CN104045712B - Monoclonal antibody that a kind of ultraviolet excision repair protein is white and application thereof - Google Patents
Monoclonal antibody that a kind of ultraviolet excision repair protein is white and application thereof Download PDFInfo
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- CN104045712B CN104045712B CN201410247368.8A CN201410247368A CN104045712B CN 104045712 B CN104045712 B CN 104045712B CN 201410247368 A CN201410247368 A CN 201410247368A CN 104045712 B CN104045712 B CN 104045712B
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Abstract
The present invention relates to the white monoclonal antibody of a kind of ultraviolet excision repair protein and application thereof, in particular to the white monoclonal antibody of (hHR23b) of ultraviolet excision repair protein, the hybridoma cell strain secreting this monoclonal antibody and the application in the clinical therapeutic efficacy of prediction hdac inhibitor of the described antibody.
Description
Technical field
The present invention relates to the monoclonal antibody of a kind of ultraviolet excision repair protein white (hHR23b), secrete the hybridoma cell strain of this monoclonal antibody and the application of described antibody.
Background technology
Histon deacetylase (HDAC) (HDAC, Histonedeacetylase) is a newer and very potential cancer target.2006, FDA have approved the new drug Vorinostat of first hdac inhibitor, obtain " breakthrough medicine " qualification, increase the weight of for treating, continue and recurrence or with cutaneous T cell lymphoma (CTCL, one non-Hodgkin lymphoma) invalid after the treatment of two kinds of systemic medication.A new step is entered with the new drug development that hdac inhibitor is target.2013, FDA gives grace for Nuo Te (Entinostat) " breakthrough medicine " certification, it is combined with exemestane, for treating after Postmenopausal Breast Cancer patient's nonsteroidal aromastase inhibitor (NSAIs) is treated, sb.'s illness took a turn for the worse, the metastatic breast cancer of local recurrence or estrogen receptor positive there is, it was shown that the FDA attention to this type of medicine.In the same year, Spectrum pharmacy submits New-type wide-spectrum hdac inhibitor Belinostat new drug application to FDA, is used for treating relapsed or stubborn lymphoma peripheral T cell.
Although the research and development of hdac inhibitor antitumor drug are like a raging fire, but the medication mechanism of related drugs is also not very clear.Therefore treated effect is relatively low, and side effect is also relatively big, is therefore mainly used in treatment such as these small group tumors of cutaneous T cell lymphoma (CTCL).Treat the cutaneous T cell lymphoma of relapse and metastasis for Vorinostat, its treated effect only has 30%；And Belinostat connects in subject relapsed or stubborn lymphoma peripheral T cell experimenter at 129, total response rate is 26%.In order to improve the curative effect of hdac inhibitor, the detection method that can effectively predict drug responses efficiency must be had to occur, the patient being suitable for using this type of medicine is obtained medical treatment in time, and the patient being not suitable for medication will not waste the time and money of necessity, it is achieved the purpose of individualized treatment.
2009, Oxonian NicholasB.LaThangue teaches, adopt Loss-of-Function screening in genome range, it was found that the expression of hHR23b (the white Rad23B of ultraviolet excision repair protein) can affect the apoptosis of various hdac inhibitor induction.Next year, they have delivered further research, it was shown that, the expression of hHR23b is the Vorinostat key molecule treating cutaneous T cell lymphoma.In the second stage of clinical sample of Vorinostat, the patient of hHR23b high expressed, response rate is 71.7%.And in the patient of the low expression of hHR23b, response rate only has 41.7%, there is significant difference.The Belinostat delivered in 2012 equally treats in the clinical second phase result of primary hepatocarcinoma, in the patient of hHR23b high expressed, has the patient of 58% to have therapeutic effect.And in the patient of the low expression of hHR23b, only the patient of 14% is effective in cure.This has absolutely proved that hHR23b is likely to a universal Tumor marker, and it is possibly used for the predicting tumors patient therapeutic effect for hdac inhibitor, thus reasonably instructing clinical application.
Summary of the invention
The present invention relates to a kind of hHR23b monoclonal antibody (anti-hHR23b)
Monoclonal antibody anti-hHR23b of the present invention is the hybridoma cell strain secretion being by preserving number, described hybridoma cell strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, depositary institution address 3, numbering CGMCCNo.8883, preservation date on May 7th, 2014, specific name is mouse hybridoma cell strain.
The invention still further relates to described monoclonal antibody anti-hHR23b application in detection target protein hHR23b.
The invention still further relates to described monoclonal antibody anti-hHR23b application in the clinical therapeutic efficacy of prediction hdac inhibitor, described application includes but not limited to, described monoclonal antibody anti-hHR23b is used to detect the expression of hHR23b albumen in the section of target patient tumor tissue pathology, the treatment of hdac inhibitor is responded better by high expressed patient, and described hdac inhibitor is preferably Vorinostat.
The invention still further relates to described monoclonal antibody and hybridoma and judge that the hdac inhibitor of tumor patient treats the application in the test kit of response and the diagnostic kit prepared by the method in preparation.Described tumor includes but not limited to carcinoma of prostate, epidermis scale cancer, chronic myelocytic leukemia, acute T-cell leukemia, cerebral glioma, cervical cancer, t cell lymphoma, primary hepatocarcinoma etc..
Accompanying drawing explanation
Fig. 1, SDS-PAGE analyze the hHR23b monoclonal antibody after purification.
Fig. 2, immunoblotting (Westernblot) detect hHR23b expression in A431, K562, Jurkat, C6,3T3 and HeLa cell.The extension rate of hHR23b monoclonal antibody is 1: 10000.
The expression of hHR23b albumen in Fig. 3, Immunofluorescence test HeLa cell.
The expression of hHR23b albumen in Fig. 4, SABC detection prostate cancer tissue.
Detailed description of the invention
In following embodiment, method therefor is normal applying method if no special instructions.
Reagent, enzyme, carrier and cell strain:
(1), cell strain: HeLa (CCL-2TM)、A431(CRL-1555TM)、K562(CCL-243TM)、Jurkat(TIB-152TM)、C6(CRL-2199TM)、NIH/3T3(CRL-1658TM) and myeloma cell SP2/0 (CRL-1581TM)；
(2), laboratory animal: female BAl BIc/c mice (Hunan Si Laike Jing Da laboratory animal company limited)；
(3), enzyme and antibody reagent: the various enzymes of carrier construction process and PCR process are purchased from Fermentas, ELISA experiment antibody anti-mouseIgG/HRP (JacksonImmunoResearch) used, ELISA substrate TMB (Sigma), Reverse Transcriptase kit (Fermentas), BSA is purchased from (Yuan Heng Golden Horse bio tech ltd, Beijing)
(5), other reagent are domestic analytical pure if no special instructions.
Embodiment 1: the acquisition of the monoclonal antibody of hybridoma cell strain 5H1-A10-A7 and generation thereof.
(1), animal immune
With recombined human hHR23b albumen for antigen immune 6-8 week old female BAl BIc/c mice, carry out three inoculations according to the immunization method pre-established.
The sequential structure of antigen protein is:
MQVTLKTLQQQTFKIDIDPEETVKALKEKIESEKGKDAFPVAGQKLIYAGKILNDDTALKEYKIDEKNFVVVMVTKPKAVSTPAPATTQQSAPASTTAVTSSTTTTVAQAPTPVPALAPTSTPASITPASATASSEPAPASAAKQEKPAEKPAETPVATSPTATDSTSGDSSRSNLFEDATSALVTGQSYENMVTEIMSMGYEREQVIAALRASFNNPDRAVEYLLMGIPGDRESQAVVDPPQAASTGAPQSSAVAAAAATTTATTTTTSSGGHPLEFLRNQPQFQQMRQIIQQNPSLLPALLQQIGRENPQLLQQISQHQEHFIQMLNEPVQEAGGQGGGGGGGSGGIAEAGSGHMNYIQVTPQEKEAIERLKALGFPEGLVIQAYFACEKNENLAANFLLQQNFDED
First time every mice groin subcutaneous injection 0.2ml emulsion of immunity 2ML syringe (recombiant protein hHR23b (diluting with 1 × PBS)+100ul Freund's complete adjuvant of 100ul purification, containing 100ug antigen).
Second time immunization interval two weeks, with every mouse peritoneal injection 0.2ml emulsion of 2ML syringe (recombiant protein hHR23b (diluting with 1 × PBS)+100ul incomplete Freund's adjuvant that 100ul is purified, containing 100ug antigen).
Third time immunization interval two weeks, with every mice groin subcutaneous injection injection 0.2ml emulsion of 2ML syringe (recombiant protein hHR23b (diluting with 1 × PBS)+100ul incomplete Freund's adjuvant that 100ul is purified, containing 100ug antigen).
Surveying titer according to time of fusion arrangement, third time immunity takes blood examination after 7-10 days and surveys, and by conventional western method detection titer, selects the mice that titer reaches 1: 1000 to be used for merging.
Booster immunization merges first 3 days, carries out booster immunization to spleen mice to be taken, mouse peritoneal injection 200ul liquid (containing 60ug antigen, solvent is 1 × PBS, pH7.4).
(2), cell fusion
Adopting eyeball excise depletion method to put to death mice, sterile working takes out spleen, puts in the plate with 200 order stainless (steel) wires and grinds, prepares cell suspension.Ready homology myeloma cell SP2/0 is mixed by a certain percentage with mouse boosting cell (1: 5-1: 10), and adds short fusion agent Polyethylene Glycol (50%).Adopting HAT Selective agar medium, the selectivity carrying out hybridoma is cultivated and screening.
Hybridoma Cell Culture supernatant is detected: add in 96 hole ELISA Plate with being coated liquid (pH9.6,0.05M carbonate buffer solution) dilution for 8ug/ml by antigen by ELISA method, 50ul/ hole, put in 4 DEG C of refrigerator-freezers and be coated overnight.Discard and be coated liquid, wash plate three times with PBST (phosphate buffer, pH7.4), add confining liquid 3%BSA-PBST (adding the phosphate buffer of 3% bovine serum albumin), 100ul/ hole, hatch 1 hour for 37 DEG C.Discarding confining liquid, PBST (phosphate buffer, pH7.4) washes plate once, gets rid of in plank.Hybridoma Cell Culture supernatant is added ELISA Plate 100ul/well, is simultaneously introduced PBST (phosphate buffer) as negative control.Hatch 1 hour for 37 DEG C.Abandon supernatant, wash plate three times with washing trigger PBST, add anti-mouseIgG/HRP, 100ul/well, 37 DEG C diluted and hatch 1 hour.Abandon supernatant, wash plate three times with washing trigger PBST, add substrate TMB100ul/well, RT, lucifuge 37 DEG C, after 10-15min, add 50ul stop buffer (2mol/L sulphuric acid).Use microplate reader measures, and microplate reader is set to: 450nm colorimetric.Compare with negative control, if being exactly positive on OD value 2.5 times.Finally screening obtains the hybridoma cell strain of the best anti-hHR23b of a strain titer, and called after 5H1-A10-A7, hypotype is accredited as IgG2b.
The positive hybridoma cell strain 5H1-A10-A7 filtered out is carried out monoclonal screening (limiting dilution assay), it is thus achieved that the hybridoma cell clone of high-titer (WB thinner ratio > 1: 1000) monoclonal antibody can be produced.By hybridoma cell strain amplification culture, and frozen conservation.Described positive hybridoma cell is anti-human hHR23b hybridoma cell line 5H1-A10-A7, this cell line has been stored in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number is CGMCCNo.8883, preservation date on May 7th, 2014, specific name is mouse hybridoma cell strain.
(3) preparation of monoclonal antibody and purification
The cell strain 5H1-A10-A7 that step (2) obtains is seeded to BALB/c mouse abdominal cavity, prepares ascites, then antibody purification (obtained antibody called after anti-hHR23b) from ascites.
The purification of anti-hHR23b monoclonal antibody: use ProteinG affinity purification.The ProteinG filler good with PB solution equilibria is loaded in purification column, add the ascites through the monoclonal antibody anti-hHR23b of PB solution dilution and cross post.After end of the sample, wash pillar to stream with PB solution and wear liquid OD value < 0.01.Then with the glycine-HCI eluant solution of 0.1MpH3.0, the solution of whole eluting peak is collected.Eluent is through dialysis concentration and is replaced by PBS solution.SDS-PAGE result shows, purified antibodies purity more than 95% (referring to Fig. 1), concentration 0.8mg/ml.
Embodiment 2: Identification of Monoclonal Antibodies and application
1. the immune-blotting method of little mouse-anti hHR23b monoclonal antibody (anti-hHR23b) is identified
Extract the cell pyrolysis liquid of various tumor cell line, total protein concentration adjusts 2mg/ml, in the SDS-PAGE glue hole of loading to 12%, 20ug/ hole, by on the protein delivery in gel to pvdf membrane after electrophoresis, after 5% defatted milk powder is closed, the anti-hHR23b antibody prepared by embodiment 1 carries out immunostaining: add the anti-hHR23b of embodiment 1 step (3) preparation purification, working concentration is 1: 1000, 4 degree of overnight incubation, it is subsequently adding the Goat anti-mouse antibodies of HRP labelling, working concentration 1: 20000, 1hr is reacted in 37 degree, all there is the band (referring to Fig. 2) of 55Kd in each lysate, it is consistent with bibliographical information, prove the specific antibody that this antibody is anti-hHR23b；Antibody dilution gradient reaches 1: 1000 (antibody working concentration can reach 0.gug/ml) clear band, it is possible to detection hHR23b expression in cell.
2. dyeing immunofluorescence cell is identified
HeLa cell PBS washes twice, and 4% paraformaldehyde room temperature fixing 30min, PBS rinse, and thoroughly change 20min by 0.5%TritonX-100PBS room temperature.It is subsequently adding 5%BSAPBS to close nonspecific reaction.The anti-hHR23b monoclonal antibody 1: 100,37 degree adding embodiment 1 step (3) preparation purification hatches 30min.Rinsing, add the mountain sheep anti-mouse igg of FITC labelling, room temperature lucifuge hatches 1hr.Removing free two to resist, PBS rinses, fluorescence mountant mounting, and fluorescence microscopy Microscopic observation excites observation green florescent signal with blue light.Experimental result (Fig. 3) clear view presents obvious green fluorescence in nucleus, and Cytoplasm has faint expression, positions consistent with bibliographical information.Illustrate that the specific antibody of this anti-hHR23b may be used for Immunofluorescence test and observes expression and the location of hHR23b.
3. SABC detection
The paraffin section of prostate cancer tissue, after dewaxing aquation, adds 3%H2O2Incubated at room 5～10 minutes, to eliminate the activity of endogenous peroxydase.Adopting high pressure antigen retrieval method to carry out antigen retrieval, solution uses citrate buffer (pH6.0).After 5% Normal Goat Serum (PBS dilution) is closed, add anti-hHR23b monoclonal antibody 1: 100,37 degree and hatch 30min, PBS flushing 3 times, add the mountain sheep anti-mouse igg of HRP labelling, incubated at room 1hr.PBS rinses 5 times, adds chromogenic reagent (DAB).Tap water fully rinses, and redyes, mounting.Experimental result (Fig. 4) clear view presents obvious dyeing in nucleus, and Cytoplasm has faint dyeing, positions consistent with bibliographical information.Illustrate that the specific antibody of this anti-hHR23b may be used for SABC detection and observes hHR23b expression in the tissue and location.
Finally; it should be noted that; above example is only adapted to assist in the essence that skilled artisan understands that the present invention; it is not intended to limit the scope of the invention; the related art scheme that any those skilled in the art can obtain according to general technology knowledge and known general knowledge, broadly falls into the scope of protection of present invention.
Claims (5)
1. the monoclonal antibody for the white Rad23B of ultraviolet excision repair protein, it is characterised in that this antibody is by the hybridoma secretion that preserving number is CGMCCNo.8883.
2. secrete the hybridoma cell line of monoclonal antibody as claimed in claim 1, it is characterised in that the preserving number of described cell line is CGMCCNo.8883.
3. the monoclonal antibody described in claim 1 and the hybridoma described in claim 2 application in the preparation of the preparation detection white Rad23B of ultraviolet excision repair protein.
4. the monoclonal antibody described in claim 1 and the hybridoma described in claim 2 judge the application in the test kit of the Antibiotic FR 901228 treatment response of tumor patient in preparation, and described tumor is carcinoma of prostate, epidermis scale cancer, chronic myelocytic leukemia, acute T-cell leukemia, cerebral glioma, cervical cancer, t cell lymphoma, primary hepatocarcinoma.
5. the diagnostic kit that the method described in claim 4 prepares.
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Address after: Science Park Road Chengdu city Sichuan province 610041 No. 9 of No. 1 East Building Room 610 Applicant after: Chengdu Zenable Bioscience Co., Ltd. Applicant after: Qiu Junkang Applicant after: Nie Zimei Address before: 610200, 6, the Yellow River Road, Shuangliu industrial port, Shuangliu, Sichuan, Chengdu province 59 Applicant before: Chengdu Zenable Bioscience Co., Ltd. Applicant before: Qiu Junkang Applicant before: Nie Zimei |
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Application publication date: 20140917 Assignee: Chengdu Huiruixinyuan Biotechnology Co., Ltd. Assignor: Chengdu Zenable Bioscience Co., Ltd.|Qiu Junkang|Nie Zimei Contract record no.: 2018510000022 Denomination of invention: Monoclonal antibody of ultraviolet excision repair protein and application thereof Granted publication date: 20160629 License type: Exclusive License Record date: 20180530 |
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