CN108892724A - A kind of preparation method of Vip3Aa20 rabbit polyclonal antibody - Google Patents
A kind of preparation method of Vip3Aa20 rabbit polyclonal antibody Download PDFInfo
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- CN108892724A CN108892724A CN201810746540.2A CN201810746540A CN108892724A CN 108892724 A CN108892724 A CN 108892724A CN 201810746540 A CN201810746540 A CN 201810746540A CN 108892724 A CN108892724 A CN 108892724A
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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)
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- 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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
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- 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 kind of preparation method for the Vip3Aa20 rabbit polyclonal antibody for belonging to technical field of bioengineering, the preparation method of the polyclonal antibody includes the following steps：A) Vip3Aa20 recombinant protein is obtained by prokaryotic expression；B) animal is immunized：Japan large ear rabbit is immunized using Vip3Aa20 recombinant protein as antigen；C) bioactivity；D) polyclonal antibody purification and western verifying.The polyclonal antibody is to realize that qualitative and quantitative analysis of the insect resistance protein Vip3Aa20 in transgenic corns MIR162 lays the foundation.
Description
Technical field
The present invention relates to bioengineering fields, and in particular to a kind of preparation method of Vip3Aa20 rabbit polyclonal antibody.
Background technique
Dipel (Bacillus thuringiensis, Bt) is a kind of gram sun being widely present in soil
Property bacterium, there is high pest-resistant active soluble protein (Vegetative Insecticidal in vegetative growth phase secretion
Proteins,Vip proteins).The table of Vip3 albumen can be detected to spore phase, supernatant of bacteria solution is produced in 15 hours of inoculation
It reaches.Insect resistance protein Vip3 has extensive toxicity to lepidoptera (Lepidoptera) insect, after insect's food-taking Vip3 albumen, the egg
White through Midgut protein enzyme activation, the albumen of activation penetrates funnel, in conjunction with the epithelial cell teleblem differential protein of midgut epithelial cells
And enterobrosis is formed, to destroy the osmotic equilibrium of cell, cellular swelling is caused to crack, eventually leads to death.Therefore Vip3 base
Because having become most potential and application prospect anti insect gene in plant genetic engineering and transgenic breeding application.Wherein
Vip3Aa gene is successfully used for transgene cotton and corn.
While bringing tremendous economic interests, people also make by increasingly concern transgenosis for the rapid development of genetically modified crops
The possible not expected edible safety of object and environmental safety problem, more and more national requirements are to transgenic product
It is detected to carry out mark system.
The information disclosed in the background technology section is intended only to increase the understanding to general background of the invention, without answering
When being considered as recognizing or imply that the information constitutes the prior art already known to those of ordinary skill in the art in any form.
Summary of the invention
The purpose of the present invention is to provide a kind of preparation method of Vip3Aa20 rabbit polyclonal antibody, obtain its more and resist be
Realize that qualitative and quantitative analysis of the insect resistance protein Vip3Aa20 in transgenic corns MIR162 lays the foundation.
To achieve the above object, the present invention provides a kind of preparation method of Vip3Aa20 polyclonal antibody, including it is following
Step：
A) Vip3Aa20 recombinant protein is obtained by prokaryotic expression；
B) animal is immunized：Japan large ear rabbit is immunized using Vip3Aa20 recombinant protein as antigen；
C) bioactivity：Using indirect elisa method, gradient dilution is carried out to antibody and detects the potency of antibody and sensitive
Degree；
D) polyclonal antibody purification：Using Protein G affinity chromatography, polyclonal antibody is obtained.
Above-mentioned polyclonal antibody detects obtained potency by indirect ELISA and is followed successively by 1:512000.
The present invention also provides application of the above-mentioned polyclonal antibody in detection Vip3Aa20 insect resistance protein.
The present invention also provides above-mentioned polyclonal antibodies in the reagent of preparation ELISA method detection Vip3Aa20 insect resistance protein
Application.
The present invention also provides above-mentioned polyclonal antibodies to detect the pest-resistant egg of Vip3Aa20 in preparation ELISA double antibody sandwich method
Application in white reagent.
Beneficial effects of the present invention：The high-purity insect resistance protein that utilizing works bacterial strain of the present invention is expressed, purifying obtains
Vip3Aa20 obtains the antibody indirect ELISA potency that antiserum obtains after purification as antigen, by the way that Japan large ear rabbit is immunized
It is 1:512000, the polyclonal antibody can be with the recombination Vip3Aa20 albumen and transgenic corns of specific recognition prokaryotic expression
Endogenous Vip3Aa20 albumen in MIR162 standard items, the rabbit polyclonal antibody of Vip3Aa20 insect resistance protein are configured to transgenosis
The detection of the albumen provides substance and technical support in crop.
Detailed description of the invention
Fig. 1 is Vip3Aa20 recombinant protein SDS-PAGE electrophoresis result figure.
Fig. 2 is Vip3Aa20 recombinant protein polyclonal antibody western result figure, secondary antibody:IgG(H+L)-HRP(1:
5000)。
Fig. 3 is the SDS-PAGE electrophoresis result figure after polyclonal antibody purification.
Fig. 4 is polyclonal antibody titre.
Fig. 5 is in polyclonal antibody specific detection Vip3Aa20 recombinant protein and transgenic corns MIR162
Vip3Aa20 Western result figure.
Specific embodiment
With reference to the accompanying drawing, specific embodiments of the present invention will be described in detail, it is to be understood that guarantor of the invention
Shield range is not limited by the specific implementation.
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples, it is unless otherwise specified, commercially commercially available.
Embodiment 1
1. the preparation of immunizing antigen
(1) recombinant protein Vip3Aa20 expresses strain construction, thallus culture and cracking
The amplification of Vip3Aa20 encoding gene：Using transgenic corns MIR162 genomic DNA as template, vip3a20-NdeI-
F:Catatggctagcatgactggtggacagc and vip3a20-XhoI-R:Ctcgagctacttgatgctcacgtc is to draw
Object carries out PCR amplification with high-fidelity Fastpfu, and the PCR product expanded, will after the identification of (1.0%) agarose gel electrophoresis
Purpose band gel extraction is recycled with Universal DNA purification and recovery kit (Tiangeng), and recovery product is through restriction enzyme
Enzyme Bam HI and XhoI digestion are recycled through glue and are stayed overnight with the pET28a plasmid fragments for carrying out same digestion, glue recovery processing in 4 DEG C
Connection, conversion Trans10 competent cell (the full formula gold in Beijing), picked clones carry out bacterium colony PCR identification, and positive colony is by north
Capital six directions Huada gene company is sequenced.
It will identify that correct recombinant expression carrier pET28a-vip3Aa20 conversion e. coli bl21 (DE3) competence is thin
Born of the same parents, bacterium solution are coated on the LB plate containing kanamycins (50 μ g/mL), and 37 DEG C are incubated overnight.Next day picking Dan Ke on plate
In the LB liquid medium of grand inoculation kanamycins containing same concentrations, it is incubated overnight in 37 DEG C.Resulting expression bacterial strain bacterium solution with
50% glycerite mixes in equal volume, in -80 DEG C of cold storage.
By the recombinant protein Vip3Aa20 expression bacterial strain recovery culture of preservation, bacterium solution is coated on containing kanamycins (50 μ g/
ML on LB plate), 37 DEG C are incubated overnight.Picking monoclonal is inoculated with 37 in liquid LB (the 50 μ g/mL containing kanamycins) culture medium
It DEG C is incubated overnight.It with the inoculation of 1% inoculum concentration, cultivates in 37 DEG C to OD within second day600It is 0.8 or so, 1mM IPTG is added in 16 DEG C
Induction expression protein overnight.Induction terminates bacterium solution ice bath, and in 4 DEG C, thalline were collected by centrifugation by 4000rpm, 10min, abandons supernatant, bacterium
Body is suspended after PBS is washed with lysate (50mM Tris pH7.5,300mM NaCl, 5%Glycerol and 20mM imidazoles)；
Ultrasonication (2s on/4s off, Amp：45%, Time：3min)；4 DEG C of 15000rpm are centrifuged 1hr, collect supernatant.
(2) recombinant protein purification
Cellular lysate liquid supernatant and the Ni Sepharose 6FF Beads (GE Healthcare) through lysate balance are mixed
It closes, 2-3hrs, 3000rpm is combined to be centrifuged 3min and abandon supernatant in 4 DEG C；Protein-bonded Ni Sepharose 6FF beads warp
Cleaning solution (50mM Tris pH7.5,300mM NaCL, 5%Glycerol and 50mM imidazoles) is washed 2-3 times；Eluent is used again
(50mM Tris pH7.5,300mM NaCL, 5%Glycerol and 200mM imidazoles) elution.Collecting eluent is that albumen is thick
Pure solution.
The protein sample that affinity purification is obtained is dialysed to protein storage solution (50mM Tris pH7.5,300mM
NaCL, 5%Glycerol), and with the 200 Increase 10/30GL (GE of Superdex balanced through same solution
Healthcare it) is further purified, collects protein peak component and the purity through SDS-PAGE detection protein sample, it can by Fig. 1
, electrophoretically pure Vip3Aa20 albumen is finally obtained by gel filtration, molecular weight is about 90KD.
2. immune animal
Use the Vip3Aa20 recombinant protein of above-mentioned Quality Control qualification that the big ear of SPF grade Japan at 3 3-5 monthly ages is immunized as antigen
White rabbit (is purchased in Hubei Province Animal Experimental Study center, credit number：SCXK (Hubei Province) 2015-0018), antigen with it is isometric complete
Full Freund's adjuvant (head exempts from) and incomplete Freund's adjuvant (booster immunization) are mixed and are emulsified, and are fully mixed to Water-In-Oil state
It is immune to carry out subcutaneous multiple spot, 2-3 booster immunization in 2 weeks each immunization interval periods, carries out bioactivity later, be higher than>1:
Abdominal cavity impact is carried out after 10000 in 1 week, is directly dissolved in the antigen of immunizing dose in the PBS of 250 μ L, specific immune time and
Immunizing dose is as shown in table 1：
1 immune time of table and immunizing dose
Vip3Aa20 recombinant protein polyclonal antibody western result figure, secondary antibody:IgG(H+L)-HRP(1:5000) as schemed
Shown in 2.
3 polyclonal antibody purifications
Antiserum after collection selects Protein G- agarose affinity chromatography column purification, specific steps after pretreatment
It is as follows：
1) balance chromatographic column is washed with the PBS of 10 times of column volumes (or TBS, similarly hereinafter).
2) after the serum containing antibody or other body fluid high speed centrifugations, supernatant is mixed with isometric 2 × PBS buffer solution, is adjusted
After whole pH and ion concentration, it is slowly added to chromatographic column.
3) it is washed with PBS more than 10 times of column volumes, until efflux is detected without albumen.
4) 2 times of column volume 0.1M citric acids (Citrate Acid, pH 2.7) are added, clamp effuser, stand after five minutes
Collection is pierced by liquid, in triplicate.It measures OD280 and estimates antibody concentration, if gained antibody is more to can be used SDS-PAGE inspection
Survey purity.Also it can choose 0.1M glycine (Glycine, pH 3.0) elution.
5) the 1M Tris, pH 8.0 that 2/5 volume is added in the antibody after eluting is neutralized, and uses Millipore protein concentration pipe
It is switched to required buffer liquid, usually 2 × PBS contains 0.02%NaN3 and 1mMEDTA.
6) it is concentrated to required volume, detects purity (see Fig. 3) by SDS-PAGE, -20 DEG C of preservations avoid freezing.
4. the titration of polyclonal antibody
By purified polyclonal antibody, using recombinant protein Vip3Aa20 as antigen, its effect is detected with indirect ELISA method
Valence is measured through ELISA as shown in Figure 4, and the anti-titre of the polyclonal antibody purified is 1:512000.
5. polyclonal antibody specific detection
Transgenic corns MIR162 standard items are extracted respectively and its to convert the albumen of parental seed powder, run SDS-PAGE
Glue, transferring film use purified polyclonal antibodies as primary antibody, 680 goat anti-rabbit IgG (H+L) of Alexa FlurorTM
(Invitrogen) be secondary antibody, with Odyssey infrared740 imager (9120, Li-COR Biosciences,
Lincolin, NE) infrared scanner western testing result, as shown in Figure 5, the Anti-TNF-α physical efficiency purified is special
Identify the Vip3Aa20 and recombinant protein Vip3Aa20 in endogenous sample MIR162；
Wherein, transgenic corns MIR162 standard items (AOCS 1208-A, USA) protein extracting method:
Liquid nitrogen flash freezer is organized, is ground, 1mL (by sample size, general 0.5g adds 1-2mL) protein extract is added, 4 DEG C mix 30
Minute, (12,000rpm 4 DEG C of centrifugation 15min, take supernatant), protein extraction formula of liquid is shown in Table 2：
2 protein extraction formula of liquid of table
The aforementioned description to specific exemplary embodiment of the invention is in order to illustrate and illustration purpose.These descriptions
It is not wishing to limit the invention to disclosed precise forms, and it will be apparent that according to the above instruction, can much be changed
And variation.The purpose of selecting and describing the exemplary embodiment is that explaining specific principle of the invention and its actually answering
With so that those skilled in the art can be realized and utilize a variety of different exemplary implementation schemes of the invention and
Various chooses and changes.The scope of the present invention is intended to be limited by claims and its equivalents.
Claims (6)
1. a kind of preparation method of Vip3Aa20 rabbit polyclonal antibody, which is characterized in that include the following steps：
A) Vip3Aa20 recombinant protein is obtained by prokaryotic expression；
B) animal is immunized：White rabbit is immunized using Vip3Aa20 recombinant protein as antigen；
C) bioactivity：Using indirect elisa method, gradient dilution is carried out to antibody and detects the potency and sensitivity of antibody；
D) polyclonal antibody purification：Using Protein G affinity chromatography, polyclonal antibody is obtained.
2. preparation method according to claim 1, which is characterized in that the immune white rabbit of the Vip3Aa20 recombinant protein
For Japan large ear rabbit.
3. polyclonal antibody according to claim 2, it is characterised in that generated polyclonal antibody passes through indirect ELISA
It detects obtained potency and is followed successively by 1:512000.
4. application of the polyclonal antibody described in claim 1 in detection Vip3Aa20 insect resistance protein.
5. polyclonal antibody described in claim 1 answering in the reagent of preparation ELISA method detection Vip3Aa20 insect resistance protein
With.
6. polyclonal antibody described in claim 1 is detecting Vip3Aa20 insect resistance protein in preparation ELISA double antibody sandwich method
Reagent in application.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113493512A (en) * | 2021-07-26 | 2021-10-12 | 中国农业科学院生物技术研究所 | Preparation method of Cry3Bb rabbit polyclonal antibody |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101548011A (en) * | 2006-06-03 | 2009-09-30 | 先正达参股股份有限公司 | Corn event MIR162 |
| WO2011075588A1 (en) * | 2009-12-16 | 2011-06-23 | Dow Agrosciences Llc | COMBINED USE OF CRY1Ca AND CRY1Fa PROTEINS FOR INSECT RESISTANCE MANAGEMENT |
| CN108004215A (en) * | 2017-12-13 | 2018-05-08 | 中国农业科学院生物技术研究所 | Hybridoma cell strain and its antibody and preparation method of generation |
-
2018
- 2018-07-09 CN CN201810746540.2A patent/CN108892724A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101548011A (en) * | 2006-06-03 | 2009-09-30 | 先正达参股股份有限公司 | Corn event MIR162 |
| WO2011075588A1 (en) * | 2009-12-16 | 2011-06-23 | Dow Agrosciences Llc | COMBINED USE OF CRY1Ca AND CRY1Fa PROTEINS FOR INSECT RESISTANCE MANAGEMENT |
| CN102821596A (en) * | 2009-12-16 | 2012-12-12 | 陶氏益农公司 | Combined use of CRY1Ca and CRY1Fa proteins for insect resistance management |
| CN108004215A (en) * | 2017-12-13 | 2018-05-08 | 中国农业科学院生物技术研究所 | Hybridoma cell strain and its antibody and preparation method of generation |
Non-Patent Citations (3)
| Title |
|---|
| R C ARGÔLO-FILHO等: "Immunodetection of the toxic portion of Vip3A reveals differential temporal regulation of its secretion among Bacillus thuringiensis strains", 《JOURNAL OF APPLIED MICROBIOLOGY》 * |
| 陈建武等: "苏云金杆菌vip3 A基因的克隆、表达及杀虫活性分析", 《生物工程学报》 * |
| 韩丽珍等: "苏云金芽孢杆菌营养期杀虫蛋白VIP3研究进展", 《基因组学与应用生物学》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113493512A (en) * | 2021-07-26 | 2021-10-12 | 中国农业科学院生物技术研究所 | Preparation method of Cry3Bb rabbit polyclonal antibody |
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