of the probe is not limited, as long as completing specific hybrid, being specifically bound with purpose nucleotide sequence, is appointed What length can.The length of the probe can be as short as 25,20,15,13 or 10 bases longs.Equally, the length of the probe Degree can be grown to 60,80,100,150,300 base-pairs or longer, or even whole gene.Because different probe lengths is to hybridization Efficiency, signal specificity have different influences, and the length of the probe is typically at least 14 base-pairs, most long to be usually no more than 30 base-pairs, complementary length is optimal with 15-25 base-pair with purpose nucleotide sequence.The probe self-complementary sequences Most preferably less than 4 base-pairs, in order to avoid influence hybridization efficiency.

Further, the specific antibody of the MYLK albumen includes monoclonal antibody, polyclonal antibody.The MYLK albumen Specific antibody include complete antibody molecule, any fragment of antibody or modification (for example, chimeric antibody, scFv, Fab, F (ab ') 2, Fv etc..As long as the fragment can retain the binding ability with MYLK albumen.Antibody for protein level Preparation when well known to a person skilled in the art and the present invention can use any method to prepare the antibody.

In specific embodiments of the present invention, the primer sequence for MYLK genes is as follows：Forward primer sequence As shown in SEQ ID NO.3, reverse primer is as shown in SEQ ID NO.4.

Include but is not limited to tissue and body fluid for the MYLK genes of diagnosis of endometrial carcinoma and its source of expression product, Body fluid includes the internal liquid component that blood, tissue fluid etc. have DNA.In specific embodiments of the present invention, for diagnosing The MYLK genes of carcinoma of endometrium and its source of expression product are tissues.

The particular sequence of the MYLK genes (NC_000003.12 (123612296..123884302)) of the present invention can be in state Inquired in the public GenBank GeneBank in border.

The corresponding DNA sequence dna of mRNA sequence of the MYLK genes of the present invention is in sequence table shown in SEQ ID NO.1 DNA sequence dna.

" the MYLK albumen " of the present invention includes any functional equivalent of MYLK albumen and MYLK albumen.Described function etc. Jljl includes MYLK albumen conservative variation protein or its active fragment, or its reactive derivative, allelic variant, natural Mutant, induced mutants, can be with the protein coded by the DNA of MYLK DNA hybridization under high or low stringent condition.

Preferably, MYLK albumen is the protein with following amino acid sequences：

(1) protein being made up of the amino acid sequence in sequence table shown in SEQ ID NO.2；

(2) by the amino acid sequence shown in SEQ ID NO.2 is by the substitution of one or several amino acid residues and/or lacks Lose and/or addition and with the amino acid sequence shown in SEQ ID NO.2 have identical function as the ammonia shown in SEQ ID NO.2 Protein derived from base acid sequence.The number of the amino acid of substitution, missing or addition is usually 1-50, preferably 1-30 It is individual, more preferably 1-20, most preferably 1-10.

In specific embodiments of the present invention, the MYLK albumen is with the amino acid sequence shown in SEQ ID NO.2 The protein of row.

It is known that, conventionally, the modification of one or more amino acid does not interfere with the function of protein in a protein. Those skilled in the art can approve the amino acid that changes single amino acids or small percentage or indivedual additions to amino acid sequence, Missing, insertion, replacement are conservative modifications, and the change of wherein protein produces the protein with identity function.Function phase is provided As the Conservative substitution tables of amino acid be well known in the art.

By adding the fusion that the example for the protein that an amino acid or more amino acid are modified is MYLK albumen Albumen.Do not limited for the peptide or protein with MYLK protein fusions, as long as the fusion protein of gained retains MYLK albumen Biological activity.

The MYLK albumen of the present invention also includes the non-conservative modification to the amino acid sequence shown in SEQ ID NO.2, as long as Protein by modification remains able to retain the biological activity of MYLK albumen.It is mutated in such modifying protein Amino acid number is typically 10 or less, such as 6 or less, such as 3 or less.

In the context of the present invention, " diagnosis of endometrial carcinoma " both includes judging whether subject suffers from intrauterine Film cancer, also include judging that subject whether there is the risk with carcinoma of endometrium, in addition to predict endometrial carcinoma Prognosis, in addition to judge whether the subject with carcinoma of endometrium is recurring after treatment.

Brief description of the drawings

Fig. 1 displays are detected in MYLK genes endometrial tissues and normal endometrial tissue using RNA-seq Differential expression；

Fig. 2 displays detect the expression in MYLK genes endometrial tissues and normal endometrial tissue using QPCR Difference.

Specific embodiment

The present invention is further detailed explanation with reference to the accompanying drawings and examples.Following examples are merely to illustrate this Invention rather than limitation the scope of the present invention.The experimental method of unreceipted actual conditions in embodiment, generally according to conventional strip Part, such as Sambrook et al., molecular cloning：Laboratory manual (New York:Cold Spring HarborLaboratory Press, 1989) described in condition, or according to the condition proposed by manufacturer.

Embodiment 1 screens difference expression gene

1st, sample is collected：

Endometrial sample：Collect endometrial carcinoma, the equal underwent operative treatment of all patients, paraffin mark of performing the operation This 10.All patients are made a definite diagnosis with carcinoma of endometrium by pathologic finding.Other enter a group condition：Before all patients are admitted to hospital Any treatment was not received：Other malignant tumours are not merged；Other hormone related disorders are not merged；Complete clinical data.

10 patients with endometrial cancer, morbidity average age 57 years old.Patient's main clinical manifestation be Irregular vagina bleeding, Hypogastralgia, paramenia, neoplasm etc., also have some patientss non-evident sympton and are found in Physical Examination.Carcinoma of endometrium Sample is diagnosed as carcinoma of endometrium through HE stained slices, tectology.

Normal endometrial tissue sample：10 normal endometriums operation paraffin specimens.The patient of samples sources is suffered from Disease includes：Fibroid, the prolapse of uterus, the bulging of wing moon bright, Rectocele.

2nd, RNA is extracted

Endometrial tissue is individually placed in ceramic mortar crushed into powder under liquid nitrogen cryogenics environment, Trizol examinations are added Agent is homogenized, and takes supernatant to be taken out again through sodium acetate and 5: 1 acid phenol-chloroforms after being extracted twice through 1: 1 acid phenol-chloroform after centrifugation Carry, isometric isopropanol precipitating, precipitation is dissolved with Milli-Q water after centrifugation.

3rd, the purity analysis (NanoDrop1000 spectrophotometers) of RNA sample

NanoDrop1000 spectrophotometers detect RNA sample, the sample requirement of RNA-seq sequencings：OD260/OD280 is 1.8-2.2。

4th, the quality analysis (Agilent Technologies 2100Bioanalyzer) of RNA sample

Agilent Technologies 2100Bioanalyzer detect RNA sample quality, observation 28S rRNA and 18S Substantially, without degraded, the RNA-seq that meets that RNA Perfection Index is qualified, concentration reaches requirement cDNA library structure is sequenced in rRNA master tapes The requirement built, can be used for library construction and sequencing.

5th, high flux transcript profile is sequenced

(1) RNA-seq reads are positioned

First by low-quality read remove obtain clean read, then using TopHat v1.3.1 will clean fragment and UCSC H.sapiens reference genes groups (hg19) are matched, the index of H.sapiens UCSC hg19 editions advance structure Downloaded from TopHat homepages, and as reference gene group, when being matched using TopHat with genome, it is allowed to each read (acquiescence To 20) having multiple matching sites, most 2 mispairing.TopHat sets up possible according to exon region and GT-AG shear signals Shearing site storehouse, navigates to the read for not navigating to genome on genome according to these shearing site storehouses.We use The system default parameter of TopHat methods.

(2) transcript abundance is assessed

The read file matched is by Cufflinks v1.0.3 processing, and Cufflinks v1.0.3 are by RNA-seq pieces Hop count mesh is standardized the relative abundance for calculating transcript.FPKM values refer to it is every 1,000,000 sequencing fragment in match it is specific The segment number of the exon region of gene 1kb length.The confidential interval of FPKM estimates is calculated by Bayesian inference method. The GTF comment files for the reference that Cufflinks is used download (Homo_ from Ensembl databases sapiens.GRCh37.63.gtf)。

(3) detection of difference expression gene

The original document matched by the Ensembl GTF files of download and by TopHat is transferred to Cuffdiff, Cuffdiff re-evaluates the gene expression abundance for the transcript listed in GTF files using original matching files, detects difference table Reach.The only q values ＜ 0.01 in Cuffidff outputs, test display is considered as successfully more just differential expression.

6th, result

RNA-seq results are shown, expressing gene is had differences between normal endometrial tissue and endometrial, Wherein, the gene of up-regulation has 374, and the gene of downward has 268.Wherein, MYLK gene mRNAs water in endometrial Flat to significantly reduce, difference has statistical significance (P<0.05).

Checking of the difference expression gene of embodiment 2 in large sample

1st, sample is collected

Endometrial 50, normal endometrial tissue 45 are collected according to the method for embodiment 1.

2nd, verified in mRNA level in-site

2.1 extract tissue RNA

Step be the same as Example 1.

2.2 reverse transcription

Using Reverse Transcriptase kit, converse record synthesis cDNA is carried out to l μ g total serum IgEs with RT Buffer.Using 25 μ l Reaction system, each sample takes 1 μ g total serum IgEs as template ribonucleic acid, and following components is separately added into PCR pipe：DEPC water, 5 × inverse Transcription buffer, 10mmol/l dNTP, 0.1mmol/l DTT, 30 μm of mol/l Oligo dT, 200U/ μ l MMLVRT, template RNA.42 DEG C be incubated 1 hour, 72 DEG C 10 minutes, of short duration centrifugation.

2.3PCR

MRNA fluorescent quantitation upstream and downstream PCR primers, synthesis are designed using primer-design software Primer Premier 5.0 Primer sequence, is operated using SYBR Green PCR Master Mix kits, and specific steps by specification is operated, adopted With 25 μ l reaction systems, each sample sets 3 parallel pipes, all amplified reactions in triplicate more than can with ensure result By property.Following reaction system (as shown in table 1) is prepared, operations are carried out on ice：

The quantitative fluorescent PCR each component of table 1 and respective volume

Using GAPDH as internal reference, using SYBR Green I as fluorescent marker, in Light Cycler quantitative fluorescent PCRs The enterprising performing PCR reaction of instrument, determines purpose band, Δ Δ CT methods carry out relative quantification by melt curve analysis analysis and electrophoresis.

MYLK gene primer sequences are as follows：

Sense primer：5’-AAGTGGAGGTGTCAGATG-3’(SEQ ID NO.3)；

Anti-sense primer：5’-TCAATGTCGTAGAAGTCAGA-3’(SEQ ID NO.4).

GAPDH gene primer sequences are as follows：

Sense primer：5’-AACTCTGGTAAAGTGGATATTG-3’(SEQ ID NO.5)；

Anti-sense primer：5’-GGTGGAATCATATTGGAACA-3’(SEQ ID NO.6).

2.4 result

As a result as shown in Fig. 2 compared with normal endometrial tissue, the mRNA water of MYLK genes in endometrial Dawn aobvious downward, difference has statistical significance (P<0.05), as a result same RNA-seq.

The preparation of the carcinoma of endometrium diagnostic kit of embodiment 3

According to MYLK genes and the correlation of carcinoma of endometrium, it can be diagnosed by detecting the expression of MYLK genes Whether carcinoma of endometrium occurs, accordingly the invention provides it is a kind of based on detection MYLK gene expressions come diagnosis of endometrial carcinoma Component in kit, the diagnostic kit is as follows：SYBR Green PCR systems；Expand MYLK genes and The primer pair of GAPDH genes.Positive-the AAGTGGAGGTGTCAGATG-3 ' of sequence 5 ' of amplification MYLK genes, reverse sequence 5 '- TCAATGTCGTAGAAGTCAGA-3’；The forward primer sequence for expanding GAPDH is 5 '-AACTCTGGTAAAGTGGATATTG- 3 ', reverse primer sequences are 5 '-GGTGGAATCATATTGGAACA-3 '.SYBR Green PCR systems are included PCR buffer solutions, dNTPs, SYBR Green fluorescent dyes.PCR buffer components are：25mM KCL, 2.5mM MgCL₂、200mM (NH₄)₂SO₄。

The preparation of the carcinoma of endometrium diagnostic kit of embodiment 4

According to MYLK genes and the correlation of carcinoma of endometrium, it can be diagnosed by detecting the expression of MYLK genes Whether carcinoma of endometrium occurs, accordingly the invention provides it is a kind of based on detection MYLK gene expressions come diagnosis of endometrial carcinoma Component in kit, the diagnostic kit is as follows：SYBR Green PCRs system, amplification MYLK genes and Primer pair, the M-MLV reverse transcription systems of GAPDH genes.The positive sequence 5 ' of amplification MYLK genes- AAGTGGAGGTGTCAGATG-3 ' ,-TCAATGTCGTAGAAGTCAGA-3 ' of reverse sequence 5 '；Expand GAPDH forward primer Sequence is 5 '-AACTCTGGTAAAGTGGATATTG-3 ', and reverse primer sequences are 5 '-GGTGGAATCATATTGGAACA-3 '. SYBR Green PCRs system includes PCR buffer solutions, dNTPs, SYBR Green fluorescent dyes.PCR buffer solutions Component is：25mM KCL、2.5mM MgCL₂、200mM(NH₄)₂SO₄.M-MLV reverse transcription system components are：T repeats oligonucleotides Oligo (dT), reverse transcription reaction liquid, M-MLV reverse transcriptases, RNase inhibitor, dNTPs.Reverse transcription reaction liquid component is： 250mM Tris-HCL(pH8.3)、375mM KCL、15mM MgCL₂, 50mM DTT.RNase inhibitor is Escherichia coli table The recombinant protease of the Noncompetition inhibition RNase reached.

The preparation of the carcinoma of endometrium diagnostic kit of embodiment 5

According to MYLK genes and the correlation of carcinoma of endometrium, it can be diagnosed by detecting the expression of MYLK genes Whether carcinoma of endometrium occurs, accordingly the invention provides it is a kind of based on detection MYLK gene expressions come diagnosis of endometrial carcinoma Component in kit, the diagnostic kit is as follows：SYBR Green PCRs system, amplification MYLK genes and Primer pair, the RNA extracts reagents of GAPDH genes.Positive-the AAGTGGAGGTGTCAGATG-3 ' of sequence 5 ' of MYLK genes is expanded, - the TCAATGTCGTAGAAGTCAGA-3 ' of reverse sequence 5 '；Expand GAPDH forward primer sequence for 5 '- AACTCTGGTAAAGTGGATATTG-3 ', reverse primer sequences are 5 '-GGTGGAATCATATTGGAACA-3 '.SYBR Green PCR system includes PCR buffer solutions, dNTPs, SYBR Green fluorescent dyes.PCR buffer compositions are：25mM KCL、2.5mM MgCL₂、200mM(NH₄)₂SO₄.RNA extracts reagents include Trizol, chloroform, isopropanol, 75% ethanol.

The preparation of the carcinoma of endometrium diagnostic kit of embodiment 6

According to MYLK genes and the correlation of carcinoma of endometrium, it can be diagnosed by detecting the expression of MYLK genes Whether carcinoma of endometrium occurs, accordingly the invention provides it is a kind of based on detection MYLK gene expressions come diagnosis of endometrial carcinoma Component in kit, the diagnostic kit is as follows：SYBR Green PCRs system, amplification MYLK genes and Primer pair, M-MLV reverse transcriptions system, the RNA extracts reagents of GAPDH genes.The positive sequence 5 ' of amplification MYLK genes- AAGTGGAGGTGTCAGATG-3 ' ,-TCAATGTCGTAGAAGTCAGA-3 ' of reverse sequence 5 '；Expand GAPDH forward primer Sequence is 5 '-AACTCTGGTAAAGTGGATATTG-3 ', and reverse primer sequences are 5 '-GGTGGAATCATATTGGAACA-3 '. SYBR Green PCRs system includes PCR buffer solutions, dNTPs, SYBR Green fluorescent dyes.PCR buffer solutions Component is：25mM KCL、2.5mM MgCL₂、200mM(NH₄)₂SO₄.M-MLV reverse transcription system components are：T repeats oligonucleotides Oligo (dT), reverse transcription reaction liquid, M-MLV reverse transcriptases, RNase inhibitor, dNTPs.Reverse transcription reaction liquid component is： 250mM Tris-HCL(pH8.3)、375mM KCL、15mM MgCL₂, 50mM DTT.RNase inhibitor is Bacillus coli expression Noncompetition inhibition RNase recombinant protease.RNA extracts reagents include Trizol, chloroform, isopropanol, 75% ethanol.

The explanation of above-described embodiment is only intended to understand the method and its core concept of the present invention.It should be pointed out that for this For the those of ordinary skill in field, under the premise without departing from the principles of the invention, some improve can also be carried out to the present invention And modification, these are improved and modification will be also fallen into the protection domain of the claims in the present invention.

SEQUENCE LISTING

<110>Changning district, Shanghai healthcare hospital for women ＆ children

<120>MYLK genes as diagnosis of endometrial carcinoma mark

<160> 6

<170> PatentIn version 3.5

<210> 1

<211> 7644

<212> DNA

<213>People source

<400> 1

ggcgctgagc gagctcggag cccgcgctgt gcgcctgcgg ccggggcgcc ccgccgagcg 60

ccggtgcccc ggctcccggg ccgccttcgc cgcgcgggaa ggattcttca aaattaacag 120

aaaccaattc gggccagctg aagagaaaaa ataaaggtgg ctcccggctg cctctgctgc 180

agttcagagc aacttcagga gcttcccagc cgagagcttc aggacgcctt tcctgtccca 240

ctggcccagt tgccacaaca aacaacagag aagacggtga ccatggggga tgtgaagctg 300

gttgcctcgt cacacatttc caaaacctcc ctcagtgtgg atccctcaag agttgactcc 360

atgcccctga cagaggcccc tgctttcatt ttgccccctc ggaacctctg catcaaagaa 420

ggagccaccg ccaagttcga agggcgggaa gttttgcgaa gcagcttggt cagcctgttg 480

tttccaaaac cttaggggat agattttcag ctccagcagt ggagacccgt cctagcatct 540

ggggggagtg cccaccaaag tttgctacca agctgggccg agttgtggtc aaagaaggac 600

agatgggacg attctcctgc aagatcactg gccggcccca accgcaggtc acctggctca 660

agggaaatgt tccactgcag ccgagtgccc gtgtgtctgt gtctgagaag aacggcatgc 720

aggttctgga aatccatgga gtcaaccaag atgacgtggg agtgtacacg tgcctggtgg 780

tgaacgggtc ggggaaggcc tcgatgtcag ctgaactttc catccaaggt ttggacagtg 840

ccaataggtc atttgtgaga gaaacaaaag ccaccaattc agatgtcagg aaagaggtga 900

ccaatgtaat ctcaaaggag tcgaagctgg acagtctgga ggctgcagcc aaaagcaaga 960

actgctccag cccccagaga ggtggctccc caccctgggc tgcaaacagc cagcctcagc 1020

ccccaaggga gtccaagctg gagtcatgca aggactcgcc cagaacggcc ccgcagaccc 1080

cggtccttca gaagacttcc agctccatca ccctgcaggc cgcaagagtt cagccggaac 1140

caagagcacc aggcctgggg gtcctatcac cttctggaga agagaggaag aggccagctc 1200

ctccccgtcc agccaccttc cccaccaggc agcctggcct ggggagccaa gatgttgtga 1260

gcaaggctgc taacaggaga atccccatgg agggccagag ggattcagca ttccccaaat 1320

ttgagagcaa gccccaaagc caggaggtca aggaaaatca aactgtcaag ttcagatgtg 1380

aagtttccgg gattccaaag cctgaagtgg cctggttcct ggaaggcacc cccgtgagga 1440

gacaggaagg cagcattgag gtttatgaag atgctggctc ccattacctc tgcctgctga 1500

aagcccggac cagggacagt gggacataca gctgcactgc ttccaacgcc caaggccagc 1560

tgtcctgtag ctggaccctc caagtggaaa ggcttgccgt gatggaggtg gccccctcct 1620

tctccagtgt cctgaaggac tgcgctgtta ttgagggcca ggattttgtg ctgcagtgct 1680

ccgtacgggg gaccccagtg ccccggatca cttggctgct gaatgggcag cccatccagt 1740

acgctcgctc cacctgcgag gccggcgtgg ctgagctcca catccaggat gccctgccgg 1800

aggaccatgg cacctacacc tgcctagctg agaatgcctt ggggcaggtg tcctgcagcg 1860

cctgggtcac cgtccatgaa aagaagagta gcaggaagag tgagtacctt ctgcctgtgg 1920

ctcccagcaa gcccactgca cccatcttcc tgcagggcct ctctgatctc aaagtcatgg 1980

atggaagcca ggtcactatg actgtccaag tgtcagggaa tccaccccct gaagtcatct 2040

ggctgcacaa tgggaatgag atccaagagt cagaggactt ccactttgaa cagagaggaa 2100

ctcagcacag cctttgtatc caggaagtgt tcccggagga cacgggcacg tacacctgcg 2160

aggcctggaa cagcgctgga gaggtccgca cccaggccgt gctcacggta caagagcctc 2220

acgatggcac ccagccctgg ttcatcagta agcctcgctc agtgacagcc tccctgggcc 2280

agagtgtcct catctcctgc gccatagctg gtgacccctt tcctaccgtg cactggctca 2340

gagatggcaa agccctctgc aaagacactg gccacttcga ggtgcttcag aatgaggacg 2400

tgttcaccct ggttctaaag aaggtgcagc cctggcatgc cggccagtat gagatcctgc 2460

tcaagaaccg ggttggcgaa tgcagttgcc aggtgtcact gatgctacag aacagctctg 2520

ccagagccct tccacggggg agggagcctg ccagctgcga ggacctctgt ggtggaggag 2580

ttggtgctga tggtggtggt agtgaccgct atgggtccct gaggcctggc tggccagcaa 2640

gagggcaggg ttggctagag gaggaagacg gcgaggacgt gcgaggggtg ctgaagaggc 2700

gcgtggagac gaggcagcac actgaggagg cgatccgcca gcaggaggtg gagcagctgg 2760

acttccgaga cctcctgggg aagaaggtga gtacaaagac cctatcggaa gacgacctga 2820

aggagatccc agccgagcag atggatttcc gtgccaacct gcagcggcaa gtgaagccaa 2880

agactgtgtc tgaggaagag aggaaggtgc acagccccca gcaggtcgat tttcgctctg 2940

tcctggccaa gaaggggact tccaagaccc ccgtgcctga gaaggtgcca ccgccaaaac 3000

ctgccacccc ggattttcgc tcagtgctgg gtggcaagaa gaaattacca gcagagaatg 3060

gcagcagcag tgccgagacc ctgaatgcca aggcagtgga gagttccaag cccctgagca 3120

atgcacagcc ttcagggccc ttgaaacccg tgggcaacgc caagcctgct gagaccctga 3180

agccaatggg caacgccaag cctgccgaga ccctgaagcc catgggcaat gccaagcctg 3240

atgagaacct gaaatccgct agcaaagaag aactcaagaa agacgttaag aatgatgtga 3300

actgcaagag aggccatgca gggaccacag ataatgaaaa gagatcagag agccagggga 3360

cagccccagc cttcaagcag aagctgcaag atgttcatgt ggcagagggc aagaagctgc 3420

tgctccagtg ccaggtgtct tctgaccccc cagccaccat catctggacg ctgaacggaa 3480

agaccctcaa gaccaccaag ttcatcatcc tctcccagga aggctcactc tgctccgtct 3540

ccatcgagaa ggcactgcct gaggacagag gcttatacaa gtgtgtagcc aagaatgacg 3600

ctggccaggc ggagtgctcc tgccaagtca ccgtggatga tgctccagcc agtgagaaca 3660

ccaaggcccc agagatgaaa tcccggaggc ccaagagctc tcttcctccc gtgctaggaa 3720

ctgagagtga tgcgactgtg aaaaagaaac ctgcccccaa gacacctccg aaggcagcaa 3780

tgccccctca gatcatccag ttccctgagg accagaaggt acgcgcagga gagtcagtgg 3840

agctgtttgg caaagtgaca ggcactcagc ccatcacctg tacctggatg aagttccgaa 3900

agcagatcca ggaaagcgag cacatgaagg tggagaacag cgagaatggc agcaagctca 3960

ccatcctggc cgcgcgccag gagcactgcg gctgctacac actgctggtg gagaacaagc 4020

tgggcagcag gcaggcccag gtcaacctca ctgtcgtgga taagccagac cccccagctg 4080

gcacaccttg tgcctctgac attcggagct cctcactgac cctgtcctgg tatggctcct 4140

catatgatgg gggcagtgct gtacagtcct acagcatcga gatctgggac tcagccaaca 4200

agacgtggaa ggaactagcc acatgccgca gcacctcttt caacgtccag gacctgctgc 4260

ctgaccacga atataagttc cgtgtacgtg caatcaacgt gtatggaacc agtgagccaa 4320

gccaggagtc tgaactcaca acggtaggag agaaacctga agagccgaag gatgaagtgg 4380

aggtgtcaga tgatgatgag aaggagcccg aggttgatta ccggacagtg acaatcaata 4440

ctgaacaaaa agtatctgac ttctacgaca ttgaggagag attaggatct gggaaatttg 4500

gacaggtctt tcgacttgta gaaaagaaaa ctcgaaaagt ctgggcaggg aagttcttca 4560

aggcatattc agcaaaagag aaagagaata tccggcagga gattagcatc atgaactgcc 4620

tccaccaccc taagctggtc cagtgtgtgg atgcctttga agaaaaggcc aacatcgtca 4680

tggtcctgga gatcgtgtca ggaggggagc tgtttgagcg catcattgac gaggactttg 4740

agctgacgga gcgtgagtgc atcaagtaca tgcggcagat ctcggaggga gtggagtaca 4800

tccacaagca gggcatcgtg cacctggacc tcaagccgga gaacatcatg tgtgtcaaca 4860

agacgggcac caggatcaag ctcatcgact ttggtctggc caggaggctg gagaatgcgg 4920

ggtctctgaa ggtcctcttt ggcaccccag aatttgtggc tcctgaagtg atcaactatg 4980

agcccatcgg ctacgccaca gacatgtgga gcatcggggt catctgctac atcctagtca 5040

gtggcctttc ccccttcatg ggagacaacg ataacgaaac cttggccaac gttacctcag 5100

ccacctggga cttcgacgac gaggcattcg atgagatctc cgacgatgcc aaggatttca 5160

tcagcaatct gctgaagaaa gatatgaaaa accgcctgga ctgcacgcag tgccttcagc 5220

atccatggct aatgaaagat accaagaaca tggaggccaa gaaactctcc aaggaccgga 5280

tgaagaagta catggcaaga aggaaatggc agaaaacggg caatgctgtg agagccattg 5340

gaagactgtc ctctatggca atgatctcag ggctcagtgg caggaaatcc tcaacagggt 5400

caccaaccag cccgctcaat gcagaaaaac tagaatctga agaagatgtg tcccaagctt 5460

tccttgaggc tgttgctgag gaaaagcctc atgtaaaacc ctatttctct aagaccattc 5520

gcgatttaga agttgtggag ggaagtgctg ctagatttga ctgcaagatt gaaggatacc 5580

cagaccccga ggttgtctgg ttcaaagatg accagtcaat cagggagtcc cgccacttcc 5640

agatagacta cgatgaggac gggaactgct ctttaattat tagtgatgtt tgcggggatg 5700

acgatgccaa gtacacctgc aaggctgtca acagtcttgg agaagccacc tgcacagcag 5760

agctcattgt ggaaacgatg gaggaaggtg aaggggaagg ggaagaggaa gaagagtgaa 5820

acaaagccag agaaaagcag tttctaagtc atattaaaag gactatttct ctaaaactca 5880

aaaaaaaaaa aaaaactcaa gatagtaaaa gcacctagtg tgatagatta tcggttaggt 5940

catttgtggg ttgattcttc agaaacagca gttgatacct agcagcgtta ttgatgggca 6000

ttaatctatg ttagttggca ccttaagata ctagtgcagc tagatttcat ttagggaaat 6060

caccagtaac ttgactgacc aattgatttt agagagaaag taaccaaacc aaatatttat 6120

ctgggcaaag tcataaattc tccacttgaa tgcgctcatg aaaaataagg ccaaaacaag 6180

agttctgggc cacagctcag cccagagggt tcctggggat gggaggcctc tctctcccca 6240

ccccctgact ctagagaact gggttttctc ccagtactcc agcaattcat ttctgaaagc 6300

agttgagcca ctttattcca aagtacactg cagatgttca aactctccat ttctctttcc 6360

ccttccacct gccagttttg ctgactctca acttgtcatg agtgtaagca ttaaggacat 6420

tatgcttctt cgattctgaa gacaggtccc tgctcatgga tgactctggc ttccttagga 6480

aaatattttt cttccaaaat cagtaggaaa tctaaactta tcccctcttt gcagatgtct 6540

agcagcttca gacatttggt taagaaccca tgggaaaaaa aaaatccttg ctaatgtggt 6600

ttcctttgta aaccaggatt cttatttgtg ctgttataga atatcagctc tgaacgtgtg 6660

gtaaagattt ttgtgtttga atataggaga aatcagtttg ctgaaaagtt agtcttaatt 6720

atctattggc cacgatgaaa cagatttcaa ctgataaaga gctggagaac tccatgtact 6780

ttggaatctc ctccaagata gccagagttt aatacatctt cattctcaac actctccaaa 6840

gaacttgacc taccttatgg gttccatatt tttcttctta aatgtgcatc aatcatgcct 6900

tgcccccaac ctttaaatat attcttagac ctggtaaatg cactcagact tgcgtcttta 6960

ggaattttta actttctttc actacattgg cacttaaatt ttttctttat aaagcttttt 7020

gaaggtcata aacaaagacc ataattgatg atagacctaa tacatttcct ctgtgtgtgt 7080

gtgtaacatt ccaaatactt tttttttctt ttccactgtt tgtaaggtgc aacaatttaa 7140

tatttttaag ggacttttta agagttcctt aagaaccaat ttaaaattac ttcagtgcaa 7200

tcctacacag tatcaacatt agaattttga tattagtctt atgttatctt ccattctatt 7260

tttatctgct ttttgctgct agtttcaaac tgccagtatt tttccttttg cttttaaaat 7320

agttacaata tttttcatga tagccacagt attgccacag tttattataa taaagggttt 7380

ttatttgatt tagcgcattc aaagcttttt tctatcactt ttgtgttcag aatataacct 7440

ttgtgtgcgt gtatgttgtg tgtgtgcatg tgtggcgtat atgtgtgtta caggttaatg 7500

ccttcttgga attgtgttaa tgttctcttg gtttattatg ccatcagaat ggtaaatgag 7560

aacactacaa ctgtagtcag ctcacaattt ttaaataaag gataccacag tgcatgctgt 7620

ttgttcaaaa aaaaaaaaaa aaaa 7644

<210> 2

<211> 1738

<212> PRT

<213>People source

<400> 2

Met Gly Arg Phe Ser Cys Lys Ile Thr Gly Arg Pro Gln Pro Gln Val

1 5 10 15

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

20 25 30

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

35 40 45

Gln Asp Asp Val Gly Val Tyr Thr Cys Leu Val Val Asn Gly Ser Gly

50 55 60

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

65 70 75 80

Asn Arg Ser Phe Val Arg Glu Thr Lys Ala Thr Asn Ser Asp Val Arg

85 90 95

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

100 105 110

Glu Ala Ala Ala Lys Ser Lys Asn Cys Ser Ser Pro Gln Arg Gly Gly

115 120 125

Ser Pro Pro Trp Ala Ala Asn Ser Gln Pro Gln Pro Pro Arg Glu Ser

130 135 140

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

145 150 155 160

Val Leu Gln Lys Thr Ser Ser Ser Ile Thr Leu Gln Ala Ala Arg Val

165 170 175

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

180 185 190

Glu Glu Arg Lys Arg Pro Ala Pro Pro Arg Pro Ala Thr Phe Pro Thr

195 200 205

Arg Gln Pro Gly Leu Gly Ser Gln Asp Val Val Ser Lys Ala Ala Asn

210 215 220

Arg Arg Ile Pro Met Glu Gly Gln Arg Asp Ser Ala Phe Pro Lys Phe

225 230 235 240

Glu Ser Lys Pro Gln Ser Gln Glu Val Lys Glu Asn Gln Thr Val Lys

245 250 255

Phe Arg Cys Glu Val Ser Gly Ile Pro Lys Pro Glu Val Ala Trp Phe

260 265 270

Leu Glu Gly Thr Pro Val Arg Arg Gln Glu Gly Ser Ile Glu Val Tyr

275 280 285

Glu Asp Ala Gly Ser His Tyr Leu Cys Leu Leu Lys Ala Arg Thr Arg

290 295 300

Asp Ser Gly Thr Tyr Ser Cys Thr Ala Ser Asn Ala Gln Gly Gln Leu

305 310 315 320

Ser Cys Ser Trp Thr Leu Gln Val Glu Arg Leu Ala Val Met Glu Val

325 330 335

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

340 345 350

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

355 360 365

Ile Thr Trp Leu Leu Asn Gly Gln Pro Ile Gln Tyr Ala Arg Ser Thr

370 375 380

Cys Glu Ala Gly Val Ala Glu Leu His Ile Gln Asp Ala Leu Pro Glu

385 390 395 400

Asp His Gly Thr Tyr Thr Cys Leu Ala Glu Asn Ala Leu Gly Gln Val

405 410 415

Ser Cys Ser Ala Trp Val Thr Val His Glu Lys Lys Ser Ser Arg Lys

420 425 430

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

435 440 445

Phe Leu Gln Gly Leu Ser Asp Leu Lys Val Met Asp Gly Ser Gln Val

450 455 460

Thr Met Thr Val Gln Val Ser Gly Asn Pro Pro Pro Glu Val Ile Trp

465 470 475 480

Leu His Asn Gly Asn Glu Ile Gln Glu Ser Glu Asp Phe His Phe Glu

485 490 495

Gln Arg Gly Thr Gln His Ser Leu Cys Ile Gln Glu Val Phe Pro Glu

500 505 510

Asp Thr Gly Thr Tyr Thr Cys Glu Ala Trp Asn Ser Ala Gly Glu Val

515 520 525

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

530 535 540

Pro Trp Phe Ile Ser Lys Pro Arg Ser Val Thr Ala Ser Leu Gly Gln

545 550 555 560

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

565 570 575

His Trp Leu Arg Asp Gly Lys Ala Leu Cys Lys Asp Thr Gly His Phe

580 585 590

Glu Val Leu Gln Asn Glu Asp Val Phe Thr Leu Val Leu Lys Lys Val

595 600 605

Gln Pro Trp His Ala Gly Gln Tyr Glu Ile Leu Leu Lys Asn Arg Val

610 615 620

Gly Glu Cys Ser Cys Gln Val Ser Leu Met Leu Gln Asn Ser Ser Ala

625 630 635 640

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

645 650 655

Gly Gly Gly Val Gly Ala Asp Gly Gly Gly Ser Asp Arg Tyr Gly Ser

660 665 670

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

675 680 685

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

690 695 700

Gln His Thr Glu Glu Ala Ile Arg Gln Gln Glu Val Glu Gln Leu Asp

705 710 715 720

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

725 730 735

Asp Asp Leu Lys Glu Ile Pro Ala Glu Gln Met Asp Phe Arg Ala Asn

740 745 750

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

755 760 765

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

770 775 780

Gly Thr Ser Lys Thr Pro Val Pro Glu Lys Val Pro Pro Pro Lys Pro

785 790 795 800

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

805 810 815

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

820 825 830

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

835 840 845

Pro Val Gly Asn Ala Lys Pro Ala Glu Thr Leu Lys Pro Met Gly Asn

850 855 860

Ala Lys Pro Ala Glu Thr Leu Lys Pro Met Gly Asn Ala Lys Pro Asp

865 870 875 880

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

885 890 895

Asn Asp Val Asn Cys Lys Arg Gly His Ala Gly Thr Thr Asp Asn Glu

900 905 910

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

915 920 925

Gln Asp Val His Val Ala Glu Gly Lys Lys Leu Leu Leu Gln Cys Gln

930 935 940

Val Ser Ser Asp Pro Pro Ala Thr Ile Ile Trp Thr Leu Asn Gly Lys

945 950 955 960

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

965 970 975

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

980 985 990

Lys Cys Val Ala Lys Asn Asp Ala Gly Gln Ala Glu Cys Ser Cys Gln

995 1000 1005

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

1010 1015 1020

Glu Met Lys Ser Arg Arg Pro Lys Ser Ser Leu Pro Pro Val Leu

1025 1030 1035

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

1040 1045 1050

Thr Pro Pro Lys Ala Ala Met Pro Pro Gln Ile Ile Gln Phe Pro

1055 1060 1065

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

1070 1075 1080

Lys Val Thr Gly Thr Gln Pro Ile Thr Cys Thr Trp Met Lys Phe

1085 1090 1095

Arg Lys Gln Ile Gln Glu Ser Glu His Met Lys Val Glu Asn Ser

1100 1105 1110

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

1115 1120 1125

Cys Gly Cys Tyr Thr Leu Leu Val Glu Asn Lys Leu Gly Ser Arg

1130 1135 1140

Gln Ala Gln Val Asn Leu Thr Val Val Asp Lys Pro Asp Pro Pro

1145 1150 1155

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

1160 1165 1170

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

1175 1180 1185

Ser Tyr Ser Ile Glu Ile Trp Asp Ser Ala Asn Lys Thr Trp Lys

1190 1195 1200

Glu Leu Ala Thr Cys Arg Ser Thr Ser Phe Asn Val Gln Asp Leu

1205 1210 1215

Leu Pro Asp His Glu Tyr Lys Phe Arg Val Arg Ala Ile Asn Val

1220 1225 1230

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

1235 1240 1245

Gly Glu Lys Pro Glu Glu Pro Lys Asp Glu Val Glu Val Ser Asp

1250 1255 1260

Asp Asp Glu Lys Glu Pro Glu Val Asp Tyr Arg Thr Val Thr Ile

1265 1270 1275

Asn Thr Glu Gln Lys Val Ser Asp Phe Tyr Asp Ile Glu Glu Arg

1280 1285 1290

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

1295 1300 1305

Lys Thr Arg Lys Val Trp Ala Gly Lys Phe Phe Lys Ala Tyr Ser

1310 1315 1320

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

1325 1330 1335

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

1340 1345 1350

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

1355 1360 1365

Glu Leu Phe Glu Arg Ile Ile Asp Glu Asp Phe Glu Leu Thr Glu

1370 1375 1380

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

1385 1390 1395

Tyr Ile His Lys Gln Gly Ile Val His Leu Asp Leu Lys Pro Glu

1400 1405 1410

Asn Ile Met Cys Val Asn Lys Thr Gly Thr Arg Ile Lys Leu Ile

1415 1420 1425

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

1430 1435 1440

Val Leu Phe Gly Thr Pro Glu Phe Val Ala Pro Glu Val Ile Asn

1445 1450 1455

Tyr Glu Pro Ile Gly Tyr Ala Thr Asp Met Trp Ser Ile Gly Val

1460 1465 1470

Ile Cys Tyr Ile Leu Val Ser Gly Leu Ser Pro Phe Met Gly Asp

1475 1480 1485

Asn Asp Asn Glu Thr Leu Ala Asn Val Thr Ser Ala Thr Trp Asp

1490 1495 1500

Phe Asp Asp Glu Ala Phe Asp Glu Ile Ser Asp Asp Ala Lys Asp

1505 1510 1515

Phe Ile Ser Asn Leu Leu Lys Lys Asp Met Lys Asn Arg Leu Asp

1520 1525 1530

Cys Thr Gln Cys Leu Gln His Pro Trp Leu Met Lys Asp Thr Lys

1535 1540 1545

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

1550 1555 1560

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

1565 1570 1575

Ile Gly Arg Leu Ser Ser Met Ala Met Ile Ser Gly Leu Ser Gly

1580 1585 1590

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

1595 1600 1605

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

1610 1615 1620

Val Ala Glu Glu Lys Pro His Val Lys Pro Tyr Phe Ser Lys Thr

1625 1630 1635

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

1640 1645 1650

Cys Lys Ile Glu Gly Tyr Pro Asp Pro Glu Val Val Trp Phe Lys

1655 1660 1665

Asp Asp Gln Ser Ile Arg Glu Ser Arg His Phe Gln Ile Asp Tyr

1670 1675 1680

Asp Glu Asp Gly Asn Cys Ser Leu Ile Ile Ser Asp Val Cys Gly

1685 1690 1695

Asp Asp Asp Ala Lys Tyr Thr Cys Lys Ala Val Asn Ser Leu Gly

1700 1705 1710

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

1715 1720 1725

Gly Glu Gly Glu Gly Glu Glu Glu Glu Glu

1730 1735

<210> 3

<211> 18

<212> DNA

<213>Artificial sequence

<400> 3

aagtggaggt gtcagatg 18

<210> 4

<211> 20

<212> DNA

<213>Artificial sequence

<400> 4

tcaatgtcgt agaagtcaga 20

<210> 5

<211> 22

<212> DNA

<213>Artificial sequence

<400> 5

aactctggta aagtggatat tg 22

<210> 6

<211> 20

<212> DNA

<213>Artificial sequence

<400> 6

ggtggaatca tattggaaca 20

Claims (10)

1. detect application of the product of MYLK gene expressions in the instrument of diagnosis of endometrial carcinoma is prepared.

2. application according to claim 1, it is characterised in that the product includes：By RT-PCR, real-time quantitative PCR, Immune detection, in situ hybridization, the expression of chip or high-flux sequence detection of platform MYLK genes are with diagnosis of endometrial carcinoma Product.

3. application according to claim 2, it is characterised in that the product of the use RT-PCR diagnosis of endometrial carcinoma is at least Include the primer of a pair of specific amplified MYLK genes；The product of the use real-time quantitative PCR diagnosis of endometrial carcinoma at least includes The primer of a pair of specific amplified MYLK genes；The product of the use immune detection diagnosis of endometrial carcinoma includes：With MYLK albumen The antibody of specific binding；The product of the use in situ hybridization diagnosis of endometrial carcinoma includes：With the nucleotide sequence of MYLK genes The probe of hybridization；The product of the use chip diagnosis of endometrial carcinoma includes：Protein chip and genetic chip；Wherein, albumen core Piece includes the antibody combined with MYLK protein-specifics, and genetic chip includes the probe with the nucleic acid array hybridizing of MYLK genes.

4. application according to claim 3, it is characterised in that the production of the use real-time quantitative PCR diagnosis of endometrial carcinoma The primer for a pair of specific amplified MYLK genes that product at least include is as shown in SEQ ID NO.3 and SEQ ID NO.4.

5. a kind of instrument for diagnosis of endometrial carcinoma, it is characterised in that the instrument can be by detecting MYLK in sample The expression of gene carrys out diagnosis of endometrial carcinoma.

6. instrument according to claim 5, it is characterised in that the instrument includes chip, kit, test paper or high flux Microarray dataset.

7. instrument according to claim 6, it is characterised in that the chip includes genetic chip, protein-chip；It is described Genetic chip includes solid phase carrier and is fixed on the oligonucleotide probe of solid phase carrier, and the oligonucleotide probe includes being used for Detect the oligonucleotide probe for MYLK genes of MYLK gene transcription levels；The protein-chip include solid phase carrier with And it is fixed on the specific antibody of the MYLK albumen of solid phase carrier；The kit includes gene detecting kit and protein immunization Detection kit；The gene detecting kit includes the reagent for being used to detect MYLK gene transcription levels；The protein immunization Detection kit includes the specific antibody of MYLK albumen；The test paper includes the examination for being used to detect MYLK gene transcription levels Agent；The high-flux sequence platform includes the reagent for being used to detect MYLK gene transcription levels.

8. instrument according to claim 7, it is characterised in that the reagent of the detection MYLK gene transcription levels includes pin To the primer and/or probe of MYLK genes.

9. instrument according to claim 8, its spy is characterised by, the primer sequence for MYLK genes is as follows：Just To primer sequence as shown in SEQ ID NO.3, reverse primer is as shown in SEQ ID NO.4.

10. the instrument according to any one of claim 5-9, it is characterised in that the sample is tissue.