CN103675294A

CN103675294A - Novel tumour serum marker and application thereof

Info

Publication number: CN103675294A
Application number: CN201310611491.9A
Authority: CN
Inventors: 邵根泽; 朱柏力; 李莉; 王亚清; 王晓珍; 蔡小青; 林明; 张沙; 周柔丽; 严考文; 匡静宇; 易娟; 卢广
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2013-11-26
Filing date: 2013-11-26
Publication date: 2014-03-26
Anticipated expiration: 2033-11-26
Also published as: CN103675294B

Abstract

The invention provides a novel tumour serum marker and application thereof, particularly relates to application of autoantibodies of POTE protein being taken as tumour serum markers, further relates to application of a reagent for detecting the autoantibodies of the POTE protein in preparation of a composition for detection, auxiliary diagnosis and/or prognosis judgement of cancers, and meanwhile relates to the reagent for detecting the autoantibodies of the POTE protein, a method for detecting the autoantibodies of the POTE protein, and a method for enriching and purifying the autoantibodies of the POTE protein. Besides, the invention further relates to the POTE autoantibodies obtained after the enrichment and the purification according to the method, and application of the POTE autoantibodies.

Description

New tumour serum mark and application thereof

Technical field

The present invention relates to a kind of new tumour serum mark and application thereof, belong to biotechnology and medical domain, specifically, the present invention relates to the preparation method of the sequence of the corresponding identification antigen of autoantibody, autoantibody of a kind of new tumour serum mark POTE and the affinity column of the recombinant technique of this antigen protein, enrichment and purifying POTE autoantibody, from the method for this autoantibody of human serum enriching and purifying and and the technical method that detects human serum POTE autoantibody.The invention also discloses the purposes of POTE autoantibody and corresponding method of detection, as for the diagnosis and treatment of tumour.

Background technology

Tumour is the current second-biggest-in-the-world cause of the death.In 2008, approximately there are 1,270 ten thousand new cases, because the number of tumor mortality reaches 7,600,000.Although modern medicine develop rapidly, and also constantly drop in a large number aspect tumor research and treatment, when the later stages such as tumour generation local diffusion and far-end transfer, modern treatment means are also stymied by, and oncotherapy is produced little effect; And many patients' tumour by when diagnosis in late stage.In the past few decades, there is not too large variation in 5 of these late tumor patients years survival rates.By contrast, infantile tumour patient's prognosis will be got well a lot (Etzioni R comparatively speaking, Urban N, Ramsey S, McIntosh M, Schwartz S, Reid B, Radich J, Anderson G & Hartwell L (2003) .The case for early detection.Nat Rev Cancer3,1 – 10).The progress of modern medicine science and advanced medical procedure have significant curative effect for the treatment of the tumour, particularly tumor in situ of commitment.Therefore, modern medicine has focused on emphasis the early detection of tumour, and tumour was obtained the effective treatment before developing into late period.

Tumour early detection and discovery will or be found tumour at tumor development before tumour diffusion and transfer exactly before untreatable.In tumour, be also in original position and maybe can the healing stage find tumour, not only can reduce mortality ratio, but also tremendous economic and the burden on society that can reduce tumor incidence and bring because treating tumour.

The early diagnosis of tumour and discovery research mainly comprised with the next stage: 1, potential tumor mark is as the invention of the discovery of gene, albumen or antigen or tumor imaging instrument; 2, the tumor markers of discovery is converted into the screening implement of screening tumour, is applied to clinical; 3, to set up early diagnosis of tumor method and the assessment of application thereof.

Tumor markers refers in generation in tumour, evolution, the specific molecular being produced by tumour cell itself or produced by body reply tumour cell.These molecules can reflect existence and the progress of tumour, and often specificity appears at tumor patient, and do not exist or its expression compares with tumor patient that there were significant differences normal person.These molecules belong to protein, polypeptide, nucleic acid or micromolecular compound conventionally, mainly comprise that oncogene and product thereof, hormone, enzyme (isodynamic enzyme), tomour specific or related antigen and respective needle are to the autoantibody of these antigens, polyamines etc.Such as, the Bence-Jones albumen of finding for 1846 is applied to the diagnosis of multiple myeloma, becomes first tumor markers; The AFP finding for 1956, is applied to the diagnosis of liver cancer; Nineteen sixty-five has been found CEA, is applied to the diagnosis of colon cancer.After the human immunity that Herberman in 1978 holds at U.S. NCI and immunologic diagnosis of tumor can be gone up the concept that proposes first tumor markers, tumor markers is widely used in clinical.The tumor markers with clinical meaning of finding has at present reached kind more than 100.Desirable tumor markers should have following characteristics: (1) is highly sensitive; (2) specificity is good; (3) can position tumour; (4) with coincident with severity degree of condition, tumor size or by stages relevant; (5) can monitor the effect to oncotherapy; (6) recurrence of monitoring tumour; (7) prognosis of predicting tumors.Due to the complicacy of tumor etiology, there is no a kind of tumour is single type, therefore find that the tumor markers of " ideal " is just very difficult.Up to now, there is no the tumor markers of a kind of " ideal ".

In 100 Diagnostic Value of Several Serum Tumor Markers with clinical meaning of finding at present, having most is to be present in patient's blood, such as many tumor associated antigens (TAA) or tomour specific or carcinomebryonic antigen, TAA or autoantibody etc.The existence of the tumor markers in these serum to the early stage diagnosis and detection of tumour provide may and convenient way.These blood serum designated objects are convenient on the one hand detects, and has avoided the various nocuities of diseased region to detect, thereby has been easy to crowd to carry out extensive examination; On the other hand, some blood serum designated object, as the autoantibody of Tumor-assaciated or specific antibody, occur, and sensitivity is higher, can be used as the early diagnosis index of tumour at tumour commitment.Therefore, explored and find those before tumour occurs and the blood serum designated object of initial period, early detection and significant (the Wagner PD of diagnosis for tumour, Verma M & Srivastava S (2004) .Challenges for biomarkers in cancer detection.Ann N Y AcadSci1022,9-16).

Tumor-assaciated autoantibody is the most attractive tumor markers of a class.In normal cell early carcinomatous change process, with tumorigenic initial period, the related antigen of tumor cells expression may appear at diseased region.The immune response of body can be induced the corresponding antibodies producing for the higher titre of these molecules; they are present in blood circulation of human body system (Lu H; Goodell V, Disis ML.Humoral immunity directed against tumor associated antigens as potential biomarkers for the early diagnosis of cancer.J Proteome Res2008; 7:1388 – 94; Anderson KS, Labaer J.The sentinel within:exploiting the immune system for cancer biomarkers.J ProteomeRes2005; 4:1123 – 33).These autoantibodies have in blood that stability is high, retention long, occur the features such as morning, and are easy to be detected by measures such as immunologys.On the contrary, many tumor associated antigens, if traditional tumor markers CA-15-3, CA-19-9, CA-125 and CEA etc. are to come off from the tumor tissues compared with large to release, only have when tumor growth arrives larger volume, could in serum, reach enough concentration; Secondly, they discharge after after hemodilution concentration very low; But also may further by the immune system of body, be removed (J.-Y.Zhang, C.A.Casiano, X.-X.Peng, J.A.Koziol, E.K.L.Chan, and E.M.Tan, " Enhancement of antibody detection in cancer using panel of recombinant tumor-associated antigens, " Cancer Epidemiology Biomarkers and Prevention, vol.12, no.2, pp.136 – 143,2003).Therefore, comparatively speaking, the autoantibody of tumor associated antigen, as tumour early sign thing, has more potential value in tumour early detection, and application prospect is extensive clinically.At present, the autoantibody of a series of tumor associated antigens is found, and comprises p53, MUC-1, heat shock protein (HSP-27, HSP-60, HSP-90), HER2, c-myc, CT antigen NY-ESO-1, lipophilin B etc.; There is certain relation in generation and the prognosis of they and tumour.But the autoantibody of these molecules all lacks higher specificity and susceptibility as the index of tumour early detection and diagnosis; can not be separately for early diagnosis and prognosis evaluation (Piura E, the Piura B.Autoantibodies to tumor-associated antigens in breast carcinoma.J Oncol.2010 of tumour; 2010:264926).Cause the chief reason that these antibody specificitys and susceptibility are low to be, the corresponding tumor associated antigen molecule the non-tumor cell institute that induce these autoantibodies to produce are peculiar, at normal cell, also generally express.Therefore, find the autoantibody of tumour specific antigen molecule, for solving above specificity and sensitive question, significant for tumour early detection and diagnosis.

Tumour specific antigen is the protein molecule that in tumour cell generation evolution, expressed, most normal structures and cell are not expressed; Theoretically, such antigen can be induced the specific autoantibody that body produces.Tumour testis (Cancer-Testis, CT) antigen just belongs to such molecule.CT antigen is mainly expressed in Human germ cells, and very low or do not express at adult body cellular expression; But when cell carcinogenesis and tumour generation, this class CT antigen gene can reactivate, abnormal high level expression.Only about half of above CT antigen is encoded by X sex chromosome, therefore they be also referred to as CT-X, to distinguish over the coding CT antigen (G.Curigliano in autosome source, G.Viale, M.Ghioni et al., " Cancer-testis antigen expression in triple-negative breast cancer, " Annals of Oncology; T.Suyama, T.Shiraishi, Y.Zeng et al., " Expression of cancer/testis antigens in prostate cancer is associated with disease progression, " Prostate, vol.70, no.16, pp.1778 – 1787,2010).The expression of CT antigen is often relevant with clinical stages to tumor prognosis and pathologic grading of cancer.Recent studies have found that, some CT antigens are if NY-ESO-1, MAGE-A are at three feminine gender (ER-, PR-, and HER2-) breast cancer is expressed and is increased (G.Curigliano, G.Viale, M.Ghioni et al., " Cancer-testis antigen expression in triple-negative breast cancer, " Annals of Oncology; Grigoriadis A, Caballero OL, Hoek KS; da Silva L, Chen YT, Shin SJ; Jungbluth AA; Miller LD, Clouston D, Cebon J; Old LJ; Lakhani SR, Simpson AJ, Neville AM.CT-X antigen expression in human breast cancer.Proc Natl Acad Sci U S is A.2009; 106 (32): 13493-8).

For the autoantibody of CT antigen, in comprising breast cancer, many tumours are proved as NY-ESO-1, and combine with other tumor markerses to use and can be used for clinical tumor and early examine (C.Chapman, A.Murray, J.Chakrabarti et al., " Autoantibodies in breast cancer:their use as an aid to early diagnosis, " Annals of Oncology, vol.18, no.5, pp.868 – 873,2007).The autoantibody of change of serum C T antigen to exist for tumour early detection and diagnosis and prognosis all significant.But a ubiquitous difficult problem is, it be not immediately clear which CT antigen can induce body to produce autoantibody certain titre, detectable; What method can confirm the existence of these autoantibodies by, and how to detect these autoantibodies.In addition, the ambiguity Chu between these novel autoantibodies and tumour early detection, diagnosis and prognosis, has largely limited its clinical value.

Therefore, explore and find that CT antigen autoantibody and detection method thereof are significant.

Summary of the invention

The object of the invention is to research provides a kind of new autoantibody and related detecting method and application, for human tumor early detection, diagnosis and treatment and prognosis judgement provide foundation.

This case inventor has found the autoantibody of the protein molecule POTE of in research.

POTE is one group of histone family molecule that is only expressed in prostate, ovary, testis and placenta tissue.In human genome, 13 height POTE genes homology, different subtype are distributed in respectively on 8 different chromosomes (2,8,13,14,15,18,21, and 22).Although different subtype molecular size differs, their coded albumen has 3 to be rich in halfcystine structure territory at N end; In addition in molecule, comprise respectively in addition 4-7 ankyrin repetitive sequence and SPECTRIN sample helical structure territory.This molecule in the location of cell mainly at cytoplasma membrane and endochylema (Bera TK; Zimonjic DB, Popescu NC, Sathyanarayana BK; Kumar V; Lee B, Pastan I.POTE, a highly homologous gene family located on numerous chromosomes and expressed in prostate; ovary; testis, placenta, and prostate cancer.Proc Natl Acad Sci U S is A.2002; 99 (26): 16975-80).

Although POTE does not express at the most of vital tissue of human body, in many tumours as unconventionality expressions such as breast cancer, prostate cancer, lung cancer; Secondly; some hypotype is also expressed (Bera TK in hESC; Saint Fleur A; Lee Y, Kydd A, Hahn Y; Popescu NC; Zimonjic DB, Lee B, Pastan I.POTE paralogs are induced and differentially expressed in many cancers.Cancer Res.2006Jan1; 66 (1): 52-6; Bera TK; Saint Fleur A; Ha D; Yamada M, Lee Y, Lee B; Hahn Y; Kaufman DS, Pera M, Pastan I.Selective POTE paralogs on chromosome2are expressed in human embryonic stem cells.Stem Cells Dev.2008; 17 (2): 325-32).Therefore, in theory, POTE possesses the feature of CT antigen, and himself antibody is potential important tumor markers.

Although POTE is a CT antigen in theory, there is the possibility that induction body produces autoantibody, nobody proves and detects the existence of this protein autoantibody in blood of human body all the time.In the present invention, first by building prokaryotic expression protein, these recombinant proteins of purifying of different structure region POTE albumen and utilizing them to remove the antibody in the dissimilar Serum of Cancer Patients of the enrichment mankind as antigen, confirmed the existence of this autoantibody; Secondly, utilize the POTE fragment of the expressed purifying of the present invention, successfully set up the ELISA method that detects POTE autoantibody; Further, by the detection to normal person, tumor patient and non-Serum of Cancer Patients POTE, find that POTE specificity is present in 70-80% Serum of Cancer Patients, and in Healthy Human Serum, almost can't detect (1%), these results suggest, POTE autoantibody is a species specificity and highly sensitive tumour serum mark, can be used for examination and the early detection of tumour, and the treatment of tumour and prognosis are had to important value.

Thereby, according to experimental study of the present invention, the invention provides the autoantibody of POTE albumen in the application as in tumour serum mark.POTE autoantibody is a species specificity and highly sensitive tumour serum mark.

According to specific embodiment of the invention scheme, on the one hand, the application of the reagent that the invention provides the autoantibody that detects POTE albumen in the composition of the detection for the preparation of cancer, auxiliary diagnosis and/or prognosis judgement.The present invention confirms by experiment, by detecting the method for POTE autoantibody, comes diagnosing tumour to have than other tumour antigen detection methods to have more susceptibility and specificity as CA199, CA125, CEA etc.

According to specific embodiment of the invention scheme, preferably, the reagent of the autoantibody of described detection POTE albumen comprises: with restructuring POTE albumen or the POTE polypeptide fragment of the autoantibody specific binding of POTE albumen, or for the PCR primer of the gene DNA chain at POTE albumen described in PCR composite coding or POTE polypeptide fragment and/or its cDNA chain, or the encode gene order of described POTE albumen or POTE polypeptide fragment or the expression vector that comprises this gene order.

According to specific embodiment of the invention scheme, described and POTE polypeptide fragment POTE albumen autoantibody specific binding preferably includes:

(a) polypeptide fragment being formed by the amino acid sequence shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 or SEQ ID No.4; Or

(b) in the amino acid sequence that (a) limits through replacement, lack or add one or several amino acid and with (a) have identical function by (a) derivative polypeptide fragment;

More preferably, described and POTE polypeptide fragment POTE albumen autoantibody specific binding also can further comprise: the polypeptide fragment being comprised of the amino acid sequence shown in SEQ ID No.6, and/or the polypeptide fragment being comprised of the amino acid sequence shown in SEQ ID No.8.Or further comprise the polypeptide fragment with other POTE family members of POTE-E polypeptide fragment (as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.6 or SEQ ID No.8) height homology, for example, from the polypeptide fragment of POTE-A, POTE-B, POTE-C, POTE-D, POTE-F, POTE-G, POTE-H, POTE-I, POTE-J, POTE-M.

Described and restructuring POTE albumen POTE albumen autoantibody specific binding comprises:

(c) fusion being formed by the amino acid sequence shown in SEQ ID No.5; Or

(d) in the amino acid sequence that (c) limits through replacement, lack or add one or several amino acid and with (c) have identical function by (c) derivative albumen;

More preferably, described and restructuring POTE albumen POTE albumen autoantibody specific binding also can further comprise:

The fusion being formed by the amino acid sequence shown in SEQ ID No.7, and/or the fusion being formed by the amino acid sequence shown in SEQ ID No.9.

In the present invention, the polypeptide fragment being comprised of the amino acid sequence shown in SEQ ID No.1 is the polypeptide that POTE-E albumen 1-144 amino acids sequence forms; The polypeptide fragment being comprised of the amino acid sequence shown in SEQ ID No.2 is the polypeptide that POTE-E albumen 20-53 amino acids sequence forms; The polypeptide fragment being comprised of the amino acid sequence shown in SEQ ID No.3 is the polypeptide that POTE-E albumen 57-90 amino acids sequence forms; The polypeptide fragment being comprised of the amino acid sequence shown in SEQ ID No.4 is the polypeptide that POTE-E albumen 94-127 amino acids sequence forms.The present invention studies confirm that by experiment, the autoantibody of the POTE albumen of these polypeptide fragments in can specific binding serum, thus can be effectively by autoantibody enrichment the purifying of the POTE albumen in serum.The polypeptide fragment being comprised of the amino acid sequence shown in SEQ ID No.6 is the polypeptide that POTE-E albumen 390-524 amino acids sequence forms, and the polypeptide fragment being comprised of the amino acid sequence shown in SEQ ID No.8 is the polypeptide that POTE-E albumen 528-641 amino acids sequence forms.The autoantibody of the POTE albumen of the polypeptide fragment wherein, being formed by the amino acid sequence shown in SEQ ID No.6 in also can specific binding serum; Although the autoantibody of the POTE albumen of the polypeptide fragment being formed by the amino acid sequence shown in SEQ ID No.8 in can not specific binding serum, can with the autoantibody of other specific fragments combinations for the POTE albumen in conjunction with serum.These polypeptide fragments of the present invention are also referred to as POTE polypeptide fragment.

Restructuring POTE albumen of the present invention and POTE polypeptide fragment all can obtain by conventional method.For example, pcr amplification technology that can establishing criteria is using cDNA, mRNA or genomic DNA as template, and chooses suitable Oligonucleolide primers amplification obtain the encoding nucleotide of described POTE albumen or POTE polypeptide fragment.The nucleotide obtaining like this can be cloned in suitable carrier, by various known methods, by transformed host cell and at the host cell being converted, grow into after suitable cell density, with suitable method evoked promoter, then continue to cultivate, after cultivation completes, reclaim and purifying POTE albumen of the present invention or POTE polypeptide fragment.In the present invention, as restructuring POTE albumen, can with or not with label.For example, in a specific embodiments of the present invention, to pass through PCR method, by POTE protein molecular 1-144aa(or the 2-144aa of encoding respectively), the gene order in 390-524aa and 528-641aa region is cloned into pGEX-6P-3 carrier, builds pGEX-GST-POTE_144, pGEX-GST-POTE_390 and pGEX-GST-POTE_528 fusion protein expression vector.Again above-mentioned plasmid pGEX-GST-POTE_144, pGEX-GST-POTE_390 and pGEX-GST-POTE_528 are transformed respectively to e. coli bl21 or Rosette, cultivate and IPTG induction, express destination protein.With the cutting of Sepharose4B Glutathione affinity chromatography, Pressicion enzyme and base exchange method, purify destination protein afterwards, obtain the GST-144(SEQ ID No.5 of POTE), GST-390(SEQ ID No.7) and GST-528(SEQ ID No.9) fusion, and the polypeptide fragment 1-144aa(SEQ ID No.1 of POTEE), 390-524aa(SEQ ID No.6) and 528-641aa(SEQ ID No.8).

The nucleotide sequence of coding POTE protein molecular 1-144aa refers to SEQ ID No.10; The nucleotide sequence of coding POTE protein molecular 390-524aa refers to SEQ ID No.11; The nucleotide sequence of coding POTE protein molecular 528-641aa refers to SEQ ID No.12.

On the other hand, the present invention also provides a kind of reagent of autoantibody of the POTE of detection albumen, this reagent comprises: with restructuring POTE albumen or the POTE polypeptide fragment of the autoantibody specific binding of POTE albumen, or for the PCR primer of the gene DNA chain at POTE albumen described in PCR composite coding or POTE polypeptide fragment and/or its cDNA chain;

Preferably, described and POTE polypeptide fragment POTE albumen autoantibody specific binding comprises:

More preferably, described and POTE polypeptide fragment POTE albumen autoantibody specific binding also can further comprise:

The polypeptide fragment being formed by the amino acid sequence shown in SEQ ID No.6, and/or the polypeptide fragment being formed by the amino acid sequence shown in SEQ ID No.8.

(c) fusion being formed by the amino acid sequence shown in SEQ ID No.5; Or

On the other hand, the present invention also provides a kind of method of autoantibody of the POTE of detection albumen, and the method comprises:

Utilize the reagent of the autoantibody that detects POTE albumen, adopt ELISA method, DOT BLOT method, WESTERN BLOT method and/or immunofluorescence method to detect the existence of POTE autoantibody in blood serum sample.

In a specific embodiments of the present invention, utilize elisa technique method, detected respectively the titre that normal person's group, dissimilar tumour comprise the patients serum POTE autoantibodies such as breast cancer, oophoroma, prostate cancer, lung cancer, the cancer of the esophagus, nasopharyngeal carcinoma, cancer of the stomach, colorectal cancer, appendix myxoadenocarcinoma, laryngocarcinoma, neuroendocrine lung carcinoma, liver cancer, cancer of pancreas or the non-formula of fund suddenly lymthoma, the autoantibody of finding POTE albumen has very high sensitivity and specificity, with coincident with severity degree of condition, tumor size or by stages relevant; Can be used for the effect of monitoring to oncotherapy; Also significant to the monitoring recurrence of tumour and the prognosis of predicting tumors.

On the other hand, the present invention also provides a kind of method of autoantibody of enriching and purifying POTE albumen, and the method comprises:

Prepare immobilization affinity column: adopt curing diamido dipropyl ammonia agarose and the POTE polypeptide fragment of Thermo to mix respectively, and add the coupling of EDC coupling agent;

Utilize above chromatographic column to carry out affinity chromatography to Serum of Cancer Patients, obtain the autoantibody of POTE albumen.

According to specific embodiment of the invention scheme, in method of the present invention, for the preparation of the described POTE polypeptide fragment of immobilization affinity column, comprise one or more in the polypeptide fragment being formed by the amino acid sequence shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 or SEQ ID No.4; Preferably, described POTE polypeptide fragment also can further comprise the polypeptide fragment that the amino acid sequence shown in SEQ ID No.6 and/or SEQ ID No.8 forms.

In a specific embodiments of the present invention, fragment with the excision label of the polypeptide fragment being comprised of amino acid sequence shown in SEQ ID No.1, the polypeptide fragment being comprised of amino acid sequence shown in SEQ ID No.6, three kinds of prokaryotic expressions of polypeptide fragment of being comprised of amino acid sequence shown in SEQ ID No.8 is prepared immobilization affinity column, enriching and purifying POTE autoantibody, has proved the existence of the POTE antibody that enriching and purifying obtains according to the method described above by electrophoresis and Western blotting according to the method described above.

On the other hand, the present invention also provides the POTE autoantibody obtaining according to method enriching and purifying of the present invention.

The autoantibody that the present invention also provides described POTE albumen is for the preparation of diagnosis and/or the diagnostic preparation for the treatment of cancer or application in pharmaceutical composition.Wherein, described cancer can be breast cancer, oophoroma, prostate cancer, lung cancer, the cancer of the esophagus, nasopharyngeal carcinoma, cancer of the stomach, colorectal cancer, appendix myxoadenocarcinoma, laryngocarcinoma, neuroendocrine lung carcinoma, liver cancer, cancer of pancreas or the non-formula of fund suddenly lymthoma.Particularly, POTE autoantibody of the present invention at least can be applied to following aspect: 1, POTE antibody of the present invention (comprising its various modified antibodies) can be for various objects as passed through the methods such as immunofluorescence, Western blotting, SABC for detection of human archeocyte, human tumor cells and organize POTE family antigen particularly, thereby for diagnosing tumor, for oncotherapy provides foundation.Due to the high homology of the N of POTE family end protein matter sequence, by the purified POTE antibody obtaining of preceding method of the present invention, not only can detect cell POTE-E, and can be simultaneously for detection of other POTE family member molecules, comprise POTE-A ,-B ,-B-like,-C,-F ,-G ,-H,-I, the different subtypes such as-J and M.2, in conjunction with the feature of POTE autoantibody specific recognition POTE family antigen, and the feature of tumour cell specifically expressing POTE, by POTE autoantibody is modified, in coupling, different molecular and compound, can be for objects such as tumor-localizing detection, metastases supervision and oncotherapies as nucleic, fluorescence molecule, therapeutic protein or polypeptide equimolecular.Such as, coupling fluorescence molecule or radioactive nuclide on POTE autoantibody, can be for clinical tumor iconography as CT, PET-CT scanning etc., realize that the in situ detection of tumour, metastases are monitored, thereby be applied to diagnosing tumor, treatment and prognostic analysis etc.; In addition, utilize antibody of the present invention to there is the feature of specific recognition tumor cell surface POTE antigen, at POTE autoantibody by combinations such as the whole bag of tricks and means and antineoplastic, molecules, directed agents as these antineoplastics and molecule, target recognition of tumor cell, realization killing and wounding and removing tumour cell, thereby plays a significant role in clinical cancer therapy.

In sum, the present invention has found the autoantibody of protein molecule POTE, and its Identification and detection method is provided; The application process of this autoantibody in human tumor early detection, diagnosis and treatment and prognosis is provided simultaneously.Because POTE albumen is class CT antigen molecule, in normal structure, do not express.But POTE albumen starts to express when cell carcinogenesis and tumour generation.Theoretically, POTE may induce the immune system with excitating organism as antigen, produces the autoantibody for POTE antigen molecule.Therefore, the appearance of these autoantibodies in serum can be pointed out generation, development and the prognosis of tumour, and can be used as examination, diagnosis and the treatment analysis that a tumor markers applies to tumour, also significant to the monitoring recurrence of tumour and the prognosis of predicting tumors.

Accompanying drawing explanation

Fig. 1: the prokaryotic protein expression GST-144 of POTE fragment, GST-390, GST-528.Wherein, picture the 4th, 6 swimming lane: GST-144; The 8th swimming lane: GST-390, the 10th swimming lane: GST-528.

Fig. 2: the POTE autoantibody electrophoresis of purifying is identified.

Fig. 3 A and Fig. 3 B: the POTE autoantibody WB of purifying identifies.Wherein, Fig. 3 A: prokaryotic expression total protein.The WB of Fig. 3 B:GST purifying protein antigen affinitive layer purification POTE autoantibody detects: with GST-144, and GST-390, GST-528 is antigen; With process GST-144, GST-390, the Serum of Cancer Patients hybridization of GST-528 affinity column purifying; Result shows GST-144, and GST-390 antibody purification can specific binding antigen, but GST-528 antibody purification specificity not.

Fig. 4: the POTE autoantibody epi-position of purifying is identified.

Fig. 5: Elisa method detects human serum POTE autoantibody.

Fig. 6: DOT BLOT method detects human serum POTE autoantibody.

Fig. 7: GST-144 is that antigen WESTERN BLOT method detects blood-serum P OTE autoantibody.Wherein: P1, P8 is that Serum of Cancer Patients is; N3 is normal human serum.P1, detects POTE autoantibody in P8 serum.

Fig. 8: immunofluorescence method detects the fluorescence microscope result of POTE autoantibody.

Fig. 9: utilize elisa technique method to detect the titre of kind patients serum POTE autoantibody.

Embodiment

Below in conjunction with specific embodiment, further illustrate the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition, or the condition of advising according to manufacturer.

In following examples, all commercially available acquisitions of original reagent used and material, main agents and material are: TRIzol (Invitrogen); IPTG (Isopropyl beta-D-thiogalactopyranoside), Sepharose4B Glutathione (GE); PreScission Protease (GE); EDC(1-ethyl-3-(3-dimethylaminopropyl) carbodiimide); Solidify diamido dipropyl ammonia agarose (Thermo Scientific); Rosetta E.coli strain; PGEX-6P-3; PcDNA3.1-FLAG-HA-POTE; 96 hole ELISA Plate (Elisa plate).

The Prokaryotic expression vector construction of embodiment 1, POTE fragment antigen, expression and purifying

(1) clone of POTE gene cDNA

With TRIzol reagent, extracting HELA cell mRNA and carry out reverse transcription (RT) is cDNA.Take this cDNA as template, and pcr amplification obtains the cDNA fragment of two hypotype 2A and the 2C of POTE, and after measured, its sequence is as SEQID No.13(POTE-2A ORF sequence) and SEQ ID No.14(POTE-2C ORF sequence) as shown in.

(2) POTE polypeptide fragment Prokaryotic expression vector construction

Pass through PCR method, by POTE-E protein molecular 1-144aa(or the 2-144aa of encoding respectively) gene order (referring to SEQ ID No.10), the gene order (referring to SEQ ID No.11) in coding POTE-E protein molecular 390-524aa region and the gene order (referring to SEQ ID No.12) in coding POTE-E protein molecular 528-641aa region be cloned into pGEX-6P-3 carrier, structure pGEX-GST-POTE_144, pGEX-GST-POTE_390 and pGEX-GST-POTE_528 fusion protein expression vector.

(3) expression, the purifying of GST-POTE_144, GST-POTE_390 and GST-POTE_528 fusion

Above-mentioned plasmid pGEX-GST-POTE_144, pGEX-GST-POTE_390 and pGEX-GST-POTE_528 are transformed respectively to Escherichia coli Rosetta cultivation and IPTG induction, express destination protein.With the cutting of Sepharose4B Glutathione affinity chromatography, Pressicion enzyme and base exchange method, purify destination protein, obtain GST-144, GST-390 and the GST-528 fusion of POTE-E, and polypeptide fragment 1-144aa, 390-524aa and the 528-641aa of POTE, all carrier sequences are all identified authentication (Fig. 1) through DNA sequencing.The sequence of GST-144, GST-390 and GST-528 fusion is respectively as shown in SEQ ID No.5, SEQ ID No.7, SEQ ID No.9; Polypeptide fragment 1-144aa is as shown in SEQ ID No.1, and 390-524aa is as shown in SEQ ID No.6; 528-641aa is as shown in SEQ ID No.8.

The purifying of embodiment 2, POTE autoantibody and evaluation

In the present embodiment, in order to prove the existence of POTE autoantibody, first prepare POTE polypeptide fragment immobilization affinity column, and with this post, Serum of Cancer Patients has been carried out to enrichment and purifying, obtained POTE autoantibody.Concrete operations are as follows:

POTE polypeptide fragment immobilization affinity column preparation: (1) POTE polypeptide preparation: the GST-POTE-144 fragment (GST-144) of prokaryotic expression is used after the affine combination of Glutathione-Sepharose beads, with precission enzyme, hatch, the GST label that excision is connected with 144 polypeptide, then, it is centrifugal with 10kDa aperture super filter tube 14000rpmll that enzyme is cut potpourri, precission enzyme is removed in ultrafiltration, obtains the POTE-144(POTE-E-144 of purifying) polypeptide fragment.With same method, can obtain POTE-390(390-524aa) and POTE-528(528-641aa) polypeptide fragment.(2) the curing diamido dipropyl ammonia agarose (diaminodipropylamine immobilized to4%agarose) that adopts ThermoScientific respectively with POTE-144(1-144aa), POTE-390(390-524aa), POTE-528(528-641aa) three peptide species fragments mix respectively, and add EDC(1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) coupling agent coupling, obtain POTE polypeptide fragment immobilization affinity column.This post can be used for follow-up blood-serum P OTE autoantibody purifying.

The autoantibody purifying of POTE: utilize above chromatographic column to carry out affinity chromatography to Serum of Cancer Patients, can obtain the autoantibody of POTE, concrete purification step is as follows: (1) human serum is hatched 2 hours at 4 ℃ with Protein G-Agrose after diluting 10 times with PBS, PBS washing 3 times, with the antibody of 100mM Glycine (pH3.0) eluent elution of bound, and neutralize with the 1M TrisCl (pH8.0) of 1/10 volume.This step obtains the total antibody in human serum.(2) by coupling the Sepharose of POTE-144 polypeptide fragment mix with the total antibody of human serum obtaining in above-mentioned (1) step, hatch 2 hours for 4 ℃, then with PBS washing 3 times.With the antibody of 100mM Glycine (pH3.0) eluent elution of bound, and neutralize with the 1M TrisCl (pH8.0) of 1/10 volume.This step obtains in human serum the specific antibody for POTE-144 fragment epi-position.Because the amino acid sequence of POTE-144 is identical with other members of POTE family or height homology, the antibody that therefore above method obtains, except for POTE-E albumen, also comprises for other POTE family member molecules.

Purified gained antibody is carried out to electrophoresis evaluation, prove exist (Fig. 2) of POTE antibody.The serum total Ig G that in Fig. 2, left figure is purifying, middle graph is to utilize the POTE autoantibody of POTE-E-144 purifying, right figure is POTE-E-390, the POTE autoantibody that POTE-E-528 is purified.

With above-mentioned antibody purification respectively with Fig. 3 A) or the POTE prokaryotic expression protein of purifying (bacterium GST purifying protein: Fig. 3 B) carrying out WESTERN Western blotting is proved POTE prokaryotic expression protein (total bacterial protein:.

The evaluation of embodiment 3, POTE autoantibody epi-position

By GST-144 affinity column purifying gained POTE autoantibody from Serum of Cancer Patients, it may be the polyclonal antibody for different sequence epi-positions in the 1-144aa fragment polypeptide of POTE.In order to identify the epi-position of POTE autoantibody, in the present embodiment, pass through respectively PCR method, by the i.e. 20-53aa(SEQ ID No.2 of the Probability Area epi-position in 144 amino acid residues of POTE-E albumen n end of encoding respectively), 57-90aa(SEQ ID No.3) and 94-127aa(SEQ ID No.4) the corresponding gene order in region is cloned into pGEX-6P-3 carrier, structure pGEX-GST-POTE_20-53, _ 57-90 and _ 94-127aa fusion protein expression vector.Above-mentioned plasmid is transformed to Rosetta Escherichia coli, and the former nucleoprotein of IPTG abduction delivering is POTE-E respective segments albumen.Whether ultrasonication and cracking Escherichia coli, use the gst fusion protein by GST-144 affinity chromatography gained POTE autoantibody and these fragments to carry out Western blotting, detect and can mutually combine, thereby carry out epi-position differentiation.As shown in Figure 4, the gst fusion protein of POTE20-53aa, 57-90aa and 94-127aa can with by the purified antibody of GST-POTE-144, respond, show that human body POTE autoantibody contains the antibody for epi-positions such as 20-53aa, 57-90aa and 94-127aa.It should be noted that above three epitope sequences are closely similar.

The detection method of embodiment 4, blood-serum P OTE autoantibody

In the present embodiment, adopt following three kinds of methods to detect the POTE autoantibody in human serum:

ELISA method: utilize antigen and antibody specific combination and this feature of reaction, take the 144(1-144aa of POTE) be antigen coated 96 hole ELISA Plate, adopt the titre of Elisa method detection blood-serum P OTE autoantibody.Specific as follows: to adopt every 96 holes of mixed solution 100ul/ that comprise 144 each 3.3ug of fragment that cut the prokaryotic expression after label on GST purifying post, 4 ℃ of coating spend the night, Tecan washes plate machine 200ulPBS and washes 4 times, 10%BSA4 ℃ of 200ul every hole sealing spent the night, and PBS washes 4 times, and serum 1:1000 dilutes 4 ℃, the every hole of rear 100ul and spends the night, PBS washes 4 times, the anti-human two anti-1:5000 dilution room temperature 2h of goat, OPD method lucifuge colour developing 20min after PBS washes 4 times, microplate reader 592nm reading.Result is referring to Fig. 5.Fig. 5 represents with the coated ELISA96 orifice plate of POTE-144 fragment, 5%BSA sealing, different extension rate tumour patients and non-affinity antibody to SpA are as primary antibodie, and HRP mark goat anti-human two is anti-to be hatched, with OPD development process, develop the color, finally by microplate reader, at 592nm emission wavelength, detect.Whether being intended to detect of this experiment there is the antibody that may be combined with POTE-144 fragment and explores the suitable serum dilution ratio with larger discrimination in affinity antibody to SpA.Horizontal ordinate is serum diluting multiple, is respectively 1:10,1:50,1:300,1:900,1:2700,1:8100,16200 and blank; Ordinate is that ELISA detects numerical value, and P1～P8 is different affinity antibody to SpAs; Na is normal human serum sample.Fig. 5 result shows, the susceptibility that ELISA detects blood-serum P OTE autoantibody is very high, even approval effectively detects POTE autoantibody after 1:3000 dilution.Therefore serum dilution can arrive 1:3000 at 1:10, and has best discrimination in 1:100 to 1:1000 scope.

DOT BLOT method: with GST-144, GST-390 and the GST-528 fusion of the POTE that recombinates in embodiment 1, or polypeptide fragment 1-144aa, the 390-524aa of POTE and 528-641aa point film, respectively with normal person and Serum of Cancer Patients hybridization, identify in serum and whether have POTE autoantibody, in addition, with GST (Fig. 6) in contrast.As shown in Figure 6, GST-144 and POTE144 polypeptide (1-144aa), as antigen, can detect in Serum of Cancer Patients (P1, P8, P14) and have POTE antibody, and do not have in contrast normal human serum (N1, N12, N14); Using the 390-524aa polypeptide fragment of POTE as antigen, also can specific detection to the existence that has POTE autoantibody in tumor patient rather than normal person.But POTE528-641aa fragment is not special as antigen, cannot distinguish normal person and tumor patient.This result is consistent with the conclusion of Fig. 3, i.e. the 144(1-144aa of POTE) and 390(390-524aa) fragment specific recognition POTE autoantibody comparatively.Therefore, POTE1-144 and 390-524 fragment polypeptide are desirable antigen, can be used for ELISA method specific detection blood-serum P OTE autoantibody.

WESTERN BLOT method: with the GST-144 of GST, restructuring POTE, GST-390, GST-528 fusion carries out SDS-PAG electrophoresis, after transferring film respectively with normal person (N3) and tumor patient (P1, P8) serum hybridization, identifies in serum whether have POTE autoantibody (Fig. 7).As shown in Figure 7, GST-POTEE-144 is as antigen, can specific detection to POTE autoantibody (P1, P8) in tumour serum, but in normal human serum, (N3) cannot detect.Other two kinds of antigens, GST-390 and GST-528 effect are bad.Therefore, POTE1-144 polypeptide fragment is comparatively ideal detection POTE and other members' of family thereof expression.

Immunofluorescence method: in order to confirm purified POTE autoantibody, we have built the expression vector pcDNA31-POTE-2C-HA-FLAG(plasmid sequence of the HA-FLAG label merging with POTE-2C C end and have seen SEQ ID No.15), transfection HELA cell, heterogenous expression POTE-2C/HA-FLAG fusion in cell.Cell is fixed through 4%PFA, and after PBS washing, 0.1%Triton-X100 penetrates, sheep blood serum sealing 1 hour.Use respectively the human serum POTEE-144 antibody (1:50) of purifying and the anti-HA monoclonal antibody of mouse (1:2000, Covance) hatch 37 ℃ 2 hours, PBST washing 5 times.Hatch TRITC-IgG37 ℃ of goat-anti people FITC-IgG and goat-anti mouse and hatch 1 hour, PBST washing 5 times, mounting (containing DAPI).Fluorescence microscope.As shown in Figure 8, the cell (HA stained positive) of expressing POTE-2C/HA-FLAG fusion can be dyeed by the Serum Antibodies of POTEE-144 purifying, and the two exists location altogether.Above result shows POTE-2C albumen that can specific recognition cellular expression by the purified antibody of GST-POTEE-144, has the autoantibody of anti-POTE in reference's serum.

The detection of embodiment 5, POTE autoantibody and the effect in diagnosing tumor and prognosis thereof

In the present embodiment, utilize elisa technique method, detected respectively the titre (see figure 9) that normal person's group (100 example), dissimilar tumour comprise the patients serum POTE autoantibodies such as breast cancer (70 example), lung cancer (27 example), nasopharyngeal carcinoma (35 example), cervical carcinoma and oophoroma (12 example), cancer of the stomach (7 example), colorectal cancer (9 example), the cancer of the esophagus (12 example), liver cancer (4 example) and Fei Hejinjieshi lymph cancer (5 example).Learn by statistics check, find that this autoantibody is at breast cancer (P=<0.001), lung cancer (P=<0.001), nasopharyngeal carcinoma (P=<0.001), cervical carcinoma and oophoroma (P=0.001), colorectal cancer (P=<0.001), in the kinds of tumors patients serums such as the cancer of the esophagus (P=<0.001) and cancer of the stomach (P=<0.001), titre is significantly higher than normal human serum, the detection of prompting POTE autoantibodies may be relevant with generation and the clinical pathology of these tumours, the early detection that can be used for these tumours.POTE autoantibody is also expected to the effect of monitoring to oncotherapy; Also significant to the monitoring recurrence of tumour and the prognosis of predicting tumors.

Claims

The autoantibody of 1.POTE albumen is in the application as in tumour serum mark.
2. the application of the reagent of the autoantibody of detection POTE albumen in the composition of the detection for the preparation of cancer, auxiliary diagnosis and/or prognosis judgement;

Preferably, the reagent of the autoantibody of described detection POTE albumen comprises: with restructuring POTE albumen or the POTEE polypeptide fragment of the autoantibody specific binding of POTE albumen, or for the PCR primer of the gene DNA chain at POTE albumen described in PCR composite coding or POTE polypeptide fragment and/or its cDNA chain, or the encode gene order of described POTE albumen or POTE polypeptide fragment or the expression vector that comprises this gene order.
3. application according to claim 1 and 2, wherein, described cancer is breast cancer, oophoroma, prostate cancer, lung cancer, the cancer of the esophagus, nasopharyngeal carcinoma, cancer of the stomach, colorectal cancer, appendix myxoadenocarcinoma, laryngocarcinoma, neuroendocrine lung carcinoma, liver cancer, cancer of pancreas or the non-formula of fund suddenly lymthoma.
4. application according to claim 2, wherein,

Described and POTE polypeptide fragment POTE albumen autoantibody specific binding comprises:

(a) polypeptide fragment being formed by the amino acid sequence shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 or SEQ ID No.4; Or

(b) in the amino acid sequence that (a) limits through replacement, lack or add one or several amino acid and with (a) have identical function by (a) derivative polypeptide fragment;

More preferably, described and POTE polypeptide fragment POTE albumen autoantibody specific binding also can further comprise: the polypeptide fragment being comprised of the amino acid sequence shown in SEQ ID No.6, and/or the polypeptide fragment being comprised of the amino acid sequence shown in SEQ ID No.8; Or further comprise the polypeptide fragment from POTE-A, POTE-B, POTE-C, POTE-D, POTE-F, POTE-G, POTE-H, POTE-I, POTE-J, POTE-M with SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.6 or SEQ ID No.8 height homology;

Described and restructuring POTE albumen POTE albumen autoantibody specific binding is:

(c) fusion being formed by the amino acid sequence shown in SEQ ID No.5; Or

(d) in the amino acid sequence that (c) limits through replacement, lack or add one or several amino acid and with (c) have identical function by (c) derivative albumen;

More preferably, described and restructuring POTE albumen POTE albumen autoantibody specific binding also can further comprise:

The fusion being formed by the amino acid sequence shown in SEQ ID No.7, and/or the fusion being formed by the amino acid sequence shown in SEQ ID No.9.
5. a reagent that detects the autoantibody of POTE albumen, this reagent comprises: with restructuring POTE albumen or the POTE polypeptide fragment of the autoantibody specific binding of POTE albumen, or for the PCR primer of the gene DNA chain at POTE albumen described in PCR composite coding or POTE polypeptide fragment and/or its cDNA chain;

Preferably, described and POTE polypeptide fragment POTE albumen autoantibody specific binding is:

(a) polypeptide fragment being formed by the amino acid sequence shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 or SEQ ID No.4; Or

(b) in the amino acid sequence that (a) limits through replacement, lack or add one or several amino acid and with (a) have identical function by (a) derivative polypeptide fragment;

Described and restructuring POTE albumen POTE albumen autoantibody specific binding is:

(c) fusion being formed by the amino acid sequence shown in SEQ ID No.5; Or

(d) in the amino acid sequence that (c) limits through replacement, lack or add one or several amino acid and with (c) have identical function by (c) derivative albumen.
6. a method that detects the autoantibody of POTE albumen, the method comprises:

Utilize the reagent of the autoantibody that detects POTE albumen, adopt ELISA method, DOT BLOT method, WESTERN BLOT method and/or immunofluorescence method to detect the existence of POTE autoantibody in blood serum sample.
7. a method for the autoantibody of enriching and purifying POTE albumen, the method comprises:

Prepare immobilization affinity column: adopt curing diamido dipropyl ammonia agarose and the POTE polypeptide fragment of Thermo to mix respectively, and add the coupling of EDC coupling agent;

Utilize above chromatographic column to carry out affinity chromatography to Serum of Cancer Patients, obtain the autoantibody of POTE albumen.
8. method according to claim 7, wherein, described POTE polypeptide fragment comprises the polypeptide fragment being comprised of the amino acid sequence shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 or SEQ ID No.4; Preferably, described POTEE polypeptide fragment also comprises the polypeptide fragment being comprised of the amino acid sequence shown in SEQ ID No.6 and/or the polypeptide fragment being comprised of the amino acid sequence shown in SEQ ID No.8.
9. the autoantibody of the POTE albumen obtaining according to the method enriching and purifying described in claim 7 or 8.
The autoantibody of 10.POTE albumen is for the preparation of diagnosis and/or the treatment diagnostic preparation of cancer or the application in pharmaceutical composition, wherein, preferably, described cancer is breast cancer, oophoroma, prostate cancer, lung cancer, the cancer of the esophagus, nasopharyngeal carcinoma, cancer of the stomach, colorectal cancer, appendix myxoadenocarcinoma, laryngocarcinoma, neuroendocrine lung carcinoma, liver cancer, cancer of pancreas or the non-formula of fund suddenly lymthoma.