A p72 full-length trimer protein expression system

Abstract

The present invention provides a p72 full-length trimer protein expression system, belonging to the field of molecular biotechnology. The African swine fever virus p72 full-length trimer protein expression system of the present invention solves the problem that there is no solution for expressing soluble trimer P72 full-length protein in Escherichia coli at present. The present invention can directly express soluble trimer p72 full-length protein in Escherichia coli, and the preparation method is simple and low in cost. The obtained product can be used for related vaccine preparation or other research and development, and has broad application prospects.

Description

Technical Field

[0001] The invention relates to the field of molecular biotechnology, and in particular to a p72 full-length trimer protein expression system. Background Art

[0002] African swine fever (ASF) is an acute, severe, hemorrhagic, and highly contagious disease of pigs caused by African swine fever virus (ASFV). The p72 protein is the main structural protein of the virus encoded by the ASF B646L gene and is an important detection target. It is a trimer structure in nature and has strong immunogenicity. Therefore, the p72 protein is often used as a serological diagnostic target to construct ASFV antigen or antibody serological diagnostic methods.

[0003] In the prior art, the expression of p72 trimer protein is often achieved through a eukaryotic expression system, such as the preparation method and application of a recombinant African swine fever virus p72 trimer subunit protein disclosed in Chinese patent CN117286185A, which expresses p72 trimer protein through a baculovirus expression system; or using p72 gene fragments, such as the African swine fever virus p72 fusion protein disclosed in patent CN111234036A, and the preparation method and application thereof, which are expressed on CHO cells by fusing p72 protein fragments and GCN4 fragments, all of which have the disadvantages of low expression amount and high cost; or prokaryotic expression as a monomer protein, such as the recombinant African swine fever virus p72 subunit soluble fusion protein disclosed in patent CN111607001 B, and the preparation method and application thereof, which can directly express p72 in Escherichia coli, but it is a monomer protein. However, there is no report on the prokaryotic expression of p72 trimer full-length protein. Summary of the invention

[0004] The purpose of the present invention is to provide a p72 full-length trimer protein expression system, which can directly and solublely express the trimer p72 full-length protein in Escherichia coli.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a p72 protein recombinant vector, wherein the recombinant vector comprises an initial vector and a p72 gene;

[0007] The initial vector is pET28a, pET30a, pBAD, pcold, pQE or pKK.

[0008] The present invention also provides a B602L protein recombinant vector, the recombinant vector comprising an initial vector and a B602L gene;

[0009] The initial vector is pG-KJE8, pGro7, pKJE7, pG-Tf2 or pTf16.

[0010] The present invention also provides a recombinant vector B602L-pF16, the nucleotide sequence of the recombinant vector B602L-pF16 is shown in SEQ ID NO.1.

[0011] The present invention also provides a recombinant vector P72-pET28a, the nucleotide sequence of the recombinant vector P72-pET28a is shown in SEQ ID NO.2.

[0012] The present invention also provides a recombinant strain, wherein the recombinant strain is transformed with the above-mentioned p72 protein recombinant vector and the above-mentioned B602L protein recombinant vector.

[0013] The present invention also provides a recombinant strain, into which the recombinant strain is transformed with the recombinant vector B602L-pF16 and the recombinant vector P72-pET28a.

[0014] Preferably, the recombinant strain is induced to express by arabinose and IPTG.

[0015] Preferably, the concentration of arabinose is 0.3-0.8 mg / mL;

[0016] The concentration of the IPTG is 0.05-0.15 mM.

[0017] Preferably, the temperature for inducing expression is 13-18°C;

[0018] The time for inducing expression is 10 to 30 hours.

[0019] The present invention also provides the use of the above recombinant strain in preparing p72 full-length trimer protein.

[0020] Beneficial effects of the present invention:

[0021] The African swine fever virus p72 full-length trimer protein expression system provided by the present invention solves the problem that there is currently no solution for expressing soluble trimer P72 full-length protein in Escherichia coli. The present invention can directly express soluble trimer p72 full-length protein in Escherichia coli, and the preparation method is simple and low-cost. The obtained product can be used for related vaccine preparation or other research and development, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the plasmid map of pET28a-p72 in the present invention;

[0023] Figure 2This is the plasmid map of pTf16-B602L in the present invention;

[0024] Figure 3 The PCR reaction temperature cycle parameters used in the present invention;

[0025] Figure 4 This is the SDS-PAGE identification result diagram, in which:

[0026] M: prestained protein molecular mass standard;

[0027] 1:P72-B602L-BL21(DE3) stock solution;

[0028] 2: P72-B602L-BL21(DE3) flowthrough;

[0029] 3: P72-B602L-BL21 (DE3) 20 mM imidazole elution;

[0030] 4: P72-B602L-BL21 (DE3) eluted with 50 mM imidazole;

[0031] 5: P72-B602L-BL21 (DE3) 100 mM imidazole elution;

[0032] 6: P72-B602L-BL21 (DE3) 250 mM imidazole elution;

[0033] 7: P72-B602L-BL21 (DE3) 500 mM imidazole elution;

[0034] Figure 5 This is the result of HIS monoclonal antibody identification, in which:

[0035] M: prestained protein molecular mass standard;

[0036] 1:P72-B602L-BL21(DE3) stock solution;

[0037] 2: P72-B602L-BL21(DE3) flowthrough;

[0038] 3: P72-B602L-BL21 (DE3) 20 mM imidazole elution;

[0039] 4: P72-B602L-BL21 (DE3) eluted with 50 mM imidazole;

[0040] 5: P72-B602L-BL21 (DE3) 100 mM imidazole elution;

[0041] 6: P72-B602L-BL21 (DE3) 250 mM imidazole elution;

[0042] 7: P72-B602L-BL21 (DE3) 500 mM imidazole elution;

[0043] Figure 6 The figure below shows the identification results of P72 non-denaturing gel polymer, where the left figure shows the identification results of His monoclonal antibody and the right figure shows the identification results of positive serum. DETAILED DESCRIPTION

[0044] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0045] Example

[0046] 1.1 Selection of African swine fever p72 protein

[0047] The p72 protein encoded by the B646L gene of the African swine fever virus is the main structural protein of the African swine fever virus. Studies have shown that the B602L protein is a chaperone protein of the p72 protein, which helps the p72 protein structure to fold correctly. Existing studies have shown that most p72 trimer proteins are expressed in eukaryotes, and the p72 proteins expressed in prokaryotes are proteins with truncated or altered natural sequences. There is no report on how to express the full-length p72 trimer protein in a prokaryotic system.

[0048] 1.2 Selection of African swine fever p72 and B602L protein sequences

[0049] The present invention uses the African swine fever virus subtype, refers to the ShanDong2021 full gene sequence (GenBank: OR660089.1) as a template to amplify the full-length sequence of p72, and refers to the Synthetic construct pB602L protein (B602L) gene, complete cds. (GenBank: OL698793.1) as a template to synthesize the B602L sequence. For the convenience of subsequent detection and purification, the His tags at the N-terminus and C-terminus of the pET28a vector are retained.

[0050] In subsequent experiments, pET28a-p72 and pTf16-B602L plasmids were constructed, as shown in the following diagrams: Figures 1-2 shown.

[0051] The sequence of B602L-pF16 is as follows:

[0052]

[0053] The sequence of P72-pET28a is as follows:

[0054]

[0055] 1.3 Construction of pET28a-p72 recombinant plasmid by homologous recombination

[0056] 1.3.1 PCR amplification of target fragment

[0057] (1) Primer design and synthesis

[0058] Upstream primer: 5'-GGTGGTGGTGCTCGAGGGTACTGTAACGCAGCACAGC-3', as shown in SEQ ID NO.3.

[0059] Downstream primer: 5'-TCGCGGATCCGAATTCATGGCATCAGGAGGAGCTTTTTG-3', as shown in SE Q ID NO.4.

[0060] (2) Using the ShanDong2021 full gene sequence stored in the laboratory as a template, add 50 μL of the sample system as follows:

[0061]

[0062] PCR amplification procedures, such as Figure 3 shown.

[0063] 1.3.2 PCR product gel recovery

[0064] All centrifugations were performed at room temperature

[0065] (1) Cut the target DNA band from the agarose gel, put it into a clean centrifuge tube and weigh it. If the gel weighs 100 mg, it can be regarded as 100 uL, and so on;

[0066] (2) Add 3 times the volume of GSB solution, melt the gel in a 55°C water bath for 6-10 minutes, mix intermittently (2-3 minutes) to ensure that the gel block is completely melted. When the gel is completely melted, observe the color of the solution. If the color is purple, add an appropriate amount of 3M sodium acetate (pH5.2) to adjust the color to the same as GSB (yellow). (To increase the amount of DNA recovered, add 1 volume of isopropanol to the melted gel solution. If the gel weight is 100 mg, add 100uL of isopropanol).

[0067] (3) After the melted gel solution cools to room temperature (the centrifuge column has a weak ability to bind DNA at high temperatures), add it to the centrifuge column and let it stand for 1 minute. Centrifuge at 10,000 × g for 1 minute and discard the effluent.

[0068] (4) Add 650u of WB solution, centrifuge at 10,000×g for 1 min, and discard the flow-through;

[0069] (5) Centrifuge at 10,000 × g for 1-2 minutes to completely remove residual WB;

[0070] (6) Place the centrifuge column in a clean centrifuge tube, open the cover and let it stand for 1 minute to allow the residual ethanol to evaporate. Add 30-50ul EB or deionized water (pH>7.0) to the center of the column (preheat EB or deionized water in a 60-70℃ water bath for better results) and let it stand at room temperature for 1 minute.

[0071] (7) Centrifuge at 10,000 × g for 10 minutes to elute the DNA, and store the eluted DNA at -20°C.

[0072] 1.3.3 Double restriction digestion of vector

[0073] (1) Mark the 1.5 mL EP tubes to be used, add samples and mix according to the following table: 50 μL reaction system

[0074]

[0075]

[0076] (2) Place the 1.5 mL EP tube in step (1) in a constant temperature water bath at the optimal temperature for the corresponding enzyme and bathe for 2-3 hours. Gel recovery of double enzyme digestion product: Take out the above double enzyme digestion system and perform agarose gel electrophoresis to recover the DNA fragments therein, using the same method as the gel recovery of PCR product in 1.3.1.2.

[0077] 1.3.4 Homologous recombination ligation

[0078] (1) Prepare several clean 1.5 mL EP tubes, label them, and place them in an EP tube rack for later use.

[0079] (2) Add sample to a 1.5 mL EP tube and mix thoroughly according to the table below.

[0080]

[0081] 1.3.5 Conversion reaction

[0082] (1) Quickly add 10 μL of ligation reaction solution into 100 μL of BL21(DE3) competent cells, pipette to mix, and place on ice for 30 min;

[0083] (2) Take out the sample tube, place it in a 42°C water bath for 90 seconds, and then immediately place it in an ice bath for 2 minutes;

[0084] (3) Take out the sample tube, add 800 μL of liquid LB medium to the sample tube in a clean bench, and then place the sample tube in a 37°C constant temperature shaker at 220 rpm for 1 h;

[0085] (4) Plate coating: Take out the sample tube in step (3), centrifuge at 8000 rpm for 2 min at room temperature, remove 600 μL of supernatant, use the remaining supernatant to resuspend the bacteria at the bottom of the tube, add the resuspended bacterial solution to the center of the corresponding transformation plate, and use a bacterial spreading stick to evenly spread the bacterial solution in the center of the transformation plate.

[0086] (5) Place the transformation plate in step (4) upright in a biochemical constant temperature incubator and incubate at 37°C for 1 hour.

[0087] The cells were cultured for 15 h.

[0088] (6) Observe the transformation results.

[0089] 1.3.6 Plasmid extraction and double enzyme digestion identification

[0090] 1.3.6.1 Plasmid extraction

[0091] (1) Use a 10 μL pipette tip to pick a single clone from the transformation plate and transfer it to 5 mL of LB liquid medium containing kanamycin resistance, and shake the culture overnight at 37°C and 220 rpm;

[0092] (2) Transfer the bacterial solution to a 1.5 mL EP tube and centrifuge at room temperature, 12,000 rpm, for 2 min, and discard the supernatant;

[0093] (3) Add 150 μL of plasmid extraction reagent P1 buffer to the EP tube in step (2) to thoroughly suspend the bacteria;

[0094] (4) Add 150 μL of P2 buffer to the solution in step (3), and immediately gently invert the centrifuge tube 6-12 times to mix thoroughly to lyse the bacteria;

[0095] (5) Add 350 μL P3 buffer to the solution in step (4) and immediately gently invert the centrifuge tube 12-20 times to mix thoroughly. At this time, a flocculent precipitate will appear;

[0096] (6) Centrifuge the solution from step (5) at room temperature, 12000 rpm, for 2 min;

[0097] (7) Move the supernatant solution in step (6) to the center of the adsorption column, centrifuge at room temperature, 12000 rpm, 30 s, and discard.

[0098] Collect the liquid in the tube;

[0099] (8) Add 300 μL of PWT rinse solution to the center of the adsorption column, centrifuge at room temperature, 12000 rpm, 30 s, and discard the liquid in the collection tube;

[0100] (9) Empty adsorption column, centrifuge at room temperature, 12000 rpm, 1 min.

[0101] (10) Place the adsorption column in a clean 1.5 mL centrifuge tube, add 50 μL of elution buffer TB to the center of the adsorption membrane, let stand at room temperature for 2 min, and centrifuge at room temperature, 12,000 rpm, for 30 s. Save the DNA solution in the tube.

[0102] 1.3.6.2 Double enzyme digestion identification

[0103] Mark the 1.5mL EP tubes to be used and add samples according to the following table: 20μL reaction system

[0104]

[0105] (2) Place the 20 μL reaction system in the EP tube in step (1) in a 37°C constant temperature water bath for 2 h.

[0106] (3) Perform agarose gel electrophoresis on the double enzyme digestion system sample in step (2) to check whether the size of the inserted fragment is

[0107] Correct; the correctly constructed plasmid was named pET28a-p72.

[0108] 1.4 Construction of pTf16-B602L by homologous recombination

[0109] 1.4.1 PCR amplification of B602L target fragment

[0110] (1) Primer design and synthesis

[0111] Upstream primer: 5'-TTTTGAGGTAACAAGCATATGGCTGAATTTAATATAGATGAGTTACTAAAGAACGT-3', as shown in SEQ ID NO.5.

[0112] Downstream primer: 5'-GTTATGCTGCGTAAACTCGAGTTACAGTTCCGCTTTGG-3', as shown in SEQ ID NO.6.

[0113] (2) Using the complete gene sequence of B602L stored in the laboratory as a template, add 50 μL of the sample system as follows:

[0114]

[0115] Reaction temperature cycle parameters such as Figure 3 shown.

[0116] Total 50 PCR amplification procedures:

[0117] 1.4.2 Gel recovery of target fragment PCR product

[0118] The gel was recovered in the same manner as in 1.3.2, and the recovered fragments were collected in 1.5 mL EP tubes and stored at 4°C.

[0119] 1.4.3 PCR amplification of pTf16 vector fragment

[0120] (1) Primer design and synthesis

[0121] Upstream primer: 5'-TTTACGCAGCATAACGCGCT-3', as shown in SEQ ID NO.7.

[0122] Downstream primer: 5'-CTTGTTACCTCAAAAAATCACAGTGCT-3', as shown in SEQ ID NO.8.

[0123] (2) Using the pTf16 gene stored in the laboratory as a template, add 50 μL of the sample system as follows:

[0124]

[0125] Reaction temperature cycle parameters such as Figure 3 shown.

[0126] PCR amplification procedure:

[0127] 1.4.4 Gel recovery of vector PCR products

[0128] The gel was recovered in the same manner as in 1.3.2, and the recovered fragments were collected in 1.5 mL EP tubes and stored at 4°C.

[0129] 1.4.5 Homologous recombination ligation

[0130] (1) Prepare several clean 1.5 mL EP tubes, label them, and place them in an EP tube rack for later use.

[0131] (2) Add sample to a 1.5 mL EP tube and mix thoroughly according to the table below.

[0132]

[0133] 1.4.6 Conversion reaction

[0134] 1.4.6.1 Preparation of pET28a-p72-BL21(DE3) competent cells

[0135] (1) When the OD600 of the cultured bacteria reaches 0.6-0.7, place the culture solution in an ice bath and cool for 15 minutes;

[0136] (2) Centrifuge at 4000 g for 5 min at 4°C to collect bacteria and discard the supernatant;

[0137] (3) If the volume of bacteria before centrifugation is 50 ml, proceed with the subsequent operation; if it is another volume, proceed with the subsequent operation after conversion according to the proportion;

[0138] (4) Gently resuspend the bacterial pellet with 20 ml of pre-cooled Super Competent Preparation Reagent A. Be very gentle and slow when suspending, otherwise it will affect the effect;

[0139] (5) Ice bath for 15 minutes;

[0140] (6) Centrifuge at 4000 g for 5 min at 4°C to collect the bacteria and discard the supernatant;

[0141] (7) Gently resuspend the bacterial pellet with 2 ml of pre-cooled Super Competent Preparation Reagent B. Be very gentle and slow when suspending, otherwise it will affect the effect;

[0142] (8) Ice bath for 15 minutes;

[0143] (9) Aliquot the volume on an ice bath and dispense into 100 μL / tubes as needed.

[0144] 1.4.6.2 Transformation of pTf16-B602L Ligation Product

[0145] (1) Quickly add 10 μL of the ligation reaction solution to 100 μL of the competent cells prepared in 1.4.6.2, pipette and mix well, and place on ice for 30 min;

[0146] (2) Take out the sample tube, place it in a 42°C water bath for 90 seconds, and then immediately place it in an ice bath for 2 minutes;

[0147] (3) Take out the sample tube, add 800 μL of liquid LB medium to the sample tube in a clean bench, and then place the sample tube in a 37°C constant temperature shaker at 220 rpm for 1 h;

[0148] (4) Plate coating: Take out the sample tube in step (3), centrifuge at 8000 rpm for 2 min at room temperature, remove 600 μL of supernatant, use the remaining supernatant to resuspend the bacteria at the bottom of the tube, add the resuspended bacterial solution to the center of the corresponding transformation plate, and use a bacterial spreading stick to evenly spread the bacterial solution in the center of the transformation plate.

[0149] (5) Place the transformation plate prepared in step (4) upright in a biochemical constant temperature incubator and incubate at 37°C for 1 hour. Then invert the transformation plate and incubate for 15 hours.

[0150] (6) Observe the transformation results.

[0151] 1.4.7 PCR identification of bacterial fluid

[0152] Use a 10 μL pipette tip to pick a single clone from the transformation plate and transfer it to 5 mL of LB liquid medium containing kanamycin resistance, and shake the culture overnight at 37°C and 220 rpm;

[0153] The selected monoclones were identified by bacterial liquid PCR according to the PCR methods in 1.3.1 and 1.4.1 respectively.

[0154] The correctly identified E. coli was named pET28a-p72-pTf16-B602L-BL21(DE3)

[0155] 1.5 Protein expression

[0156] 1.5.1 Inducible expression

[0157] (1) In a clean bench, add 200 mL of liquid LB medium, 200 μL of kanamycin, 2 mL of pET28a-p72-pTf16-B602L-BL21 (DE3) bacterial solution, and arabinose to a final concentration of 0.5 mg / mL into a 500 mL conical flask sterilized by autoclave;

[0158] (2) Culture at 37°C and 220 rpm for 2 h;

[0159] (3) Measure the OD600nm of the culture medium using a micro-spectrophotometer. When it reaches about 0.6, add IPTG to a final concentration of 0.1 mM.

[0160] (4) Induce culture at 16°C and 180 rpm for 12 h.

[0161] 1.5.2 Analysis of recombinant protein solubility

[0162] (1) Centrifuge the induced expression bacterial solution at 4000 rpm for 15 min to collect the bacterial precipitate;

[0163] (2) Resuspend the collected bacterial precipitate in Binding buffer (pH = 7.4) at a ratio of 1:10 (m / v) according to the mass of the bacterial precipitate;

[0164] (3) Place the resuspended bacteria in an ice-water mixture and subject them to ultrasonic disruption. One cycle is 5 seconds of operation and 5 seconds of rest, and the operation time is 10 minutes.

[0165] (4) Place the disrupted bacterial solution in an ultracentrifuge and centrifuge at 4°C and 12,000 g for 15 min. Collect the supernatant and aspirate as much as possible without collecting the precipitate (the supernatant can also be filtered through a 0.22 μm filter to remove the precipitate);

[0166] (5) The precipitate was then washed with an equal volume of Binding buffer (pH = 7.4) to eliminate the influence of the residual supernatant on the precipitate, and then the precipitate was resuspended with an equal volume of Binding buffer (pH = 7.4);

[0167] (6) 80 μL of supernatant and precipitate suspension were taken out respectively, and then 20 μL of 5× loading buffer was added to the supernatant and precipitate suspension, and the mixture was placed in a metal bath at 99°C for 10 min for denaturation treatment. After SDS-PAGE gel electrophoresis, the protein solubility was identified by Coomassie Brilliant Blue staining and Western Blot. The results are as follows: Figures 4-5 shown.

[0168] Depend on Figure 4 and Figure 5 It can be seen that the full-length trimer of the recombinant protein provided by the present invention is expressed in prokaryotes and purified by Ni column to obtain the target protein with a purity of 95%. The specific operation steps are as follows:

[0169] SDS-PAGE identification

[0170] (1) Collecting bacteria: After the induction culture is completed, the bacterial solution is centrifuged at 4°C and 4000 rpm for 15 min, the supernatant is discarded, and the bacteria are resuspended in 20 mL of Binding buffer (pH = 7.2). The resuspended bacteria are placed at 4°C for ultrasonic disruption.

[0171] (2) Collection of supernatant: After bacterial cell disruption, centrifuge at 4°C, 12,000 rpm for 15 min, collect the supernatant and discard the precipitate. Take 100 μL of the supernatant as the supernatant sample after disruption for SDS-PAGE detection.

[0172] (3) Equilibration of nickel column: After shaking Ni Sepharose 6 Fast Flow, take 2 mL and add it to the gravity purification column. First, equilibrate it with deionized water filtered through a 0.22 μm filter membrane (all subsequent solutions are filtered) for 5 column volumes, and then equilibrate it with Binding buffer (pH = 7.2) for 5 column volumes.

[0173] (4) Binding of p72 protein to Ni Sepharose 6 Fast Flow: The equilibrated Ni Sepharose 6 Fast Flow filler was extracted and added to the supernatant collected in (2), and the mixture was allowed to bind at 4°C and 50 rpm for 2 h.

[0174] (5) Elution of protein: After the binding is completed, the supernatant in the column is allowed to flow out naturally. Wash buffer 1, Wash buffer 2, Wash buffer 3, Wash buffer 4, and Wash buffer 5 are used in order from low to high imidazole concentrations to wash the column to elute the impurities and the target protein. Coomassie Brilliant Blue G-250 protein indicator is used to determine the elution effect of each wash buffer. First, 80 μL of Coomassie Brilliant Blue G-250 protein indicator solution is added to each well of the 96-well V-shaped plate. Then, 20 μL of the column outflow is taken at regular intervals and added to the V-shaped plate. If the column turns blue, it means that protein has flowed out. After the indicator does not change color, the next concentration of wash buffer is used for elution. After each solution is added, the column is slightly turned upside down to fully combine the filler and the wash buffer to elute the sample bound to the filler. 80 μL of the collected flow-through and the wash buffer 1 to 5 liquids are taken to prepare the sample.

[0175] (6) Add 20 μL of 5× loading buffer to each of the samples collected above, denature at 99°C for 10 min, perform SDS-PAGE electrophoresis, and identify the protein by staining with Coomassie Brilliant Blue. The protein eluate with high purity is dialyzed into Binding buffer (pH=7.2), and then concentrated using a 10 kDa 50 mL Merck ultrafiltration tube and stored at 4°C for later use. Figure 5 Results The results of SDS-PAGE analysis of each purified component showed that the target protein was eluted at 250 mM and 500 mM imidazole, and the target protein with a purity of 95% was obtained.

[0176] The purified p72 protein was identified by Western blot, and the binding ability of the expressed protein with ASFV positive standard serum was detected. The specific test process is as follows:

[0177] (1) Sample treatment: Take 80 μL of purified p72 protein, add 20 μL of 5× loading buffer to the protein, and denature at 99°C for 10 min;

[0178] (2) SDS-PAGE electrophoresis: Use the Yazyme protein gel preparation kit to prepare 12.5% ​​SDS-PAGE protein gel, install it in a fixed position in the electrophoresis tank, add SDS-PAGE electrophoresis buffer to submerge the protein gel, remove the comb on the SDS-PAGE, add 20 μL of sample to each gel hole according to the experimental needs, close the electrophoresis tank cover, set the constant voltage mode, and perform electrophoresis at 150 V for 50 min;

[0179] (3) Transfer: Soak the PVDF membrane of the cut size in methanol for about 5 minutes, take it out and put it in the transfer solution to balance for 1 minute, use the Bio-Rad Semi-Dry Transfer Rapid Transfer Instrument to transfer the membrane, open the lid of the transfer tank, place the transfer thick filter paper soaked in the transfer solution in the order of "filter paper - PVDF membrane - SDS-PAGE gel - filter paper", cover the lid, tighten the knob to fix it, put it in the Bio-Rad Semi-Dry Transfer Rapid Transfer Instrument for transfer, and transfer for 15 minutes;

[0180] (4) Blocking: After successful transfer, the transferred membrane is blocked with Western blot blocking solution at room temperature and 45 rpm for 1 h or at 4°C overnight. After blocking, the remaining blocking solution on the membrane is washed with 1× TBST. Each washing time is 5 min, and the membrane is washed 5 times in total;

[0181] (5) Incubation with primary antibody: After washing the membrane, the membrane was treated with African swine fever positive serum prepared with Western blot blocking solution at a ratio of 1:5000, and the binding was carried out at 4°C overnight or at room temperature for 1 hour; then, the residual primary antibody on the membrane was washed with 1×TBST to eliminate the interference of unbound primary antibody on the results. The membrane was washed for 5 minutes each time, and the membrane was washed 5 times in total;

[0182] (6) Incubation with secondary antibody: After washing the membrane, HRP-labeled goat anti-swine IgG prepared in a Western blot blocking solution at a ratio of 1:5000 was used to bind to the PVDF membrane incubated with the positive serum. The binding was carried out at room temperature for 30 min. Then, the residual secondary antibody on the membrane was washed with 1× TBST to eliminate the interference of the residual secondary antibody on the results. The membrane was washed for 5 min each time, and the membrane was washed 5 times in total.

[0183] (7) Development: After washing the membrane, evenly add the ECL basic chemiluminescence detection kit substrate onto the membrane, place it in a GE developer and select automatic exposure for color analysis to identify the expression of the target protein.

[0184] according to Figure 4 The identification results showed that the target protein specifically bound to the African swine fever standard positive serum and was identified as African swine fever p72 protein.

[0185] Depend on Figure 6 It can be seen that the trimer is formed under natural conditions through WB identification, and the size is about 250Kd. The specific experimental plan is as follows:

[0186] (1) Sample treatment: Take 80 μL of purified p72 protein and add 20 μL of 5× non-reducing protein loading buffer to the protein without heat treatment;

[0187] (2) SDS-PAGE electrophoresis: Use the Yazyme protein gel preparation kit to prepare 12.5% ​​SDS-PAGE protein gel, install it in a fixed position in the electrophoresis tank, add SDS-PAGE electrophoresis buffer to submerge the protein gel, remove the comb on the SDS-PAGE, add 20 μL of sample to each gel hole according to the experimental needs, close the electrophoresis tank cover, set the constant voltage mode, and perform electrophoresis at 150 V for 50 min;

[0188] (3) Transfer: Soak the PVDF membrane of the cut size in methanol for about 5 minutes, take it out and put it in the transfer solution to balance for 1 minute, use the Bio-Rad Semi-Dry Transfer Rapid Transfer Instrument to transfer the membrane, open the lid of the transfer tank, place the transfer thick filter paper soaked in the transfer solution in the order of "filter paper - PVDF membrane - SDS-PAGE gel - filter paper", cover the lid, tighten the knob to fix it, put it in the Bio-Rad Semi-Dry Transfer Rapid Transfer Instrument for transfer, and transfer for 15 minutes;

[0189] (4) Blocking: After successful transfer, the transferred membrane is blocked with Western blot blocking solution at room temperature and 45 rpm for 1 h or at 4°C overnight. After blocking, the remaining blocking solution on the membrane is washed with 1× TBST. Each washing time is 5 min, and the membrane is washed 5 times in total;

[0190] (5) Incubation with primary antibody: After washing the membrane, the membrane was treated with African swine fever positive serum prepared with Western blot blocking solution at a ratio of 1:5000, and the binding was carried out at 4°C overnight or at room temperature for 1 hour; then, the residual primary antibody on the membrane was washed with 1×TBST to eliminate the interference of unbound primary antibody on the results. The membrane was washed for 5 minutes each time, and the membrane was washed 5 times in total;

[0191] (6) Incubation with secondary antibody: After washing the membrane, HRP-labeled goat anti-swine IgG prepared in a Western blot blocking solution at a ratio of 1:5000 was used to bind to the PVDF membrane incubated with the positive serum. The binding was carried out at room temperature for 30 min. Then, the residual secondary antibody on the membrane was washed with 1× TBST to eliminate the interference of the residual secondary antibody on the results. The membrane was washed for 5 min each time, and the membrane was washed 5 times in total.

[0192] (7) Development: After washing the membrane, evenly add the ECL basic chemiluminescence detection kit substrate onto the membrane, place it in a GE developer and select automatic exposure for color analysis to identify the expression of the target protein.

[0193] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A p72 protein recombinant vector, characterized in that: The recombinant vector comprises an initial vector and a p72 gene; The initial vector is pET28a, pET30a, pBAD, pcold, pQE or pKK.

2. A B602L protein recombinant vector, characterized in that: The recombinant vector includes an initial vector and a B602L gene; The initial vector is pG-KJE8, pGro7, pKJE7, pG-Tf2 or pTf16.

3. A recombinant vector B602L-pF16, characterized in that: The nucleotide sequence of the recombinant vector B602L-pF16 is shown in SEQ ID NO.

1.

4. A recombinant vector P72-pET28a, characterized in that: The nucleotide sequence of the recombinant vector P72-pET28a is shown in SEQ ID NO.

2.

5. A recombinant strain, characterized in that: The recombinant strain is transformed with the p72 protein recombinant vector according to claim 1 and the B602L protein recombinant vector according to claim 2.

6. A recombinant strain, characterized in that: The recombinant strain is transformed with the recombinant vector B602L-pF16 described in claim 3 and the recombinant vector P72-pET28a described in claim 4.

7. The recombinant strain according to claim 5 or 6, characterized in that The recombinant strain was induced to express by arabinose and IPTG.

8. The recombinant strain according to claim 7, characterized in that The concentration of arabinose is 0.3-0.8 mg / mL; The concentration of the IPTG is 0.05-0.15 mM.

9. The recombinant strain according to claim 8, characterized in that The temperature for inducing expression is 13-18°C; The time for inducing expression is 10 to 30 hours.

10. Use of the recombinant strain according to any one of claims 5 to 9 in the preparation of p72 full-length trimer protein.

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