CN102838657A - Human tumor marker TBRG4 (transforming growth factor beta regulator 4) polypeptide and application thereof - Google Patents

Abstract

The invention belongs to the fields of biotechnology and medicine, and relates to new human tumor marker TBRG4 (transforming growth factor beta regulator 4) polypeptide, a polynucleotide sequence for coding the TBRG4 and a method for producing the protein through a recombinant technology. The invention also relates to application of the TBRG4 protein and the coding sequence of the TBRG4 protein and a medicinal composition comprising a TBRG4 protein antagonist. Experiments show that the TBRG4 polypeptide, the coding sequence of the TBRG4 polypeptide and an expression product of the gene can be hardly detected in normal cells and tissues, but are extremely highly expressed in tumor cells and tissues; and the expression is in obvious positive correlation with the level, the differentiation degree and the prognosis of tumor. The result shows that the TBRG4 is a specific tumor marker which can be used as a marker for prevention and early detection of breast carcinoma or a tumor treatment target.

Description

A kind of human tumor marker thing TBRG4 polypeptide and uses thereof

Technical field

The invention belongs to biotechnology and medical field, relate to a kind of new human tumor marker thing TBRG4 polypeptide (transforming growth factor beta regulator), the polynucleotide sequence of coding TBRG4 and produce this proteic method through recombinant technology.The invention still further relates to the purposes of TBRG4 albumen and encoding sequence thereof.

Background technology

At present one of problem of the most critical of tumor research is promptly sought early detection, diagnosis typing, and treatment monitoring and prognosis, and as the biomarker of drug targets.Biomarker (biomarker) is meant can be by objective measurement, indication normal physiological or pathologic process, or body is for physics, chemistry, the Biological indicators of drug reaction.Research shows that cell changes into the process of tumour from standard state, and a series of variations can take place the intracellular protein express spectra.Tumor markers be exactly tumour take place with evolution in, by the synthetic release of tumour cell or host one type of material to the release of tumor response property.They have certain specificity and sensitivity, can be as the monitoring index in the Clinics and Practices.

At present, being used to clinical tumor markers also quite lacks.The mark auditing standards of various countries is slightly different.China mainly still is with reference to the standard of western developed countries such as the U.S. in the tumor markers application facet.With the U.S. is example, and annual ASCO (American Society of Clinical Oncology) can propose guidance instruction to the used biomarker of clinical tumor.The mark that can be used for mammary cancer as 2007 comprises ER/PR, HER2, and uPA/PAI1 and multiparameter gene expression analysis method (Oncotype DM, MammaPrint etc.), and detailed suggestion working conditions and scope is provided.Remaining is like Ki67, Cyclin D, and Cyclin E, p27, p21, Proteomic analysis etc. are insufficient because of evidence, are not proposed use (referring to the ASCO webpage).Can see that admitting that these marks are improving patient's diagnosis, the effect while in the treatment, the sickness rate and the lethality rate of tumour patient are still in rising trend generally at present.This phenomenon requires the more better discovery and the clinical applications of tumor markers.

Summary of the invention

The purpose of this invention is to provide a kind of new human tumor marker thing; Be specifically related to the polypeptide of human tumor marker thing TBRG4, this polypeptide comprises: the polypeptide of the aminoacid sequence of SEQ ID NO:1 or its conservative property variation polypeptide or its active polypeptide or its reactive derivative; Its coding nucleic acid (RNA) has the sequence of SEQ ID NO:5.

Another object of the present invention provides the above-mentioned RNA of coding, picodna sequence (DNA) the SEQ IDNO:6 of polypeptide.

Another object of the present invention provides the preparation method of above-mentioned RNA and polypeptide, and this RNA, the purposes of polypeptide and its encoding sequence.

Among the present invention, described RNA can produce through the mode of PCR; Specifically, can adopt the primer among the SEQ ID NO:3 right, through the polymerase chain reaction, obtaining sequence is the RNA product of SEQ ID NO:5; Said polypeptide can be produced through the mode of chemosynthesis; This RNA and polypeptide all can be used for helping to detect the existence of tumour.

The present invention the results showed, has obtained the new gene that can be used as tumor markers; This expression of gene product comprises RNA SEQ ID NO:4 and Protein S EQ ID NO:2, and described polynucleotide SEQ ID NO:4 coding has the albumen of the aminoacid sequence of SEQID NO:2; Described expression product normal cell with the tissue in almost detect less than, tumour cell with the tissue in very high expression is but arranged; And the rank of described expression amount and tumour, differentiation degree, and patient's prognosis all is obvious positive correlation; Result of study shows, described TBRG4 is a special tumor markers, has accomplished the present invention on this basis.

Specifically, the present invention relatively from breast cancer cell lines same patient, different grade malignancies, has obtained a series of differential expression protein through systematically; Said TBRG4 be one of them new in tumour the protein of specifically expressing; Through quantitative PCR and western blotting method, the result shows, mRNA and the protein expression level of TBRG4 all apparently higher than normal cell are in the high tumor cell line of grade malignancy; Simultaneously, find TBRG4 albumen high expression level in the tumour patient tissue, expression level very low (as shown in Figure 1) then in the healthy tissues through antibody checking; Further; Method through immunohistochemical methods; The present invention experimentizes to 62 examples tissue normal and the breast cancer tumour patient, and the result shows that not seeing in the normal galactophore tissue has the proteic expression of TBRG4; And then show the low expression level of TBRG4 in the early stage galactophore disease (Hyperplasia, cyclomastopathy); And it has general high expression level in malignant breast carcinomas such as infiltrative type duct carcinoma and lobular carcinoma; Above-mentioned experimental evidence shows that described TBRG4 has the latent effect as the early detection markers for breast cancer.

The present invention is through the TBRG4 in the following method test sample, and it comprises: the RNA in the extracting tissue, and reverse transcription becomes cDNA, with the primer sequence quantitative pcr amplification of SEQ ID NO:3; Or the protein in the extracting tissue carries out immune marking reaction with specific antibody, detects protein level; Or, to paraffin-embedded tissue, carry out the immunohistochemical methods test with specific antibody.Described specific antibody is and TBRG4 protein-specific bonded antibody.

The present invention is according to TBRG4 cross expressing in tumour, and suppress its expression and whether can suppress the growth of tumor check: the shRNA to TBRG4 has been synthesized in design, but and it is cloned in slow virus (lenti-viral) carrier of abduction delivering; The result shows that the expression that suppresses TBRG4 can significantly suppress growth of tumor; Said result confirms that further TBRG4 can be as the target of oncotherapy.

In of the present invention, simulation is provided, has promoted, picked up the active compound of anti-people TBRG4, and the compound that suppresses the expression of people TBRG4.Preferably, this compound is people TBRG4 encoding sequence or its pulsating small molecules interference RNA.Be cloned in the pLKO.1. carrier pLKO.1-shTBRG4. that this carrier is gone into packing cell 293T to produce virus with the packaging plasmid corotation described small molecules shRNA.With virus infection tumour cell to be measured, the result shows that the growth of tumour cell to be measured obviously slows down.

TBRG4 albumen and the encoding sequence thereof that the present invention relates to; Can be used for prevention, the early detection of tumour, the judgement of prognosis; And the preparation above-mentioned TBRG4 albumen that contains safe and effective amount is picked up the pharmaceutical composition of anti-agent (like antibody and antisense nucleic acid, RNAi etc.) and pharmaceutically acceptable carrier.

Description of drawings

Fig. 1 has shown the expression of TBRG4 of the present invention in breast cancer cell line and tissue,

Wherein, A: with the quantitative reverse transcription PCR that the special primer of TBRG4 carries out, the result carries out stdn with crt gene GAPDH and representes, 16N among the figure, HME are the normal breast epithelial cell, and all the other are breast cancer cell; B: detect TBRG4 albumen with immune marking method in the mammary gland cell, cell identifies like A; C: in normal and canceration mammary tissue sample, detect TBRG4 albumen with immunoblotting; D: in organization chip, detect TBRG4 albumen with immunohistochemical methods, figure is for normally, the example of carcinoma in situ and invasive carcinoma; On: normal; In: DCIS; Down: invasive carcinoma.

Fig. 2 shows that suppressing TBRG4 expresses the speed of growth and the clonality that can significantly reduce breast cancer cell,

Wherein, A: use three TBRG4 stable inhibition strain cell and corresponding contrast to clone and form experiment, formed clone is through violet staining and counting; B: use three TBRG4 stable inhibition strain cell and corresponding contrast to carry out the MTT experiment, each cell strain growth tendency every day is shown in left hand view, and right side side western blot figure is represented each stable expression level that suppresses TBRG4 in cell strain.

Fig. 3 shows that suppressing TBRG4 expresses the travelling speed that can significantly reduce breast cancer cell, wherein, uses three TBRG4 stable inhibition strain cell and corresponding contrast to carry out the cell migration experiment, and the cell of wearing film dyes through crystallization.

Embodiment

In first aspect of the present invention, the TBRG4 polypeptide of new isolated differential expression is provided, it comprises: polypeptide or its conservative property variation polypeptide or its active polypeptide or its reactive derivative with SEQ ID NO:1 aminoacid sequence.

Embodiment 1: the TBRG4 polypeptide that obtains differential expression

To separate the breast cancer cell line 16N (normally) that obtains from a patient, the protein among NT (TIS) and the MT2 (metastatic tumour) is with lysate (8M urea, 2.5M thiourea; 65mM DTT; 4% (w/v) CHAPS, 0.5% (v/v) Biolytes pH 3-10, protease inhibitor cocktail) extracting; Quantitative (RC DC Protein Assay Kit; Bio-rad company) appearance is carried out the one dimension isoelectrofocusing earlier on the back, then carries out two-dimentional SDS-PAGE after the completion, finishes back silver and dyes to show each protein site.Compare the gained differential protein spot through decolouring through PDQuest software, dried glue, in-gel digestion is put forward steps such as peptide, uses the liquid chromatography isolated polypeptide again.Isolated polypeptide is identified by LTQ-Orbitrap (Thermo Electron company) mass spectrograph.TBRG4 is one of differential protein that identifies, and it is low the expression in 16N, and high expression level in tumor cell line NT and MT2, bioinformatic analysis show that it is the localized kinase protein of a plastosome.

In second aspect of the present invention, the polynucleotide of the isolating said polypeptide of encoding are provided; SEQ ID NO:4.

Embodiment 2: the full-length gene that from breast cancer cell line, obtains coding TBRG4

Extract the total RNA in the MT2 breast cancer cell line with Trizol reagent (Invitrogen company), total RNA reverse transcription is become cDNA with PrimeScript 1st strand cDNA Synthesis Kit (TaKaRa company); With synthetic cDNA is template; Design contains TBRG4 clone gene the forward primer 5 '-CAGGGATCCATGGCAGCTCACCTGGTAAA-3 ' and the reverse primer 5 '-GACTCTAGATCACTTGGCCAGCTCCTC-3 ' of restriction enzyme site, carries out the PCR reaction with warm start Taq archaeal dna polymerase (TaKaRa company), and reaction conditions is the preparatory sex change of 98 degree 2 minutes; 95 degree are 10 seconds then; 55 degree annealing 2 minutes, 72 degree carried out extension in 40 seconds, reacted 30 circulations; PCR product and pcDNA3.0-HA-FLAG empty carrier are carried out BamHI and Xba I substep single endonuclease digestion respectively, are connected and the transformation experiment of DH5 α bacterium; Obtain the positive colony of TBRG4 gene transformation, treat that dna sequencing confirms that gene order obtains the pcDNA3.0-HA-FLAG-TBRG4 carrier after errorless.

In the third aspect of the invention, simulation is provided, has promoted, picked up the active compound of anti-people TBRG4, and the compound that suppresses the expression of people TBRG4.Preferably, this compound is people TBRG4 encoding sequence or its pulsating small molecules interference RNA.Be cloned in the pLKO.1. carrier pLKO.1-shTBRG4. that this carrier is gone into packing cell 293T to produce virus with the packaging plasmid corotation described small molecules shRNA.With virus infection tumour cell to be measured, the result shows that the growth of tumour cell to be measured obviously slows down.

Embodiment 3:TBRG4 small molecules interference RNA suppresses growth of tumor and migration

Use the Public TRC Portal and the BLOCK-iT of Invitrogen company of Broad Institute website simultaneously ^TMThe online design of RNAi Designer TBRG4 shRNA; Comprehensively both Search Results obtain six pairs of shRNA sequences to TBRG4 gene different positions; With connecting into the pLKO.1 carrier through Age I and EcoR I double digestion after the synthetic oligo annealing, the picking positive colony is the pLKO.1-shTBRG4 carrier.Based on this lentiviral vectors, the present invention with itself and packaging plasmid psPAX2 and pMD2.G cotransfection HEK-293T cell with packaging virus; Transfection was collected substratum after 48 hours, was slow virus stoste after removing residual cell; Viral liquid is mixed the cell that the back adds the logarithmic phase growth with polybrene, hatch after 12 hours and abandon viral liquid, add the puromycin screening after 24 hours and promptly get the stable cell strain cell that suppresses of TBRG4.

The immunoblotting of learning from else's experience checking suppresses effect three stable cell strain cell (sh3 that suppress of TBRG4 preferably; Sh4; Sh5) and corresponding pLKO.1 control cells; Density with 2000 cells in every hole is inoculated in 96 orifice plates, whenever uses mtt assay afterwards at a distance from 24 hours or directly carries out cell counting measurement cell growth condition; The result shows that the inhibition that TBRG4 expresses has significantly reduced the speed of growth of cell.Inoculate in above-mentioned three kinds of TBRG4 stable inhibition cell and control cells to six orifice plate with the cell concentration in 500 or 250 every holes, three use violet staining to weigh its clonality after all around.The result shows that the expression of reducing TBRG4 has significantly reduced the clonality (as shown in Figure 2) of breast cancer cell.

Use stable cell and the control cells of suppressing of above-mentioned three kinds of TBRG4 equally, inoculate every hole 50000 cells to the upper strata cell of Transwell device (Corning), the while contains the substratum of 10%FBS to attract cell-penetrating in lower floor's adding.Downcut film after 8 hours, and carry out violet staining, the observed result of microscopically shows that the inhibition of TBRG4 has significantly reduced the transfer ability of cell (as shown in Figure 3).

In fourth aspect of the present invention, the carrier and the virus that contain above-mentioned small molecules interference RNA are provided.The production process of carrier and virus details in embodiment 3.

Aspect the of the present invention the 5th, the method that whether has TBRG4 in the test sample is provided, it comprises: the RNA in the extracting tissue, reverse transcription becomes cDNA, carries out quantitative pcr amplification with design synthetic primer sequence (SEQ ID NO:3).Another method that detects protein level is: the protein in the extracting tissue, carry out immune marking reaction with specific antibody, and detect protein level; Or, to paraffin-embedded tissue, carry out the immunohistochemical methods test with specific antibody.The method that detects tumour also is provided, has comprised: sample is carried out above-mentioned quantitative PCR test, immune marking test or immunohistochemical methods test, the individuality that the result is higher than background level suffers from tumour or tumor susceptibility is higher than normal population.

Embodiment 4: the mRNA level of quantitative PCR detection TBRG4

Albumen corresponding sequence and the gene order information on the NCBI website (accession number:NM_004749.2) according to mass spectrum identifies design the TBRG4 primer with primer3 software (http://frodo.wi.mit.edu/primer3/input.htm).The upstream primer sequence that obtains is 5 '-TCCGGCTCTCTCACTTGCTGT-3 ', and the downstream primer sequence is 5 '-GGTACCATGCCAGACCGAGGC-3 '.Three kinds of isoform to TBRG4 during design of primers reflect the mRNA level of TBRG4 in the cell fully to guarantee qPCR result.The confidential reference items of quantitative PCR are the GAPDH gene.The upstream primer of used GAPDH gene is 5 '-cgagatccctccaaaatcaa-3 ', and downstream primer is 5 '-ttcacacccatgacgaacat-3 '.Extract total RNA with Trizol, survey concentration and the purity of RNA through spectrophotometer (260nm/280nm).Carry out reverse transcription (TaKaRa) with total RNA of 3 μ g and oligo dT primer through PrimeScript 1st strand cDNA Synthesis Kit.In 25 μ l quantitative PCR reaction systems, add 75ng cDNA template, 1 * SYBR Premix Ex Taq (Perfect Real Time; TaKaRa) and the primer of each 200nM.(Bio-rad company) carries out amplified reaction with iQ5 quantitative PCR appearance.Response procedures is: 95 ℃ of initial sex change 10 seconds, and two-step approach is carried out 40 circulations, and 95 ℃, sex change in 5 seconds, 60 ℃ of annealing were extended 20 seconds.Under the relative quantification pattern, carry out the data quantitative analysis with iQ5 (Bio-Rad) v2.0 version software.Figure 1A is a result of quantitative PCR experiment, uses clone RNA, finds normal cell 16N, and the expression level of TBRG4 mRNA is very low among HMEC and the MCF10A, and tumour cell, like NT, MT2, its expression amount is significantly higher than normal cell among MCF7 and the MDA231.

Embodiment 5: immunoblotting and immunohistochemical methods detect TBRG4 albumen

Use the antibody (rabbit against human T BRG4 antibody, PTGLAB company) of TBRG4, in cell and tissue, carry out immunoblotting and immunohistochemical experiment.In immunoblotting,, transfer to then on the pvdf membrane (Amersham Bioscience company) with the about 30 μ g total proteins of the SDS-PAGE gel separation of 10% concentration.With TBST damping fluid (0.1%Tween 20 for 137mM NaCl, 20mM Tris-HCl pH7.6) the sealing pvdf membrane that contains 5%BSA, with TBRG4 antibody incubated at room one hour or 4 degree incubated overnight, washing, HRP link coupled two anti-hatching.Washed film adds that hypersensization luminescent solution (Amersham Bioscience) develops, through the LAS-3000 imaging system form images (Fuji, Japan).(Fuji company, Japan) software is quantitative to the band of immunoblotting to use Multi Gauge V3.0.Figure 1B is the result of immunoblot experiment in clone.β-actin antibody (mouse-anti people β-actin antibody, PTGLAB company) is as the contrast of last appearance.Experiment shows, at the normal breast epithelial cell, and 16N, among HMEC and the MCF10A, TBRG4 is in very low expression level, and at breast cancer cell MCF7, MDA231, among NT and the MT2, the expression level of TBRG4 significantly increases.

In immunohistochemical experiment, through formalin fixed and paraffin-embedded organization chip available from US Biomax company (CA, U.S.A.Catalog:TMA-BR480).Section is repaired antigen through 0.01M citrate buffer (pH 6.0) poach after dewaxing and aquation.Spending night with the TBRG4 one anti-4 of dilution in 1: 100 hatches.HRP polymer detection system by anti-rabbit detects again, the imaging of Olympus BX51 microscopic system.Fig. 1 C example normal, the immunohistochemical staining of DCIS and invasive carcinoma.By two researchists painted organization chip is marked, each sample is divided into nothing by staining power, and is low, in, high fourth gear, corresponding 0-3 branch respectively; According to stained area marking, 0-5% is 0 minute again, and 5-25% is 1 minute, and 25-50% is 2 minutes, and 50-75% is 3 minutes, is 4 minutes more than 75%.Intensity is divided and area divides addition to be final score.The gained data use Student ' s paired t-test and Spearman rank sum test method to carry out the significance,statistical check.The result shows, does not express TBRG4 albumen in the healthy tissues, and it is galactophore disease in early days the earliest, has part to express in the cyclomastopathy.And in infiltrative type duct carcinoma and lobular carcinoma, TBRG4 exists general high expression level, and its expression level increases progressively with the rising of mammary cancer grade.The result shows that TBRG4 can be as the mark of mammary cancer prevention and early detection.

Aspect the of the present invention the 6th, the purposes of polypeptide of the present invention and encoding sequence is provided; Purposes comprises with the polypeptide immune animal and obtains the antibody to polypeptide, again the expression through antibody detection protein; Can pass through blood, tissue, and other body fluid (like the mammary gland imbibition, nipple aspirate) detect.The effect of encoding sequence comprises through its design primer, probe, and siRNA, and shRNA etc. are used to detect DNA, and the expression of RNA suddenlys change and because inhibition of gene expression reaches the result of treatment that suppresses tumor growth.Said purposes is described in embodiment 3,4,5 to some extent.

Aspect the of the present invention the 7th, a kind of drug regimen is provided, the people TBRG4 of the present invention that it contains safe and effective amount picks up anti-agent and pharmaceutically acceptable carrier; Preferably picking up anti-agent is the small molecules interference RNA sequence; These pharmaceutical compositions can be treated tumour.Concrete is, with the pLKO.1-shTBRG4 of pLKO.1 vector construction one-tenth, and with the defective virus of its generation.These results describe in embodiment 3 to some extent.

In other aspects of the present invention because disclosing of the technology of this paper is conspicuous to those skilled in the art.

SEQUENCE LISTING

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tcctcagcca cctcacccat ttcccacctc ccagttcctt gatggagccg gtggagaagg 180

aacgagcatc tactccctac atagagaagc aggtggacca cctcatcaag aaggccacaa 240

ggccagagga gctcctggag ctacttggtg gcagtcacga cttggacagc aatcaagcag 300

caatggtact tatccggctc tctcacttgc tgtctgagaa gccagaagat aaaggcttgc 360

tcatacagga tgcccacttt catcaacttc tctgtctgct caacagtcag attgcctcgg 420

tctggcatgg taccctctcg aagctgctgg gaagcctgta tgctctgggc atccccaagg 480

cctcaaggag ctgcagtcgg tggagcagga ggtccgctgg cgcatgcgga agctcaagta 540

caagcacctg gccttcctgg cagagtcctg tgccaccctc tcacaggagc agcactcgca 600

ggagctgctg gctgagctgc tcacacacct ggaaaggcgt tggacagaaa ttgaagattc 660

ccacacatta gtgaccgtca tgatgaaggt gggacacctc tcggagccac taatgaaccg 720

cctggaagac aagtgcctgg agttggtgga gcactttggc cccaatgagc tgcggaaggt 780

gctggtgatg ctggcagctc agagccggcg gtccgtgccc ttgctgcggg ccatctccta 840

ccacctggtg cagaagccct tctctctgac gaaagatgtg ctcttggacg tggcctatgc 900

ctatggcaaa ctcagctttc accagaccca ggtgtcccag cgcctggcca ccgacctgct 960

atccctcatg cccagcctga cttctggtga ggtggcccac tgtgccaagt ccttcgcctt 1020

actcaagtgg ctcagcctgc ccctgtttga ggcctttgcc cagcacgtcc tgaacagagc 1080

gcaggacatc accctgcccc acctgtgcag cgtacttctg gcttttgcgc gtctgaactt 1140

ccatccagac caagaggatc agttcttcag cctggtacat gagaagctgg ggtcagagct 1200

gccaggcctg gagccagccc tgcaggtgga cctggtgtgg gccctgtgtg tgctgcagca 1260

ggcacgggaa gcagagctgc aagccgtcct ccaccctgaa tttcacatcc aatttctagg 1320

gggcaagtct cagaaggatc agaacacctt ccagaagctg ctccacatca acgccactgc 1380

cctgctggag taccccgagt actcgggtcc ccttctgcct gcctcggctg tggcccctgg 1440

gccctcagcc cttgacagga aggtgacccc cctgcaaaag gagctgcagg agacgctgaa 1500

ggggctgctg gggagcgccg acaagggcag cctcgaggtg gccacgcagt atggctgggt 1560

gctggatgct gaggtgctgc tggacagtga cggcgagttt ctgcccgtaa gggactttgt 1620

ggcacctcac cttgcccagc caactgggag ccagtcacca cctccagggt ctaagaggct 1680

agcgttcttg cggtgggagt tccccaactt caacagccga agcaaggact tgctgggtcg 1740

ctttgttctg gcccggcgac acatagtggc tgcaggcttc ctgatagtgg acgtcccatt 1800

ctatgagtgg ctggaactca agtctgaatg gcagaaaggc gcctacctca aggacaagat 1860

gcgcaaagcg gtggctgagg agctggccaa gtga 1894

Claims (11)

1. a human tumor marker thing TBRG4 polypeptide is characterized in that this polypeptide comprises: the polypeptide of the aminoacid sequence of SEQ ID NO:1 or its conservative property variation polypeptide or its active polypeptide or its reactive derivative; Its coding nucleic acid has the sequence of SEQ ID NO:5.

2. a TBRG4 albumen is characterized in that, this albumen has the aminoacid sequence of SEQ ID NO:2.

3. isolating polynucleotide is characterized in that, described polynucleotide have the sequence of SEQ ID NO:4, the albumen of its coding claim 2.

4. a DNA is characterized in that, has the structure of SEQ ID NO:6, the RNA and the polypeptide of its coding claim 1.

5. a compound is characterized in that, this compound is picked up anti-agent for picking up anti-people TBRG4 activity, and it contains people TBRG4 encoding sequence or its pulsating small molecules interference RNA of claim 1.

6. a pharmaceutical composition is characterized in that, it contains the compound and the pharmaceutically acceptable carrier of the claim 5 of safe and effective amount.

7. an ability and claim 1 or the described TBRG4 protein-specific of claim 2 bonded antibody.

8. the method for TBRG4 in the test sample is characterized in that it comprises: the RNA in the extracting tissue, and reverse transcription becomes cDNA, with the primer sequence quantitative pcr amplification of SEQ ID NO:3; Or the protein in the extracting tissue carries out immune marking reaction with specific antibody, detects protein level; Or, to paraffin-embedded tissue, carry out the immunohistochemical methods test with specific antibody.

9. the TBRG4 polypeptide of claim 1 and encoding sequence thereof detect the purposes in tumor markers or the oncotherapy target in preparation.

10. the albumen of claim 2 and encoding sequence thereof detect the purposes in tumor markers or the oncotherapy target in preparation.

11. by the purposes of claim 9 or 10, wherein said tumour is a mammary cancer.

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