CN116333073A - Prokaryotic expression and polyclonal antibody preparation method and detection of wheat head germination resistance related gene TaAFP - Google Patents

Abstract

The invention discloses prokaryotic expression of a wheat head germination resistance related gene TaAFP gene and preparation and detection of a polyclonal antibody. Analyzing and synthesizing codons to construct into a prokaryotic expression vector pET-28a based on a gene sequence of TaAFP; the recombinant protein is expressed and purified by procaryon, and a polyclonal antibody of His-TaAFP protein is prepared by immunizing Balb/C mice. Westernblot detection shows that the TaAFP polyclonal antibody can be specifically combined with purified TaAFP protein, which indicates that the antibody preparation is successful. By applying the invention, the existence of TaAFP protein in wheat, the distribution of each tissue and the research of interaction protein thereof can be rapidly detected under laboratory conditions, thereby laying a foundation.

Description

Prokaryotic expression and polyclonal antibody preparation method and detection of wheat head germination resistance related gene TaAFP

Technical Field

The invention relates to prokaryotic expression of wheat head germination resistance related gene TaAFP recombinant protein, polyclonal antibody preparation and detection.

Background

The sprouting of wheat ears refers to the phenomenon that seeds sprout on ears when the wheat ears are subjected to a humid or overcast environment before harvesting, and the phenomenon is a worldwide disaster problem. The germination of wheat ears can cause a series of biochemical reactions to occur in the kernels, especially the activities of enzymes such as carbohydrate degrading enzymes, proteolytic enzymes and the like are increased, and storage substances in embryo and endosperm are degraded, so that the processing quality of the wheat ears can be influenced, and the yield can be seriously influenced. Different wheat varieties have different spike germination resistance mechanisms, and the spike germination of the wheat is controlled by multiple genes, and physiological and biochemical characteristics and spike morphological characteristics, such as seed dormancy characteristics, alpha-amylase activity, spike structure, seed coat color and the like, have obvious influence on the spike germination. Therefore, how to control the sprouting of the ears when the wheat is mature and obtain an excellent variety of the wheat with the sprouting resistance of the ears becomes an important content for improving the variety of the wheat at home and abroad, and the identification method, the genetic mechanism, the resistance mechanism and the like of the wheat are widely studied.

In molecular biological research of wheat head germination resistance, the head germination resistance related gene attracts great attention. At present, candidate genes which are identified to control seed dormancy and are related to spike germination resistance are ABI3, FUS3, LECI, vp-1 and the like, and the genes have double functions of inhibiting germination and promoting embryo maturation related processes. Wherein Vp-1 is the main transcriptional regulator that regulates embryo development, promotes embryo maturation and dormancy. ABI3 is homologous to Vp-1 in Arabidopsis thaliana, and is a homologous gene to ABI 5. ABI3 and ABI5 regulate seed maturation and germination through interactions during ABA signaling, and promote expression of embryonic dehydration tolerance genes at the maturation stage. ABI5 is an important transcription factor of the ABA signal transduction pathway, and accumulation of proteins thereof is mainly induced by ABA to increase transcriptional activity and increase stability of proteins; its stability and activity are related to phosphorylation. AFP (ABI 5 binding protein) acts as a negative regulator of the ABA signal transduction pathway, interacts with ABI5 and acts upstream of ABI5, promoting degradation of ABI5 protein, preventing excessive accumulation of ABI5, and further weakening the ABA signal pathway, possibly closely related to the regulation mechanism of promoting embryo germination and inhibiting seed dormancy.

1. In the current molecular biology research on wheat head germination resistance, the techniques of molecular marking, RT-PCR, qRT-PCR, westernblot and the like are utilized, wherein the molecular marking, qRT-PCR is more applied. The invention successfully prepares the polyclonal antibody by prokaryotic expression of the TaAFP coding region, and can detect the existence of the TaAFP protein by obtaining the antibody through Westernblot, which lays a solid foundation for the subsequent detection of the distribution of the TaAFP protein in various tissues and the like.

Disclosure of Invention

The invention aims to provide a method for prokaryotic expression of wheat spike resistance related gene TaAFP and preparation and detection of polyclonal antibody, and provides a rapid and efficient method for detection of TaAFP protein, distribution thereof and the like.

In order to solve the problems, the invention adopts the following technical scheme: prokaryotic expression and purification of wheat head germination resistance related gene TaAFP are used for preparing polyclonal antibody by immunizing Balb/C mice.

The prokaryotic expression of the wheat head germination resistance related gene TaAFP is to add enzyme cutting sites BamHI and EcoRI, his tag and stop codon synthetic gene into the TaAFP coding gene, construct the prokaryotic expression vector pET-28a to obtain pET-28a-TaAFP recombinant plasmid, the amino acid sequence of the synthetic gene is shown as SEQ ID No.1, and the nucleotide is shown as SEQ ID No. 2.

Recombinant proteins were obtained according to the prokaryotic induction expression described above and purified. Purifying and recovering protein by using His tag purification resin (Ni-NTA) affinity chromatography column: the supernatant containing the TaAFP protein obtained by induction expression is passed through a Ni-NTA affinity chromatography column, then the protein is eluted by using 100mM imidazole concentration elution buffer solution with the volume of 10mL, and the eluted protein is collected and subjected to SDS-PAGE gel electrophoresis, so that the purified TaAFP recombinant protein is obtained by detection.

And (3) preparing polyclonal antibodies by immunizing mice with the purified TaAFP recombinant protein. The method is that purified TaAFP recombinant protein is used as antigen, a subcutaneous multipoint injection method is adopted to immunize Balb/C mice, freund complete adjuvant with the same amount as the protein is added for emulsification for the first time, and the Balb/C male mice are subcutaneously injected in a multipoint way; immunization was performed 3 times at 15 day intervals; and adding Freund's incomplete adjuvant with the same amount as the protein into the mixture for subsequent immunization; and after the last immunization for one week, collecting mouse carotid artery antiserum, and purifying to obtain the wheat TaAFP polyclonal antibody.

The wheat TaAFP polyclonal antibody is prepared by the method.

And (3) western-blot detection of the wheat TaAFP polyclonal antibody.

The method for detecting the western-blot is as follows:

(1) Subjecting the purified TaAFP recombinant protein of claim 3 to SDS-PAGE gel electrophoresis with a concentration of 5% gel and a concentration of 12% gel;

(2) After electrophoresis, cutting gel to proper size, and soaking in transfer buffer solution for 2min; soaking filter paper and PVDF membrane which are cut in advance and matched with gel in size for 5min, and then transferring to a membrane transfer buffer solution for soaking for 2min; the semi-dry transfer membrane method is utilized, and an anode carbon plate, filter paper, PVDF membrane, gel, filter paper and a cathode carbon plate are respectively arranged from bottom to top, so that the filter paper, the PVDF membrane and the gel are ensured to be aligned accurately, air bubbles are removed, a power supply is connected, a constant voltage of 14V is realized, and the transfer membrane is carried out for 52min.

(3) After the film transfer is finished, the power supply is turned off, the film is taken out, and is washed by 1 XPBST, and the film is washed for 5min at the temperature of 27 ℃ at 50 rpm/min; subsequently, the mixture was blocked with 5% skim milk at 27℃for 2h at 50 rpm/min; discarding the blocking solution, diluting the TaAFP polyclonal antibody of claim 5 at 1:500, adding and incubating at 27 ℃ at 60rpm/min for 2h; recovering the primary antibody, and washing the membrane 3 times by using 1 XPBST, and washing for 10min at 50rpm/min at 27 ℃; HRP-labeled rabbit anti-mouse IgG antibody was used as per 1:5000 dilution, adding and incubating at 27 ℃ at 60rpm/min for 1.5h; the secondary antibody was recovered, and ECL-developed solution was added thereto for observation.

(4) The 1 XPBST is 10mM Na3PO4,150mM NaCl,0.05%Tween-20, pH7.4; the transfer buffer is 2% commercial 50×transfer buffer, 20% methanol, and the rest is supplemented with distilled water.

The invention utilizes prokaryotic expression induction and purification to obtain TaAFP recombinant protein, adopts a subcutaneous multipoint injection method to immunize Balb/C mice, prepares and obtains a wheat TaAFP polyclonal antibody, carries out Western blot detection on the antibody, and can detect the TaAFP protein by using the antibody. The invention utilizes prokaryotic expression induction and purification to obtain TaAFP protein as immune antigen, and overcomes the problem that the full-length sequence is difficult to express normally. The prepared wheat TaAFP polyclonal antibody detects the TaAFP protein by using a Western blot technology, which proves that the antibody can be well used for detecting the TaAFP protein in wheat. The TaAFP polyclonal antibody provided by the invention can be used for rapidly and efficiently detecting the existence of TaAFP protein in wheat, distributing each tissue and screening the related research of interaction protein thereof, thereby laying a technical foundation.

Drawings

FIG. 1-double restriction identification of BamHI and EcoRI of recombinant proteins: m is Marker DL 12000;1 is pET-28a-TaAFP double enzyme cutting result.

FIG. 2 SDS-PAGE of expressed forms of recombinant proteins: the A diagram is the induction recombinant bacterium precipitation (M is the protein molecular weight standard; 1 is 0.4mmol/L IPTG induction recombinant bacterium precipitation; 2 is 0.8mmol/L IPTG induction recombinant bacterium precipitation; 3 is 1.2mmol/L IPTG induction recombinant bacterium precipitation); panel B shows the supernatant of the induced recombinant bacterium (M is the molecular weight standard of the protein; 1 is 0.4mmol/LIPTG induced recombinant bacterium supernatant; 2 is 0.8mmol/L IPTG induced recombinant bacterium supernatant; 3 is 1.2mmol/LIPTG induced recombinant bacterium supernatant).

FIG. 3 SDS-PAGE of purified recombinant proteins: m is a protein molecular weight standard; 1 is a nickel column purified TaAFP recombinant protein.

FIG. 4 shows a wheat TaAFP polyclonal antibody Westernblot detection pattern: m is a protein molecular weight standard; 1 is a prokaryotic expression purified TaAFP protein.

Detailed Description

The invention is further specifically illustrated by the following description in conjunction with the specific embodiments.

1. Synthesis of wheat head germination resistance related gene TaAFP, construction of prokaryotic expression vector and identification

According to the gene sequence of TaAFP, the coding region of the gene is analyzed by software to obtain the codon sequence of TaAFP, the coding sequence of 963 amino acids is coded, and the predicted molecular weight is 33.83kDa.

The cleavage site of the prokaryotic expression vector pET-28a is analyzed, and the TaAFP coding region is provided with two cleavage sites of BamHI and EcoRI, and is provided with His tag for gene synthesis and construction into the prokaryotic expression vector pET-28a (sent to Anhui general biological Co., ltd. Www.generalbiol.com). The amino acid sequence of the synthetic gene is shown as SEQ ID No.1, and the nucleotide is shown as SEQ ID No. 2.

The constructed prokaryotic expression vector is transformed into escherichia coli competent DH5 alpha (purchased from Sigma reagent company) by a thermal shock method, positive clones are selected and inoculated into LB liquid medium, plasmids are extracted, double enzyme digestion identification is carried out, and gel electrophoresis separation is carried out (figure 1), so that the prokaryotic expression vector is proved to be successfully constructed.

2. Prokaryotic induction expression and purification of wheat TaAFP recombinant protein

Prokaryotic induction expression of recombinant proteins:

(1) E.coli BL21 (preserved in the laboratory) was transformed with recombinant plasmids pET-28-TaAFP and pET-28a empty vector, positive clones were selected, single colonies were picked and inoculated into sterile liquid LB medium containing antibiotic kanamycin (50 mg/ml), and cultured overnight at 37℃for 180 r/min.

(2) The next day, take out at 1:100 volumes of the culture medium are inoculated into sterile liquid LB culture medium containing corresponding antibiotics, the culture is continued until the OD600 is between 0.7 and 0.8, and isopropyl-beta-D-thiogalactopyranoside (IPTG) is added to induce protein expression. Wherein, three final concentrations of 0.4mmol/L, 0.8mmol/L and 1.2mmol/L are selected by IPTG, and pET-28a empty plasmid is used for comparison, and protein induction expression is carried out to determine the optimal inducer concentration.

(3) Centrifugation at 5000r/min for 15min at 4deg.C, cells were collected, resuspended in 20ml of 1 XPBS buffer per gram of cells, sonicated, and centrifuged at 18000r/min for 40min at 4deg.C, the pellet and supernatant were collected, respectively, and SDS-polyacrylamide gel electrophoresis (SDS-PAGE) detected to confirm that the TaAFP recombinant protein was present as inclusion bodies (FIG. 2) and approximately 36kDa in size, and subsequent massive induction was performed at an optimal inducer IPTG final concentration of 0.6 mmol/L.

Purification of TaAFP recombinant protein:

the recombinant protein was induced in a large amount by 1L volume, and the cells were collected. The purification is to use His tag purification resin (Ni-NTA) affinity chromatography column to purify and recycle protein, the inclusion body obtained by induced expression is crushed by ultrasonic, supernatant containing TaAFP protein is passed through the Ni-NTA affinity chromatography column, then the protein is eluted by 100mM imidazole concentration elution buffer with the volume of 10mL, and the eluted protein is collected and subjected to SDS-PAGE gel electrophoresis (figure 3), and the purified TaAFP recombinant protein is obtained by detection.

3. Preparation of wheat TaAFP polyclonal antibody

Immunized mice were 5 total Balb/C male mice (purchased from university of inner mongolia) 5 weeks old. The purified recombinant protein is subjected to protein concentration measurement by using a BCA protein quantitative kit (purchased from Sigma reagent company) and has the concentration of 1.41mg/mL, and can be used for immunizing antigens, and a subcutaneous multipoint injection method is adopted to immunize Balb/C mice, wherein Freund complete adjuvant with the same amount as the protein is added for emulsification for the first time, and the Balb/C male mice are subjected to subcutaneous multipoint injection; immunization was performed 3 times at 15 day intervals; and adding Freund's incomplete adjuvant with the same amount as the protein into the mixture for subsequent immunization; and after the last immunization for one week, collecting mouse carotid artery antiserum, and purifying to obtain the wheat TaAFP polyclonal antibody.

4. Western blot detection method of wheat TaAFP polyclonal antibody

(1) Carrying out SDS-PAGE gel electrophoresis on the purified TaAFP recombinant protein, wherein the concentration of concentrated gel is 5% and the concentration of separation gel is 12%;

(2) After electrophoresis, cutting gel to proper size, and soaking in transfer buffer solution for 2min; soaking filter paper and PVDF membrane which are cut in advance and matched with gel in size for 5min, and then transferring to a membrane transfer buffer solution for soaking for 2min; the semi-dry transfer membrane method is utilized, and an anode carbon plate, filter paper, PVDF membrane, gel, filter paper and a cathode carbon plate are respectively arranged from bottom to top, so that the filter paper, the PVDF membrane and the gel are ensured to be aligned accurately, air bubbles are removed, a power supply is connected, a constant voltage of 14V is realized, and the transfer membrane is carried out for 52min.

(3) After the film transfer is finished, the power supply is turned off, the film is taken out, and is washed by 1 XPBST, and the film is washed for 5min at the temperature of 27 ℃ at 50 rpm/min; subsequently, the mixture was blocked with 5% skim milk at 27℃for 2h at 50 rpm/min; discarding the blocking solution, diluting the TaAFP polyclonal antibody of claim 5 at 1:500, adding and incubating at 27 ℃ at 60rpm/min for 2h; recovering the primary antibody, and washing the membrane 3 times by using 1 XPBST, and washing for 10min at 50rpm/min at 27 ℃; HRP-labeled rabbit anti-mouse IgG antibody was used as per 1:5000 dilution, adding and incubating at 27 ℃ at 60rpm/min for 1.5h; the secondary antibody was recovered and added to ECL chromogenic solution (Millipore, WBKLS 0100) and observed (fig. 4), which antibody was able to specifically bind TaAFP protein.

(4) The 1 XPBST is 10mM Na3PO4,150mM NaCl,0.05%Tween-20, pH7.4; the transfer buffer is 2% commercial 50×transfer buffer, 20% methanol, and the rest is supplemented with distilled water.

Sequence listing

INNER MONGOLIA AGRICULTURAL University

Prokaryotic expression of wheat head germination resistance related gene TaAFP and polyclonal antibody preparation

Claims (7)

1. A prokaryotic expression method of a wheat head germination resistance related gene TaAFP comprises the steps of transferring a recombinant plasmid pET-28a-TaAFP containing a TaAFP coding gene into escherichia coli BL21 to perform prokaryotic expression of a TaAFP protein; the recombinant plasmid is prepared by constructing a prokaryotic expression vector pET-28a by adding a nucleotide sequence of TaAFP protein and enzyme cutting sites BamHI and EcoRI, a His tag and a stop codon synthetic gene, wherein the amino acid sequence of the synthetic gene is shown as SEQ ID No.1, and the nucleotide is shown as SEQ ID No. 2.

2. The recombinant protein obtained by prokaryotic expression according to claim 1 is subjected to purification by purifying and recovering the protein by using His tag purification resin (Ni-NTA) affinity chromatography column: the supernatant containing the TaAFP protein obtained by induction expression is passed through a Ni-NTA affinity chromatography column, then the protein is eluted by using 100mM imidazole concentration elution buffer solution with the volume of 10mL, and the eluted protein is collected and subjected to SDS-PAGE gel electrophoresis, so that the purified TaAFP recombinant protein is obtained by detection.

3. Purified recombinant protein is prepared according to the method of claims 1-2.

4. A preparation method of a recombinant protein polyclonal antibody of a wheat head germination resistance related gene TaAFP uses the purified recombinant protein as an antigen, adopts a subcutaneous multipoint injection method to immunize a Balb/C mouse, adds Freund complete adjuvant with the same amount as the protein for emulsification for the first time, and carries out subcutaneous multipoint injection on the Balb/C male mouse; immunization was performed 3 times at 15 day intervals; and adding Freund's incomplete adjuvant with the same amount as the protein into the mixture for subsequent immunization; and after the last immunization for one week, collecting mouse carotid artery antiserum, and purifying to obtain the wheat TaAFP polyclonal antibody.

5. A wheat TaAFP polyclonal antibody prepared by the method of claim 4.

6. Westernblot detection based on the wheat TaAFP polyclonal antibody of claims 4-5.

7. The Westernblot detection method of claim 6:

(1) Subjecting the purified TaAFP recombinant protein of claim 3 to SDS-PAGE gel electrophoresis with a concentration of 5% gel and a concentration of 12% gel;

(2) After electrophoresis, cutting gel to proper size, and soaking in transfer buffer solution for 2min; soaking filter paper and PVDF membrane which are cut in advance and matched with gel in size for 5min, and then transferring to a membrane transfer buffer solution for soaking for 2min; the semi-dry transfer membrane method is utilized, and an anode carbon plate, filter paper, PVDF membrane, gel, filter paper and a cathode carbon plate are respectively arranged from bottom to top, so that the filter paper, the PVDF membrane and the gel are ensured to be aligned accurately, air bubbles are removed, a power supply is connected, a constant voltage of 14V is realized, and the transfer membrane is carried out for 52min.

(3) After the film transfer is finished, the power supply is turned off, the film is taken out, and is washed by 1 XPBST, and the film is washed for 5min at the temperature of 27 ℃ at 50 rpm/min; subsequently, the mixture was blocked with 5% skim milk at 27℃for 2h at 50 rpm/min; discarding the blocking solution, diluting the TaAFP polyclonal antibody of claim 5 at 1:500, adding and incubating at 27 ℃ at 60rpm/min for 2h; recovering the primary antibody, and washing the membrane 3 times by using 1 XPBST, and washing for 10min at 50rpm/min at 27 ℃; HRP-labeled rabbit anti-mouse IgG antibody was used as per 1:5000 dilution, adding and incubating at 27 ℃ at 60rpm/min for 1.5h; the secondary antibody was recovered, and ECL-developed solution was added thereto for observation.

(4) The 1 XPBST is 10mM Na3PO4,150mM NaCl,0.05%Tween-20, pH7.4; the transfer buffer is 2% commercial 50×transfer buffer, 20% methanol, and the rest is supplemented with distilled water.

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