CN102816235B - Preparation method of hybrid tumor of monoclonal antibody inhibiting common bacillus thuringiensis CryI, and application of monoclonal antibody thereof - Google Patents
Preparation method of hybrid tumor of monoclonal antibody inhibiting common bacillus thuringiensis CryI, and application of monoclonal antibody thereof Download PDFInfo
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Abstract
The invention relates to a broad-spectrum monoclonal antibody aiming at a bacillus thuringiensis (Bt) CryI protein common in gene transformation, and a preparation method thereof. The invention aims at providing a novel monoclonal antibody which can be used for detecting whether Bt protein exists in natural transgenic plants (paddy rice, cotton, corn, and the like). A special antigen for the monoclonal antibody is prepared through the coupling of a polypeptide designed according to an amino acid sequence of the common Bt CryI protein and carrier protein. A subtype of the monoclonal antibody is IgG2a, and an affinity constant reaches 3.84*10<9>. The monoclonal antibody can identify a plurality of proteins in a CryI family, and can be used in detections of various Bt CryI proteins. The invention also provides a hybrid tumor cell line 70647s-9 which secret the monoclonal antibody. The collection number of the cell line is CGMCC No.4856. The cell line can stably secret the monoclonal antibody with high titer.
Description
Technical field
The present invention relates to a kind of can with the protein bound antibody of exogenous gene expression in transgenic plant and hybridoma cell line thereof.Particularly, the invention provides the monoclonal antibody of Su Yun gold bud pole bacterium CryI albumen common in a kind of anti-transgenic plant, can be used for the test kit that preparation detects CryI albumen common in transgenic plant, belong to field of biological detection.
Background technology
Su Yun gold bud pole bacterium (Bacillus thuringiensis) is called for short Bt, belongs to the Gram-positive edaphic bacillus, is distributed widely in soil, dust, waters, desert, vegetation, insect corpse.In the sporulation process, Su Yun gold bud pole bacterium can produce a large amount of parasporal crystals, the crystalline protein with high degree of specificity insecticidal activity, consists of.This protein is commonly called insecticidal crystal protein (ICP) or delta-endotoxin.ICP exists with the form of parent toxin usually, and under the insect midgut alkaline environment, parent toxin is degraded to fragment and the receptors bind of 60kDa left and right.The protein that is incorporated into acceptor is inserted on cytolemma thereupon and forms perforation, makes the ionic equilibrium in cytolemma pericentral siphon and film chamber destroyed, causes that cellular swelling even breaks, thereby causes the death of insect.The selection insecticidal properties of Bt insecticidal crystal protein is to be determined by the specific receptors on insect gastrointestinal epithelial cells surface.The albumen produced due to the Bt bacterium has killing ability, and people produce a kind of microbial preparation through cultivating the Bt bacterium--the Bt sterilant.Its good disinsection effect of Bt sterilant, to the person poultry safety, to natural enemy, without injury, and resistance is given birth in difficult labour, has been widely used in preventing and treating the crop pests aspect.Along with continuous utilization and the development of Bt sterilant, the Bt gene of take is cultivated the new genetic engineering technique with plant of self insect-resistance as foreign gene and is in succession risen.
Along with development and the commercial kind thereof of transgenic Bt crops constantly increases, the security of genetically modified organism itself and they become one of hot issue of international community and numerous common people's extensive concern to the potential threat of human health and ecotope.Comprise that the increasing country of China formulates and implemented the pressure sign system of genetically modified food.Therefore, the scientific management of transgenic product and application need to obtain the support of transgenic product and composition detection technology thereof.At present, the most frequently used method of transgenic product and composition detection has two classes: a class is the detection technique for its exogenous nucleic acid composition; Another kind of is immunological analysis method for its exogenous protein composition.Detection method based on DNA can only reflect on nucleic acid level whether the transgenosis sample contains foreign DNA, and can not detect its foreign gene is that this detection technique just there is no guiding significance to the resistance that turns Bt in reticent or expression.For the product of some transgenic plant, DNA can decompose and be difficult to detect in the course of processing, now, need to utilize enzyme-linked immunosorbent assay to detect genetically modified component.Immunological analysis method based on foreign protein, adopt the antigen antibody reaction of high degree of specificity, realizes that to the transgenic plant sample fast, detect accurately and efficiently its key problem in technology is the antibody that preparation has high degree of specificity.
For genetically modified Bt ICP gene, be mainly the gene of CryI family, wherein the most frequently used is Cry1Ab, Cry1Ac, Cry1Ah, Cry1C and Cry1A (fusion rotein be comprised of Cry1Ab first half section and Cry1Ac second half section).Although also do not discharged in China at present with Cry1Ah and the genetically modified kind of Cry1C, likely discharge in the future, external also likely flows into.Bt antibody in the domestic and foreign literature report, no matter be many anti-or monoclonal antibodies, basically only identify single Bt CryI gene, can not be identified in various common Bt CryI genes in transgenic plant simultaneously, can only be for detection of the transgenic event of single CryI gene.As the antibody in the patent No. application number patent that is 00510130058.9, only for the polypeptide of one section 16 amino-acid residue in Cry1A, purpose is for specific recognition Cry1A albumen.We are through the sequence alignment discovery, and this section peptide sequence also only appears in Cry1Ab and Cry1Ac, in Cry1Ah and Cry1C, has sequence difference, and the antibody of preparation can not be identified Cry1Ah and Cry1C accordingly.Other take the monoclonal antibody that complete recombinant C ry1Ab albumen is prepared as immunogen, do not using in advance and identify multiple toxin protein as design considerations, also its epitope is not confirmed, so its ability of identifying multiple toxin protein can't be confirmed (Zhang Xiao, record bright, Wang Yun, Wen Shuan, Liu Xianjin, indirect elisa method detects the foundation of bacillus thuringiensis (Bt) Cry1Ab toxalbumin antibody method, the Nanjing agriculture journal, 2010,26 (4)).If can prepare the wide spectrum monoclonal antibody for the common Bt CryI of transgenic plant albumen, just can utilize a reagent to detect the transgenic event of multiple CryI gene, thereby greatly save testing cost.
Summary of the invention
First purpose of the present invention be to provide a kind of can be for wide spectrum monoclonal antibody hybridoma of Bt CryI albumen common in transgenic plant and preparation method thereof.
Second purpose of the present invention is to provide the preparation method of the monoclonal antibody that a species specificity is good, this antibody capable specific combination Bt recombinant antigen Cry1A, Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ah, Cry1C and natural antigen.
The 3rd purpose of the present invention be to provide a kind of can be for laboratory or Fields detection farm crop blade whether with the detection reagent of common Bt CryI gene.
The technique means that the technical solution problem adopts
After the Characterization of antigenic epitopes of the present invention to Bt ICP gene C ry1Ab, Cry1Ac, Cry1Ah, Cry1c and Cry1A commonly used in transgenic plant, select existing immunogenicity again 13 amino-acid residues of homology as the sequence of synthetic polypeptide.Using synthetic polypeptide and carrier protein couplet as immunizing antigen, immune Balb/c mouse.Through cytogamy, recombinant C ry1A screening and cloning, obtain the positive hybridoma cell system of efficient secrete monoclonal antibody.
Utilize this hybridoma cell line to carry out the ascites preparation with mouse, Protein A/G post affinitive layer purification ascites, obtain mouse monoclonal antibody.Measure its subclass and affinity costant with elisa technique.Detect the ability of the CryI albumen in this antibody specific recognition restructuring and transgenic paddy rice with the immune marking (WB) experiment.
Particularly, the present invention relates to the following aspects:
1. a monoclonal antibody, the mouse hybridoma cell that to it is characterized in that by preserving number be CGMCC 4856 is that 70647s-9 produces.
2. monoclonal antibody claimed in claim 1, is characterized in that the antigen that its immune mouse is used is that polypeptide and carrier protein couplet with amino acid residue sequence of SEQ ID No:1 in sequence table are obtained.
3. monoclonal antibody claimed in claim 1, is characterized in that it is mouse IgG 2a hypotype monoclonal antibody.
4. monoclonal antibody claimed in claim 1, is characterized in that the multiple restructuring Su Yun gold of its identification bud pole bacterium CryI albumen.
5. monoclonal antibody claimed in claim 1, is characterized in that it is for immunoblotting, the CryI albumen in enzyme linked immunosorbent detection transgenic plant (paddy rice, cotton, corn etc.).
6. monoclonal antibody claimed in claim 1, is characterized in that can be used as the preparation of detection reagent for the transgenic plant detection test kit.
7. a mouse hybridoma cell is 70647s-9, and preserving number is CGMCC 4856, and it is characterized in that can stably excreting monoclonal antibody claimed in claim 1.
Advantage of the present invention and beneficial effect
(1) hybridoma (70647s-9 that the present invention obtains, protecting a surname number be CGMCC 4856) monoclonal antibody of secretion generation, can identify recombinant protein c ry1Ab, Cry1Ac, Cry1Ah, Cry1C and Cry1A, there is the characteristic that detects multiple common transgenosis Bt CryI albumen, than the antibody of identifying single Bt CryI gene, there is wider purposes.
(2) monoclonal antibody that the hybridoma (70647s-9, preserving number is CGMCC 4856) that the present invention obtains produces and the CryI albumen from the protein extract of transgenic paddy rice blade be combined with extremely strong specificity and susceptibility.
(3) hybridoma (70647s-9 that the present invention obtains, preserving number is CGMCC 4856) monoclonal antibody that produces can be applicable to detection and the examination that the immune marking (Western b1otting), double-antibody sandwich elisa, indirect ELISA, antibody chip such as prepare at the transgenic plant, specificity and highly sensitive, have very high use value.
The accompanying drawing explanation
Fig. 1: the sequence alignment of Bt CryI gene commonly used
The WB detected result of Fig. 2: 70647-S9 to restructuring Cry1Ab, Cry1Ac and Cry1A albumen
The Western Blot detected result of Fig. 3: 70647-S9 to transgenic paddy rice
Embodiment
Below in conjunction with chart and the concrete mode of implementing, the present invention being further elaborated, so that those skilled in the art can more clearly learn technical scheme of the present invention, is not limitation of the present invention.
The preparation of embodiment 1 immunizing antigen
Utilize software analysis Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ah, Cry1C and Cry1A protein sequence, in conjunction with its homology (Fig. 1), antigenicity, wetting ability, surperficial accessibility and secondary structure, select wherein not only to there is immunogenicity but also there is the polypeptide fragment that 12 amino-acid residues (seeing SEQ IDNo:1 in sequence table) of sequence homology form, synthetic with solid-phase synthesis, and carry out coupling with maleimide method and carrier proteins.Concrete steps are as follows:
The mcKLH (Thermo-Fisher company) of the sulfo-GMBS of 10mg/ml (PIERCE company) and 10mg/ml is mixed with the ratio of 1: 5, after being placed under room temperature on shaking table and slowly shaking up 30min, the centrifugal 5min of 12000rpm.Get supernatant, through sephadex
tMthe carrier protein KLH-sulfo-GMBS that G-25Fine (GE company) separated and collected activates, be added drop-wise in the polypeptide solution of 10mg/ml to wait mass ratio, and at room temperature rotation mixes 3h (or 4 ℃ of rotations are spent the night) and gets final product.
The preparation of embodiment 2 recombinant C ry1A albumen
One, gene clone
By after gene order (GenBank:ACF32736.1) synthetic gene of coding Cry1A, by the method for PCR, at 5 ' and 3 ' of this antigen-4 fusion protein gene, hold and add respectively NcoI and BamHI restriction enzyme site.The PCR product reclaims after agarose gel electrophoresis separates, and carries out NcoI and the BamHI enzyme is cut to the antigen-4 fusion protein gene that reclaims with for the plasmid vector pET-BPI expressed respectively, and electrophoresis reclaims again, with the T4DNA ligase enzyme, connects.Connect product and transform competent escherichia coli cell BL21, the clone's inoculation on the picking flat board, extract plasmid DNA, carries out the PCR evaluation.PCR shows that the clone of the antigen-4 fusion protein gene positive carries out sequencing analysis, the Cry1A albumen of the right-on clone of sequence for expressing restructuring.
Two, protein expression and purifying
The bacterium that spends the night of single bacterium colony being cultivated in the ratio of 1: 100 is forwarded to 100ml LB substratum, adds the kantlex that final concentration is 50 μ g/ml, and 37 ℃ of shaking culture are to OD
600be 0.6～0.8.The IPTG that adds 0.1mol/L, 8h is cultivated in 25 ℃ of concussions, ultrasonication after the receipts bacterium.This recombinant protein, with histidine-tagged, is used the Ni post to carry out the affinity purification of protein.After carrying out wash-out with the imidazoles solution of different concns, each component and stream are worn to loading respectively and carry out the SDS-PAGE separation detection, the purity of recombinant C rylA albumen is more than 90%, and concentration is about 1-1.5mg/mL, can meet the requirement of antibody screening and evaluation.
The foundation of embodiment 3 hybridoma cell lines
One, immunity
By the Freund's complete adjuvant for polypeptide of coupling in embodiment 1 (Sigma company) emulsification, immune 4-6 week female Balb/c mouse in age (being provided by Military Medical Science Institute), every mouse of abdominal part hypodermic 6 points, dosage is 60 μ g/.Once, antigen is used the non-Freund's complete adjuvant of Fu Shi (Sigma company) emulsification to every 14 days booster immunizations, and dosage is 30 μ g/.Within after the 3rd booster immunization 7 days, with indirect ELISA (wavelength 450nm), detect in mice serum and resist immunogenic many anti-tiring, the highest mouse of tiring is impacted immunity with tail vein injection, and antigen mixes with physiological saline, and dosage is 50 μ g/.
Two, cytogamy
Aseptic preparation immunity mouse boosting cell suspension up to standard, mix centrifugal 1500rpm, 5min with 5: 1 ratios with murine myeloma cell sp2/0 (ATCC).After abandoning supernatant, centrifuge tube is put into 37 ℃ of water-baths, slowly adds the PEG1500 (Roche company) of 1ml in 1 minute, and stirs cell.After standing 1min, add the IMDM (Sigma company) of 10ml serum-free in warm water, mix centrifugal 1000rpm, 5min.After abandoning supernatant, add careful cell is blown and beaten of 10ml serum (PAA company), and add the thymocyte of 5ml mixing 10xHAT (Sigma company), mix.The semisolid medium that adds again 25ml to contain 2.1% Nitrocellulose (Sigma company) fully mixes, and then pours into uniformly in 20 Tissue Culture Dishs.Tissue Culture Dish is put in wet box, put into 37.℃ 5%CO
2in incubator, cultivate.
Three, choose the clone
Merging latter 7 days clone cells, to roll into a ball big or small density moderate, under anatomical lens, draws round, real, large cloning cluster and squeeze in 96 well culture plates that are ready in advance substratum, puts into 37 ℃ of 5%CO
2in incubator, cultivate.
Four, ELISA screening positive hybridoma cell
After 3 days, cell concentration accounts for greatly floorage 2/3, gets 100 μ l supernatants and carries out respectively the ELISA screening with immunogen and synthetic polypeptide.Positive colony changes liquid fully, adds the perfect medium of 200 μ l containing feeder cell and 1%HT (Sigma company).Carry out two days later ELISA screening for the second time, positive colony proceeds to 24 orifice plates that are ready in advance substratum (containing feeder cell and HT) and cultivates.Get 100 μ l supernatants after five days and carry out ELISA screening for the third time, positive colony successively proceeds to 6 orifice plates and Tissue Culture Flask enlarged culturing frozen.
One, ascites preparation
The logarithmic phase cell washs with serum free medium and has hanged, counting～5 * 10
5, 1ml.The cell abdominal injection suspended is used the mouse of paraffin oil sensitization in advance.Start to collect ascites after 7 days.The ascites of taking out is in 4 ℃ of centrifugal 4000rpm, 10min.Ascites in the middle of careful sucking-off is collected in centrifuge tube, 4 ℃ or-20 ℃ of preservations.
Two, the purifying of monoclonal antibody
With HiTrap rProteinAFF (GE company) affinity chromatography by specification antibody purification from ascites.SDS-PAGE glue is identified purity, and the Bradford method is measured concentration.The antibody of purifying is stored in-20 ℃.
One, subgroup identification
With the coated sheep anti-mouse igg (Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge) of 100mM PBS (pH7.4) dilution, to 0.5 μ g/ml, every hole adds 100 μ l, 4 ℃, spends the night.Be emptied liquid, wash 3 times with the PBS (PBS-T) containing 0.05%Tween, every hole adds 200 μ l confining liquids (containing the PBS of 2%BSA and 3% sucrose), hatches 1h for 37 ℃.Be emptied liquid, clean 3 times with PBS-T.Every hole adds 0.1ml hybridoma supernatant, hatches 1h for 37 ℃.Being emptied liquid cleans 3 times with PBS-T.Sheep anti mouse (κ with 1: 1000 dilution HRP mark of confining liquid, λ) sheep anti mouse (the IgM of antibody or 1: 2000 dilution HRP mark, IgGl, IgG2a, IgG2b, IgG3, IgA) antibody (Southern Biotech company) the every hole of 0.1ml adds respectively in suitable hole, hatches 1h for 37 ℃.Be emptied liquid, clean 3 times with PBS-T.Every hole adds 50 μ l containing 0.15%ABTS (SouthernBiotech company) and 0.03%H
2o
2citrate buffer solution (PH4.0) carry out color reaction, measure the OD value under the 405nm wavelength in 10-20min.The result demonstration, monoclonal antibody of the present invention is IgG2a type mouse resource monoclonal antibody.
Two, affinity costant is measured
Coated synthetic polypeptide, coated concentration is 2 μ g/ml, 100 μ l/ holes, 4 ℃ of coated spending the night, PBS-T washes 3 times.Every hole adds 37 ℃ of sealing 2h of 200 μ l confining liquid, and PBS-T washes 3 times.The monoclonal antibody of purifying in embodiment 4, since 1: 200 2 times of gradient dilution, the contrast that blanks of last 1 hole, hatched 1h for 37 ℃, and PBS-T washes 3 times.Sheep anti mouse two dilutions in anti-1: 20000 of HRP mark, every hole 100 μ l, hatch 1h for 37 ℃, and PBS-T washes 3 times.Every hole adds 100 μ l containing 0.1%TMB (Sigma company) and 0.03%H
2o
2citric acid-phosphoric acid buffer colour developing 10min, add 50 μ l 0.5M sulphuric acid soln termination reactions.Measure the light absorption value of wavelength 450nm by microplate reader.Draw the curve of the corresponding antibody dilution multiple of OD value, find out >=extension rate A corresponding to 1/2 " platform OD value ".Utilizing following formula to calculate affinity costant is 3.8 * 10
9.
Three, monoclonal antibody atopic
Select Bt recombinant C ry1Ab, Cry1Ac, Cry1A albumen, detect the identification specificity of monoclonal antibody of the present invention by the method for immunoblotting.
The immunoblot experiment process is as follows: every kind of about 100ng of albumen loading, carry out 12% polyacrylamide gel electrophoresis.The gel protein band is transferred to according to a conventional method to (Millipore company) on pvdf membrane in Bio-Rad electrotransfer system.Film is placed in containing 4 ℃ of the TBS-T confining liquids of 5% skim-milk and spends the night.Add 4 ℃ of overnight incubation of monoclonal antibody 70647-S9 (dilution in 1: 1000).After washing film with TBS-T, add the sheep anti mouse two anti-(Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge) of dilution in 1: 5000, incubated at room 1 hour.TBST washes film again, adds the super quick nitrite ion of ECL (Puli's Lay company), with ImageQuant ECL instrument (GE company), catches the colour developing image.
What Fig. 2 showed is the immune marking result of monoclonal antibody 70647-S9 to recombinant protein.This antibody can be identified Cry1Ab, Cry1Ac, Cry1A albumen, and other irrelevant recombinant proteins of nonrecognition.
The effect of embodiment 6 monoclonal antibodies of the present invention
Extract albumen from transgenosis and non-transgenic rice leaf, by the method for the immunoblotting described in embodiment 5, detect the effect of monoclonal antibody of the present invention in detecting transgenic plant.
What Fig. 3 showed is the immune marking result of monoclonal antibody 70647-S9 to transgenic paddy rice.This antibody can detect the transgenic paddy rice leaf protein extract through 100 times of dilutions, and the non-transgenic paddy rice is not detected, and the specificity detected and sensitivity are all very high.
Claims (7)
1. a monoclonal antibody, the mouse hybridoma cell that to it is characterized in that by preserving number be CGMCC4856 is that 70647s-9 produces.
2. monoclonal antibody claimed in claim 1, is characterized in that the antigen that its immune mouse is used is that polypeptide and carrier protein couplet with amino acid residue sequence of SEQ ID No:1 in sequence table are obtained.
3. monoclonal antibody claimed in claim 1, is characterized in that it is mouse IgG 2a hypotype monoclonal antibody.
4. monoclonal antibody claimed in claim 1, is characterized in that the multiple restructuring bacillus thuringiensis CryI albumen of its identification.
5. monoclonal antibody claimed in claim 1, is characterized in that it is for immunoblotting, the CryI albumen in the enzyme linked immunosorbent detection transgenic plant.
6. monoclonal antibody claimed in claim 1, is characterized in that the preparation for the transgenic plant detection test kit as detection reagent.
7. a mouse hybridoma cell is 70647s-9, and preserving number is CGMCC4856, and it is characterized in that can stably excreting monoclonal antibody claimed in claim 1.
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| CN105198990B (en) * | 2015-10-12 | 2018-10-30 | 江苏省农业科学院 | Antibody and the preparation method and application thereof for the detection of Bt Cry1 toxoid wide spectrums |
| CN105693856A (en) * | 2016-04-25 | 2016-06-22 | 江苏省农业科学院 | Monoclonal antibody, cell strain secreting monoclonal antibody, preparation method and application |
| CN105884892B (en) * | 2016-06-28 | 2019-08-13 | 江苏省农业科学院 | A kind of detection of Bt Cry toxin wide spectrum albumen and its encoding gene and application |
| CN108314711B (en) * | 2018-02-28 | 2021-05-28 | 吉林省农业科学院 | Cry1C recombinant protein with immunogenicity, isolated nucleic acid molecule and application thereof |
| CN109781992B (en) * | 2018-12-27 | 2021-04-06 | 中国农业科学院生物技术研究所 | Colloidal gold immunochromatographic assay rapid test card for insect-resistant protein Cry1C |
| CN110294803B (en) * | 2019-05-27 | 2020-12-01 | 中国农业科学院植物保护研究所 | Monoclonal antibody of Cry1Ah1 protein and application thereof |
| CN116047047B (en) * | 2022-10-27 | 2024-01-26 | 中国农业科学院生物技术研究所 | Kit and method for quantitative detection of insect-resistant protein Cry1F enzyme-linked immunity |
| CN117143832B (en) * | 2023-11-01 | 2024-02-13 | 中国农业科学院生物技术研究所 | Insect-resistant protein Cry1Ah hybridoma cell strain, antibody produced by same and application thereof |
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