CN112920271A - Polyclonal antibody of cabbage type rape drought-resistant gene BnatZF1A and preparation method thereof - Google Patents
Polyclonal antibody of cabbage type rape drought-resistant gene BnatZF1A and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biochemistry, and particularly relates to a polyclonal antibody of a cabbage type rape drought-resistant gene BnatZF1A and a preparation method thereof. The invention designs primer pairs BnTZF1A-RRF and BnTZF1A-TZFR by using the DNA fragment of the cabbage type rape drought-resistant gene BnTZF1A, constructs a recombinant prokaryotic expression vector, transforms expression cells to perform protein expression, obtains a target product obtained after purifying a protein expression culture, performs animal immunity induced antibody generation, and finally purifies the antibody to obtain the polyclonal antibody of the cabbage type rape drought-resistant gene BnTZF 1A. The prepared polyclonal antibody of the cabbage type rape drought-resistant gene BnatZF1A has good specificity.
Description
Technical Field
The invention belongs to the technical field of biochemistry, and particularly relates to a polyclonal antibody of a cabbage type rape drought-resistant gene BnatZF1A and a preparation method thereof.
Background
The invention discloses a BnaTZF1A gene in cabbage type rape and an overexpression vector CaMV35S of the BnaTZF1A gene, which is transformed into cabbage type rape XY15 (Xiang oil 15), wherein the BnaTZF1A gene is overexpressed in the cabbage type rape, so that the drought resistance of the cabbage type rape is enhanced, but antibodies related to the cabbage type rape gene BnaTZF1 drought resistance A protein are lacked at the present stage.
Disclosure of Invention
The invention aims to provide a rabbit anti-BnaTZF 1A polyclonal antibody which is specifically combined and reacted with a cabbage type rape drought-resistant related factor BnaTZF1A protein and a preparation method thereof.
The technical scheme of the invention is as follows:
a preparation method of a polyclonal antibody of a cabbage type rape drought-resistant gene BnatZF1A comprises the following steps:
(1) selecting a nucleic acid sequence of a protein fragment with strong antigenicity in a cabbage type rape drought-resistant gene BnaTZF1A, designing a primer pair BnTZF1A-RRF and BnTZF1A-TZFR for cloning, and constructing a recombinant prokaryotic expression vector;
(2) transforming the recombinant prokaryotic expression vector in the step (1) into an expression cell for protein expression to obtain a protein expression culture;
(3) purifying the protein expression culture obtained in the step (2), performing animal immunity induction antibody generation on a target product obtained by purification, and finally purifying the antibody to obtain the polyclonal antibody of the cabbage type rape drought-resistant gene BnatZF 1A.
Preferably, the coding nucleic acid sequence in step (1) is shown as SEQ ID NO: 1.
Preferably, the BnTZF1A-RRF sequence in the step (1) is shown as SEQ ID NO:2, and the BnTZF1A-TZFR sequence is shown as SEQ ID NO: 3.
Preferably, the recombinant prokaryotic expression vector in the step (1) is a pET28a-SUMO-JT vector.
Preferably, the expression cell of step (2) is Rosetta (DE 3).
Preferably, the step (3) of purifying the protein expression culture comprises the following steps: resuspending the protein expression culture with 60ml of precooled NTA-0 buffer solution, ultrasonically crushing the protein expression culture, controlling the power to be 300W, ultrasonically treating for 4s, pausing for 4s, and ultrasonically treating for 99 times; centrifuging at 20000g4 deg.C for 30min, collecting supernatant and precipitate respectively, resuspending with 40 μ l 1X SDS-PAGE sample buffer, boiling water bathing for 5min, collecting 10ul, performing protein electrophoresis, collecting the remaining supernatant and precipitate at 0-7 deg.C, performing electrophoresis, and purifying with affinity chromatography resin.
A polyclonal antibody of cabbage type rape drought-resistant gene BnatZF 1A.
The invention selects the coding nucleic acid sequence of the protein fragment with strong antigenicity in the cabbage type rape drought-resistant gene BnaTZF1A, designs a primer to clone BnTZF1A-RRF and BnTZF1A-TZFR, constructs a recombinant prokaryotic expression vector, transforms an expression cell to express protein to obtain a protein expression culture, uses a target product obtained by purifying the protein expression culture to carry out animal immunity induced antibody generation, finally purifies an antibody to obtain a polyclonal antibody of the cabbage type rape drought-resistant gene BnaTZF1A, and the obtained polyclonal antibody of the cabbage type rape drought-resistant gene BnaTZF1A has high specificity and is subjected to WB quality control.
Drawings
FIG. 1 is a graph showing the results of small amounts of protein expression.
FIG. 2 is an electrophoretic protein detection map after the protein is expressed in large quantities.
FIG. 3 is an SDS-PAGE electrophoresis of antibodies.
Fig. 4 is a WB diagram for antibody validation.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 cloning of cabbage type rape drought-resistant gene BnatZF1A and construction of prokaryotic expression vector thereof
The nucleic acid sequence SEQ ID NO 1 of the BnatZF1A protein fragment with strong antigenicity obtained by the analysis of bioinformatics software DNAStar, and the upstream Primer pair is designed by the software Primer 5.0 to construct a prokaryotic expression vector, which comprises the following specific operations:
A. designing a primer pair BnTZF1A-RRF and BnTZF1A-TZFR by using a nucleic acid sequence of a protein fragment with strong antigenicity of the cabbage type rape drought-resistant gene BnTZF 1A; wherein the DNA fragment of BnatZF1A is shown as SEQ ID NO 1; the BnTZF1A-RRF sequence is shown as SEQ ID NO. 2, and the BnTZF1A-TZFR sequence is shown as SEQ ID NO. 3;
B. PCR amplification, 200ul system, PCR amplification of target fragment, product recovery.
PCR amplification of 200ul system: is configured according to the following system
After the addition and mixing, the amplification was performed according to the following PCR conditions:
C. connection and transformation
Connecting the PCR product with pET28a-SUMO-JT vector for 2h under the action of ligase;
transformation into TOP10 host cells;
inverting the flat plate, culturing in a constant-temperature incubator at 37 ℃, and allowing colonies to appear after 12-16 hours;
D. screening and identification of recombinants
Subpackaging 40ul of LB culture medium without antibiotics into PCR tubes, picking 8 single colonies of each clone, uniformly mixing the single colonies with the PCR tubes, and placing the single colonies in a shaking table at 37 ℃ for 220r/2 h; taking 2ul of each tube as a template to perform bacteria liquid PCR, and if the result is positive, sending to sequencing identification;
E. and (5) sequencing and identifying.
Example 2 protein expression purification
1. Small amount of expression and identification
A. Transforming the obtained correct recombinant expression plasmid into expression competent cells Rosetta (DE 3);
B. culturing in 37 deg.C incubator by inversion overnight;
C. positive single colonies were picked and transferred to 3ml LB medium containing antibiotics and cultured overnight at 37 ℃ at 200 rpm. Adding 30ul of overnight cultured bacterial liquid into 3ml of LB culture medium, and keeping the temperature at 37 ℃ and 200 rpm;
D. culturing until OD value is 0.6; adding glycerol into the residual bacterial liquid, and storing at-80 deg.C;
E. adding IPTG to the final concentration of 1mmol/L for induction, setting a control group, and inducing at 37 ℃ and 200rpm for 3 h;
200ul of the bacterial liquid is taken, 12000g of the bacterial liquid is centrifuged for 30s, the supernatant is discarded, the bacterial liquid is re-suspended by 40 mul of 1X SDS-PAGE sample buffer solution, 10ul of the bacterial liquid is taken for protein electrophoresis detection after boiling water bath for 5 min.
The results are shown in FIG. 1, lane 1: comparison; lane 2: marker (116, 66.2, 45, 35, 25, 18.4, 14.4kDa from top to bottom) lane 3: after induction. (Note: the size of the expression of the target protein indicated by the arrow in the figure)
2. Protein mass expression and purification
A. Inoculating 20ul of strain stored at-80 deg.C into 20ml LB medium containing antibiotic, and culturing at 37 deg.C under 200rpm overnight; 2ml of overnight-cultured bacterial liquid was added to 2000ml of LB medium, cultured at 37 ℃ and 200rpm until the OD value became 0.6, IPTG was added to a final concentration of 1mmol/L, and cultured at 37 ℃ and 200rpm for 3 hours.
B. Collecting a large amount of cultured bacteria liquid, subpackaging, centrifuging at 6000g for 10min, and discarding the supernatant; resuspending the thallus with 60ml of precooled NTA-0 buffer solution, ultrasonically crushing the thallus, controlling the power to be 300W, ultrasonically treating for 4s, pausing for 4s, and ultrasonically treating for 99 times; centrifuging at 20000g4 deg.C for 30min, collecting supernatant and precipitate respectively, resuspending with 40 μ l 1X SDS-PAGE sample buffer, boiling water bathing for 5min, taking 10ul for protein electrophoresis detection, collecting the remaining supernatant and precipitate at 0-7 deg.C for use, and performing electrophoresis detection;
C. purifying with affinity chromatography resin.
The results are shown in FIG. 2: 3177-F1-1-pET28a-KSI (fragment of interest 50-290aa), size: the theoretical size is predicted to be 42kDa, and the observed size is about 42 kDa.
Example 3 animal immunization
A. Antigen emulsification, Freund's complete adjuvant primary immunization, Freund's incomplete adjuvant boosting (secondary to five immunization), 0.5mg of each immunization;
B. new Zealand big ear white rabbit, subcutaneous multiple immunity, each immunity interval 2 weeks;
C. blood sampling detection is carried out after immunization for the fourth time for one week;
D. one week after the fifth immunization, a large number of blood samples were collected.
Example 4 potency assay
ELISA indirect method step:
A. antigen 2ug/ml, 100ul coating per well, 4 degree overnight;
B. sealing with 5% skimmed milk powder at 37 deg.C for 2 hr;
C. serum was diluted in duplicate (blank serum was used as negative control) at 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000 using dilutions, 100ul per well;
D. incubate at 37 ℃ for one hour;
E. secondary antibody HRP-labeled goat anti-rabbit IgG (1:50000), and incubating at 37 ℃ for 30 minutes;
F. adding TMB for developing for 20 minutes;
G. and (4) terminating and reading.
Potency data:
antiserum potency
Example 5 antibody purification and purity testing
1. Affinity chromatography for purification
A. Washing a ProG affinity chromatography column by 10 times of the volume of a column bed with water, and washing by 10 times of the volume of the column bed with sodium acetate buffer;
B. centrifuging the serum/ascites sample at 4 deg.C and 12000rpm for 10min, collecting supernatant, and filtering;
C. mixing 1 part of serum with 4 parts of sodium acetate buffer solution, loading at the speed of 0.5ml/min, and collecting penetration;
D. after the sample loading is finished, the solution is continuously washed by sodium acetate buffer until the solution is detected to be colorless by G250 (one drop is 100ulG 250);
E. eluting with glacial acetic acid, buffering, washing column bed, collecting eluate, and rapidly adjusting pH to neutral with saturated sodium carbonate;
F. washing with water 10 times the volume of the column bed, sealing the column with 10ml NaCl-sodium azide buffer, and standing at 4 deg.C;
G. ultrafiltering and concentrating the elution peak to equal volume of serum, and filling into a dialysis bag;
H. taking out a sample, centrifuging at 12000rpm for 10min at 4 ℃, collecting supernatant, temporarily storing at 4 ℃, and performing SDS-PAGE detection;
I. purity and concentration were estimated from the electrophoresis results, labeled, and the purified antibody was stored at-20 ℃.
The detection results are shown in fig. 3: SDS-PAGE of antibody (98% pure)
Example 6 antibody WB detection
WB procedure, experimental conditions:
A. protein sample loading, 12% electrophoresis 80V-30 min, 120V-60 min;
B. transferring the semi-dry transfer film for 60mA for 80 min;
C. sealing 5% PBST skimmed milk at 25 deg.C for 2 h;
D. incubating primary antibody for 1:2000 (5% PBST skimmed milk powder) dilution, and incubating for 1h at 25 ℃;
E. PBST4 washes 10 min;
F. the secondary antibody is 500005 percent PBST skimmed milk powder, and is incubated for 1h at 25 ℃;
G. PBST4 washes 10 min;
H. ECL chemiluminescence X-ray film development imaging;
antibody results are shown in figure 4:
and (4) conclusion: immunizing two rabbits with antigen expressed by Escherichia coli; WB verifies that the expression corresponding to the expression system of the sample large intestine is 50-290aa, and the expression corresponding to the expression system is connected with pET28a-SUMO-JT vector, the theoretical size of the fusion protein is about 42kDa, the result of the antigen WB is about 42kDa, and the WB quality control is passed from the viewpoint of the specificity of the result band.
Sequence listing
<110> Hunan agriculture university
<120> polyclonal antibody of cabbage type rape drought-resistant gene BnatZF1A and preparation method thereof
<141> 2021-02-01
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<170> SIPOSequenceListing 1.0
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tactccggta cggcgtgccc tgagtttcgc aaaggcggtt gcaaaaaagg cgacgcgtgt 180
gagttctctc acggcgtttt cgagtgttgg cttcatccgg cgcgttatcg gactcagccg 240
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Claims (7)
1. A preparation method of a polyclonal antibody of a cabbage type rape drought-resistant gene BnatZF1A is characterized by comprising the following steps:
(1) selecting a coding nucleic acid sequence of a protein fragment with strong antigenicity in a cabbage type rape drought-resistant gene BnaTZF1A, designing a primer pair BnTZF1A-RRF and BnTZF1A-TZFR for cloning, and constructing a recombinant prokaryotic expression vector;
(2) transforming the recombinant prokaryotic expression vector in the step (1) into an expression cell for protein expression to obtain a protein expression culture;
(3) purifying the protein expression culture obtained in the step (2), performing animal immunity induction antibody generation on a target product obtained by purification, and finally purifying the antibody to obtain the polyclonal antibody of the cabbage type rape drought-resistant gene BnatZF 1A.
2. The method according to claim 1, wherein the nucleic acid sequence encoded in step (1) is represented by SEQ ID NO 1.
3. The preparation method of claim 1, wherein the BnTZF1A-RRF sequence in the step (1) is shown as SEQ ID NO:2, and the BnTZF1A-TZFR sequence is shown as SEQ ID NO: 3.
4. The method according to claim 1, wherein the recombinant prokaryotic expression vector in step (1) is pET28a-SUMO-JT vector.
5. The method according to claim 1, wherein the expression cells in the step (2) are Rosetta (DE 3).
6. The method according to claim 1, wherein the step (3) of purifying the protein expression culture comprises the steps of: resuspending the protein expression culture with 60ml of precooled NTA-0 buffer solution, ultrasonically crushing the protein expression culture, controlling the power to be 300W, ultrasonically treating for 4s, pausing for 4s, and ultrasonically treating for 99 times; centrifuging at 20000g4 deg.C for 30min, collecting supernatant and precipitate respectively, resuspending with 40 μ l 1X SDS-PAGE sample buffer, boiling water bathing for 5min, collecting 10ul, performing protein electrophoresis, collecting the remaining supernatant and precipitate at 0-7 deg.C, performing electrophoresis, and purifying with affinity chromatography resin.
7. A polyclonal antibody produced by the production method according to any one of claims 1 to 6.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103468712A (en) * | 2013-08-20 | 2013-12-25 | 华中农业大学 | Application of GhTZF1 gene to reinforcement on drought resistance of plants and senility delay |
| CN103805613A (en) * | 2014-02-27 | 2014-05-21 | 中国科学院青岛生物能源与过程研究所 | Zinc finger protein and application thereof |
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