CN102532317B - The monoclonal antibody of PES1 and application thereof - Google Patents
The monoclonal antibody of PES1 and application thereof Download PDFInfo
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Abstract
The present invention relates to PES1 monoclonal antibody or derive from this antibody can the bioactive fragment of specific binding PES1, the hybridoma that described monoclonal antibody is CGMCC No.3646 or CGMCC No.3647 by the preserving number of China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation is secreted.Monoclonal antibody of the present invention has higher sensitivity and specificity, and identifiable design endogenous PES1 albumen also can be used for various immunology detection; The invention still further relates to the hybridoma cell line of this antibody of secretion, comprise the test kit of described antibody or its bioactive fragment, antitumor drug and detection reagent, described antibody or its bioactive fragment are in the application detecting PES1 protein level in sample, and the application of described antibody in auxiliary diagnosis tumour, monitoring tumour and/or tumour patient Index for diagnosis.
Description
Technical field
The present invention relates to field of immunology, relate to particularly for PES1 monoclonal antibody or derive from this antibody can the bioactive fragment and preparation method thereof of specific binding PES1, and secrete the hybridoma cell line of this antibody, the invention still further relates to the test kit, antitumor drug and the detection reagent that comprise described antibody or its bioactive fragment, described antibody or its bioactive fragment are in the application detecting PES1 protein level in sample, and the application of described antibody in auxiliary diagnosis tumour, monitoring tumour and/or tumour patient Index for diagnosis.
Background technology
The sickness rate of malignant tumour continues the gesture in rising in recent years in the world.Because the morbidity of malignant tumour is comparatively hidden mostly, there is no manifest symptom in early days, often reach an advanced stage when patient goes to a doctor, and manyly show close patient at clinical traditional pathology and TNM by stages, its prognosis also has larger difference, reflects that malignant tumour occurs, the complicacy of development.Still lack at present and can be used for diagnosing or the desirable tumor markers of Index for diagnosis.As the mark CEA in decades as digestive tract tumor, in some non-neoplastic diseases are as gastritis, liver cirrhosis, ulcerative colitis, also can have rising, its specificity is unsatisfactory.Therefore, find and occur to malignant tumour, develop relevant tumor markers and/or tumour antigen, all significant, simultaneously also significant to tumorigenic molecular mechanism to the clinical diagnosis of malignant tumour, supervision PD and judging prognosis.
Pescadillo gene finds in the research retrovirus Zebrafish Embryo defect of bringing out, this gene all has expression yeast, mouse and the mankind, and high conservative, be named as YPHI/Nop7p, Pes1 and PES1 respectively, the pescadillo homology of its small mouse and people is up to 89%.PES1 gene (GenBank Accession No.NM_014303) is positioned human chromosome 22q12.1, and containing 11 exons and 10 introns, 588 amino acid of encoding, express the albumen that molecular weight is about 70kD.PES1 albumen tool nuclear localization signal sequence, C end has a little ubiquitination sample modified protein (small ubiquitin-like modifyer, SUMO) site, there is in 315th ~ 412aa region a typical mammary cancer sensitive Protein 1C end (breast cancer susceptibility protein 1C-terminal, BRCT) structural domain.There are some researches show, many molecules with BRCT structural domain participate in DNA reparation, restructuring and the control of cell cycle.
Research shows, in zebrafish development, pescadillo transgenation can cause the growth of the organs such as head, eyes, liver to be subject to obvious suppression; In yeast cell pescadillo gene undergo mutation after its growth be subject to obvious suppression, cell is blocked in G1 or the G2 phase, shows that pescadillo plays an important role in cell proliferation and cell cycle progression.Cell proliferation comprises the process that cell volume increases and cell fission two is coordinated mutually, and last process mainly carries out ribosomal synthesis, and the efficiency of its synthesis is subject to the regulation and control of rna plymerase i and Function protein thereof; Fissional, need active cell cycle program, numerous protein molecular energy participates in this two processes simultaneously.Research shows that PES1 albumen is key protein matter very important in the generation of ribonucleoprotein, in the mitotic later stage, it also participates in the formation of early stage kernel, after mitotic division terminates, it participates in the formation of the precursor of ribosome, and PES1 sudden change or defect cause cell-cycle arrest.
Research shows, the mouse liver of adult is after Partial Resection, and residual parenchyma breeds rapidly to recover normal liver volume, and the Pescadillo expression level that these are in multiplicative stage cell in 16 ~ 48 hours raises rapidly.The Pescadillo of exogenous overexpression can the inoblast of transformation of human and mouse, makes it that anchorage independence growth occur in soft agar.PES1 finds that there is overexpression in many tumour cells, as mammary cancer, glioma, prostate cancer and cancer of the stomach etc.
Above-mentioned research show PES1 and cell propagation, transform, cancerate closely related, and play important function in the generation and evolution of tumour.
Mostly be to utilize the polyclonal antibody of PES1 to carry out about the research of PES1 in prior art, have no the report of PES1 monoclonal antibody.The poor specificity of polyclonal antibody, high for the background that develops the color during immunohistochemical methods, quality is wayward, is difficult to promote in clinical application.
Clinical in high specificity, PES1 monoclonal antibody that susceptibility is high at present, and the hybridoma of monoclonal antibody described in high-titer can be secreted, to contribute to clinical diagnosis, the Index for diagnosis of tumour, and guide clinical treatment.Meanwhile, monoclonal antibody can be used as the important tool of PES1 biological function research, for experiments such as Western blot, immunoprecipitation, immunocytochemistries.
Summary of the invention
An object of the present invention is to provide a kind of PES1 monoclonal antibody or derive from this antibody can the bioactive fragment of specific binding PES1;
Another object of the present invention is to provide the hybridoma cell line secreting monoclonal antibody of the present invention;
Another object of the present invention is a kind of test kit detecting PES1 level.
Another object of the present invention is to provide monoclonal antibody of the present invention or its bioactive fragment for the preparation of the application detected in the reagent of PES1 level in sample or test kit.
Another object of the present invention is to provide monoclonal antibody of the present invention or its bioactive fragment for the preparation of the application in the reagent of auxiliary diagnosis tumour, monitoring tumour and/or tumour patient Index for diagnosis or test kit.
Another object of the present invention is to provide a kind of antitumor drug.
Another object of the present invention is to provide a kind of detection reagent of PES1 level.
On the one hand, the invention provides a kind of PES1 monoclonal antibody or derive from this antibody can the bioactive fragment of specific binding PES1, the hybridoma that described monoclonal antibody is CGMCC No.3646 or CGMCC No.3647 by the preserving number of China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation is secreted.The present inventor is by great many of experiments and enrich technical experience and obtain monoclonal antibody of the present invention (monoclonal antibody).Through qualification, the subclass of monoclonal antibody of the present invention is IgG1.
Monoclonal antibody of the present invention has higher sensitivity and specificity, identifiable design endogenous PES1 albumen also can be used for the immunology detection such as immunoblotting, immunoprecipitation, immunocytochemistry, immunofluorescence, immunohistochemistry (immunohistochemical methods), can be applicable to the detection of PES1 protein expression level in clinical tissue sample, with the prognosis of the transfer tendency of predicting tumors and patient; Can be applicable to the detection of PES1 albumen in doubtful or tumour (as mammary cancer, glioma, prostate cancer or cancer of the stomach) patient body fluid, to help the diagnosis of tumour; Also can improve the Sensitivity and Specificity of diagnosing tumor with the agents detecting other tumor markers, also contribute to dynamically observing tumor progression, for the treatment plan of individuation provides reference frame; Also also can be used for correlative study and the drug development of PES1.
On the other hand, the invention provides a kind of hybridoma cell line secreting monoclonal antibody of the present invention, it was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica) on 03 05th, 2010 with preserving number CGMCC No.3646 or CGMCC No.3647.Hybridoma cell line of the present invention has the ability of stronger secrete monoclonal antibody, can stablize, secrete monoclonal antibody of the present invention in a large number.
Those of ordinary skill in the art are known, PES1 albumen directly can be adopted as antigen, obtain monoclonal antibody of the present invention.Also PES1 albumen of the present invention and known label can be formed recombinant protein as antigen, obtain responding property of PES1, reactive monoclonal antibody of the present invention is not had to label.In an embodiment of the invention, the present invention obtains the GST-PES1 fusion rotein of purifying by prokaryotic expression, and obtains the monoclonal antibody of PES1 albumen by hybridoma technology.
Therefore, the present invention also provides the preparation method of monoclonal antibody of the present invention, present invention also offers the method for the monoclonal antibody described in preparation, the method comprises: with fusion rotein GST-PES1 immune mouse, get splenocyte and the myeloma cell fusion of mouse, filter out and can secrete responding property of PES1, Triptide thioltransferase be there is no to the hybridoma of reactive monoclonal antibody, be preferably CGMCC No.3646 of the present invention or CGMCC No.3647 hybridoma, then hybridoma injected suitable Mammals and obtain antibody from animal ascites.The method is only exemplary, and such as can use the same method other Mammalss of immunity, rat, rabbit, cavy or hamster etc.
In an embodiment of the invention, with the cDNA of PES1 gene for template, by the cDNA fragment of PCR method amplification PES1 gene.Pcr amplification product is cloned on prokaryotic expression carrier pGEX4T-1, builds Prokaryotic Recombinant Plasmids pGEX4T-1-PES1, and at expression in escherichia coli.Select mono-clonal positive bacteria, a large amount of abduction delivering GST-PES1 albumen purifying.With reference to B lymphocyte hybridoma technology (the Kohler G that Kohler and Milstein sets up, Milstein C, Continuous cultures of fused cells secreting antibody of predefined specificity.Nature.1975Aug 7; 256 (5517): 495-7), the GST-PES1 protein immunization BALB/c mouse of the present invention's purifying.The splenocyte of immune mouse and myeloma cell SP2/0 are merged, prepares hybridoma.Positive cell clone is screened by ELISA method.The present inventor, through test of many times, obtains many strains positive cell clone.Analysis and Identification is carried out to the monoclonal antibody secreted by these positive colonies, find hybridoma cell strain CGMCCNo.3646 or the CGMCC No.3647 of two strain secrete monoclonal antibodies, PES1 monoclonal antibody 3B1 and the 1H5 of its secretion have higher sensitivity and specificity, and identifiable design endogenous PES1 albumen also can be used for the immunology detection such as immunoblotting, immunoprecipitation, immunocytochemistry, immunofluorescence, immunohistochemistry (immunohistochemical methods); Can be applicable to the detection of PES1 protein expression level in clinical tissue sample.
On the other hand, the invention provides a kind of test kit detecting PES1 level, it contains monoclonal antibody of the present invention or its active fragments; Preferably described test kit is also containing second antibody with for the enzyme that detects or fluorescence or radio-labeled thing, and damping fluid; Preferred described second antibody is the anti-antibody (sheep anti-mouse igg that such as this area is conventional) of monoclonal antibody of the present invention or anti-monoclonal antibody of the present invention or resisting of the anti-PES1 for preparing by technology known in the art more.
Can be radio isotope, fluorescent chemicals, vitamin H and enzyme etc. as the fluorescence of detection signal and/or radio-labeled.Described enzyme comprises, but be not limited to, horseradish peroxidase (horseradish Peroxidase, HRP), alkaline phosphatase (alkaline phosphatease, AP), glucose oxidase, beta-D-galactosidase and urase etc., preferred horseradish peroxidase and/or alkaline phosphatase.Those of ordinary skill in the art are known, for different enzymes, can use different substrates.The substrate of HRP effect includes, but not limited to O-Phenylene Diamine (OPD), tetramethyl benzidine (TMB), ABTS, diaminobenzidine (diamino benzidine, DAB) etc.The substrate of alkaline phosphatase generally adopts red (the alkaline phosphatase-red of p-nitrophenyl phosphoric acid ester (p-NPP), alkaline phosphatase, AP-Red, also known as Fast Red, red soon), AP also has fluorescent substrate (phosphatase 24-methyl umbrella ketone).Test kit of the present invention can be used for detecting the PES1 expression level in the tumour such as mammary cancer, glioma, prostate cancer or cancer of the stomach and kinds cancer clone and serum, and then for the auxiliary diagnosis of above-mentioned kinds cancer and relevant experimental study and guiding clinical treatment.
In another embodiment of the present invention, described test kit is for detecting the expression level of PES1 in histocyte, particularly to the detection of PES1 in the tumor tissues such as mammary cancer, glioma, prostate cancer or cancer of the stomach, it comprises monoclonal antibody of the present invention or its active fragments, also comprises anti-antibody for detecting monoclonal antibody of the present invention or active fragments and enzyme or fluorescent marker and corresponding damping fluid; In a preferred embodiment of the invention, described histocyte is from the biopsy cells of tumor tissues or culturing cell or is built system culturing cell.
On the other hand, the invention provides monoclonal antibody of the present invention or the application of its bioactive fragment in the reagent preparing PES1 level in detection sample or test kit.Detection method used comprises all immunization methods that can use antibody of the present invention, such as Western Blot, immunoprecipitation, immunohistochemical methods, immunofluorescence etc.Be preferably immunoprecipitation.Monoclonal antibody of the present invention can detect the expression level of the PES1 of any sample, and preferred described sample is individual test subjects (as human body) humoral sample or cell protein extracting solution; Can be the biofluid from individual test subjects and tissue sample (such as breast cancer tissue, samples of human glioma, prostate cancer tissue or stomach organization).
On the other hand, the invention provides monoclonal antibody of the present invention or its bioactive fragment for the preparation of the application in the reagent of auxiliary diagnosis tumour, monitoring tumour and/or tumour patient Index for diagnosis and/or test kit.The anti-PES1 monoclonal antibody of the present invention that can be used alone carries out auxiliary diagnosis tumour, monitoring tumour and/or tumour patient Index for diagnosis, also can the reagent of combine detection tumor markers known in the art, to improve auxiliary diagnosis tumour, monitoring tumour and/or tumour patient Index for diagnosis further.
On the other hand, the invention provides a kind of antitumor drug, this antitumor drug contains monoclonal antibody of the present invention or its bioactive fragment.PES1 monoclonal antibody of the present invention can with PES1 specificity, be efficiently combined.Therefore, give the bioactive fragment that can be combined with PES1 protein-specific of tumour patient monoclonal antibody of the present invention or this antibody, the activity of PES1 in possible inhibition tumor cell, thus play the effect of Tumor suppression infiltration, transfer.Therefore, the present invention also provides one to treat the method for animal (comprising people) tumour, comprises monoclonal antibody of the present invention or its bioactive fragment of the treatment of animals significant quantity giving needs treatment." tumour " used herein or " cancer " are not particularly limited, as long as relevant to the process LAN of PES1, are preferably mammary cancer, glioma, prostate cancer or cancer of the stomach.
The preparation method of described medicine can be any method well known to those skilled in the art, such as, by monoclonal antibody of the present invention or its bioactive fragment and suitable mixed with excipients.Described vehicle is the vehicle that this area routine is prepared for antibody drug, as water, salt solution, buffer reagent etc.The route of administration of described medicine can be the conventional administration route of any antibody drug, such as, and intravenous injection, subcutaneous injection, intra-tumoral injection etc.
On the other hand, the invention provides a kind of detection reagent, this detection reagent contains monoclonal antibody of the present invention or its bioactive fragment.The preparation method of described detection reagent can be any method well known to those skilled in the art, such as, mix, monoclonal antibody of the present invention or its bioactive fragment and suitable carrier as water, salt solution, buffer reagent etc.Detection reagent of the present invention can be applicable to the detection of PES1 protein expression level in clinical tissue sample, the prediction transfer tendency of infantile tumour and the prognosis of patient; Also can be applicable to the detection of infantile tumour patients serum PES1 protein expression, dynamic detects the change of PES1 protein level in tumour progression process simultaneously, provides reference frame to the treatment plan of clinicians make individuation.
Accompanying drawing explanation
Fig. 1: carry out quantitative analysis with 1B7 antibody, 1H5 antibody and 3B1 antibody that SDS-PAGE glue carries out purification and identify its purity.Result display purifying to 1B7 antibody, 1H5 antibody and 3B1 antibody, SDS-PAGE glue has no foreign protein band, and visible Dispersal risk purity is higher, all can reach more than 95%; Quantitative analysis is known prepares purifying 1B7 antibody 2mg/mL × 4mL, 1H5 antibody 3mg/mL × 3.5mL and 3B1 antibody 2mg/mL × 5mL respectively.
Fig. 2: analyzed tiring of 1B7 antibody, 1H5 antibody and 3B1 antibody by the microtiter plate of GST-PES1 recombinant protein with ELISA method with bag.Wherein, be negative control with bag by the microtiter plate of GST.Antibody purification concentration being respectively 2 μ g/ml is added in microtiter plate, and with mouse anti-GST antibody for antibody positive contrasts, with normal mice IgG for antibody negative controls.Result display 1B7 antibody, 1H5 antibody and a 3B1 antibody specific combination GST-PES1, with other albumen as GST no cross reaction.
Fig. 3: the expression detecting endogenous PES1 in protokaryon Protein G ST-PES1 and SGC-7901 ags cell by the method for Western-Blot.12%SDS-PAGE electrophoresis, GST-PES1 every hole applied sample amount is 100ng (the every hole 100ng of GST purifying protein is negative control), ags cell total protein applied sample amount 50 μ g, primary antibodie is 1B7 antibody, 1H5 antibody and 3B1 antibody concentration 1 μ g/ml (normal mice IgG is negative control) detect.Result display 1B7 antibody, 1H5 antibody and 3B1 antibody can be combined with GST-PES1, and not in conjunction with GST (picture A).Apply the expression (picture B) that 1B7 antibody, 1H5 antibody and 3B1 antibody can detect endogenous PES1 in ags cell simultaneously.
Fig. 4: immunoprecipitation (IP) detects the expression of PES1 in ags cell.Result is pointed out, and antibody 3B1 can be used for immunoprecipitation, and monoclonal antibody 1B7 and 1H5 then can not carry out immunoprecipitation.
Fig. 5: carry out immunocytochemistry to detect the expression of PES1 in human stomach cancer cell line AGS with 1B7 antibody, 1H5 antibody and 3B1 antibody.The entoblast colour developing of result display AGS is brown color, illustrates that PES1 is expressed in ags cell strain.
Fig. 6: the location detecting PES1 in people's gastrointestinal cancer cell strain ags cell by the method for immunofluorescence.Primary antibodie is 1B7 antibody, 1H5 antibody or 3B1 antibody, and two resist the fluorescence two for FITC mark to resist, and apply DAPI dyes nucleus simultaneously.Result green fluorescence is mainly distributed in entoblast, thus prompting PES1 is mainly positioned entoblast.
Fig. 7: the expression detecting PES1 in cancer of the stomach and human esophageal carcinoma chip with ImmunohistochemistryMethods Methods.Do not express PES1 in result display cancer of the stomach Carcinoma side normal tissue and esophageal carcinoma Carcinoma side normal tissue, and can detect that PES1 expresses in corresponding cancerous tissue.
Fig. 8: application Western-Blot detects the expression of PES1 in cancer of the stomach and colorectal carcinoma far-end healthy tissues, cancer beside organism and cancerous tissue.12%SDS-PAGE electrophoresis, cancer of the stomach and colorectal carcinoma far-end healthy tissues, cancer beside organism and cancerous tissue lysate every hole applied sample amount are 50 μ g, conventional SDS-PAGE electrophoresis, transferring film, close after, add primary antibodie, be respectively 3B1 antibody or internal reference albumen β-Actin antibody carries out conventional WesternBlot detection.In result display cancer of the stomach and colorectal carcinoma cancerous tissue, comparatively respective distal end healthy tissues and cancer beside organism obviously raise for the expression of PES1, wherein there is PES1 high expression level in 14 routine cancerous tissues in 18 routine cancer of the stomach pairing tissues, there are 2 routine PES1 to express no significant difference in different lesions tissue, have 2 routine PES1 at far-end healthy tissues and cancer beside organism's comparatively cancerous tissue high expression level (picture A); There is PES1 high expression level in 10 routine cancerous tissues in 14 routine colorectal carcinoma pairing tissues, have 4 routine PES1 in different lesions tissue, express no significant difference (picture B).Microorganism for patented procedure preserves:
(1) the hybridoma cell strain α PES1-2-3B1 of monoclonal antibody 3B1 of the present invention is secreted
Preservation date: on 03 05th, 2010;
Depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC);
Deposit number: CGMCC No.3646;
Classification And Nomenclature: the hybridoma cell line secreting anti-Pescadillo (PES1) monoclonal antibody.
(2) the hybridoma cell strain α PES1-1-1H5 of monoclonal antibody 1H5 of the present invention is secreted
Preservation date: on 03 05th, 2010;
Depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC);
Deposit number: CGMCC No.3647;
Classification And Nomenclature: the hybridoma cell line secreting anti-Pescadillo (PES1) monoclonal antibody.
Embodiment
In order to more clearly understand essence of the present invention, coordinate accompanying drawing to further describe the present invention below by specific embodiment, but the present invention is not therefore subject to any restriction.The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition is as " Molecular Cloning: A Laboratory the guide " (work such as the third edition [U.S.] Pehanorm Brookers in 2002, Science Press) described in condition, or according to the condition that manufacturer advises.
The preparation of embodiment 1, anti-human PES1 monoclonal antibody
1, the preparation of PES1 antigen
Design 5 ' end sense primer 5 '-CGCGGATCCTTCCGCCTTTACCAGTTGC-3 ' (SEQ ID NO:1) and 3 ' end antisense primer 5 '-CCGGAATTCTCACTCCGGCCTTGCCTTCTTG-3 ' (SEQ ID NO:2) (primer is synthesized by Shanghai Sheng Gong bio-engineering corporation) of PES1 gene (GenBank Accession No.NM 014303) cDNA fragment respectively, and introduce BamH I and EcoR I restriction enzyme site in primer two ends respectively.Extract gastric carcinoma cells AGS total serum IgE with Trizol (purchased from Invitrogen company), reverse transcription becomes cDNA, and applies above-mentioned primer and carry out pcr amplification PES1cDNA fragment, and product length is 1062bp (SEQ ID NO:3).
Be connected after PCR primer is all cut with EcoR I enzyme with BamH I respectively with pGEX-4T1 carrier (purchased from Amersham Biosciences company), this connection product conversion is entered e. coli strain bl21 (purchased from Tian Gen biochemical technology company limited) and picking mono-clonal carries out enzyme cuts qualification, send order-checking simultaneously.The SD sequence downstream of prokaryotic expression carrier pGEX-4T-1 is exactly glutathione sulfydryl transferase (GST) gene, and the external source PES1 gene fragment of clone is then connected with gst gene.When genetic expression, expression product is the syzygy GST-PES1 of GST and PES1 gene fragment product, to facilitate the purifying of PES1.
The BL21 bacterial strain selecting the correct positive colony that checks order carries out a small amount of abduction delivering of GST-PES1 recombinant protein.Get abduction delivering in a small amount and be accredited as a large amount of abduction deliverings that positive bacterial strain carries out GST-PES1 recombinant protein.The abduction delivering condition of contriver to PES1 is explored, and use e. coli strain bl21, temperature is 37 DEG C, and the concentration of inductor IPTG is 0.25mmol/L, and induction time is 5 hours.With this understanding, the expression amount of GST-PES1 is the highest, and GST-PES1 is mainly present in expression bacterium with inclusion bodies.In the precipitation of the centrifugal rear acquisition of every 200mL bacterium liquid, add 10mL 0.5%Triton X-100/PBS suspended bacterial; The ultrasonic 50s of ice bath interval, broken bacterium; Centrifugal (10000g, 4 DEG C, 10min); Abandon supernatant, add 10ml 2% sodium deoxycholate in precipitation, ice bath interval ultrasonic 50s, rocked at room temperature 30min, centrifugal (10000g, 4 DEG C, 10min), abandons supernatant; 500 μ l sex change liquid (Urea8M are added in precipitation, DTT 10mM, Tris-HCl pH8.5 50mM, above-mentioned materials is all purchased from Amresco company), and add the abundant sex change dissolution precipitation of a small amount of 10M NaOH, room temperature shakes 3h gently, after liquid change is limpid, centrifugal (10000g, 4 DEG C, 10min), precipitation is abandoned; Supernatant liquor after sex change is added renaturation solution (the GSH 5mM of 9 times of volumes, GSSG 2mM, EDTA 5mM, Tris-HCl pH8.5 50mM, above-mentioned materials is all purchased from Amresco company), limit edged stirs, after room temperature renaturation 1h, 4 DEG C are continued renaturation 4h, centrifugal (10000g, 4 DEG C, 5min); Supernatant 2000ml, 25mMTris-HCl pH 7.4 dialysed overnight, period, changes liquid once, secondary daily sucrose embedding is concentrated, and 10%SDS-PAGE detects its content and purity, and visible GST-PES1 purity is more than 90%, its content is 0.8mg/mL, packing ,-80 DEG C of storages.
2, the preparation of PES1 monoclonal antibody
(1) mouse immune
A () antigen: the GST-PES1 fusion rotein of prokaryotic expression, during immunity, antigen amount is 50mg//time, and it is 200mL that a cumulative volume is often only injected in back, tail vein injection 50mL.
(b) animal: 6 ~ 8 week age female Balb/c mouse, body weight 20g/ is (purchased from dimension tonneau China Experimental Animal Center) only.
C () immunization method: before initial immunity, gets the tail vein 2mL of Balb/c mouse as negative control; During initial immunity by the complete Freund's adjuvant (available from Sigma) of GST-PES1 fusion rotein quantitative for purifying and equivalent fully mixing littlely drop in indiffusion in water, dorsal sc multiple spot immunity Balb/c mouse until get one; After surrounding, carry out first time booster immunization, the incomplete Freund's adjuvant (available from Sigma) of the fusion rotein of purifying and equivalent is mixed emulsification, same dorsal sc multiple spot immunity; After 7 ~ 14 days, get mouse tail vein blood and detect antibody titer by ELISA method, as antibody titer reaches 10
-410
-5, carry out antigen impact with tail intravenous immunisations, merge after 3 ~ 4 days.
(2) cytogamy
The preparation of (a) feeder cell: get two normal kunming mices, disconnected neck gets blood (its serum is normal mouse serum), alcohol-pickled 5min sterilization, cut off abdominal cavity skin, expose peritonaeum, peritonaeum is cut an osculum, draws the appropriate selective d MEM nutrient solution (purchased from Hyclone company) containing HAT (available from Sigma) and rinse abdominal cavity, collect abdominal cavity feeder cell for subsequent use.
The preparation of (b) myeloma cell SP2/0: in fusion recovery the last week SP2/0 cell, cultivate in the DMEM nutrient solution of the foetal calf serum containing 15% deactivation.Merge and change liquid the day before yesterday, make SP2/0 be in good growth conditions, get 2 ~ 3 bottles of SP2/0 cells be closely paved with, blowing afloat cell with 15mL serum-free medium, to be placed in 50mL aseptic plastic centrifuge tube for subsequent use.
The preparation of (c) splenocyte: will immunized mice be impacted, disconnected neck gets blood (its serum is positive polyvalent antibody), alcohol-pickled sterilization, by mouse left lateral position, skin and peritonaeum are cut off in layering, expose abdominal cavity, win spleen, be placed in the plate filling serum-free DMEM nutrient solution, cut off peripheral adipose and manadesma, then spleen is put on the woven wire (200 order) of plate, add the DMEM having a small amount of serum-free, break coating, cross net with piston extruding splenocyte, collect the splenocyte after net in glass test tube; Vertical standing several minutes; In sucking-off, confluent monolayer cells puts into 50mL aseptic plastic test tube gently.
(d) cleaning SP2/0 cell and splenocyte: by the SP2/0 cell of above-mentioned collection and splenocyte centrifugal, 800 ~ 1000g, 3 ~ 5min, abandon supernatant, knock gently and bottom test tube, make the cell mass of centrifugation disperse, blow and beat cell gently with the DMEM of serum-free, 800 ~ 1000g, centrifugal 3 ~ 5min, repeats once.
E () merges: SP2/0 cell and splenocyte are mixed, the ratio of splenocyte and SP2/0 cell is made to be about 10: 1,800 ~ 1000g, centrifugal 3 ~ 5min, abandon supernatant, exhaust with aseptic filter paper bar the cultivation raffinate of centrifugal tube wall, knocks gently and make the cell mass of centrifugation disperse bottom test tube.In 37 DEG C of water-baths, slowly added in cell by 1mL 50%PEG, limit edged stirs gently, add in 1min, continue to stir 30s, leave standstill 1min, 1.5mL serum-free DMEM nutrient solution is added in cell in 1min, action is as far as possible soft, limit edged stirs, and stops merging, more slowly adds the serum-free DMEM nutrient solution of 3mL, finally, nutrient solution is added to 20mL.800 ~ 1000g, centrifugal 3 ~ 5min, abandons supernatant, selects nutrient solution to stir loose sedimentation cell gently with the HAT containing 20% foetal calf serum on a small quantity, makes it to suspend, and is added to the 80mL containing abdominal cavity feeder cell prepared and contains in the HAT selection nutrient solution of 20% foetal calf serum.Finally spread 96 orifice plates, 100mL/ hole, in 37 DEG C, 5%CO
2cultivate in incubator.
(3) enzyme-linked immunosorbent assay (ELISA) screens specific monoclonal antibody:
GST, GST-PES1 fusion rotein of prokaryotic expression is diluted to 5mg/mL with coating buffer respectively, wraps by 96 orifice plates, 50mL/ hole, and 4 DEG C are spent the night.0.05%Tween20/PBS washes twice.Skim-milk (PBS preparation) the 200mL/ hole of 4%, room temperature 2h or 4 DEG C spends the night, and 0.05%Tween20/PBS washes twice, and-70 DEG C save backup.Detecting step is as follows:
A () gets hybridoma supematant, 50mL/ hole, room temperature 1h.
B () 0.05%Tween-20/PBS washes 5 times.
C () adds the sheep anti-mouse igg (1: 3000) of HP mark, 50mL/ hole, room temperature 45min.
D () 0.05%Tween-20/PBS washes 5 times.
E () adds OPD substrate reactions liquid, 100mL/ hole, and lucifuge develops the color
F add stop buffer 50mL/ hole after () colour developing fully, observations also detects OD492nm by microplate reader.
(4) positive colony screening and subclone
The screening of (a) positive colony
After merging, 6th ~ 8 days visible significantly cell colonies, calculate cell confluency, and select nutrient solution entirely to change liquid with the HAT containing 20% foetal calf serum.Merge and within latter about 10 days, get hybridoma supematant and carry out ELISA screening, choose and only carry out subcloning with GST-PES1 reacting positive with the cell clone of GST reaction negative.
(b) subcloning (Subclone)
Adopt limiting dilution assay.Carry out ELISA screening again after 7th ~ 10 days after general subclone, subclone is next time carried out to positive colony, subclone like this 3 ~ 5 times, be the positive, enlarged culturing until all by inspection clone, build strain, collect supernatant, frozen.Because methotrexate is comparatively large to the toxicity of cell, in the process of carrying out subclone, HAT nutrient solution is changed gradually into HT (available from Sigma) nutrient solution.
After building strain, HT nutrient solution can be changed to Nostoc commune Vanch liquid.
Repeatedly subclone is carried out to the hybridoma of the positive.Choose clone's enlarged culturing of strong positive, build strain.
In the present invention, by the anti-PES1 monoclonal antibody called after 3B1 wherein secreted by a strain, and this hybridoma cell strain is α PES1-2-3B1 cell strain, this hybridoma cell strain was preserved in (address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms's common micro-organisms center on 03 05th, 2010, Institute of Microorganism, Academia Sinica), deposit number: CGMCC No.3646; Anti-PES1 monoclonal antibody called after 1H5 secreted by another strain, and this hybridoma cell strain is α PES1-1-1H5 cell strain, this hybridoma cell strain was preserved in (address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms's common micro-organisms center on 03 05th, 2010, Institute of Microorganism, Academia Sinica), deposit number: CGMCC No.3647; Anti-PES1 monoclonal antibody called after 1B7 secreted by another strain, and this hybridoma cell strain is 1B7 cell strain.1B7 cell, α PES1-1-1H5 cell or α PES1-2-3B1 cell are inoculated in 10 week age in the Bal b/c mouse peritoneal of pristane process, when ascites is formed and increases belly special bulge, put to death mouse get ascites, it includes a large amount of 1B7, α PES1-1-1H5 or α PES1-2-3B1 antibody.
The purifying of embodiment 2, qualification 1B7 antibody, 1H5 antibody and 3B1 antibody
With the subclass (Subclass of antibody test kit available from Sigma) of conventional ELISA method qualification 1B7 antibody, 1H5 antibody and 3B1 antibody, confirm that it is IgG1, therefore select ProteinG Sepharose-4B post to carry out purifying.The each 10mL of ascites is collected after 12000 × g is centrifugal in Example 1, avoid grease and draw supernatant, 5 times are diluted to balance buffer, by 0.45 μm of frit, purifying is carried out in application Purify protein purification instrument (purchased from GE company), by the ascites of dilution with the flow velocity loading of 1mL/min, after balance buffer (PBS) washes away foreign protein, with the acidic effluent liquid of pH2.6 (100mmol/L Glycin-HCl (pH 2.6)) wash-out, collect the component of OD280 > 0.5, finally add 100 μ l 2M Tris-base neutralizers in every milliliter to neutralize, to keep antibody stabilization, by frozen in-80 DEG C for the antibody packing of purifying, part adds 50% glycerine, 4 DEG C of preservations, separately get the capable 12%SDS-PAGE glue of 4 μ l carry out quantitatively and identify its purity.Result is shown in Figure 1, purifying to 1B7 antibody, 1H5 antibody and 3B1 antibody, SDS-PAGE glue has no foreign protein band, visible Dispersal risk purity higher (all can reach more than 95%), quantitative analysis is known prepares purifying 1B7 antibody 2mg/mL × 4mL, 1H5 antibody 3mg/mL × 3.5mL and 3B1 antibody 2mg/mL × 5mL respectively, and the output producing antibody from another aspect visible embodiment 1 small mouse ascites all can reach more than 1mg/mL.
The specificity analyses of embodiment 3, mensuration 1B7 antibody, 1H5 antibody and 3B1 antibody
With the PBS damping fluid dialysis 1B7 antibody of purifying, 1H5 antibody and 3B1 antibody, by the concentration of ultraviolet spectrophotometer at 280nm place mensuration antibody.
1B7 antibody, 1H5 antibody or 3B1 antibody that concentration is 2 μ g/ml is added respectively in the microtiter plate of GST-PES1 albumen bag quilt, and the antibody of same concentration is added in the microtiter plate of GST albumen bag quilt as negative control, and with normal mouse IgG (purchased from Zhong Shan biotech firm) for antibody positive contrast, incubation at room temperature 1 hour; Afterwards, with 0.05%Tween-20/PBS washing reaction hole 3 times, 2 times, PBS washing reaction hole; Add the antibody (purchased from Jackson Research Laboratories company) of the sheep anti-mouse igg of horseradish peroxidase-labeled again, incubation at room temperature 1 hour; Use 0.05%Tween-20/PBS washing reaction hole 3 times afterwards, 2 times, PBS washing reaction hole; Then in reacting hole, add horseradish peroxidase substrate OPD, color development at room temperature, after 30 minutes, adds stop buffer (12.5%H
2sO
4).The absorbance at OD492 place is measured by microplate reader.Result is shown in Figure 2,1B7 antibody, 1H5 antibody and 3B1 antibody specific combination GST-PES1, and with GST no cross reaction.
Embodiment 4, Western-Blot detect the expression of PES1
1, Western-Blot detects GST-PES1
Specifically carry out according to following operation:
1) get the SDS-PAGE electrophoresis of GST-PES1 and the GST albumen capable 12% of purifying, every porin applied sample amount is 100ng.
2) stop when tetrabromophenol sulfonphthalein reaches the most lower edge of glue, carry out electrotransfer.
3) after transferring film terminates, ponceau is dyeed, and detects transferring film effect and carries out mark.
4) 5% skim-milk is closed, and 4 DEG C are spent the night.
5) add primary antibodie 1B7 antibody, 1H5 antibody or 3B1 antibody, concentration is 1 μ g/ml, normal mice IgG is negative control, room temperature 1 hour.
6) 0.1%Tween-20/PBS washs 7 times.
7) antibody of the sheep anti-mouse igg of horseradish peroxidase-labeled is added, incubation at room temperature 40 minutes.
8) 0.1%Tween-20/PBS washs 7 times, and PBS washs 1 time.
9) luminous development.Result refers in Fig. 3 shown in picture A, display 1B7 antibody, 1H5 antibody and 3B1 antibody specific combination GST-PES1, and with GST no cross reaction.
2, in Western-Blot detection ags cell, endogenous PES1 expresses.
Cell pyrolysis liquid RIPA (Tris (pH7.4), 150mM NaCl, 1%Triton X-100,1%sodiumdeoxycholate, 0.1%SDS) cracking SGC-7901 ags cell (purchased from Chinese Academy of Sciences's cell bank), extract total protein of cell, the SDS-PAGE electrophoresis of row 12%, every porin applied sample amount is 50 μ g.All the other steps are the same.
Result refers in Fig. 3 shown in picture B, and display 1B7 antibody, 1H5 antibody and 3B1 antibody capable detect the expression of endogenous PES1 in SGC-7901 AGS.
Embodiment 5, immunoprecipitation (IP) detect the expression of PES1 in ags cell
Carry out according to following operation in the present embodiment:
1, cell pyrolysis liquid RIPA cracking ags cell, extracts total protein of cell.
2, get 200 μ g total protein of cell and hatch 2h with monoclonal antibody 1B7 antibody, 1H5 antibody or 3B1 antibody hybridoma supernatant 0.5ml 4 DEG C respectively.
3, add Protein G Sepharose 4Fast Flow (GE Healthcare, Uppsala, Sweden), hatch 2h for 4 DEG C.
4, after reaction mixture PBST washs 3 times, the SDS-PAGE electrophoresis of row conventional 12%.
5, after transferring film is closed, carry out conventional Western blot with monoclonal antibody 3B1 antibody for primary antibodie and detect.Result is shown in Figure 4, and monoclonal antibody 3B1 antibody can react with the PES1 of native conformation in ags cell, and monoclonal antibody 1B7 antibody and monoclonal antibody 1H5 antibody then can not carry out immunoprecipitation.
The expression of PES1 in embodiment 6, Immuncytochemical detection human stomach cancer cell line AGS
Carry out according to following operation in the present embodiment:
1, ordinary method prepares the cell climbing sheet of human stomach cancer cell line AGS, and cold acetone/methyl alcohol (volume ratio 1: 1) is fixed.
2, creep plate is soaked in 3%H
2o
2(PBS dilution) interior 10 minutes, to remove endogenous peroxydase.
3, PBS washs creep plate 2 times.Then 1%BSA (PBS dilution) is used to close 30 minutes at 37 DEG C.Drip 1B7 antibody, 1H5 antibody or 3B1 antibody (antibody concentration is 2 μ g/mL), react 1 hour at 37 DEG C.
4, PBS washs creep plate 3 times, drips antibody working fluid (the DAKO ENVISION of the sheep anti-mouse igg of horseradish peroxidase-labeled
tM+ System HRP Mouse), react 1 hour at 37 DEG C.
5, wash creep plate 3 times with PBS, develop the color 20 minutes in the solution of horseradish peroxidase substrate DAB.
6, use PBS color development stopping, cover on slide glass by counter for creep plate, basis of microscopic observation, to occur in cytoplasm that brown yellow granule shape is for the positive.
Result is shown in Figure 5, occurs brown color in display ags cell core, illustrates and utilizes 1B7 antibody, 1H5 antibody or 3B1 antibody capable ags cell strain expression PES1 to be detected.
Embodiment 7, the location of Immunofluorescence test PES1 in stomach cancer cell AGS
Carry out according to following operation in the present embodiment:
1, ordinary method prepares the cell climbing sheet of human stomach cancer cell line AGS, and cold acetone/methyl alcohol (volume ratio 1: 1) is fixed.
2, creep plate is soaked in 3%H
2o
2(PBS dilution) interior 10 minutes, to remove endogenous peroxydase.
3, PBS washs creep plate 2 times.Then 1%BSA (PBS dilution) is used to close 30 minutes at 37 DEG C.Drip 1B7 antibody, 1H5 antibody or 3B1 antibody (antibody concentration is 2 μ g/mL), react 1 hour at 37 DEG C.
4, creep plate is washed 3 times with PBS.
5, two anti-(purchased from the Zhong Shan biotech firm) 1: 200 of dropping FITC mark dilutes and makes working fluid, reacts 40min under room temperature.
6, creep plate is washed 3 times with PBS.
7,1 μ g/mL DAPI transfect cell core 5min is dripped.
8, creep plate is washed 3 times with PBS.
9,50% glycerine mounting, fluorescence microscopy Microscopic observation is taken a picture.
Result is shown in Figure 6, ags cell kernel place visible green fluorescence, and blue-fluorescence is the nucleus (its empty cavity position is entoblast) that DAPI dye.Result display PES1 is mainly positioned in entoblast.
Embodiment 8, immunohistochemical methods detect the expression of PES1 in cancer of the stomach and human esophageal carcinoma chip
Carry out according to following operation in the present embodiment:
1, people's cancer of the stomach and human esophageal carcinoma chip (purchased from Shaanxi Chao Ying company) is chosen.Carry out dewaxing treatment with dimethylbenzene to section, graded ethanol makes section aquation, with PBS washing slice 2 times.
2, section is soaked in 3%H
2o
2(PBS dilution) interior 10 minutes, to remove endogenous peroxydase.Then PBS washing slice is used 3 times.
3, section is soaked in the 0.1M edta buffer liquid of pH9.0, uses pressure kettle to carry out conventional high-pressure 90 seconds, carry out antigen retrieval.After slice naturally cools to room temperature, cut into slices 2 times with deionized water wash, then use PBS washing slice 2 times.
4, dripped by 5% skim-milk (PBS dilution) in section, at 37 DEG C, incubation 1 hour, carries out antigen blockade.
5, in section, the 3B1 antibody of suitable concentration is dripped, 4 DEG C of overnight incubation.Then PBS washing slice is used 3 times.
6, in section, drip antibody working fluid (the DAKO ENVISION of the sheep anti-mouse igg of horseradish peroxidase-labeled
tM+ System HRP Mouse), incubation 1 hour at 37 DEG C.
7, use PBS washing slice 2 times, section is soaked in and develops the color 20 minutes in the solution of horseradish peroxidase substrate DAB.
8, use PBS color development stopping, with phenodin, section is redyed.By 1% hydrochloride alcohol differentiation section, gradient alcohol dehydration, dimethylbenzene is transparent, then by cover glass lid on tissue sections, and basis of microscopic observation.
Result is shown in Figure 7, does not express PES1 in display cancer of the stomach or oesophagus Carcinoma side normal tissue, and has the high expression level of PES1 in cancer of the stomach or human esophageal carcinoma.
Embodiment 9, Western-Blot detect the expression of PES1 in cancer of the stomach and colorectal carcinoma far-end healthy tissues, cancer beside organism and cancerous tissue
Carry out according to following operation in the present embodiment:
1) cancer of the stomach and colorectal carcinoma far-end healthy tissues, cancer beside organism and cancerous tissue block (from Beijing Tumour Hospital's sample storehouse) each 250mg is got, be ground into powder under liquid nitrogen freezing condition, add cell pyrolysis liquid RIPA (Tris (pH7.4), 150mM NaCl, 1%Triton X-100,1%sodium deoxycholate, 0.1%SDS), fully dissolving organizes powder, 12000rpm, 10min centrifuging and taking supernatant, is respective organization lysate.BCA method is adopted to carry out quantitatively Tissue lysates.The SDS-PAGE electrophoresis of row 12%, every porin applied sample amount is 50 μ g.
2) stop when tetrabromophenol sulfonphthalein reaches the most lower edge of glue, carry out electrotransfer.
3) after transferring film terminates, ponceau is dyeed, and detects transferring film effect and carries out mark.
4) 5% skim-milk is closed, and 4 DEG C are spent the night.
5) add primary antibodie 3B1 antibody, concentration is 1 μ g/ml, and positive mouse IgG is negative control, and β-Actin antibody is tissue protein internal reference standard, room temperature 1 hour.
6) 0.1%Tween-20/PBS washs 7 times.
7) antibody of the sheep anti-mouse igg of horseradish peroxidase-labeled is added, incubation at room temperature 40 minutes.
8) 0.1%Tween-20/PBS washs 7 times, and PBS washs 1 time.
9) luminous development.
Result is shown in Figure 8, in cancer of the stomach and colorectal carcinoma cancerous tissue, comparatively respective distal end healthy tissues and cancer beside organism obviously raise for the expression of PES1, wherein there is PES1 high expression level in 14 routine cancerous tissues in 18 routine cancer of the stomach pairing tissues, there are 2 routine PES1 to express no significant difference in different lesions tissue, have 2 routine PES1 at far-end healthy tissues and cancer beside organism's comparatively cancerous tissue high expression level (picture A); There is PES1 high expression level in 10 routine cancerous tissues in 14 routine colorectal carcinoma pairing tissues, have 4 routine PES1 in different lesions tissue, express no significant difference (picture B).
Claims (11)
1. a monoclonal antibody of PES1, the hybridoma that described monoclonal antibody is CGMCC No.3646 by the preserving number of China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation is secreted.
2. secrete the hybridoma cell line of monoclonal antibody according to claim 1, its preserving number is CGMCC No.3646.
3. detect a test kit for PES1 level, it contains monoclonal antibody according to claim 1.
4. test kit according to claim 3, wherein, described test kit is also containing second antibody with for the enzyme that detects or fluorescence or radio-labeled thing, and damping fluid; The anti-antibody of monoclonal antibody or resisting of anti-PES1 described in the monoclonal antibody that the hybridoma that described second antibody is CGMCC No.3647 for the preserving number by the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms is secreted or anti-claim 1 more.
5. test kit according to claim 3, this test kit is for detecting the expression level of PES1 in histocyte, and it also comprises and requires the anti-antibody of monoclonal antibody described in 1 and enzyme or fluorescent marker and corresponding damping fluid for test right.
6. monoclonal antibody according to claim 1 is for the preparation of the application detected in the reagent of PES1 level in sample or test kit.
7. application according to claim 6, wherein, described sample is humoral sample or the cell protein extracting solution of individual test subjects; The humoral sample of described individual test subjects is from breast cancer tissue, samples of human glioma or gastric cancer tumor tissue.
8. the monoclonal antibody of claim 1 is for the preparation of the application in auxiliary diagnosis tumour, monitoring tumour and/or the reagent of tumour patient Index for diagnosis or test kit; Described tumour is mammary cancer, glioma or cancer of the stomach; Described reagent or test kit are used in immunoprecipitation detection.
9. application according to claim 8, wherein, described reagent or test kit are used for the level by detecting PES1 in humoral sample or cell protein extracting solution, assist the diagnosis of tumour and the process of monitoring tumour.
10. an antitumor drug, this antitumor drug contains the monoclonal antibody of claim 1; Described tumour is mammary cancer, glioma or cancer of the stomach.
The detection reagent of 11. 1 kinds of PES1 levels, it contains monoclonal antibody according to claim 1.
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Non-Patent Citations (4)
| Title |
|---|
| Kinoshita等.Pescadillo, a novel cell cycle regulatory protein abnormally expressed in malignant cells, 《The journal of biological chemistry.《The journal of biological chemistry》.2010,第276卷(第9期),第6656-6665页. * |
| 利用不同方法检测RNAi抑制人pescadillo基因的表达;李杰萍 等;《细胞与分子免疫学杂志》;20071231(第11期);全文 * |
| 张浩 等.Pescadillo 抗体的制备及其表达研究.《中国科学 C辑:生命科学》.2007,第37卷(第2期),第135-142页. * |
| 核仁蛋白PES1可能受c-Jun转录调节在胃肠肿瘤中高表达;冯勤 等;《中国病理生理学会受体、肿瘤和免疫专业委员会联合学术会议》;20100416;第45页 * |
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