CN113493511A - Preparation method of Cry1Fa rabbit polyclonal antibody - Google Patents

Preparation method of Cry1Fa rabbit polyclonal antibody Download PDF

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Publication number
CN113493511A
CN113493511A CN202110845412.5A CN202110845412A CN113493511A CN 113493511 A CN113493511 A CN 113493511A CN 202110845412 A CN202110845412 A CN 202110845412A CN 113493511 A CN113493511 A CN 113493511A
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cry1fa
polyclonal antibody
preparation
protein
antibody
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金芜军
刘卫晓
孟丽霞
苗朝华
宛煜嵩
黄卫红
董美
王迪
高进
文婷婷
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Sanya National Academy Of Southern Propagation Chinese Academy Of Agricultural Sciences
Biotechnology Research Institute of CAAS
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Sanya National Academy Of Southern Propagation Chinese Academy Of Agricultural Sciences
Biotechnology Research Institute of CAAS
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
    • C07K16/065—Purification, fragmentation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/32—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Bacillus (G)
    • G01N2333/325—Bacillus thuringiensis crystal protein (delta-endotoxin)

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Abstract

The invention discloses a preparation method of a Cry1Fa rabbit polyclonal antibody, belonging to the technical field of biological engineering, and the preparation method of the polyclonal antibody comprises the following steps: a) obtaining a Cry1Fa recombinant protein fragment through prokaryotic expression; b) the Cry1Fa recombinant protein fragment is used as an antigen to immunize a white rabbit with a Japanese big ear; c) detecting the titer; d) polyclonal antibody purification and western validation. The polyclonal antibody lays a foundation for realizing qualitative and quantitative detection of the insect-resistant protein Cry1Fa in transgenic crops.

Description

Preparation method of Cry1Fa rabbit polyclonal antibody
Technical Field
The invention relates to the field of bioengineering, and particularly relates to a preparation method of a Cry1Fa rabbit polyclonal antibody.
Background
Bacillus thuringiensis (Bt) is a crystalline protein secreted by gram-positive bacteria widely present in soil and having high insect-resistant activity. The insect-resistant protein Cry1Fa has wide toxicity to lepidopteran (Lepidotera) insects, and when the insects eat Cry1Fa protein, the protein is activated by midgut protease, and the activated protein penetrates peritrophic membrane, is combined with epithelial cell apical membrane specific protein of midgut cells and forms intestinal perforation, so that the osmotic balance of the cells is destroyed, the cells are caused to swell and crack, and finally the death is caused. Therefore, the Cry1Fa gene becomes the insect-resistant gene with the most potential and application prospect in plant genetic engineering and transgenic breeding application. Wherein Cry1Fa gene has been successfully used for transgenic crops.
While the rapid development of transgenic crops brings great economic benefits, people are also paying more attention to the problems of unpredictable edible safety and environmental safety possibly brought by the transgenic crops, and more countries require the detection of transgenic products to implement an identification system.
The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.
Disclosure of Invention
The invention aims to provide a preparation method of a Cry1Fa rabbit polyclonal antibody, and the polyclonal antibody obtained by the preparation method lays a foundation for realizing qualitative and quantitative detection of an insect-resistant protein Cry1Fa in transgenic crops.
In order to achieve the aim, the invention provides a preparation method of a Cry1Fa polyclonal antibody, which comprises the following steps:
a) antigen preparation: obtaining a Cry1Fa recombinant protein fragment through prokaryotic expression;
b) immunizing animals: the Cry1Fa recombinant protein fragment is used as an antigen to immunize a white rabbit with a Japanese big ear;
c) and (3) potency detection: carrying out gradient dilution on the antibody and detecting the titer and sensitivity of the antibody by adopting an indirect ELISA method;
d) and (3) purifying the polyclonal antibody: and (3) obtaining the polyclonal antibody by adopting Protein G affinity chromatography.
The titer of the polyclonal antibody detected by indirect ELISA was 4.9 ng/mL.
The invention also provides application of the polyclonal antibody in detection of Cry1Fa insect-resistant protein.
The invention also provides application of the polyclonal antibody in preparation of a reagent for detecting Cry1Fa insect-resistant protein by an ELISA method.
The invention has the beneficial effects that: the invention uses high-purity insect-resistant protein Cry1Fa obtained by expression and purification of engineering strains as an antigen, and obtains an antibody indirect ELISA titer of 4.9ng/mL after antiserum is purified by immunizing white rabbits with big ears of Japan, the polyclonal antibody can specifically recognize recombinant Cry1Fa protein expressed by pronucleus and endogenous Cry1Fa protein in transgenic cotton, and the construction of the rabbit polyclonal antibody of Cry1Fa insect-resistant protein provides material and technical support for detection of the protein in transgenic crops.
Drawings
FIG. 1 is a diagram of SDS-PAGE electrophoresis result of Cry1Fa recombinant protein fragment.
FIG. 2 is a diagram showing the result of SDS-PAGE electrophoresis after the purification of the polyclonal antibody.
FIG. 3 is the polyclonal antibody titer.
FIG. 4 is a graph showing the Western results of the polyclonal antibody specific detection of Cry1Fa in transgenic cotton.
Detailed Description
The following detailed description of the present invention is provided in conjunction with the accompanying drawings, but it should be understood that the scope of the present invention is not limited to the specific embodiments.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1
1. Preparation of immune antigens
(1) Recombinant protein Cry1Fa fragment expression strain construction, thallus culture and lysis
Cry1Fa recombinant protein fragment amino acid sequence:
ELTRDIFTDPIFLLTTLQKYGPTFLSIENSIRKPHLFDYLQGIEFHTRLRPGYFGKDSFNYWSGNYVETRPSIGSSKTITSPFYGDKSTEPVQKLSFDGQKVYRTIANTDVAAWPNGKVYLGVTKVDFSQYDDQKNETSTQTYDSKRNNGHVSAQDSIDQLPPETTDEPLEKAYSHQLNYAECFLMQDRRGTIPFFTWTHRSVDFFNTIDAEKITQLPVVKAYALSSGASIIEGPGFTGGNLLFLKESSNSIAKFKVTLNSAALLQRYRVRIRYASTTNLRLFVQNSNNDFLVIYINKTMNKDDDLTYQTFDLATTNSNMGFSGDKNELIIGAESFVSNEKIYIDKIEFIPVQL。
performing PCR amplification by using high-fidelity Fastpfu, identifying the amplified PCR product by (1.0%) agarose gel electrophoresis, cutting and recovering a target band by gel, recovering the product by using a Universal DNA purification recovery kit (Tiangen), performing enzyme digestion on the recovered product by using restriction enzymes Bam HI and Xho I, recovering the product by gel, connecting the recovered product with a pET28a plasmid fragment subjected to the same enzyme digestion and gel recovery treatment at 4 ℃ overnight, transforming a Trans10 competent cell (Beijing holotype gold), selecting clones, performing colony PCR identification, and sequencing positive clones by Beijing Liuhe Huada GenBank.
Coli BL21(DE3) competent cells transformed with the correctly identified recombinant expression vector pET28a-Cry1Fa were plated on LB plates containing kanamycin (50. mu.g/mL) and cultured overnight at 37 ℃. The next day, a single clone was picked from the plate and inoculated into liquid LB medium containing the same concentration of kanamycin, and cultured overnight at 37 ℃. The obtained expression strain bacterial liquid and a 50% glycerol solution are mixed uniformly in equal volume and are frozen and stored at the temperature of minus 80 ℃.
The stored recombinant protein Cry1Fa expression strain is subjected to recovery culture, and a bacterial solution is spread on an LB plate containing kanamycin (50 mu g/mL) and cultured overnight at 37 ℃. Single colonies were picked and inoculated in liquid LB (kanamycin 50. mu.g/mL) medium overnight at 37 ℃. The next day, the cells were inoculated at 1% inoculum size and cultured at 37 ℃ to OD600About 0.8, 1mM IPTG was added to induce protein expression overnight at 16 ℃. After induction, the bacterial liquid is subjected to ice bath, the thalli is centrifugally collected at 4 ℃, 4000rpm for 10min, the supernatant is discarded, and the thalli is washed by PBS and suspended by lysis solution; ultrasonication (2s on/4s off, Amp: 45%, Time: 3 min); centrifuging at 15000rpm for 1hr at 4 deg.C, and collecting supernatant.
(2) Recombinant protein purification
Mixing the supernatant of the thallus lysate with Ni Sepharose 6FF Beads (GE Healthcare) balanced by the lysate, combining for 2-3hrs at 4 ℃, centrifuging at 3000rpm for 3min, and discarding the supernatant; washing the protein-bound Ni Sepharose 6FF beads with washing solution for 2-3 times; then eluting with eluent. Collecting the eluent to obtain the crude pure protein solution.
Dialyzing the protein sample obtained by affinity purification to a protein storage solution, further purifying by Superdex 200 Increate 10/30GL (GE healthcare) balanced by the same solution, collecting protein peak components, detecting the purity of the protein sample by SDS-PAGE, obtaining the protein by the graph 1, and finally obtaining the electrophoretically pure Cry1Fa protein through gel filtration, wherein the molecular weight is about 40 KDa.
2. Immunizing animals
Immunizing 2 SPF-level Japanese big ear white rabbits (purchased from research center of experimental animals in Hubei province, with the quality control qualified Cry1Fa recombinant protein fragment as antigen, wherein the license number is SCXK (Eo) 2015-0018), mixing the antigen with equal volume of complete Freund's adjuvant (prime) and incomplete Freund's adjuvant (boost), emulsifying, fully mixing until the water-in-oil state is subjected to subcutaneous multipoint immunization, carrying out 2-3 times of boost immunization, carrying out 2 weeks at each immunization interval, carrying out titer detection, carrying out abdominal impact within 1 week after more than 1:10000, directly dissolving the antigen of immunization dose into 250 mu L PBS, and the specific immunization times and immunization dose are shown in Table 1:
TABLE 1 immunization times and immunization doses
Figure BDA0003180646960000051
Figure BDA0003180646960000061
3 polyclonal antibody purification
The collected antiserum is purified by a Protein G-agarose affinity chromatography column after being pretreated, and the specific steps are as follows:
1) the equilibrium column was washed with 10 column volumes of PBS (or TBS, the same applies below).
2) After high speed centrifugation of serum or other body fluid containing antibody, the supernatant is mixed with equal volume of 2 XPBS buffer solution, and after pH and ion concentration are adjusted, the mixture is slowly added into a chromatographic column.
3) Washing with PBS of more than 10 column volumes until the effluent is free of protein.
4) Add 2 column volumes of 0.1M citric Acid (Citrate Acid, pH 2.7), clamp the effluent tube, stand for 5 minutes, collect the transudate, repeat three times. OD280 was determined to estimate the antibody concentration, and if more antibody was obtained, the purity could be checked by SDS-PAGE. 0.1M Glycine (Glycine, pH 3.0) was also selected for elution.
5) The eluted antibody was added to 2/5 volumes of 1M Tris, pH 8.0 for neutralization, and switched to the desired buffer using Millipore protein concentration tubes, typically 2 XPBS containing 0.02% NaN3 and 1mM EDTA.
6) Concentrating to desired volume, and detecting purity by SDS-PAGE (see FIG. 2), and storing at-20 deg.C to avoid freezing.
4. Potency assay for polyclonal antibodies
The titer of the purified polyclonal antibody is detected by an indirect ELISA method by taking the recombinant protein Cry1Fa as an antigen, and the anti-titer of the purified polyclonal antibody is 4.9ng/mL by ELISA determination according to a graph shown in FIG. 3.
5. Polyclonal antibody specificity detection
Respectively extracting transgenic cotton and protein of the transgenic cotton which is used for transforming parent leaves, running SDS-PAGE gel, using a purified polyclonal antibody as a primary antibody for membrane transformation, using Alexa FlurorTM 680goat anti-rabbitIgG (H + L) (Invitrogen) as a secondary antibody, using an Odyssey infrared740 image (9120, Li-COR Biosciences, Lincolin, NE) infrared scanner western detection result, and knowing through figure 4, the purified polyclonal antibody can specifically identify Cry1Fa and recombinant protein Cry1Fa in an endogenous sample;
the method for extracting the transgenic cotton leaf protein comprises the following steps:
quickly freezing tissue with liquid nitrogen, grinding, adding 1mL (generally 0.5g and 1-2mL) of protein extract, mixing at 4 deg.C for 30 min, (centrifuging at 12,000rpm at 4 deg.C for 15min, collecting supernatant), and making into protein extract with formula shown in Table 2:
TABLE 2 protein extract formula
Composition (I) Dosage of
1M Tris,pH 7.5 500uL
1M NaCL 1.5mL
0.5M EDTA 20uL
50％glycerol 2mL
10％SDS 1mL
Double distilled water (DDH)2O) Is prepared into 10mL
Protease inhibitors (Roche) Adding a tablet when using
1mM PMSF (phenylmethylsulfonyl fluoride, Sigma) Adding 50uL of the mixture during use
The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the invention and various alternatives and modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.
Sequence listing
<110> institute of biotechnology of Chinese academy of agricultural sciences
China southern research institute of China academy of agricultural sciences
<120> preparation method of Cry1Fa rabbit polyclonal antibody
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 354
<212> PRT
<213> amino acid (Artificial Sequence)
<400> 1
Glu Leu Thr Arg Asp Ile Phe Thr Asp Pro Ile Phe Leu Leu Thr Thr
1 5 10 15
Leu Gln Lys Tyr Gly Pro Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg
20 25 30
Lys Pro His Leu Phe Asp Tyr Leu Gln Gly Ile Glu Phe His Thr Arg
35 40 45
Leu Arg Pro Gly Tyr Phe Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly
50 55 60
Asn Tyr Val Glu Thr Arg Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr
65 70 75 80
Ser Pro Phe Tyr Gly Asp Lys Ser Thr Glu Pro Val Gln Lys Leu Ser
85 90 95
Phe Asp Gly Gln Lys Val Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala
100 105 110
Ala Trp Pro Asn Gly Lys Val Tyr Leu Gly Val Thr Lys Val Asp Phe
115 120 125
Ser Gln Tyr Asp Asp Gln Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp
130 135 140
Ser Lys Arg Asn Asn Gly His Val Ser Ala Gln Asp Ser Ile Asp Gln
145 150 155 160
Leu Pro Pro Glu Thr Thr Asp Glu Pro Leu Glu Lys Ala Tyr Ser His
165 170 175
Gln Leu Asn Tyr Ala Glu Cys Phe Leu Met Gln Asp Arg Arg Gly Thr
180 185 190
Ile Pro Phe Phe Thr Trp Thr His Arg Ser Val Asp Phe Phe Asn Thr
195 200 205
Ile Asp Ala Glu Lys Ile Thr Gln Leu Pro Val Val Lys Ala Tyr Ala
210 215 220
Leu Ser Ser Gly Ala Ser Ile Ile Glu Gly Pro Gly Phe Thr Gly Gly
225 230 235 240
Asn Leu Leu Phe Leu Lys Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys
245 250 255
Val Thr Leu Asn Ser Ala Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile
260 265 270
Arg Tyr Ala Ser Thr Thr Asn Leu Arg Leu Phe Val Gln Asn Ser Asn
275 280 285
Asn Asp Phe Leu Val Ile Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp
290 295 300
Asp Leu Thr Tyr Gln Thr Phe Asp Leu Ala Thr Thr Asn Ser Asn Met
305 310 315 320
Gly Phe Ser Gly Asp Lys Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe
325 330 335
Val Ser Asn Glu Lys Ile Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val
340 345 350
Gln Leu

Claims (6)

1. A preparation method of Cry1Fa rabbit polyclonal antibody is characterized by comprising the following steps:
a) antigen preparation: obtaining a Cry1Fa recombinant protein fragment through prokaryotic expression;
b) immunizing animals: the Cry1Fa recombinant protein fragment is used as an antigen to immunize a white rabbit;
c) and (3) potency detection: carrying out gradient dilution on the antibody and detecting the titer and sensitivity of the antibody by adopting an indirect ELISA method;
d) and (3) purifying the polyclonal antibody: and (3) obtaining the polyclonal antibody by adopting Protein G affinity chromatography.
2. The preparation method according to claim 1, wherein the rabbit immunized with the Cry1Fa recombinant protein fragment is a Japanese big ear rabbit.
3. Polyclonal antibody according to claim 2, characterized in that the produced polyclonal antibody has a titer of 4.9ng/mL as determined by indirect ELISA.
4. Use of a polyclonal antibody according to claim 1 for detecting Cry1Fa anti-insect protein.
5. Use of the polyclonal antibody of claim 1 in the preparation of a reagent for detecting Cry1Fa anti-insect protein by ELISA.
6. Use of the polyclonal antibody of claim 1 in the preparation of a reagent for detecting Cry1Fa insect-resistant protein by an ELISA double-antibody sandwich method.
CN202110845412.5A 2021-07-26 2021-07-26 Preparation method of Cry1Fa rabbit polyclonal antibody Pending CN113493511A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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

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