CN105693856A - Monoclonal antibody, cell strain secreting monoclonal antibody, preparation method and application - Google Patents
Monoclonal antibody, cell strain secreting monoclonal antibody, preparation method and application Download PDFInfo
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- CN105693856A CN105693856A CN201610259391.8A CN201610259391A CN105693856A CN 105693856 A CN105693856 A CN 105693856A CN 201610259391 A CN201610259391 A CN 201610259391A CN 105693856 A CN105693856 A CN 105693856A
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- C—CHEMISTRY; METALLURGY
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
- C07K16/1278—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Bacillus (G)
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- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
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- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
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Abstract
The invention provides a monoclonal antibody secreted by a hybridoma cell strain 2F8 with the preservation number being CCTCC NO: C201659. According to the antibody, the 2F8 bacterial strain is inoculated in BALB/c mice pre-processed by paraffin oil, ascites is prepared through an in-vivo induction method and collected, the collected ascites is subjected to Protein G column purification, then the monoclonal antibody is obtained and can be used for recognizing Bt Cry1Aa, Cry1Ab and Cry1Ac and has no cross reaction with Bt Cry1B, Cry1C and Cry1F toxoid, the monoclonal antibody can be used for universal Bt CrylA toxoid detection, operation is easy and convenient, and the monoclonal antibody is suitable for application and popularization.
Description
Technical field
The present invention relates to a kind of monoclonal antibody and secrete hybridoma cell strain 2F8, this antibody preparation method and the application of this monoclonal antibody, belonging to biological technical field。
Background technology
BtCry1A toxoid is produced in its Sporulation stage by thuringiensis (Bt), and it is a kind of insecticidal proteins, and Lepidoptera and coleoptera class insect larvae is effective。After it is absorbed by sensitive insect larva, the activated protein of 65kDa size it is cracked under the effect of alkaline environment and midgut proteinase, and be combined with the special receptor being present in intestinal cell, cause the formation of hole and the cracking of midgut epithelial cell, ultimately result in dead larvae。On the other hand, because lacking the receptor of BtCry1A toxoid albumen in mammal body, so BtCry1A toxoid is considered the mankind and domestic animal to be safe。At present, BtCry1A toxoid gene is extensively introduced in genetically modified crops, and some comprise the genetically modified crops of BtCry1A toxoid gene, and the cultivated area such as Oryza sativa L., Cotton Gossypii, Semen sojae atricolor and Semen Maydis etc. increases in the world year by year。What turn BtCry1A toxoid gene pest-resistant crop constantly occurs and obtains large-area popularization while producing economic, society and ecological benefits, and it is to the risk of ecological safety and the potential safety hazard of the mankind and other mammal is also received publicity gradually。At present, the BtCry1A toxoid gene groups being employed is more and more many, the government safety supervision brought and the limitation of the technology of food processing enterprises raw material management and control are more and more obvious, especially time gene test can not obtain promising result in for some netically modified foods, contratoxin expression product safe screen check survey just aobvious particularly urgent。
Mainly adopting the antibody test technology for single toxin for the detection method of toxin protein in transgenic product, owing to needing to prepare corresponding antibody for different toxin, and antibody preparation process is tediously long, cost is high, it is impossible to meet the requirement of wide spectrum examination detection。Therefore exploitation BtCry1A toxoid wide spectrum screening immunoassay technology is the work having very much using value。
Mainly nucleic acid and protein analysis is concentrated on currently for the detection method of toxin protein in transgenic product。Be widely used in the detection of Bt gene based on the method for DNA-PCR, it has the sensitivity of height。But, PCR method needs by the specific instrument of veteran human users, not only consuming time and be not suitable for field quick detection。On the other hand, detection method based on protein, the shortcoming that enzyme-linked immunosorbent assay test (ELISA) overcomes the former, have high specificity, highly sensitive, sample pre-treatments is simple, cost is low, is applicable to the advantages such as on-the-spot batch detection, has been applied to multiple field such as food, medical treatment。In ELISA process, the specificity of antibody, sensitivity etc. will directly affect testing result, so to set up the immunology detection technology for BtCry1A toxoid, it is necessary to first prepare the universal monoclonal antibody of high-quality anti-BtCry1A toxoid albumen。Existing a lot of commercial ELISA detection kit in the market, but the not test kit of three kinds of BtCry1A classes of wide spectrum detection, but BtCry1Aa and BtCry1Ab/Cry1Ac detection kit, therefore providing a kind of the antibody of three kinds of BtCry1A toxoids of specific recognition can just seem particularly urgent。
Summary of the invention
For the problems referred to above, the hybridoma 2F8 that the present invention provides a strain preserving number to be CCTCCC201659, the monoclonal antibody 2F8 of its secretion can be used for the total amount detection of BtCry1A toxoid, and the present invention is achieved in that
A kind of monoclonal antibody, it is that its variable region of heavy chain of this monoclonal antibody has the aminoacid sequence shown in sequence table SEQ IDNO.1 by the hybridoma cell strain 2F8 secretion that preserving number is CCTCCNO:C201659；Variable region of light chain has the aminoacid sequence shown in SEQ ID NO.2。
Further, antibody of the present invention is obtained by: be inoculated in the BALB/c mouse body processed with paraffin oil in advance by the hybridoma cell strain 2F8 that preserving number is CCTCCNO:C201659, ascites is prepared with the internal method that induces, by ProteinG column purification ascites after collection, namely obtain described monoclonal antibody。
Further, the monoclonal antibody of the present invention application in detection BtCry1A toxoid。
One strain preserving number is the hybridoma cell strain 2F8 of CCTCCNO:C201659。
Hybridoma cell strain 2F8 provided by the invention can be used for preparing high-titer anti-BtCry1A toxoid general purpose single clonal antibody, and the titer to three kinds of toxin that mouse ascites antibody ELISA immuning adsorpting analysis (ELISA) method records is all up to 2.56 × 106, i.e. ascites antibody dilution 2.56 × 106Times time OD450Value remains above 1.0；This anti-BtCry1A toxoid general purpose single clonal antibody is highly sensitive, general purpose single clonal antibody may identify which BtCry1Aa, Cry1Ab, Cry1Ac, and BtCry1B, Cry1C, Cry1F toxoid no cross reaction, can be used for the detection of general BtCry1A toxoid, easy and simple to handle, it is suitable for popularization and application。
Accompanying drawing explanation
Fig. 1 is the 3 d structure model schematic diagram of three kinds of toxin of BtCry1Aa, Cry1Ab and Cry1Ac；
Fig. 2 is monoclonal antibody 2F8 indirect ELISA testing result schematic diagram。
Fig. 3 is BtCry1A toxoid double crush syndrome standard curve。
Detailed description of the invention
Below by way of specific embodiment, technical scheme is described further。
The preparation method relating to solution in embodiment:
(1) phosphate buffer (PBS, pH=7.4):
Collocation method: NaCl8.0g, KCl0.2g, Na2HPO4·12H2O2.9g, KH2PO40.2g, adds distilled water and is settled to 1L；
(2) cleaning mixture (PBST): containing the PBS of 0.05%Tween-20；
(3) confining liquid (MPBS): containing the PBS of 3% defatted milk powder；
(4) citrate buffer (CPBS, substrate buffer solution, pH5.5):
C6H7O8(citric acid) 21g, Na2HPO4·12H2O71.6g, adds distilled water and is settled to 1L；
(5) substrate: by TMB and 25 of 10mg/mL μ L0.65%H2O2It is dissolved in 9.875mLCPBS, now with the current；
(6) tetramethyl benzidine (TMB) mother solution:
Weigh 10mg tetramethyl benzidine and be dissolved in 1mL dimethyl sulfoxide, 4 DEG C of preservations；
(7) stop buffer (2MH2SO4):
The concentrated sulphuric acid (18M) taking 11.8mL98% is dissolved in 80mL distilled water, and after cooling, constant volume is to 100mL；
(8) carbonate buffer solution (CBS):
Na2CO31.59g；NaHCO32.93g, constant volume is to 1L；
Involved reagent in embodiment:
Not formula Freund's complete adjuvant and Fu Shi Freund's incomplete adjuvant are all purchased from Sigma company；
BtCry class standard toxin is all purchased from You Long bio tech ltd, Shanghai；
In embodiment, mice used is 8 week old left and right BALB/c female mices。
Aminoacid sequence involved in embodiment:
SEQIDNO.1:MALEVKLVESGPSLVQPSQSLSITCTVSGFSLTNFGVHWVRQSPGK GLEWLGVIWRGGNTDYNAAFMSRLSITRDNSKRQIFFKMDSLQADDTAIYYCAKPL IYYGNYGYFDVWGAGTTVTVSS；
SEQIDNO.2:DIVLTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQ PPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSE GGPSWS-NELWLHHL；
Embodiment 1: the preparation of hybridoma cell strain 2F8
By mixed immunity former (three kinds of toxin mixed in equal amounts of Cry1Aa, Cry1Ab and Cry1Ac) that concentration is 1mg/mL and not formula Freund's complete adjuvant mixed in equal amounts, emulsifying is water in oil emulsion, with 100 μ g/ dose immunization mice only；Once every two weeks booster immunizations, replacing Freund's complete adjuvant to mix with immunogen with incomplete Freund's adjuvant during booster immunization, third time and the 4th immunity terminate one week after blood sampling and survey titer, when titer reaches serum-dilution 10000 times, OD later450When value is more than 1.0, according still further to conventional hybridization tumor integration technology (referring to Wu Jianxiang etc., " development of Pyricularia oryzae monoclonal antibody and the shadow noon to note fields thereof " microorganism journal, 2000,40 (6): 638-645) merge with murine myeloma cell SP2/0；Then indirect elisa method screening cells and supernatant is adopted, positive colony hybridoma wells is selected to carry out sub-clone, and with limiting dilution assay continuous cloning 2-3 time, final screening obtains a strain of hybridoma strain, applicant by it from called after hybridoma cell strain 2F8。
Described indirect elisa method step is as follows:
(1) it is coated: by the concentration being coated liquid CBS and coating antigen BtCry1Aa, Cry1Ab and Cry1Ac being diluted to 4 μ g/mL, being added separately in 96 hole ELISA Plate (three kinds of toxin are separately coated), every hole 100 μ L, 4 DEG C are coated overnight；
(2) close: PBST washes plate three times, add the MPBS of 3%, 250 μ L/ holes, put in 37 DEG C of incubators and hatch 2h；
(3) culture supernatant is added: PBST washes plate three times, draw the culture supernatant 100 μ L of cell hole to be detected, join in 96 hole ELISA Plate, put in 37 DEG C of incubators and hatch 1h, set negative serum and positive serum (1/2000 application of sample) and the comparison of acellular hole culture supernatant simultaneously；
(4) add ELIAS secondary antibody: PBST washes plate three times, add the sheep anti-mouse igg (purchased from KPL company, working concentration 1/5000, diluent is MPBS) of HRP labelling, every hole 100 μ L, put in 37 DEG C of incubators and hatch 1h；
(5) colour developing: PBST washes plate four times, adds substrate (TMB-carbamide peroxide), every hole 100 μ L, hatches 15min in 37 DEG C of incubators；
(6) terminate: add 2MH2SO4, every hole 50 μ L；
(7) result judges: measure OD by microplate reader450Value, the hole of P/N >=2.1 is judged to positive hole。
The hybridoma cell strain 2F8 of acquisition is preserved in China typical culture collection center (ChinaCenterforTypeCultureCollection, CCTCC) by applicant, address: Wuhan City, Hubei Province Wuhan University；Postcode 430072；Deposit number is CCTCCNo.C201659, preservation date on March 22nd, 2016, systematic name hybridoma cell strain 2F8。
Embodiment 2: anti-BtCry1A toxoid general purpose single clonal antibody 2F8 prepares purification and sensitivity technique
1, the hybridoma cell strain 2F8 obtained to mice Inoculation embodiment 1, utilizes the internal method that induces to prepare ascites, specifically comprises the following steps that
Purging gently by well-grown hybridoma, centrifugal collection, with the resuspended counting of RPMI-1640 basic culture solution, cell quantity controls 1～2 × 106Within the scope of individual/mL, lumbar injection is in advance with the mice that paraffin oil is pretreated, 2 mices of every strain cell infusion, every mice 0.5mL。The health status of close observation mice and sign of ascites as, about about 10 days, mouse web portion started to expand, and when ascites is many as far as possible, drew neck to put to death mice, absorption ascites of cutting open the belly。
The ascites centrifugal 10min of 5000rpm at 4 DEG C that will collect, removes fat and hemocyte etc., adds equivalent glycerol, in-20 DEG C of Refrigerator stores in ascites after centrifugation。
2, ascites ProteinG post step 1 obtained is purified, and purification step by specification operates, and after purification, the concentration of monoclonal antibody 2F8 is 1.1mg/mL。
3, the mensuration of antibody sensitivity: adopt indirect elisa method to be measured。
Coating antigen is respectively adopted BtCry1Aa, Cry1Ab, Cry1Ac, six kinds of toxin of Cry1B, Cry1C and Cry1F, and it is 4 μ g/mL that concentration is；
Antibody is the concentration obtained in step (2) is 1.1mg/mL monoclonal antibody 2F8 solution, and with PBS, antibody is diluted 8000 times, 16000 times, 32000 times, 64000 times, 128000 times and 256000 times successively respectively；
Sensitivity technique specifically comprises the following steps that
(1) it is coated: being coated in 96 hole ELISA Plate respectively by antigen BtCry1Aa, Cry1Ab, Cry1Ac, six kinds of toxin proteins of Cry1B, Cry1C and Cry1F, 100 μ L/ holes, 4 DEG C are coated overnight；
(2) close: PBST washes plate three times, add the MPBS of 3%, 250 μ L/ holes, put in 37 DEG C of incubators and hatch 2h；
(3) add antibody: PBST washes plate three times, add the monoclonal antibody after dilution by 100 μ L/ holes respectively, simultaneously replace monoclonal antibody as negative control using PBS；
(4) add ELIAS secondary antibody: PBST washes plate three times, add the sheep anti-mouse igg (purchased from KPL company, working concentration 1/5000, diluent is MPBS) of HRP labelling, every hole 100 μ L, put in 37 DEG C of incubators and hatch 1h；
(5) colour developing: PBST washes plate four times, adds substrate (TMB-carbamide peroxide), every hole 100 μ L, hatches 15min in 37 DEG C of incubators；
(6) terminate: add 2MH2SO4, every hole 50 μ L；
(7) result judges: measure OD by microplate reader450Value；
BtCry1Aa, the 3 d structure model schematic diagram of three kinds of toxin of Cry1Ab and Cry1Ac is as shown in Figure 1, antibody titer measurement result is as shown in Figure 2, as seen from Figure 2, negative control value is respectively less than 0.1, when antibody dilutes 256000 times, the OD to three kinds of toxin of BtCry1Aa, Cry1Ab and Cry1Ac450It is above 1.0, it was shown that monoclonal antibody is all higher to the titer of three kinds of toxin, and active with Cry1B, Cry1C and Cry1F no cross reaction, it is possible to it is applied to the total amount detection of BtCry1A toxoid。
Embodiment 3: the preparation of the anti-BtCry1A toxoid polyclonal antibody of enzyme mark
Specifically comprise the following steps that
(1) preparation of anti-BtCry1A toxoid polyclonal antibody
Using after three kinds of toxin mixed in equal amounts of BtCry1Aa, Cry1Ab and Cry1Ac as immunogen immune new zealand white rabbit 2, during first immunisation, after immunogen that concentration is 1mg/mL and the mixing of Fu Shi Freund's complete adjuvant 1:1 ratio by volume, carrying out immunity in back multi-point injection mode, immunizing dose is 600 μ g/；Once every two weeks booster immunizations, replacing Freund's complete adjuvant to mix with immunogen with incomplete Freund's adjuvant during booster immunization, third time immunity terminates one week after blood sampling and surveys titer, and titer reaches ideal value (serum-dilution 10000 times and OD later450Value is more than 1.0) time, ear vein injection is not added with the BtCry1A toxoid mixture 600 μ g of Freund adjuvant and carries out impacting immunity, and heart takes whole blood subsequently, 4 DEG C place overnight, next day centrifuging and taking serum, antiserum is after saturated ammonium sulphate purification, it is stored in-20 DEG C, for follow-up HRP labelling
(2) the HRP labelling of anti-BtCry1A toxoid polyclonal antibody
1. weigh 5mgHRP and be dissolved in 1mL distilled water, in supernatant, add the newly configured 0.1MNaIO of 0.2mL4Solution, lucifuge reaction 20min under room temperature。
2. above-mentioned solution is loaded in bag filter, under 4 DEG C of conditions, dialysed overnight in 1mMpH4.4 sodium-acetate buffer。Meanwhile, anti-BtCry1A toxoid polyclonal antibody 0.01MpH9.5 carbonate buffer solution dialysed overnight step (1) obtained。
3. adding 20 μ L0.2MpH9.5 carbonate buffer solutions in the above-mentioned solution of dialysed overnight, making the pH of hydroformylation HRP raise is 9.0-9.5, adds the IgG(after 10mg dialysis immediately after in 1mL0.01M carbonate buffer solution)。The 0.1mL 4mg/mLNaBH newly joined is added after room temperature lucifuge stirring 2h4, mixing, place 2h for 4 DEG C。
4. above-mentioned solution is loaded in bag filter, 0.15MpH7.4PBS dialysed overnight。
5. under agitation it is added dropwise over isopyknic saturated ammonium sulfate solution, places 1h for 4 DEG C。
6. the centrifugal 30min of 3000rpm, abandons supernatant, and precipitation semi-saturation ammonium sulfate is washed twice, and finally precipitation is dissolved in 0.15MPBS。
7. last dialysis in the PB of 0.15MpH7.4, removes ammonium ion and namely obtains the anti-BtCry1A toxoid polyclonal antibody of enzyme mark, and concentration is 2mg/mL, presses 1:5000 dilution with PBS during use。
Embodiment 4 monoclonal antibody 2F8 application in detection BtCry1A toxoid
The anti-BtCry1A toxoid general purpose single clonal antibody 2F8 that hybridoma cell strain 2F8 secretes is used for the interpolation recovery test of BtCry1A toxoid, specifically includes following steps:
1. it is coated: monoclonal antibody 2F8 PBS being diluted 32000 times and is coated 96-hole ELISA Plate, 100 μ L/ holes, 4 DEG C are coated overnight, make formation insolubilized antibody；
2. close: PBST washes plate three times, add the MPBS of 3%, 250 μ L/ holes, put in 37 DEG C of incubators and hatch 2h；
3. antigen is added: after PBST washes plate three times, being separately added into concentration is 0,10,25,50 and the BtCry1A standard solution (three kinds of toxin mixed in equal amounts of BtCry1Aa, Cry1Ab and Cry1Ac, CBS buffer) of 100ng/mL, 100 μ L/ holes, hatch 1h, make antigen fully react with the antibody on solid phase carrier, form solid phase antigen antibody complex；
4. enzyme labelled antibody is added: PBST washes plate three times, remove other and be not associated with material, (enzyme labelled antibody concentration is 2mg/mL to the anti-BtCry1A toxoid polyclonal antibody of enzyme mark that addition embodiment 3 obtains, 1:5000 dilution is pressed with PBS) during use, 100 μ L/ holes, hatching 1h, make formation insolubilized antibody determined antigen-enzyme labelled antibody sandwich complex, washing removes unconjugated enzyme labelled antibody；
5. colour developing: PBST washes plate four times, adds substrate (TMB-carbamide peroxide), every hole 100 μ L, hatches 5min in 37 ° of C incubators；
6. terminate: add 2MH2SO4, every hole 50 μ L；
7. result judges: measure OD by microplate reader450Value；
8. drawing standard curve, as it is shown on figure 3, curvilinear equation: y=0.0019x+0.077；R2=0.9625；
9. add recovery test: weigh the rice leaf after pulverizing (1g/ part) and be placed in 10mL centrifuge tube, be added to the standard BtCry1A toxoid (three kinds of toxin mixed in equal amounts of Cry1Aa, Cry1Ab and Cry1Ac) of 1 μ g/g, 0.5 μ g/g and 0.1 μ g/g respectively。Under sample room temperature, concussion 10min is placed in 4 DEG C of refrigerators and stands overnight。Second day, adding the 1mL CBS buffer containing 0.05% tween 20 in sample, under room temperature, 2h is extracted in concussion。Subsequently, mixture 3000rpm is centrifuged after 10min, supernatant CBS dilute 10 times and is directly used in double crush syndrome detection。It is shown that the TIANZHU XINGNAO Capsul in rice leaf sample is followed successively by 103.6%, 107.6%, and 106.9%。
The above is only the preferred embodiment of the present invention; it should be pointed out that, for those skilled in the art, under the premise without departing from the principles of the invention; can also making some improvements and modifications, these improvements and modifications also should be regarded as protection scope of the present invention。
SEQUENCELISTING
<110>Jiangsu Province Agriculture Science Institute
<120>a kind of monoclonal antibody and secrete the cell strain of this monoclonal antibody, preparation method and application
<130>2
<160>2
<170>PatentInversion3.3
<210>1
<211>124
<212>PRT
<213>synthetic
<400>1
MetAlaLeuGluValLysLeuValGluSerGlyProSerLeuValGln
151015
ProSerGlnSerLeuSerIleThrCysThrValSerGlyPheSerLeu
202530
ThrAsnPheGlyValHisTrpValArgGlnSerProGlyLysGlyLeu
354045
GluTrpLeuGlyValIleTrpArgGlyGlyAsnThrAspTyrAsnAla
505560
AlaPheMetSerArgLeuSerIleThrArgAspAsnSerLysArgGln
65707580
IlePhePheLysMetAspSerLeuGlnAlaAspAspThrAlaIleTyr
859095
TyrCysAlaLysProLeuIleTyrTyrGlyAsnTyrGlyTyrPheAsp
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ValTrpGlyAlaGlyThrThrValThrValSerSer
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AspIleValLeuThrGlnSerProAlaSerLeuAlaValSerLeuGly
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GlyTyrSerTyrMetHisTrpAsnGlnGlnLysProGlyGlnProPro
354045
ArgLeuLeuIleTyrLeuValSerAsnLeuGluSerGlyValProAla
505560
ArgPheSerGlySerGlySerGlyThrAspPheThrLeuAsnIleHis
65707580
ProValGluGluGluAspAlaAlaThrTyrTyrCysGlnHisIleArg
859095
GluLeuThrArgSerGluGlyGlyProSerTrpSerAsnGluLeuTrp
100105110
LeuHisHisLeu
115
Claims (4)
1. a monoclonal antibody, it is by the generation of the hybridoma cell strain 2F8 secretion that preserving number is CCTCCNO:C201659。
2. the preparation method of monoclonal antibody as claimed in claim 1, it specifically comprises the following steps that and is inoculated in by described hybridoma in the BALB/c mouse body processed with paraffin oil in advance, ascites is prepared with the internal method that induces, by ProteinG column purification ascites after collection, namely obtain described monoclonal antibody。
3. monoclonal antibody application in detection BtCry1A toxoid as claimed in claim 1。
4. a strain preserving number is the hybridoma cell strain 2F8 of CCTCCNO:C201659。
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111363722A (en) * | 2020-04-28 | 2020-07-03 | 江苏省农业科学院 | Monoclonal antibody of bacillus thuringiensis Cry2A toxin and application thereof |
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