A polyclonal antibody against the drought-resistant gene BnaTZF1A of Brassica napus and a preparation method thereof

Abstract

The present invention belongs to the field of biochemical technology, and specifically relates to a polyclonal antibody of a drought-resistant gene BnaTZF1A of Brassica napus and a preparation method thereof. The present invention uses a DNA fragment of the drought-resistant gene BnaTZF1A of Brassica napus to design primer pairs BnTZF1A-RRF and BnTZF1A-TZFR, constructs a recombinant prokaryotic expression vector, transforms expression cells for protein expression, obtains a target product after purification of the protein expression culture, and then performs animal immunization to induce antibody production, and finally purifies the antibody to obtain a polyclonal antibody of the drought-resistant gene BnaTZF1A of Brassica napus. The prepared polyclonal antibody of the drought-resistant gene BnaTZF1A of Brassica napus has good specificity.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemistry, and in particular relates to a polyclonal antibody of a drought-resistant gene BnaTZF1A of Brassica napus and a preparation method thereof. Background Art

[0002] The name of the Chinese invention patent is "Brassica napus drought-resistant gene BnaTZF1A and its primers, expression vector, application and method for improving drought resistance" (publication number: CN110804614A), which discloses the BnaTZF1A gene in Brassica napus, and then constructs the overexpression vector CaMV35S::BnaTZF1A of the BnaTZF1A gene and transforms Brassica napus XY15 (Xiangyou 15). The BnaTZF1A gene is overexpressed in Brassica napus, resulting in enhanced drought resistance of Brassica napus. However, at this stage, there is a lack of antibodies related to the Brassica napus drought-resistant gene BnaTZF1A protein. Summary of the invention

[0003] The purpose of the present invention is to provide a rabbit anti-BnaTZF1A polyclonal antibody that specifically binds to the BnaTZF1A protein, a drought resistance-related factor of Brassica napus, and a preparation method thereof.

[0004] The technical solution of the present invention is as follows:

[0005] A method for preparing a polyclonal antibody against the drought resistance gene BnaTZF1A of Brassica napus, comprising the following steps:

[0006] (1) Select the nucleic acid sequence of the protein fragment with strong antigenicity in the drought resistance gene BnaTZF1A of Brassica napus, design primers BnTZF1A-RRF and BnTZF1A-TZFR for cloning, and construct a recombinant prokaryotic expression vector;

[0007] (2) transforming the recombinant prokaryotic expression vector in step (1) into expression cells for protein expression to obtain a protein expression culture;

[0008] (3) Purifying the protein expression culture of step (2), subjecting the purified target product to animal immunization to induce antibody production, and finally purifying the antibody to obtain a polyclonal antibody against the drought resistance gene BnaTZF1A of Brassica napus.

[0009] Preferably, the encoding nucleic acid sequence in step (1) is as shown in SEQ ID NO: 1.

[0010] Preferably, in step (1), the BnTZF1A-RRF sequence is as shown in SEQ ID NO:2, and the BnTZF1A-TZFR sequence is as shown in SEQ ID NO:3.

[0011] Preferably, the recombinant prokaryotic expression vector in step (1) is a pET28a-SUMO-JT vector.

[0012] Preferably, the expression cell in step (2) is Rosetta (DE3).

[0013] Preferably, the purification step of the protein expression culture in step (3) is as follows: resuspend the protein expression culture with 60 ml of pre-cooled NTA-0 buffer, ultrasonically disrupt the protein expression culture, control the power to 300 W, ultrasonicate for 4 s, pause for 4 s, and ultrasonicate 99 times; centrifuge at 20000g and 4°C for 30 min, collect the supernatant and precipitate respectively, resuspend with 40 μl of 1X SDS-PAGE loading buffer, boil in water bath for 5 min, take 10 ul for protein electrophoresis detection, store the remaining supernatant and precipitate at 0-7°C for standby use, detect by electrophoresis, and purify with affinity chromatography resin.

[0014] A polyclonal antibody against the drought resistance gene BnaTZF1A of Brassica napus.

[0015] The invention selects the encoding nucleic acid sequence of a protein fragment with strong antigenicity in a drought-resistant gene BnaTZF1A of Brassica napus, designs primers to clone BnTZF1A-RRF and BnTZF1A-TZFR, constructs a recombinant prokaryotic expression vector, transforms expression cells to express proteins, obtains protein expression cultures, uses the target product purified from the protein expression cultures to induce antibody production by animal immunization, and finally purifies the antibodies to obtain polyclonal antibodies to the drought-resistant gene BnaTZF1A of Brassica napus. The obtained polyclonal antibodies to the drought-resistant gene BnaTZF1A of Brassica napus have high specificity and pass WB quality control. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a diagram of the protein small-scale expression results.

[0017] Figure 2 This is a protein electrophoresis detection diagram after the protein is expressed in large quantities.

[0018] Figure 3 This is the SDS-PAGE electrophoresis diagram of the antibody.

[0019] Figure 4 This is a WB image for antibody validation. DETAILED DESCRIPTION

[0020] In order to make the purpose, 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 in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0021] Example 1 Cloning of the Brassica napus drought resistance gene BnaTZF1A and construction of its prokaryotic expression vector

[0022] The nucleic acid sequence of the protein fragment with strong antigenicity in the BnaTZF1A protein, SEQ ID NO: 1, was obtained by analyzing the bioinformatics software DNAStar. The upstream primer pair was designed using Primer 5.0 software to construct a prokaryotic expression vector. The specific operations were as follows:

[0023] A. Based on the nucleic acid sequence of the protein fragment with strong antigenicity of the drought-resistant gene BnaTZF1A of Brassica napus, the primer pair BnTZF1A-RRF and BnTZF1A-TZFR were designed; wherein the DNA fragment of BnaTZF1A is shown in SEQ ID NO:1; the sequence of BnTZF1A-RRF is shown in SEQ ID NO:2; and the sequence of BnTZF1A-TZFR is shown in SEQ ID NO:3;

[0024] B. PCR amplification, 200ul system, PCR amplification of target fragment, product recovery.

[0025] 200ul system PCR amplification: according to the following system configuration

[0026]

[0027]

[0028] After adding and mixing, amplify according to the following PCR conditions:

[0029]

[0030] C. Connection and conversion

[0031] The PCR product and pET28a-SUMO-JT vector were ligated under the action of ligase for 2 h;

[0032] Transformed into TOP10 host cells;

[0033] Invert the plate and culture it in a 37°C constant temperature incubator. Colonies will appear after 12 to 16 hours.

[0034] D. Recombinant screening and identification

[0035] Aliquot 40ul of LB medium without antibiotics into a PCR tube, pick 8 single colonies of each clone and mix them into the PCR tube, place them in a 37℃ shaker at 220r / 2 hours; take 2ul from each tube as a template for bacterial liquid PCR, and those with positive results can be sent for sequencing identification;

[0036] E. Sequencing identification.

[0037] Example 2 Protein expression and purification

[0038] 1. Small-scale expression and identification

[0039] A. Transform the obtained correct recombinant expression plasmid into expression competent cells Rosetta (DE3);

[0040] B. Incubate inverted in a 37°C incubator overnight;

[0041] C. Pick a positive single colony and transfer it to 3 ml LB medium containing antibiotics, and culture it at 37℃200rpm overnight. Take 30ul of overnight cultured bacteria and add it to 3ml LB medium, and culture it at 37℃200rpm;

[0042] D. Cultivate until the OD value is 0.6; add glycerol to the remaining bacterial solution and store it at -80℃ for later use;

[0043] E. Add IPTG to a final concentration of 1 mmol / L for induction, and set up a control group for induction at 37°C and 200 rpm for 3 h;

[0044] Take 200ul of bacterial solution, centrifuge at 12000g for 30s, discard the supernatant, resuspend the bacteria with 40μl 1X SDS-PAGE loading buffer, boil in water for 5min, and take 10ul for protein electrophoresis detection.

[0045] The results are as follows Figure 1 As shown, lane 1: control; lane 2: marker (from top to bottom 116, 66.2, 45, 35, 25, 18.4, 14.4 kDa) lane 3: after induction. (Note: The arrow in the figure indicates the expression size of the target protein)

[0046] 2. Large-scale protein expression and purification

[0047] A. Take 20ul of the bacteria stored at -80℃ and inoculate it into 20ml LB medium containing antibiotics, and culture it at 37℃ and 200rpm overnight; take 2ml of the overnight cultured bacteria solution and add it into 2000ml LB medium, and culture it at 37℃ and 200rpm until the OD value is 0.6, add IPTG to the final concentration of 1mmol / L, and culture it at 37℃ and 200rpm for 3h.

[0048] B. Collect a large amount of cultured bacterial liquid for packaging, centrifuge at 6000g for 10min, and discard the supernatant; resuspend the bacteria with 60ml pre-cooled NTA-0 buffer, and disrupt the bacteria with ultrasound. The power is controlled to 300W, ultrasound for 4s, pause for 4s, and ultrasound 99 times; centrifuge at 20000g and 4℃ for 30min, collect the supernatant and precipitate respectively, resuspend with 40μl 1X SDS-PAGE loading buffer, after boiling water bath for 5min, take 10ul for protein electrophoresis detection, and the remaining supernatant and precipitate are stored at 0-7℃ for use and electrophoresis detection;

[0049] C. Purify using affinity chromatography resin.

[0050] The results are as follows Figure 2 Shown: 3177-F1-1-pET28a-KSI (target fragment 50-290aa), size: predicted theoretical size 42kDa, observed size around 42kDa.

[0051] Example 3 Animal Immunization

[0052] A. Antigen emulsification, one immunization with Freund's complete adjuvant, and two to five booster immunizations with Freund's incomplete adjuvant, 0.5 mg per immunization;

[0053] B. New Zealand white rabbits, multiple subcutaneous immunizations, each immunization interval is 2 weeks;

[0054] C. Blood test one week after the fourth immunization;

[0055] D. Collect a large amount of blood one week after the fifth immunization.

[0056] Example 4 Titer Detection

[0057] ELISA indirect method steps:

[0058] A. Antigen 2ug / ml, 100ul per well, coated at 4°C overnight;

[0059] B. 5% skimmed milk powder sealed at 37 degrees for 2 hours;

[0060] C. Dilute the serum in series according to 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000 (blank serum was used as negative control), 100ul per well;

[0061] D. Incubate at 37 degrees for one hour;

[0062] E, secondary antibody: HRP-labeled goat anti-rabbit IgG (1:50,000), incubated at 37°C for 30 min;

[0063] F, add TMB to develop color for 20 minutes;

[0064] G. Terminate and read.

[0065] Potency data:

[0066] Antiserum titer

[0067]

[0068]

[0069] Example 5 Antibody purification and purity detection

[0070] 1. Purification by affinity chromatography

[0071] A. Take the ProG affinity chromatography column, wash it with water for 10 times the column volume, and then rinse it with sodium acetate buffer for 10 times the column volume;

[0072] B. Take serum / ascites sample, centrifuge at 12000 rpm for 10 min at 4°C, collect supernatant and filter;

[0073] C. Mix 1 part of serum with 4 parts of sodium acetate buffer, load at 0.5 ml / min, and collect the penetration;

[0074] D. After loading, continue to rinse with sodium acetate buffer until the G250 test is colorless (drop 100ul G250 one drop at a time);

[0075] E. Rinse the column bed with glacial acetic acid elution buffer, collect the elution peak, and quickly adjust the pH of the elution peak to neutral with saturated sodium carbonate;

[0076] F. Wash 10 times the volume of the column bed with water, seal the column with 10 ml NaCl-sodium azide buffer, and place at 4°C for later use;

[0077] G. Ultrafiltration concentrates the eluted peak to a volume equal to that of serum and puts it into a dialysis bag;

[0078] H. Take out the sample, centrifuge it at 4°C, 12000 rpm for 10 min, collect the supernatant, store it at 4°C, and perform SDS-PAGE detection;

[0079] I. Estimate the purity and concentration based on the electrophoresis results, label the purified antibodies, and store them at -20°C.

[0080] Test results such as Figure 3 Shown: Antibody SDS-PAGE image (purity 98%)

[0081] Example 6 Antibody WB Detection

[0082] WB steps and experimental conditions:

[0083] A. Protein loading, 12% electrophoresis 80V ~ 30min, 120V ~ 60min;

[0084] B, semi-dry transfer 60mA, 80min;

[0085] C. Block with 5% PBST skim milk at 25°C for 2h;

[0086] D. Incubate with primary antibody diluted 1:2000 (5% PBST skim milk powder) and incubate at 25°C for 1 h;

[0087] E. Wash with PBST 4 times*10min;

[0088] F, secondary antibody 1:500005% PBST skim milk powder, incubated at 25°C for 1 h;

[0089] G. Wash with PBST 4 times*10min;

[0090] H, ECL chemiluminescent X-ray film development imaging;

[0091] Antibody results Figure 4 As shown:

[0092] Conclusion: Two rabbits were immunized with the antigen expressed by E. coli. The WB verified that the corresponding expression region of the sample's intestinal expression system was 50-290aa, which was connected to the pET28a-SUMO-JT vector. The theoretical size of the fusion protein was about 42kDa, and the antigen WB result was about 42kDa. Judging from the specificity of the result band, the WB quality control passed. Sequence Listing <110> Hunan Agricultural University <120> A polyclonal antibody against the drought-resistant gene BnaTZF1A of Brassica napus and a preparation method thereof <141> 2021-02-01 <160> 3 <170> SIPOSequenceListing 1.0 <210> 1 <211> 306 <212> DNA <213> Brassica napus L <400> 1 ttccggatgt acgaatttaa agtgaggcga tgcactcgtg gccggagcca cgactggacg 60 gagtgccctt acgctcatcc cggagagaaa gctcgccgtc gtgatccgag gaagtatcat 120 tactccggta cggcgtgccc tgagtttcgc aaaggcggtt gcaaaaaagg cgacgcgtgt 180 gagttctctc acggcgtttt cgagtgttgg cttcatccgg cgcgttatcg gactcagccg 240 tgtaaagacg gcggtaactg tcgccgtcgt gtatgtttct tcgctcattc gccggatcag 300 atcagg 306 <210> 2 <211> 48 <212> DNA <213> Artificial Sequence <400> 2 tggaggccga attcccgggg atccttccgg atgtacgaat ttaaagtg 48 <210> 3 <211> 45 <212> DNA <213> Artificial Sequence <400> 3 cggccgctgc aggtcgacgg atcccctgat ctgatccggc gaatg 45

Claims

1. A method for preparing a polyclonal antibody against the drought resistance gene BnaTZF1A of Brassica napus, characterized in that: The preparation method comprises the following steps: (1) Select the nucleic acid sequence encoding the protein fragment with strong antigenicity in the drought resistance gene BnaTZF1A of Brassica napus, design primers BnTZF1A-RRF and BnTZF1A-TZFR for cloning, and construct a recombinant prokaryotic expression vector; (2) transforming the recombinant prokaryotic expression vector in step (1) into expression cells for protein expression to obtain a protein expression culture; (3) purifying the protein expression culture in step (2), subjecting the purified target product to animal immunization to induce antibody production, and finally purifying the antibody to obtain a polyclonal antibody against the drought resistance gene BnaTZF1A of Brassica napus; The encoding nucleic acid sequence in step (1) is shown as SEQ ID NO: 1; In step (1), the BnTZF1A-RRF sequence is shown in SEQ ID NO: 2, and the BnTZF1A-TZFR sequence is shown in SEQ ID NO: 3; The expression cell in step (2) is Rosetta (DE3).

2. The preparation method according to claim 1, characterized in that: The purification step of the protein expression culture in step (3) is as follows: resuspend the protein expression culture with 60 ml of pre-cooled NTA-0 buffer, ultrasonically disrupt the protein expression culture, control the power to 300 W, ultrasonicate for 4 seconds, pause for 4 seconds, and ultrasonicate 99 times; centrifuge at 20000g and 4°C for 30 minutes, collect the supernatant and precipitate respectively, resuspend with 40 μl of 1X SDS-PAGE loading buffer, boil in water for 5 minutes, take 10 ul for protein electrophoresis detection, store the remaining supernatant and precipitate at 0-7°C for standby use, detect by electrophoresis, and purify with affinity chromatography resin.

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