CN102816235A - 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 protein bound antibody of exogenous gene expression in the transgenic plant and hybridoma cell line thereof.Particularly, the invention provides the proteic monoclonal antibody of Su Yun gold bud pole bacterium CryI common in a kind of anti-transgenic plant, can be used for preparing and detect the common proteic test kit of CryI in the 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, the insect corpse.In the gemma forming process, Su Yun gold bud pole bacterium can produce a large amount of parasporal crystals, is made up of the crystalline protein with high degree of specificity insecticidal activity.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 and the receptors bind about 60kDa.The protein that is incorporated into acceptor is inserted on the cytolemma thereupon and forms perforation, and the ionic equilibrium in cytolemma pericentral siphon and film chamber is destroyed, and causes cellular swelling even breaks, thereby cause the death of insect.The selection insecticidal properties of Bt insecticidal crystal protein is to be determined by the surperficial specific receptors of insect gastrointestinal epithelial cells.Because the albumen that the Bt bacterium is produced has killing ability, 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, does not have injury to natural enemy, and resistance is given birth in difficult labour, has been widely used in preventing and treating the crop pests aspect.Along with the continuous utilization and the development of Bt sterilant, be that foreign gene is cultivated new genetic engineering technique with plant of self insect-resistance and risen in succession with the Bt gene.
Along with continuous development and the commercial kind thereof of trans Bt gene plant 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 more and more countries of China formulates and implemented the pressure sign system of genetically modified foodGMF.Therefore, the scientific management of transgenic product and application need 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 types: one type is the detection technique to its exogenous nucleic acid composition; Another kind of is the immunological analysis method that is directed against its exogenous protein composition.Detection method based on DNA can only reflect on nucleic acid level whether the transgenic sample contains foreign DNA, can not detect its foreign gene and be in silence or expression, and this detection technique does not just have guiding significance to the resistance of changeing Bt.For the product of some transgenic plant, DNA can decompose in the course of processing and be difficult to detect, and at this moment, need utilize enzyme-linked immunosorbent assay to detect genetically modified component.Based on the immunological analysis method of foreign protein, adopt the antigen antibody reaction of high degree of specificity, realize 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.
Being used for genetically modified Bt ICP gene mainly is the gene of CryI family, and that wherein the most frequently used is Cry1Ab, Cry1Ac, Cry1Ah, Cry1C and Cry1A (by half section fusion rotein of forming with the Cry1Ac second half section before the Cry1Ab).Also do not obtain discharging in China at present though have the genetically modified kind of Cry1Ah and Cry1C, might discharge in the future, external also might flow into.Bt antibody in the domestic and foreign literature report; No matter be many anti-or monoclonal antibodies; Basically only discern single Bt CryI gene, can not be identified in various common Bt CryI genes in the transgenic plant simultaneously, can only be used to detect the transgenic event of single CryI gene.Like patent No. application number be antibody in 00510130058.9 the patent only to the polypeptide of one section 16 amino-acid residue among the Cry1A, purpose is for specific recognition Cry1A albumen.We find that through sequence alignment this section peptide sequence also only appears among Cry1Ab and the Cry1Ac, in Cry1Ah and Cry1C, has sequence difference, and the antibody of preparation can not be discerned Cry1Ah and Cry1C in view of the above.Other are the monoclonal antibody that immunogen preparing goes out with complete recombinant C ry1Ab albumen, not to discern multiple toxin protein as design considerations, also its epitope are not confirmed in advance; Therefore its ability of discerning multiple toxin protein can't confirm (Zhang Xiao, it is bright to record, Wang Yun; Wen Shuan, Liu Xianjin, indirect elisa method detect the foundation of bacillus thuringiensis (Bt) Cry1Ab toxalbumin antibody method; Nanjing agricultural journal, 2010,26 (4)).If can prepare proteic wide spectrum monoclonal antibody to the common Bt CryI of transgenic plant, just can utilize a reagent to detect the transgenic event of multiple CryI gene, detect cost thereby practice thrift greatly.
Summary of the invention
First purpose of the present invention provides a kind of ability to proteic wide spectrum monoclonal antibody hybridoma of Bt CryI common in the transgenic plant and preparation method thereof.
Second purpose of the present invention provides the good MONOCLONAL ANTIBODIES SPECIFIC FOR method of a specific specificity, this antibody capable specific combination Bt recombinant antigen Cry1A, Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ah, Cry1C and natural antigen.
The 3rd purpose of the present invention provides a kind of laboratory or field of can being used for and detects the detection reagent whether the farm crop blade has common Bt CryI gene.
The technique means that the technical solution problem is adopted
After the Characterization of antigenic epitopes of the present invention to Bt ICP gene C ry1Ab, Cry1Ac, Cry1Ah, Cry1c and Cry1A commonly used in the transgenic plant, select the existing immunogenicity sequence of 13 synthetic polypeptide of amino-acid residues conduct of homologous again.With 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 obtains mouse monoclonal antibody.Measure its subclass and affinity costant with elisa technique.Detect the proteic ability of CryI in this antibody specific recognition reorganization and the transgenic paddy rice with the immune marking (WB) experiment.
Particularly, the present invention relates to the following aspects:
1. monoclonal antibody is characterized in that by preserving number being that the mouse hybridoma cell of CGMCC 4856 is that 70647s-9 produces.
2. the described monoclonal antibody of 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 the sequence table are obtained.
3. the described monoclonal antibody of claim 1 is characterized in that it is a mouse IgG2a hypotype monoclonal antibody.
4. the described monoclonal antibody of claim 1 is characterized in that the multiple reorganization Su Yun gold of its identification bud pole bacterium CryI albumen.
5. the described monoclonal antibody of claim 1 is characterized in that it is used for immunoblotting, the CryI albumen in the enzyme linked immunosorbent detection transgenic plant (paddy rice, cotton, corn etc.).
6. the described monoclonal antibody of claim 1 is characterized in that can be used as the preparation that detection reagent is used 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 the described monoclonal antibody of stably excreting claim 1.
Advantage of the present invention and beneficial effect
(1) hybridoma (70647s-9 of the present invention's acquisition; Protecting a surname number be CGMCC 4856) monoclonal antibody of secretion generation; Can discern recombinant protein c ry1Ab, Cry1Ac, Cry1Ah, Cry1C and Cry1A; Have the proteic characteristic of the multiple common transgenic Bt CryI of detection, have wider purposes than the antibody of discerning single Bt CryI gene.
(2) monoclonal antibody of hybridoma (70647s-9, preserving number the are CGMCC 4856) generation of the present invention's acquisition and proteic extremely strong specificity and the susceptibility of being combined with of CryI from the protein extract of transgenic paddy rice blade.
(3) hybridoma (70647s-9 of the present invention's acquisition; Preserving number is CGMCC 4856) monoclonal antibody that produces can be applicable to detection and the examination of transgenic plant such as the immune marking (Western b1otting), double-antibody sandwich elisa, indirect ELISA, antibody chip preparation; Specificity and highly sensitive has very high use value.
Description of drawings
Fig. 1: the sequence alignment of Bt CryI gene commonly used
Fig. 2: 70647-S9 is to reorganization Cry1Ab, Cry1Ac and the proteic WB detected result of Cry1A
Fig. 3: 70647-S9 is to the Western Blot detected result of transgenic paddy rice
Embodiment
Mode below in conjunction with chart and practical implementation is done further elaboration to the present invention, 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 have immunogenicity but also have the polypeptide fragment that 12 amino-acid residues (seeing SEQ IDNo:1 in the sequence table) of sequence homology are formed; Synthesize with solid-phase synthesis, and carry out coupling with maleimide method and carrier proteins.Concrete steps are following:
With the mcKLH (Thermo-Fisher company) of the sulfo-GMBS (PIERCE company) of 10mg/ml and 10mg/ml with 1: 5 mixed, place under the room temperature slowly shake up 30min on the shaking table after, the centrifugal 5min of 12000rpm.Get supernatant, through sephadex
TMThe good carrier protein KLH-sulfo-GMBS of G-25Fine (GE company) separated and collected activation is added drop-wise in the polypeptide solution of 10mg/ml to wait mass ratio, at room temperature rotates mixing 3h (or 4 ℃ of rotations are spent the night) and gets final product.
The proteic preparation of embodiment 2 recombinant C ry1A
One, gene clone
Behind gene order (GenBank:ACF32736.1) synthetic gene with coding Cry1A, the method through PCR is held at 5 ' and 3 ' of this antigen-4 fusion protein gene and is added NcoI and BamHI restriction enzyme site respectively.The recovery after the agarose gel electrophoresis separation of PCR product is carried out NcoI to antigen-4 fusion protein gene that reclaims and the plasmid vector pET-BPI that is used to express respectively and the BamHI enzyme is cut, and electrophoresis reclaims once more, connects with the T4DNA ligase enzyme.Connect product transformed into escherichia coli competent cell BL21, DNA is extracted in the clone's inoculation on the picking flat board, carries out PCR and identifies.PCR shows that antigen-4 fusion protein gene male clone carries out sequencing analysis, and the right-on clone of sequence is used for the Cry1A albumen of express recombinant.
Two, protein expression and purifying
The bacterium of single bacterium colony being cultivated in 1: 100 ratio that spends the night is forwarded to 100ml LB substratum, and adding final concentration is the kantlex of 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 behind the receipts bacterium.This recombinant protein has histidine-tagged, uses the Ni post to carry out proteinic affinity purification.After carrying out wash-out with the imidazoles solution of different concns, each component and stream worn go up appearance respectively and carry out the SDS-PAGE separation detection, the proteic purity of recombinant C rylA is more than 90%, and concentration is about 1-1.5mg/mL, can satisfy the requirement of antibody screening and evaluation.
The foundation of embodiment 3 hybridoma cell lines
One, immunity
With link coupled polypeptide among the embodiment 1 with Freund's complete adjuvant (Sigma company) emulsification, immune 4-6 week female Balb/c mouse in age (providing) by Military Medical Science Institute, every mouse of abdominal part hypodermic 6 points, dosage be 60 μ g/ only.Per 14 days booster immunizations once, antigen uses the non-Freund's complete adjuvant of Fu Shi (Sigma company) emulsification, dosage be 30 μ g/ only.The highest mouse of tiring was impacted immune with tail vein injection to resist immunogenic many anti-tiring in indirect ELISA (wavelength 450nm) the detection mice serum in 7 days behind the 3rd booster immunization, and antigen is used the saline water mixing, and dosage is 50 μ g/.
Two, cytogamy
The mouse boosting cell suspension that sterile preparation immunity is up to standard, with murine myeloma cell sp2/0 (ATCC) with 5: 1 mixed, centrifugal 1500rpm, 5min.Centrifuge tube is put into 37 ℃ of water-baths after abandoning supernatant, in 1 minute, slowly adds the PEG1500 (Roche company) of 1ml, and stirs cell.After in warm water, leaving standstill 1min, add the IMDM (Sigma company) of 10ml serum-free, mixing, 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), mixing.Add 25ml again and contain the abundant mixing of semisolid medium of 2.1% Nitrocellulose (Sigma company), pour into uniformly then in 20 Tissue Culture Dishs.Tissue Culture Dish is put in the wet box, put into 37.℃ 5%CO
2Cultivate in the incubator.
Three, choose the clone
Merging back 7 days clone cells, to roll into a ball big or small density moderate, under anatomical lens, draws round, real, big cloning cluster and squeeze in 96 well culture plates that are ready to substratum in advance, puts into 37 ℃ of 5%CO
2Cultivate in the incubator.
Four, ELISA screening positive hybridoma cell
After 3 days, cell concentration accounts for floorage 2/3 greatly, gets 100 μ l supernatants and carries out the ELISA screening respectively with immunogen and synthetic polypeptide.Positive colony changes liquid fully, adds the perfect medium that 200 μ l contain feeder cell and 1%HT (Sigma company).Carry out the ELISA screening second time two days later, positive colony changes 24 orifice plates that are ready to substratum (containing feeder cell and HT) in advance over to and cultivates.Get 100 μ l supernatants after five days and carry out ELISA screening for the third time, positive colony changes 6 orifice plates and Tissue Culture Flask enlarged culturing and frozen one by one over to.
One, ascites preparation
The logarithmic phase cell is with the serum free medium washing and hang counting～5 * 10
5, 1ml.The cell abdominal injection that suspends is used the mouse of Yellow Protopet 2A sensitization in advance.Begin to collect ascites after 7 days.The ascites of taking out is in 4 ℃ of centrifugal 4000rpm, 10min.Careful sucking-off intermediary ascites is collected in the centrifuge tube 4 ℃ or-20 ℃ of preservations.
Two, Purification of Monoclonal Antibodies
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.Antibody purified is stored in-20 ℃.
One, subgroup identification
Encapsulate sheep anti-mouse igg (Bioisystech Co., Ltd of China fir Golden Bridge in Beijing) to 0.5 μ g/ml with 100mM PBS (pH7.4) dilution, every hole adds 100 μ l, 4 ℃, spends the night.The sky liquid that inclines is washed 3 times with the PBS (PBS-T) that contains 0.05%Tween, and every hole adds 200 μ l confining liquids (PBS that contains 2%BSA and 3% sucrose), hatches 1h for 37 ℃.The sky liquid that inclines cleans 3 times with PBS-T.Every hole adds 0.1ml hybridoma supernatant, hatches 1h for 37 ℃.The sky liquid that inclines cleans 3 times with PBS-T.Sheep anti mouse (κ, λ) sheep anti mouse (IgM, the IgGl of antibody or 1: 2000 dilution HRP mark with confining liquid dilution in 1: 1000 HRP mark; IgG2a, IgG2b, IgG3; IgA) antibody (Southern Biotech company) the every hole of 0.1ml adds respectively in the suitable hole, hatches 1h for 37 ℃.The sky liquid that inclines cleans 3 times with PBS-T.Every hole adds 50 μ l and contains 0.15%ABTS (SouthernBiotech company) and 0.03%H
2O
2Citrate buffer solution (PH4.0) carry out coupling reaction, measure the OD value under the 405nm wavelength in the 10-20min.The result shows that monoclonal antibody of the present invention is an IgG2a type mouse resource monoclonal antibody.
Two, affinity costant is measured
Encapsulate synthetic polypeptide, encapsulating concentration is 2 μ g/ml, 100 μ l/ holes, and 4 ℃ encapsulate and spend the night, and PBS-T washes 3 times.Every hole adds 37 ℃ of sealings of 200 μ l confining liquids 2h, and PBS-T washes 3 times.The monoclonal antibody of purifying among the embodiment 4, since 1: 200 2 times of gradient dilution, the contrast that blanks of last 1 hole was 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 are hatched 1h for 37 ℃, and PBS-T washes 3 times.Every hole adds 100 μ l and contains 0.1%TMB (Sigma company) and 0.03%H
2O
2Hydrocerol A-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 with ELIASA.The draw curve of the corresponding antibody dilution multiple of OD value is found out >=the corresponding extension rate A of 1/2 " platform OD value ".Utilizing 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 with the method for immunoblotting.
The immunoblot experiment process is following: the about 100ng of appearance on every kind of albumen, carry out 12% polyacrylamide gel electrophoresis.In Bio-Rad electrotransfer system, the gel protein band is transferred to (Millipore company) on the pvdf membrane by ordinary method.Place the TBS-T confining liquid that contains 5% skim-milk to spend the night for 4 ℃ film.Add 4 ℃ of incubated overnight of monoclonal antibody 70647-S9 (dilution in 1: 1000).After washing film with TBS-T, add the sheep anti mouse two anti-(Bioisystech Co., Ltd of China fir Golden Bridge in Beijing) of dilution in 1: 5000, incubated at room 1 hour.TBST washes film once more, adds the ultra quick colour developing liquid of ECL (Puli's Lay company), and (GE company) catches the colour developing image with ImageQuant ECL instrument.
That Fig. 2 shows is the immune marking result of monoclonal antibody 70647-S9 to recombinant protein.This antibody can be discerned Cry1Ab, Cry1Ac, Cry1A albumen, and other irrelevant recombinant proteins of nonrecognition.
The effect of embodiment 6 monoclonal antibodies of the present invention
From transgenic and non-transgenic rice leaf, extract albumen, detect the effect of monoclonal antibody of the present invention in detecting transgenic plant with the method for the immunoblotting described in the embodiment 5.
That Fig. 3 shows 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 specificity that detects and sensitivity are all very high.
Claims (7)
1. monoclonal antibody is characterized in that by preserving number being that the mouse hybridoma cell of CGMCC 4856 is that 70647s-9 produces.
2. the described monoclonal antibody of 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 the sequence table are obtained.
3. the described monoclonal antibody of claim 1 is characterized in that it is a mouse IgG2a hypotype monoclonal antibody.
4. the described monoclonal antibody of claim 1 is characterized in that the multiple reorganization Su Yun gold of its identification bud pole bacterium CryI albumen.
5. the described monoclonal antibody of claim 1 is characterized in that it is used for immunoblotting, the CryI albumen in the enzyme linked immunosorbent detection transgenic plant (paddy rice, cotton, corn etc.).
6. the described monoclonal antibody of claim 1 is characterized in that can be used as the preparation that detection reagent is used 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 the described monoclonal antibody of stably excreting claim 1.
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| CN105198990A (en) * | 2015-10-12 | 2015-12-30 | 江苏省农业科学院 | Antibody for broad-spectrum detection of Bt Cry1 toxoids as well as preparation method and application thereof |
| 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 |
| CN105884892A (en) * | 2016-06-28 | 2016-08-24 | 江苏省农业科学院 | Protein used for broad spectrum detection of Bt (bacillus thuringiensis) Cry toxins as well as coding gene and application thereof |
| CN108314711A (en) * | 2018-02-28 | 2018-07-24 | 吉林省农业科学院 | A kind of cry1C recombinant proteins with immunogenicity, the nucleic acid molecules of separation and its application |
| CN109781992A (en) * | 2018-12-27 | 2019-05-21 | 中国农业科学院生物技术研究所 | A kind of colloidal gold immunochromatographimethod quick measuring card of insect resistance protein Cry1C |
| CN110294803A (en) * | 2019-05-27 | 2019-10-01 | 中国农业科学院植物保护研究所 | The monoclonal antibody and its application of Cry1Ah1 albumen |
| CN116047047A (en) * | 2022-10-27 | 2023-05-02 | 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 |
| CN117143832A (en) * | 2023-11-01 | 2023-12-01 | 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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| CN105198990A (en) * | 2015-10-12 | 2015-12-30 | 江苏省农业科学院 | Antibody for broad-spectrum detection of Bt Cry1 toxoids as well as preparation method and application thereof |
| 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 |
| CN105884892A (en) * | 2016-06-28 | 2016-08-24 | 江苏省农业科学院 | Protein used for broad spectrum detection of Bt (bacillus thuringiensis) Cry toxins as well as coding gene and application thereof |
| CN108314711A (en) * | 2018-02-28 | 2018-07-24 | 吉林省农业科学院 | A kind of cry1C recombinant proteins with immunogenicity, the nucleic acid molecules of separation and its application |
| CN109781992A (en) * | 2018-12-27 | 2019-05-21 | 中国农业科学院生物技术研究所 | A kind of colloidal gold immunochromatographimethod quick measuring card of insect resistance protein Cry1C |
| 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 |
| CN110294803A (en) * | 2019-05-27 | 2019-10-01 | 中国农业科学院植物保护研究所 | The monoclonal antibody and its application of Cry1Ah1 albumen |
| CN116047047A (en) * | 2022-10-27 | 2023-05-02 | 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 |
| 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 |
| CN117143832A (en) * | 2023-11-01 | 2023-12-01 | 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 |
| 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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