CN117286185A - Preparation method and application of recombinant African swine fever virus p72 trimer subunit protein - Google Patents

Abstract

The invention discloses a preparation method and application of recombinant African swine fever virus p72 trimer subunit protein, wherein the amino acid sequence of the protein is shown as SEQ ID NO.3, and the preparation method is as follows: 1) Cloning, transforming and infecting sf9 cells and/or Highfive5 cells with the codon optimized African swine fever virus p72 protein and the codon optimized pB602L protein expression genes to obtain recombinant baculovirus containing African swine fever virus p72 subunit protein expression genes; 2) Performing amplification culture on the recombinant baculovirus, then fermenting, and collecting a culture solution; 3) And (3) purifying the culture solution in the fermentation culture step 2) to obtain the recombinant African swine fever virus p72 subunit soluble protein. The invention can provide the African swine fever structural protein p72 trimer subunit protein which can be produced in a large-scale industrialized mode, and the preparation method is simple, low in cost, high in purity and capable of easily removing the B602L protein.

Description

A method for preparing a recombinant African swine fever virus p72 trimer subunit protein and its application

Technical Field

The present invention belongs to the technical field of veterinary biological products and relates to a large-scale preparation method and application of African swine fever p72 protein.

Background Art

African swine fever (ASF) is an acute, febrile, highly contagious disease of pigs, with a morbidity and mortality rate of up to 100%. Domestic pigs and wild boars are the only mammalian hosts of ASFV natural infection, which has a huge impact on animal husbandry.

Domestic and foreign scholars have done a lot of research on African swine fever, but the research found that the conventionally prepared inactivated African swine fever vaccine is not effective, while the natural attenuated vaccine or recombinant attenuated vaccine has a good protective effect, but poor safety and is easy to cause virus shedding. At present, there is no effective vaccine to prevent African swine fever and no drug to treat the disease in the world. The development and production of new vaccines are urgently needed to prevent African swine fever.

African swine fever virus (ASFV) is a DNA virus with an envelope. The average diameter of the virus particle is 200nm, with an icosahedral symmetric structure and a glycolipid-containing envelope covering the surface. The viral genome is a double-stranded linear DNA with a size of 170-190kb. The entire genome has about 150 ORFs, encoding 150-200 proteins. From the outside to the inside, there are five layers of structure: the outer envelope structure, the outer capsid protein, the inner envelope structure, the inner capsid protein, and the viral nucleic acid. The P72 protein is the most important structural protein of the outer capsid on the surface of African swine fever. Its protein content accounts for about 32% of the protein content of the virus particle. The protein is encoded by the B646L gene of the ASFV virus and has a molecular weight of about 32kD. Studies have found that the p72 protein is relatively conservative and has stable antigenicity. It is also related to the process of the virus entering the host cell. Antibodies against p72 can inhibit the binding of the virus to macrophages. Therefore, p72 is a good protective antigen. At present, there are already P72 proteins that can be successfully expressed in prokaryotic systems, but the expression yield is low and exists in the form of inclusion bodies (see the invention patent with publication number CN103805615 for details). The structure folding is incorrect and cannot meet the development of genetic engineering subunit vaccines. The pB602L protein is a viral non-structural protein encoded by the B602L gene. Current research has found that this protein is a non-structural protein in the late stage of viral replication. As a molecular chaperone, it promotes the correct folding of the capsid protein p72 protein. If the pB602L protein is missing, the capsid protein p72 cannot be folded correctly, resulting in changes in the virus assembly process and the inability to form an icosahedron, and the virus cannot be correctly assembled into viral particles. Therefore, pB602L is very critical to the correct folding of the p72 protein, so if you want to successfully express the p72 protein in large quantities, you must express pB602L at the same time. Liu et al. used HEK293 to simultaneously express p72 and B602L to obtain a trimeric p72 protein, and for the first time demonstrated the structure of p72 (Structure of the African swine fever virus majorcapsid protein p72, 2019). However, this method cannot be mass-produced in HEK293, and it remains a challenge for subsequent development. Patent CN111363016A, article (Expression and purification of p72 trimers as subunit vaccine candidate, 2020), etc. disclose a method for producing recombinant P72 in yeast. Although yeast operation has a short fermentation cycle and low cost relative to eukaryotic cells, the p72 protein produced by this method is not as high as 50 mg/L, and p72 is impure and B602L cannot be removed. It is not conducive to the subsequent evaluation of subunit vaccines.

Summary of the invention

In order to solve the above technical problems of the prior art, the present invention provides a recombinant African swine fever p72 subunit protein, wherein the African swine fever virus p72 subunit protein is mainly a p72 subunit protein with a trimer structure, wherein the p72 subunit protein with a trimer structure is an African swine fever virus structural protein, and its amino acid sequence is shown in SEQ ID NO.3, and the p72 subunit protein with a trimer structure accounts for no less than 75% of the total p72 subunit protein. The recombinant African swine fever p72 subunit protein of the present invention has high purity, almost no B602L, and is basically a trimer structure.

In a preferred technical solution of the present invention, the p72 subunit protein with a trimer structure accounts for no less than 80% of the total p72 subunit protein content.

In a preferred technical solution of the present invention, the p72 subunit protein with a trimer structure accounts for no less than 85% of the total p72 subunit protein.

In the preferred technical solution of the present invention, the p72 subunit protein of the African swine fever virus accounts for no less than 80% of the total protein content, and the pB602L subunit protein is no more than 5%. The present invention also provides a method for preparing the p72 subunit protein of the African swine fever virus, which can effectively remove B602L.

In the preferred technical scheme of the present invention, the present invention provides an optimized nucleotide sequence of OPTI-p72 that can express p72 protein in an insect-baculovirus expression system. In order to efficiently express P72 protein in insect cells sf9 or High Five5, the present invention analyzes the p72 gene based on the existing published sequence on GenBank: FR682468.1. First, the codon preference of the codons encoded by the p72 gene in insect cell expression is optimized, and then the GC content, mRNA, and repeat sequence of the p72 gene are optimized in insect cell stability. The nucleotide sequence of the final optimized OPTI-p72 is shown in SEQ ID NO 1.

In the preferred technical scheme of the present invention, the optimized OPTI-p72 cannot obtain a large amount of soluble expression p72 directly in the insect baculovirus expression system. In order to obtain a large amount of soluble P72 protein, the present invention uses a dual expression vector pFastBacDual to simultaneously express the OPTI-p72 nucleotide sequence and the OPTI-pB602L nucleotide sequence in the same vector. The gene coding sequence of the OPTI-pB602L chaperone protein is shown in SEQ ID NO 4.

In a preferred technical solution of the present invention, the optimized OPTI-p72 nucleotide sequence and OPTI-pB602L nucleotide sequence can also be cloned into the pFastBac1 vector respectively.

In order to facilitate the purification of the fusion protein by affinity chromatography, according to the preferred technical solution of the present invention, a tag selected from FLAG, poly-His, c-myc, and Strep-tagⅡ is connected to the amino terminus or carboxyl terminus of the amino acid sequence shown in SEQ ID NO.1.

In the preferred technical solution of the present invention, in order to efficiently and soluble express P72 protein in insect baculovirus, the present invention introduces the OPTI-p72 nucleotide sequence into a baculovirus expression vector. The expression vector can be any baculovirus expression vector, and the expression vector is specifically but not limited to a pFastBacDual expression vector and a pFastBac1 expression vector. Preferably, the expression vector is a pFastBacDual vector.

In the preferred technical scheme of the present invention, in order to efficiently and solublely express P72 protein in insect baculovirus, p72 and B602L must be co-expressed; the co-expression refers to placing p72 and B602L in an expression vector for screening and preparing into a virus, or placing them in different expression vectors to screen the two viruses to co-infect sf9 cells or HighFive cells for fermentation to express p72 protein.

According to another aspect of the present invention, the present invention also provides a large-scale preparation method of African swine fever p72 subunit protein, which can effectively remove B602L. The large-scale preparation method comprises the following steps:

1) Preparation of recombinant baculovirus: clone, transform, and infect sf9 cells and/or HighFive5 cells with the codon-optimized African swine fever virus p72 protein and the codon-optimized pB602L protein expression gene to obtain a recombinant baculovirus containing the African swine fever virus p72 subunit protein expression gene;

2) Fermentation culture: expanding the recombinant baculovirus and then fermenting it, and collecting the culture fluid;

3) Subunit protein purification: The culture fluid described in the fermentation culture step 2) is purified to obtain the recombinant African swine fever virus p72 subunit soluble protein.

In the preferred technical scheme of the present invention, in step 1), the codon-optimized African swine fever virus p72 protein and the codon-optimized pB602L protein expression genes are cloned, transformed, and infected with sf9 cells and/or HighFive5 cells to simultaneously clone the codon-optimized African swine fever virus p72 protein and the codon-optimized pB602L protein expression genes into the pFastBac1 transfer vector, cloned, transformed, infected with sf9 cells and/or HighFive5 cells, and then fermented, cultured and purified to obtain a recombinant baculovirus rBac-p72-pB602L containing the African swine fever virus p72 subunit protein expression gene.

In the preferred technical scheme of the present invention, in step 1), the codon-optimized African swine fever virus p72 protein and the codon-optimized pB602L protein expression genes are cloned, transformed, and infected with sf9 cells and/or HighFive5 cells to clone the codon-optimized African swine fever virus p72 protein and the codon-optimized pB602L into the pFastBac1 transfer vector, respectively, and cloned, transformed, and infected with sf9 cells to obtain the recombinant baculovirus rBac-p72 containing the African swine fever virus p72 subunit protein expression gene and the recombinant baculovirus rBac-pB602L containing the African swine fever virus pB602L subunit protein expression gene.

The large-scale preparation method described above is characterized in that the p72 nucleotide sequence is shown in SEQ ID NO.1. The pB602L nucleotide sequence is shown in SEQ ID NO.4.

According to the large-scale preparation method of the present invention, the operation steps of step 1) are: first, the codon-optimized African swine fever virus p72 protein expression gene is cloned into the EcoRI/HindIII site of pFastBacDual to construct pFastBacDual-p72, and then the codon-optimized African swine fever virus pB602L protein expression gene is cloned into the XhoI/KpnI site of the pFastDual-p72 transfer vector, cloned and transformed into DH10Bac Escherichia coli, blue-white spot screening is performed to obtain Bacmid-p72-pB602L, and sf9 cells are infected to obtain the protein expression gene containing African swine fever virus p7 Alternatively, the codon-optimized African swine fever virus p72 protein and pB602L are cloned into the EcoRI/HindIII site of the pFastBac1 transfer vector, respectively, and Bacmid-P72 and Bacmid-pB602L are obtained by cloning and transforming DH10Bac, and blue-white screening, and sf9 cells are infected to obtain the recombinant baculovirus rBac-p72 containing the African swine fever virus p72 subunit protein expression gene and the recombinant baculovirus rBac-pB602L containing the African swine fever virus pB602L subunit protein expression gene.

Preferably, the baculovirus expression system is a eukaryotic insect baculovirus expression system.

In a preferred technical solution of the present invention, in step 2), the method for expanding the culture of the recombinant baculovirus comprises:

The recombinant baculovirus was transfected into sf9 cells and cultured for 72 hours to obtain the P1 generation recombinant baculovirus;

The first-generation recombinant baculovirus was transfected into sf9 cells and cultured for 72 hours to obtain the P2-generation recombinant baculovirus;

Repeat the steps of transfecting sf9 cells to obtain the Pn generation recombinant baculovirus;

wherein n is a natural number;

The cells used for fermentation culture in step 2) are sf9 cells and/or HighFive5 cells with a cell density of 1×106-2.5×106/ml, and the virus used for infection is rBac-p72-pB602L, which is inoculated on sf9 or HighFive at an inoculation amount of 0.2-2 at a multiplicity of infection (MOI); or rBac-p72 and rBac-pB602L are inoculated on sf9 or HighFive at the same time at an inoculation amount of 0.1-1 at a multiplicity of infection (MOI).

Specifically, the fermentation reactors include 1L, 5L, 15L, 50L, and 200L, and the culture method adopts batch culture or batch fed-batch culture.

The fermentation broth and cell lysate in step 2) were collected and purified using a nickel column and an anti-flag affinity chromatography column. During the purification process, we unexpectedly found that when the purity of p72 trimer reached more than 75%, B602L was easily and effectively removed.

The recombinant p72 protein obtained by the method can be stably stored at 4°C and -20°C, and the protein is safe for immunizing animals and can produce high-titer p72 antibodies.

Compared with the prior art, the expression sequence, expression vector and corresponding purification preparation method disclosed in the present invention overcome the defects in the prior art and solve the problem that p72 protein cannot be directly expressed in large quantities and the yield is low. The present invention can directly express p72 in an insect baculovirus system, overcome many problems in the prior art, and has a simple preparation method, high expression amount and low cost.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the plasmid enzyme cleavage map; Figure 1A, pFastBacDual-p72-B602L plasmid enzyme cleavage map; Figure 1B, pFastBac1-p72 plasmid enzyme cleavage map; Figure 1C, pFastBac1-B602L plasmid enzyme cleavage map.

Figure 2 shows the results of plasmid double enzyme digestion identification: M is DNA Marker: DL10000 Marker; Figure 2A, 1-3 are pFastBacDual-p72-B602L plasmid HindIII/EcoRI digestion, the band sizes are 6756bp and 2044bp respectively, the plasmid digestion is correct and the digestion identification construction is correct; Figure 2B, 1-4 are pFastBac1-p72 plasmids 1-4 EcoRI/HindIII double digestion, the band sizes are 4693bp and 1959bp respectively, the plasmid digestion is correct; Figure 2C, 1-2 are pFastbac1-B602L plasmid HindIII-/EcoRI double digestion, the band sizes are 4963bp and 1723bp respectively, the plasmid digestion is correct.

Figure 3 shows the western-blot results of sf9 cells infected with baculovirus expressing recombinant p72. M is a protein marker, 1 is the supernatant of sf9 cells not infected with baculovirus; 2 is the supernatant of cell lysis infected with rBac-p72 alone, with very low expression and only weak expression; 3 is the supernatant of sf9 cells co-infected with rBac-p72 and rBac-B602L; 4 is sf9 cells infected with rBac-p72-B602L. The expression level in the supernatant of cell lysis infected with rBac-p72 alone is very low and only weak expression, and the expression level in sf9 cells co-infected with rBac-p72 and rBac-B602L is higher, indicating that the expression of p72 is promoted in the presence of B602L.

Figure 4 shows the molecular sieving results of recombinant p72: the peak volume of peak 1 is 8.69 ml, the peak volume of peak 2 is 10.57 ml, the molecular weights are both greater than 670 kDa, and they are the purified p72 multimers; the peak volume of peak 3 is 15.39 ml, and the molecular weight is between 158 kDa and 440 kDa, and it is the purified p72 trimer (the size of p72 trimer is about 232 kDa).

Figure 5a shows the SDS-PAGE purification test results of recombinant p72: 1 is the protein marker, 2 is the purified p72 protein.

Figure 5b shows the SDS-PAGE purification detection results of recombinant p72 and B602L: 1 is the protein marker, 2 is the purified p72 protein, and 3 is the purified B602L protein.

Figure 6 shows the results of Western-blot detection after p72 protein purification: 1 is protein marker, 2 is p72 protein.

DETAILED DESCRIPTION

The present invention will be further described below in conjunction with the accompanying drawings and embodiments. The embodiments of the present invention are only used to illustrate the technical solutions of the present invention, but are not intended to limit the present invention.

The strains, plasmids and reagents used in the examples of the present invention are all commercially available products.

Example 1 Expression and preparation of p72 protein

1.1 Selection of African swine fever p72 protein

The structural protein p72 of African swine fever is a polypeptide encoded by the B646L gene. Studies have shown that the correct folding of the p72 protein requires the participation of the pB602L protein. If the pB602L protein is missing, the expression of the p72 protein will be significantly reduced. Existing reports do not show that the p72 protein can be effectively expressed and produced at a high level. There is currently no report on the large-scale soluble expression and purification of the protein in a rod-shaped expression system, which is also an important technical problem to be solved by the present invention.

1.2 Codon optimization of African swine fever p72 and pB602L proteins

Our laboratory used the subtype of African swine fever virus strain and the complete genome sequence of Georgia 2007/1 (GenBank: FR682468.1) as a template to optimize the nucleotide sequence of B602L encoding the African swine fever p72 and pB602L proteins, and obtained the OPTI-p72 sequence and OPTI-pB602L sequence, as shown in SEQ ID NO.1 and SEQ ID NO.4. For the convenience of subsequent detection and purification, a flag antibody tag was introduced at the N-terminus of P72 and an HA tag was introduced at the C-terminus of B602L. The sequence synthesis work was commissioned to Nanjing GenScript Biotechnology Co., Ltd.

1.3 Construction of pFastBacDual-p72-pB602L recombinant plasmid

1.3.1 PCR amplification of target fragment OPTI-p72

1.3.1.1 PCR reaction

(1) Primer design and synthesis

Upstream primer: 5'-CG GAATTC GACTACAAAGACCATGACGGT-3'

Downstream primer: 5'-GAC AAGCTT AGGTAGAGTACCTCAGCAC-3'

(2) Using the optimized OPTI-P72 as the template, add 50 μL of the sample system as shown in the following table:

PCR amplification procedure:

1.3.1.2 Gel recovery of PCR products

(1) Label the sample collection EP tube, adsorption column, and collection tube;

(2) Weigh the marked empty EP tube and record the value;

(3) Carefully cut the single target DNA band from the agarose gel using a scalpel and place it into a clean 1.5 mL centrifuge tube;

(4) Add 600 μL PC buffer to the 1.5 mL centrifuge tube in step (3) and place in a 50°C water bath for about 5 min, gently turning the centrifuge tube upside down to ensure that the gel block is fully dissolved;

(5) Column equilibration: Add 500 μL of equilibration solution BL to the adsorption column CB2 (the adsorption column is placed in the collection tube in advance), centrifuge at 12,000 rpm/min for 1 min, pour out the waste liquid in the collection tube, and put the adsorption column back into the collection tube;

(6) Add the solution obtained in step (5) to the adsorption column CB2, let it stand for 2 min, centrifuge at 10,000 rpm/min for 30 s, pour out the waste liquid in the collection tube, and then place the adsorption column CB2 into the collection tube;

(7) Add 600 μL of PW buffer to the adsorption column, let it stand for 3 min, centrifuge at 10,000 rpm/min for 30 s, pour out the waste liquid in the collection tube, and place the adsorption column CB2 in the collection tube;

(8) Repeat step (7);

(9) Centrifuge the empty adsorption column at 12,000 rpm/min for 2 min to remove as much rinse solution as possible, and place the adsorption column at room temperature for 10 min to dry thoroughly;

(10) Place the adsorption column CB2 in the collection tube, add 50 μL of Elution buffer (preheated at 65 °C) to the middle of the adsorption membrane, let it stand for 3 min, and centrifuge at 12,000 rpm/min for 2 min;

(11) Take out the centrifuge tube in step (10) from the centrifuge, discard the adsorption column CB2 in the middle, cover the centrifuge tube with a cap, and retain the DNA sample in the centrifuge tube;

(12) Store the DNA sample in step 11 at 4°C and prepare agarose gel electrophoresis to identify the recovered DNA fragments.

1.3.2 Double restriction enzyme digestion reaction of PCR products and vectors

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

(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 products: Take out the above double enzyme digestion system and perform agarose gel electrophoresis to recover the DNA fragments therein. The method is the same as the gel recovery of PCR products in 1.2.1.

1.3.3 Ligation reaction

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

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

(3) After adding samples according to the table in step (2), place each 10 μl reaction system in a 16°C low-temperature cooling liquid circulation machine and water bath for 10-16 hours;

(4) Take out the EP tube in step (3) and place it in a 65°C water bath for 15 min;

(5) Take out the EP tube from step (4) and store it at 4°C.

1.3.4 Conversion reaction

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

(2) Take out the sample tube, place it in a 42°C water bath for 100 seconds, and then immediately place it in an ice bath for 2 minutes;

(3) Take out the sample tube, add 600 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/min for 1 h;

(4) Plate coating: Take out the sample tube in step (3), centrifuge at 8,000 rpm/min 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, place the resuspended bacterial solution in 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.

(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.

(6) Observe the transformation results.

1.3.5 Plasmid extraction and double enzyme digestion identification

1.3.5.1 Plasmid extraction

(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 ampicillin resistance, and shake the culture overnight at 37°C and 220 rpm/min;

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

(3) Add 250 μL of plasmid extraction reagent P1 buffer to the EP tube in step (2) to thoroughly suspend the bacteria;

(4) Add 250 μL P2 buffer to the solution in step (3), immediately gently invert the centrifuge tube 5-10 times to mix, and let stand at room temperature for 2-4 minutes;

(5) Add 350 μL P3 buffer to the solution in step (4), and immediately gently invert the centrifuge tube 5-10 times to mix; let stand at room temperature for 2-4 minutes;

(6) Centrifuge the solution from step (5) at room temperature, 14,000 rpm/min, for 10 min;

(7) Transfer the supernatant solution from step (6) to the center of the adsorption column, centrifuge at room temperature, 12,000 rpm/min, 30 s, and discard the liquid in the collection tube;

(8) Add 500 μL of Buffer DW1 to the center of the adsorption column, centrifuge at room temperature, 12,000 rpm/min, 30 s, and discard the liquid in the collection tube;

(9) Add 500 μL of wash solution to the center of the adsorption column, centrifuge at room temperature, 12,000 rpm/min, 30 s, pour out the liquid in the collection tube, and repeat once;

(10) Empty adsorption column, centrifuge at room temperature, 12,000 rpm, 2 min.

(11) Place the adsorption column in a clean 1.5 mL centrifuge tube, add 30 μL of Elution Buffer to the center of the adsorption membrane, let stand at room temperature for 5 min, and centrifuge at room temperature, 12,000 rpm, for 2 min. Save the DNA solution in the tube.

1.3.5.2 Double enzyme digestion identification

(1) Label the 1.5mL EP tubes to be used and add samples according to the following table: 20μL reaction system

(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.

(3) The double enzyme digestion system sample in step (2) was subjected to agarose gel electrophoresis to check whether the inserted fragment size was correct; the correctly constructed plasmid was pFastBacDual-p72.

(4) Using the optimized OPTI-B602L as a template, according to the method of constructing pFastBacDual-p72 in 1.3 above, B602L can be easily cloned into the XhoI and KpnI restriction sites of pFastBacDual-p72 to construct the final vector pFastBacDual-p72-B602L. The schematic diagram of vector construction is shown in Figure 1. pFastBacDual-p72-B602L was digested and verified, and the experimental results are shown in Figure 2A: 1-3: pFastBacDual-p72-B602L plasmid was digested with HindIII/EcoRI, and the band sizes were 6756bp and 2044bp respectively. The plasmid was digested correctly and the digestion was confirmed to be correct.

(4) Select the clone with the correct insert and send it to a sequencing company for sequencing. The plasmid with the correct sequencing result will be saved for future use.

1.4 Construction of pFastBac1-p72 and pFastBac1-B602L vectors

Using the experimental method described in 1.3, it is easy to clone the optimized opti-p72 and opti-B602L genes into the EcoRI and HindIII restriction sites of pFastBac1, respectively, to construct pFastBac1-p72 and pFastBac1-B602L. The experimental results are shown in Figures 2B and 2C: Figure 2B, 1-4 is pFastBac1-p72 plasmid 1-4 EcoRI/HindIII double digestion, the band sizes are 4693bp and 1959bp, respectively, and the plasmid digestion is correct; Figure 2C, 1-2 is pFastbac1-PB602L plasmid HindIII-/EcoRI double digestion, the band sizes are 4963bp and 1723bp, respectively, and the plasmid digestion is correct.

1.5 Preparation of recombinant baculovirus rBac-p72-B602L expressing p72 protein

1) The vector pFastBacDual-p72-B602L expressing African swine fever p72 recombinant protein described in Example 1.3 was transformed into Escherichia coli DH10 Bac and cultured, and the positive plasmid containing the recombinant expression of p72 and B602L genes was screened and extracted and named Bacmid-p72-B602L.

2) The recombinant bacmid plasmid Bacmid-p72-B602L obtained in the previous step was transfected into sf9 cells using liposome transfection to obtain P1 recombinant baculovirus, named rBac-p72-B602L.

3) The same method can be used to obtain recombinant baculovirus rBac-p72 expressing p72 alone and rBac-B602L expressing B602L alone.

1.6 Western-blot detection of p72 expressed by recombinant baculovirus rBac-p72-B602L

The recombinant baculovirus rBac-p72-B602L or rBac-p72 or rBac-p72 and rBac-B602L prepared in 1.5 above were co-infected with sf9 cells at an MOI of 0.1, cultured for 72 hours, and the cells and supernatant were collected respectively. The cell and supernatant samples were subjected to SDS-PAGE electrophoresis, transferred to the membrane, blocked with 5% skim milk powder for 1 hour, rinsed with PBST for 3-5 times, diluted with anti-flag mouse monoclonal antibody at 1:5000, incubated on a shaker at room temperature for 2 hours, rinsed with PBST for 3-5 times, incubated with HRP-labeled secondary antibody goat anti-mouse at room temperature for 1 hour, rinsed with PBST for 3-5 times, and developed with supersensitive ECL luminescent liquid. The detection results are shown in Figure 3. The expression level in the supernatant of cell lysis infected with rBac-p72 alone was very low and only weak, while the expression level in the supernatant of cell lysis infected with rBac-p72-B602L and co-infected with rBac-p72 and rBac-B602L was higher, indicating that the expression of p72 was promoted in the presence of B602L.

1.7 Purification of African swine fever p72 protein

During the preparation of p72 purification, by optimizing the purification conditions, we unexpectedly found that when the purity of p72 trimer reached more than 75%, the content of B602L would be greatly reduced and B602L could not be detected on SDS-PAGE.

The recombinant baculovirus obtained in 1.6 was used to infect High FIVE cells at an MOI of 1. After culturing for three days, the virus supernatant and cells were harvested.

1.7.1 Purification

Cell disruption: 100 g of harvested cells (thawed in advance) were added to 1000 ml of Buffer A according to the ratio (1 g of cells: 10 mL of lysis buffer) and resuspended. After stirring and mixing at 2-8°C (no solid blocks) for 1 hour, centrifuged at 25,000 × g (4°C) for 10 minutes, and the supernatant was taken as the sample. The harvested cell supernatant was filtered through a 0.45 μm filter membrane and also used as the sample.

1) Column equilibration: Take 50 ml of anti-DYKDDDDK G1 affinity filler and load it into an empty chromatography column. Use Buffer A to equilibrate for 2 to 3 column volumes (CV) at a flow rate of 10 mL/min.

2) Load the supernatant after cell disruption or filtered culture supernatant at a flow rate of 2 mL/min and collect the flow-through

3) Wash the endotoxin with 30CV of Washing buffer for at least 2 hours.

4) Equilibrate and wash the column with 15CV of Buffer A.

5) Elution: Use Buffer B to elute the protein at a flow rate of 15 ml/min. Immediately after elution, neutralize with neutralizing solution (1 ml of elution solution: 15 μl of neutralizing solution).

6) Dialysis: Add 5% glycerol (mass-volume ratio) to the eluted sample for dialysis.

7) Sterile filtration In a biological safety cabinet, filter through a 0.22 μm low protein binding syringe filter, or filter a large amount of protein solution through a sterilized 0.22 μm filter membrane Nalgene filter, and store the filtered protein solution samples in a -80°C refrigerator.

Protein concentration and purity determination: The protein concentration was determined by the BCA method, and the protein yield was calculated based on the volume of the supernatant taken during purification and the total amount of protein obtained after purification. For example, in this embodiment, the cell supernatant taken was 1000 ml, the volume of protein obtained after purification was 100 ml, and the concentration was 1 mg/mL. The protein yield was calculated to be about 100 mg/L; the purity was detected by the SDS-PAGE method, and the purity could reach more than 90%, as shown in Figure 4. It is suitable for large-scale production.

Buffer A: 50 mM Tris, 500 mM NaCl. Dissolve completely in ultrapure water, adjust pH to 8.0 ± 0.01, filter through 0.8 μm membrane and store at 2-8 °C.

Washing buffer: 50mM Tris, 500mM NaCl. Dissolve completely with ultrapure water, adjust pH to 8.0±0.01, filter through 0.8μm membrane and store at 2-8℃.

Buffer B: 100 mM glycine, 500 mM NaCl. Dissolve completely in ultrapure water, adjust pH to 3.5 ± 0.01, filter through 0.8 μm membrane and store at 2-8 °C.

Neutralization solution: 1M Tris-HCl. After completely dissolving with ultrapure water, adjust the pH to 9.0±0.01, filter through a 0.8μm membrane, and store at 2-8°C.

Dialysis mother solution: 50 mM Tris, 500 mM NaCl, after completely dissolving with ultrapure water, adjust the pH value to 8.0 ± 0.01, filter through 0.8 μm membrane and store at 2-8 °C.

Dialysate: 50 mM Tris, 500 mM NaCl, 10% glycerol. After completely dissolving with ultrapure water, adjust the pH to 8.0 ± 0.01, filter through a 0.8 μm membrane, and store at 2-8°C.

1.7.2 Molecular sieve column detection

1.7.2.1 SuperoseTM 6Increase 10/300GL column equilibration

Balance 2 column volumes with ultrapure water and drain out the ethanol preservation solution; then balance 2 column volumes with the mobile phase at a flow rate of 0.4 mL/min and a pressure control within 1.8 MPa.

1.7.2.2 Sampling

0.5 mL of protein (concentration 0.5 mg/mL) was injected using an injection loop, the flow rate was 0.4 mL/min, and the pressure was controlled at 1.8 MPa.

1.7.2.3 Operation

After the injection is completed, change the inject state to the load state, run, the flow rate is 0.4mL/min, and collect samples after the peak, 1mL/tube.

The molecular sieve results are shown in Figure 4: From the protein molecular sieve peak results, it can be seen that the peak volume of peak 1 is 8.69 ml, the peak volume of peak 2 is 10.57 ml, and the molecular weight is greater than 670 kDa, which is the purified p72 polymer; the peak volume of peak 3 is 15.39 ml, and the molecular weight is between 158 kDa and 440 kDa, which is the purified p72 trimer (the size of p72 trimer is about 232 kDa).

As can be seen from the figure, the percentage of peak 3 area (49.8966) to the total area (58.3792) is 85%, which means that 85% of the purified p72 protein is a trimer before further optimization of the buffer system, which is consistent with the predicted analysis.

1.8 Identification of African swine fever p72 protein

1.8.1 SDS-PAGE detection

The protein purified in Example 1.7 was subjected to SDS-PAGE detection. The p72 protein concentration in the sample used was 2 μg/well. The results are shown in Figure 5a: It can be calculated from the figure that the SDS-PAGE purity of the purified p72 protein is 85%, and the molecular weight is about 97 kD.

1.8.2 Western Blot Detection

The protein purified in Example 1 was subjected to WESTERN-BLOT detection, the transfer time was 1 hour, the antibody used was Antiflag-Tag Mouse, the dilution ratio was 1:2000, the incubation time was 90 minutes, and the results are shown in Figure 6: It can be seen from the results in the figure that the purified p72 protein can effectively bind to the antibody.

1.8 Immunogenicity experiment of recombinant p72 protein

1.8.1 Vaccine preparation

1.8.1.1 Prepare in the ratio of oil adjuvant:water phase (v:v) = 54:46.

1.8.1.2 Antigen preparation: The purified recombinant African swine fever p72 protein is sterilized and filtered through a 0.22μm filter membrane, and the concentration and purity are tested for later use.

1.8.1.3 Preparation of aqueous phase: Dilute the p72 protein to an appropriate concentration using 1XPBS according to the p72 protein content in the vaccine and stir for 10 minutes to mix it thoroughly.

1.8.1.4 Preparation of oil phase: According to the water-oil ratio in 1.9.1.1, measure an appropriate amount of ISA 201VG adjuvant.

1.8.1.5 Emulsification: Emulsification requires the oil phase temperature to be 33±1℃. Turn on the agitator at a speed of 350rpm/min. Add the water phase to the oil phase at a uniform speed under stirring conditions and continue stirring for 10 minutes to allow the water phase and oil phase to be fully mixed and emulsified into a two-way oil emulsion vaccine.

1.8.1.6 Stabilization: After emulsification, turn off the agitator and place the emulsified vaccine at 20℃ to stabilize for 1 hour.

1.8.1.7 Packaging and storage: Pack according to immunization requirements and store at 2-8℃ for future use after passing the inspection.

1.8.2 Immunogenicity test

1.8.2.1 Immunity experiments

Take 10 healthy New Zealand white rabbits of about 6 weeks old and 2-3 kg, randomly divide them into 2 groups, 5 in each group, and use the vaccine prepared in 1.9.1 for immunization experiment. Five rabbits in the experimental group are immunized; 5 rabbits in the control group are immunized with PBS. Blood is collected before immunization, 14 days after the first immunization, and 14 days after the second immunization, and the serum is separated for ELISA detection of antibodies.

1.8.2.2ELISA test

(1) Coating: Dilute the purified p72 protein to 0.5 μg/ml with coating solution (50 mM carbonate buffer, pH 9.5), add 100 μl/well to a 96-well plate, seal with sealing film and place in a refrigerator at 4°C overnight;

(2) Washing: After taking the ELISA plate out of the refrigerator, wash the plate 5 times with PBST;

(3) Blocking: Add 200 μl of blocking solution (5% skim milk) to each well, seal with sealing film and incubate at 37°C for 2 h;

(4) Washing: Same as 2;

(5) Serum dilution: The pre-immunization serum, the serum 14 days after the first immunization, and the serum 14 days after the second immunization of the rabbits immunized with p72 protein were diluted with blocking solution. The pre-immunization serum was diluted 100 times (10 μl serum + 990 μl serum diluent), and the post-immunization serum was diluted 5000 times (first 100 times (10 μl serum + 990 μl serum diluent), and then 50 times (20 μl (100x) + 980 μl serum diluent);

(6) Loading 2x dilution samples: Take the ELISA plate from step 4, take 200l of diluted serum and add it to the first well in the direction of protein coating (vertical dilution is added to the first horizontal row of wells (1 to 12 wells), horizontal dilution is added to the first vertical column of wells (A to H wells)), and add 100l of serum diluent to each of the remaining wells. Then use a multichannel pipette to take 100ml of sample from the first well and add it to the second well to mix, and then dilute in turn. Vertical dilution stops at well G, and horizontal dilution stops at well 11 (after well G and well 11 are mixed, take 100l of diluted sample and discard; well H in the vertical direction and wells 12 in the horizontal direction are blank control wells)

(7) Washing: same as (2);

(8) Sample addition: Add diluted serum and use blocking solution as a negative control, incubate at 37°C for 1 h;

(9) Washing: same as (2);

(10) Add secondary antibody: Add 100 μl of diluted HRP-labeled goat anti-rabbit IgG secondary antibody (dilution ratio 1:5000) to each well and incubate at 37°C for 0.5 h;

(11) Washing: same as (2);

(12) Color development: Add 100 μl of TMB color development solution to each well in the dark and incubate at 37°C for 10 min.

(13) Termination: Add 50 μl of stop solution (2M H ₂ SO ₄ ) to each well to terminate the reaction;

(14) Detection: Measure the OD value of the sample at a wavelength of 450 nm and analyze the data;

(15) The results are shown in the following table: the coated p72 protein can specifically bind to the serum of rabbits immunized with p72 protein, and the antibody titer before immunization and the control group is 1:50-200; 14 days after the first immunization, the antibody titer of the immunized group increased significantly to 1:25600-1:51200, and 14 days after the second immunization, the antibody titer of the immunized group was 1:128000-1:512000. This shows that p72 protein can be used as an antigen for Elisa kits, and the serum after immunization can specifically bind to p72 protein, laying the foundation for the later development of a diagnostic kit for detecting African swine fever infection and immunity and as a candidate antigen for subunit vaccine.

The present invention is illustrated by the above examples, however, it should be understood that the present invention is not limited to the specific embodiments and embodiments described herein. The purpose of including these specific embodiments and embodiments here is to help those skilled in the art to practice the present invention. Any person skilled in the art will readily make further improvements and perfections without departing from the spirit and scope of the present invention, and therefore the present invention is only limited by the content and scope of the claims of the present invention, which is intended to cover all alternatives and equivalents included in the spirit and scope of the present invention as defined by the appended claims.

Claims (6)

1. A method for preparing recombinant African swine fever virus p72 trimer subunit protein, characterized in that the preparation method includes the following steps: 1) Preparation of recombinant baculovirus: The codon-optimized African swine fever virus p72 protein and the codon-optimized African swine fever virus pB602L protein expression gene are cloned, transformed, and infected into sf9 cells and/or HighFive5 cells, to Obtain a recombinant baculovirus containing the p72 subunit protein expression gene of African swine fever virus; 2) Fermentation culture: the recombinant baculovirus is expanded and cultured, and then fermented, and the culture fluid is collected; 3) Subunit protein purification: Purify the culture medium described in step 2) of the fermentation culture, and obtain the recombinant African swine fever virus p72 subunit soluble protein after purification; Wherein, the amino acid sequence of the African swine fever virus p72 protein is shown in SEQ ID NO.3, and the amino acid sequence of the African swine fever virus B602L protein is shown in SEQ ID NO.6. 2. The preparation method according to claim 1, characterized in that, in step 1), the codon-optimized African swine fever virus p72 protein and the codon-optimized African swine fever virus pB602L protein expression gene After cloning, transforming, and infecting sf9 cells and/or HighFive5 cells, the codon-optimized African swine fever virus p72 protein and the codon-optimized African swine fever virus pB602L protein expression gene are simultaneously cloned into the pFastBac1 transfer vector. After cloning, Transform and infect sf9 cells and/or HighFive5 cells, and then ferment, culture and purify to obtain the recombinant baculovirus rBac-p72-pB602L containing the African swine fever virus p72 subunit protein expression gene. 3. The preparation method according to claim 1, characterized in that, in step 1), the codon-optimized African swine fever virus p72 protein and the codon-optimized African swine fever virus pB602L protein expression gene After cloning, transforming, and infecting sf9 cells and/or High Five5 cells, the codon-optimized African swine fever virus p72 protein and the codon-optimized African swine fever virus pB602L were cloned into the pFastBac1 transfer vector respectively. After cloning, transformation, and Infect sf9 cells to obtain recombinant baculovirus rBac-p72 containing the expression gene of African swine fever virus p72 subunit protein and recombinant baculovirus rBac-pB602L containing the expression gene of African swine fever virus pB602L subunit protein. 4. The preparation method according to claim 1, characterized in that, in step 2), the method for expanding the culture of the recombinant baculovirus includes: The recombinant baculovirus was transfected into sf9 cells and cultured for 72 hours to obtain the P1 generation recombinant baculovirus; The first-generation recombinant baculovirus was transfected into sf9 cells and cultured for 72 hours to obtain the P2-generation recombinant baculovirus; Repeat the steps of transfecting sf9 cells to obtain Pn generation recombinant baculovirus; Where n is a natural number; The cells fermented and cultured in step 2) are sf9 cells and/or HighFive5 cells, the cell density is 1×106-2.5×106/ml, the virus used for infection is rBac-p72-pB602L, according to the multiplicity of infection (MOI): Inoculate 0.2-2 onto sf9 or HighFive; or rBac-p72 and rBac-pB602L, inoculate onto sf9 or HighFive at the same time according to an inoculation volume with a multiplicity of infection (MOI) of 0.1-1. 5. The preparation method according to claim 1, characterized in that, the African swine fever virus p72 subunit protein is connected to poly-His, A tag among FLAG, c-myc, and Strep-tagⅡ. 6. Application of the African swine fever virus p72 subunit protein obtained according to the preparation method of any one of claims 1 to 5 in the preparation of diagnostic reagents for the African swine fever p72 protein or the African swine fever p72 subunit vaccine.