Preparation method and application of porcine circovirus type 2 virus-like particles

Abstract

The invention provides a preparation method and application of porcine circovirus type 2 Cap protein virus-like particles. The preparation method comprises the following steps: 1) preparing a porcine circovirus type 2 Cap protein solution; the porcine circovirus type 2 Cap protein solution is a monomer and / or oligomer of Cap protein, and the amino acid sequence of the porcine circovirus type 2 Cap protein solution is as shown in SEQ ID NO.2; and 2) self-assembling in a slightly acidic buffer solution to form VLPs. The preparation method provided by the invention has the following advantages: 1) protein soluble expression is realized, renaturation is not needed, and VLP particles can be obtained in a slightly acidic buffer solution; and 2) high-purity uniform VLP particles can be obtained without complex operations such as gradient centrifugation, and large-scale production and purification application are facilitated.

Description

Technical Field

[0001] The invention belongs to the technical field of veterinary biological products and relates to a method for preparing porcine circovirus type 2 virus-like particles. Background Art

[0002] Porcine circovirus 2 (PCV2) is a major member of the genus Circovirus in the family Circoviridae. It is the main pathogen causing postweaning multisystemic wasting syndrome (PMWS), porcine respiratory disease syndrome (PRDC), reproductive failure, congenital tremor, porcine dermatitis and nephropathy syndrome (PDNS), etc. The disease was first reported in Canada. PMWS is also known as porcine circovirus disease (PCVD) in Europe and porcine circovirus associated disease (PCVAD) in America. The mortality rate ranges from 10% to 40%. Since it first occurred in Canada in 1991, it has spread rapidly to pig farms in various countries and has become one of the important diseases in the world's pig industry, causing serious economic losses to the pig industry.

[0003] PCV2 is the smallest virus discovered so far. The virus particle has a simple structure, with only nucleic acid and capsid protein, but no envelope. The Cap protein encoded by its ORF2 is the only structural protein of the virus. Studies have shown that PCV2 Cap protein can self-assemble to form virus-like particles (VLPs). The VLPs do not contain viral nucleic acid, but do have a capsid structure similar to that of the virus, and can act as an antigen to induce an immune response in the body, thereby producing antibodies.

[0004] Virus-like particles (VLPs) are hollow particles with a similar structure to natural viruses that are self-assembled in vitro by recombinant viral capsid proteins using recombinant expression methods. VLP is an ideal recombinant protein antigen for preparing viral vaccines. It has the following advantages: ① It does not contain viral nucleic acid and has no infection risk of live attenuated virus vaccine or inactivated virus vaccine; ② It is highly similar to the external structure of natural viruses and therefore has similar immunogenicity to viral particles; ③ It has the activity of pathogen-associated molecular patterns (PANP) and can activate innate immune responses; ④ Its morphology and size are conducive to the uptake of antigen-presenting cells, especially dendritic cells; ⑤ It can be presented by MHC class I and class II molecules to activate CD4+ helper T cells and CD8+ cytotoxic T lymphocytes; ⑥ It can activate B cells to produce high-titer antibodies and stimulate T cell-independent IgM responses; ⑦ It can be used to deliver Toll-like receptor (TLR) agonists and T cell epitopes; ⑧ It can have adjuvant function itself; ⑨ It can be used as a VLP display platform to present other antigens.

[0005] CN201911246097.3 discloses a VLP of a truncated porcine circovirus PCV2 Cap protein, which is expressed in Escherichia coli to obtain inclusion bodies, which are then renatured and replaced with liquid and then concentrated to obtain VLPs. The process is complex, requires renaturation, and the VLPs obtained are of uneven size.

[0006] CN201910161371.0 discloses a VLP of the full-length Cap of porcine circovirus subtype 2d, which is soluble expressed in Escherichia coli, precipitated by saturated ammonium sulfate, and then subjected to continuous sucrose density gradient centrifugation to obtain a crude Cap protein, and then purified Cap protein VLP is obtained by a monoclonal antibody column. This method requires gradient centrifugation and purification by a monoclonal antibody column, which is complex and not suitable for industrial production.

[0007] The inventors of the present invention have found that the purification methods for porcine circovirus type 2 virus-like particles in the prior art often use precipitation method, gradient centrifugation, ion exchange chromatography, hydrophobic chromatography, molecular exclusion chromatography, etc., which have the problems of cumbersome steps, complex processes, high production costs, low purification efficiency, and low assembly efficiency of virus-like particles. In addition, the quality of VLPs is uneven and VLPs aggregates are often present, which has a certain impact on the quality control of large-scale vaccine production in the later stage. Summary of the invention

[0008] In order to solve the above technical problems of the prior art, the inventors of the present invention unexpectedly discovered that self-assembly in a slightly acidic buffer system has higher efficiency, and its assembly efficiency is inversely proportional to the pH value. The lower the pH value, the higher the assembly efficiency, but the higher the aggregation degree of VLPs. The present invention provides a method for preparing porcine circovirus type 2 Cap protein virus-like particles, the preparation method comprising the following steps:

[0009] 1) preparing a porcine circovirus type 2 Cap protein solution; and

[0010] 2) Autonomous assembly to form VLPs in slightly acidic buffer;

[0011] Wherein, the porcine circovirus type 2 Cap protein solution in step 1) is a monomer and / or oligomer solution of the Cap protein, and the amino acid sequence of the Cap protein is shown in SEQ ID NO.2.

[0012] In the preferred technical solution of the present invention, in step 1), the purity of the porcine circovirus type 2 Cap protein solution is not less than 80%, and the Cap protein concentration is not higher than 37 mg / ml. The amount of the monomer and / or oligomer of the Cap protein in the total Cap protein is preferably not less than 80%.

[0013] In a preferred technical solution of the present invention, the porcine circovirus type 2 Cap protein has a tag selected from the group consisting of poly-His, FLAG, c-myc, and HA connected to the amino terminus or carboxyl terminus of the amino acid sequence shown in SEQ ID NO.2.

[0014] In a preferred technical solution of the present invention, the method for preparing the porcine circovirus type 2 Cap protein solution comprises the following steps:

[0015] 4-1) cloning the coding gene sequence of porcine circovirus type 2 Cap protein as shown in SEQ ID NO.1 after codon optimization into a prokaryotic expression vector to obtain a recombinant plasmid containing the porcine circovirus type 2 Cap protein coding gene;

[0016] 4-2) transforming the recombinant plasmid containing the porcine circovirus type 2 Cap protein encoding gene into competent Escherichia coli cells to obtain a recombinant expression strain;

[0017] 4-3) culturing and screening the recombinant expression strains in step 2) to obtain highly expressing strains;

[0018] 4-4) fermenting and culturing the highly expressing strain described in step 3), and inducing with IPTG to obtain bacteria that highly express Cap protein; and

[0019] 4-5) Take the bacterial cells in step 4), resuspend them in a lysis buffer, break them by high-pressure homogenization, collect the supernatant by centrifugation, and purify them by affinity chromatography to obtain a high-purity Cap protein eluate.

[0020] In a preferred technical solution of the present invention, in step 4-1), the prokaryotic expression vector is pET28a.

[0021] In a preferred technical solution of the present invention, in step 4-2), the Escherichia coli strain is selected from one of BL21 (DE3), BL21 star (DE3), and Arctic Express (DE3).

[0022] In a preferred technical solution of the present invention, in step 4-5), the affinity chromatography is one of nickel affinity chromatography, Flag affinity chromatography, and Strep column affinity chromatography. More preferably, the affinity chromatography is nickel affinity chromatography.

[0023] In the preferred technical scheme of the present invention, it also includes adding saturated ammonium sulfate to the purified Cap protein eluate in step 4-5) to precipitate the Cap protein; then adding a slightly acidic buffer to the Cap protein precipitate to redissolve it to obtain a Cap protein solution; wherein the ammonium sulfate saturation is 30-50%. More preferably, the ammonium sulfate saturation is 40%, wherein the ammonium sulfate precipitation is mainly for concentrating and removing imidazole in the eluate to increase the protein concentration; membrane exchange can also be used for concentration, removal of imidazole and increase of protein concentration.

[0024] In a preferred technical solution of the present invention, the slightly acidic buffer is selected from phosphate-citrate buffer and phosphate-carbonate buffer. More preferably, the slightly acidic buffer is phosphate-citrate buffer.

[0025] In a preferred technical solution of the present invention, in step 2), the slightly acidic buffer is a buffer with a pH of 3.5-6.5. More preferably, the slightly acidic buffer is a buffer with a pH of 5.0-6.0.

[0026] According to another aspect of the present invention, the present invention also provides a use of porcine circovirus type 2 virus-like particles obtained by the preparation method of porcine circovirus type 2 virus-like particles in the preparation of vaccines for diagnosing, preventing and treating porcine circovirus.

[0027] Compared with the prior art, the preparation method of the present invention has the following beneficial effects: 1) the protein is expressed soluble and no renaturation is required, and VLP particles can be obtained in a slightly acidic buffer; 2) high-purity and uniform VLP particles can be obtained without complex operations such as gradient centrifugation, which is convenient for large-scale production and purification applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the molecular sieve result of the sample after elution with nickel column;

[0029] Figure 2 The molecular sieve result is pH=6.5;

[0030] Figure 3 The molecular sieve result is pH=6.0;

[0031] Figure 4 The result is pH = 5.5 molecular sieve;

[0032] Figure 5 The molecular sieve result is pH = 5.0;

[0033] Figure 6 The result is pH = 4.5 molecular sieve;

[0034] Figure 7 The molecular sieve result is pH = 4.0;

[0035] Figure 8 The molecular sieve result is pH=3.0;

[0036] Fig. 9 is the molecular sieve result of sample 1;

[0037] Fig.10 is the molecular sieve result of sample 2;

[0038] Fig.11 is the molecular sieve result of sample 3;

[0039] Fig.12 is the molecular sieve result of sample 4;

[0040] Fig.13 is the molecular sieve result of sample 5;

[0041] Fig.14 is the molecular sieve result of sample 6;

[0042] Fig.15 The molecular sieve results are for 150 mM NaCl;

[0043] Fig.16 The molecular sieve results are for 500 mM NaCl;

[0044] Fig.17 Transmission electron microscopy was used to observe the formation of virus-like particles. DETAILED DESCRIPTION

[0045] 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.

[0046] The strains, plasmids and reagents used in the examples of the present invention are all commercially available products.

[0047] Example 1: Preparation method of porcine circovirus type 2 Cap protein

[0048] The preparation method of the Cap protein used in the present invention mainly refers to Journal of Biotechnology 220 (2016) 78-85, which is as follows:

[0049] 1.1. Selection of Cap protein sequence of porcine circovirus type 2

[0050] The cap protein, a surface structural protein of porcine circovirus, consists of 233 amino acids in total, and the 1-40 amino acids at its N-terminus are an arginine-rich nuclear localization signal peptide sequence. In order to better express the cap protein, we chose to express 41 (K)-233 (P) aa as shown in SEQ ID NO.2 for recombinant protein design.

[0051] 1.2. Codon optimization and construction of porcine circovirus type 2 Cap protein

[0052] The nucleic acid sequence encoding the cap protein was codon-optimized and synthesized as shown in SEQ ID NO.1. The synthesized sequence was subcloned into the NcoI and XhoI restriction sites of pET28a. The construction method was based on the third edition of Molecular Cloning Experiment Guide (translated by Huang Peitang et al., Science Press). The constructed recombinant plasmid was named pET28a-opti-Cap.

[0053] 1.3. Expression of Porcine Circovirus Type 2 Cap Protein

[0054] The recombinant plasmid pET28a-opti-Cap was transformed into competent Escherichia coli, and a strain efficiently expressing the Cap protein was obtained by pressure resistance screening. The strain was amplified and cultured, and the bacteria were obtained by IPTG induction.

[0055] 1.4. Purification of Porcine Circovirus Type 2 Cap Protein

[0056] 1.4.1 Solution preparation

[0057] (1) Lysis buffer: 50 mM NaH2PO4, 500 mM NaCl, 20 mM imidazole, 0.5% Triton X-114, 0.05% Tween-20, pH 8.0.

[0058] (2) Wash Buffer: 50mM NaH2PO4, 500mM NaCl, 20mM imidazole, 1.0% Triton X-114, 0.05% Tween-20, pH 7.4.

[0059] (3) Buffer A: 50mM NaH2PO4, 500mM NaCl, 0.05% Tween-20, pH 7.4.

[0060] (3) Buffer B: 50mM NaH2PO4, 500mM NaCl, 60mM imidazole, 0.05% Tween-20, pH 7.4.

[0061] (5) Buffer C: 50 mM NaH2PO4, 500 mM NaCl, 500 mM imidazole, 10% glycerol, 0.05% Tween-20, pH 6.5.

[0062] 1.4.2. Resuspension and disruption of bacterial cells

[0063] Weigh a certain amount of bacteria, resuspend them in lysis buffer, break them with a homogenizer, and collect the supernatant by centrifugation.

[0064] 1.4.3. Nickel column purification

[0065] (1) Column equilibration: Take the nickel affinity filler and load it into an empty chromatography column. Equilibrate it with ultrapure water for 2 to 3 CV (column volume), drain the concentrated ethanol storage solution, and then equilibrate it with the lysis solution for 2 to 3 CV.

[0066] (2) Loading: Load the supernatant after lysis, set an appropriate flow rate according to the volume of the nickel column, and collect the flow-through.

[0067] (3) Wash: Wash the column with 50 column volumes (CV) of Wash buffer.

[0068] (4) Washing: Wash the column with 20 column volumes (CV) of Buffer A.

[0069] (5) Washing: Elute the impurities with Buffer B and collect the flow-through until no blue color appears when detected by Coomassie Brilliant Blue G250.

[0070] (6) Elution: Elute the target protein with Buffer C and collect the effluent until there is no blue color in the Coomassie Brilliant Blue G250 test. The collected effluent is the Cap protein eluate. The molecular sieve results are as follows: Figure 1 shown.

[0071] Example 2: Purification and concentration of Cap protein

[0072] 1. Ammonium sulfate precipitation method

[0073] Solution preparation

[0074] Saturated ammonium sulfate: add 800g of ammonium sulfate per liter of cooled ultrapure water, stir until it no longer dissolves, and then store at 2-8°C until crystals precipitate. The supernatant is the saturated ammonium sulfate solution.

[0075] Ammonium sulfate precipitation: add saturated ammonium sulfate solution to the Cap protein solution in a dropwise manner to make the saturation of ammonium sulfate reach 40%. The Cap protein precipitates and the precipitate is collected by centrifugation.

[0076] 2. Dialysis fluid exchange

[0077] The Cap protein eluate obtained in Example 1 was poured into a dialysis bag, and dialyzed at least 1000 times with 1×PBS; and then concentrated by ultrafiltration to obtain a Cap protein concentrate.

[0078] Example 3: Effect of pH on VLPs assembly

[0079] 3.1. Solution preparation

[0080] (1) Reconstitution solution 1: 50 mM Na2HPO4, 150 mM NaCl, 0.05% Tween-20, 10% glycerol, 0.05% Tween-20, pH 3.0.

[0081] (2) Reconstitution solution 2: 50 mM Na2HPO4, 150 mM NaCl, 0.05% Tween-20, 10% glycerol, 0.05% Tween-20, pH 4.0.

[0082] (3) Reconstitution solution 3: 50 mM Na2HPO4, 150 mM NaCl, 0.05% Tween-20, 10% glycerol, 0.05% Tween-20, pH 4.5.

[0083] (4) Reconstitution solution 4: 50 mM Na2HPO4, 150 mM NaCl, 0.05% Tween-20, 10% glycerol, 0.05% Tween-20, pH 5.0.

[0084] (5) Reconstitution solution 5: 50 mM Na2HPO4, 150 mM NaCl, 0.05% Tween-20, 10% glycerol, 0.05% Tween-20, pH 5.5.

[0085] (6) Reconstitution solution 6: 50 mM Na2HPO4, 150 mM NaCl, 0.05% Tween-20, 10% glycerol, 0.05% Tween-20, pH 6.0.

[0086] (7) Reconstitution solution 7: 50 mM Na2HPO4, 150 mM NaCl, 0.05% Tween-20, 10% glycerol, 0.05% Tween-20, pH 6.5.

[0087] (8) Reconstitution solution 8: 50 mM Na2HPO4, 150 mM NaCl, 0.05% Tween-20, 10% glycerol, 0.05% Tween-20, pH 7.0.

[0088] (9) Reconstitution solution 9: 50 mM Na2HPO4, 500 mM NaCl, 0.05% Tween-20, 10% glycerol, 0.05% Tween-20, pH 5.5.

[0089] 3.2. Redissolution: Take the ammonium sulfate precipitate and use resolution solution 1 to resolution solution 8 respectively for redissolution. Centrifuge and take the supernatant as the purified virus-like particles. The results are shown in Table 1. The molecular sieve results are shown in Table 1. Figure 2-8 shown.

[0090] Table 1

[0091]

[0092]

[0093] From Table 1 and Figure 2-8 It can be seen that pH is the main factor affecting the formation of VLPs by Cap. From pH 6.5 to 3.0, the lower the pH, the higher the proportion of VLPs formed, but the lower the pH, the higher the proportion of VLPs aggregates formed.

[0094] 3.3 Molecular sieve: Take the ammonium sulfate precipitate and re-dissolve it with different volumes of re-solution 5 to obtain re-solutions of different concentrations. The samples were injected for molecular sieve detection. The results are shown in Table 2. The molecular sieve results are as follows: Figure 9-14 shown.

[0095] Table 2

[0096] Serial number Protein concentration (mg / ml) VLPs aggregates VLPs monomer Protein monomers or oligomers 1 37 10.35％ 82.44％ 7.21％ 2 9.0 9.00％ 65.44％ 25.55％ 3 4.1 5.03％ 50.57％ 44.40％ 4 2.8 4.96％ 40.14％ 54.90％ 5 1.5 3.35％ 23.99％ 72.66％ 6 0.4 0％ 14.27％ 85.73％

[0097] From Table 2 and Figure 9-14 It can be seen that the concentration also affects the formation of VLPs by Cap. The higher the concentration, the higher the proportion of VLPs formed and the proportion of VLPs aggregates is also relatively higher.

[0098] Example 4: Effect of Purity on VLPs

[0099] According to the method of Example 2, different batches of Cap protein concentrates with different purities were obtained, and a Cap protein solution with a concentration of 9 mg / ml was obtained using reconstitution solution 5 to investigate the effects of different protein purities on VLPs assembly, as shown in Table 3 below.

[0100] Table 3

[0101] SDS-PAGE purity Molecular sieve analysis of VLPs proportion 90％ 68.26& 80％ 65.44％ 60％ 31.12％ 40％ 10.05

[0102] It can be seen from Table 3 that when the purity is above 80%, the effect on the assembly of VLPs is not very large. When the purity is below 80%, the assembly rate drops sharply as the purity decreases.

[0103] Example 5: Effect of salt concentration on VLPs

[0104] The ammonium sulfate precipitate was re-dissolved with the re-solution 6 and re-solution 9 of different NaCl concentrations in Example 3 to obtain re-solutions of different salt concentrations. The samples were injected for molecular sieve detection. The results are shown in Table 4. The molecular sieve results are as follows: Figure 15-16 shown.

[0105] Table 4

[0106] Serial number NaCl concentration Protein concentration (mg / ml) VLPs aggregates VLPs monomer Protein monomers or oligomers 1 150mM 9.0 9.00％ 65.44％ 25.55％ 2 500mM 9.0 10.89％ 64.85％ 24.27％

[0107] From Table 4 and Figure 15-16 It can be seen that the salt concentrations of 150 mM NaCl and 500 mM NaCl have little effect on the formation of VLPs.

[0108] Example 6, VLP stability detection

[0109] In a biological safety cabinet, filter through a 0.22μm low protein binding syringe filter, or filter a large amount of protein solution through a sterilized 0.22μm filter membrane Nalgene filter, and store the filtered protein solution sample in a -80℃ refrigerator. Dilute the protein with PBS to 500ug / ml, a total of 10ml, and divide it into 2 portions, each 5ml; one portion is placed in a -20℃ refrigerator, and the other portion is placed in a 4℃ refrigerator. Samples are taken once in the first week, second week, fourth week, eighth week, and twelfth week, 1ml each time, and the samples are taken continuously for 5 times; after each sampling, the protein concentration is tested with BCA, and the results are shown in Table 5 below:

[0110] Table 5

[0111]

[0112] As can be seen from Table 5, judging from the changes in protein concentration, the protein remained basically stable during the two groups of experiments.

[0113] Example 6: Electron microscopy observation

[0114] The porcine circovirus type 2 virus-like particles prepared in the present invention (i.e., the virus-like particle samples obtained after re-dissolution) were observed using a transmission electron microscope. After copper mesh adsorption and negative staining with phosphotungstic acid, the formation of virus-like particles was observed using a transmission electron microscope. Fig.17 As shown, the virus-like particles formed were intact and uniform in size.

[0115] Example 7: Vaccine formulation

[0116] Take an appropriate amount of Cap protein for proper dilution, take 9 parts of antigen solution and 1 part of aqueous adjuvant GEL 02, slowly add the adjuvant to the antigen solution, the aqueous adjuvant: antigen (w:w) = 1:9, so that the final concentration of Cap protein in the vaccine is 40μg per ml. Slowly add the adjuvant to the antigen solution, stirring while adding, stirring at 300r / min for 20 minutes, and then store in separate devices at 2-8℃.

[0117] Example 8: Immunogenicity

[0118] Fifteen healthy piglets aged 14 to 21 days with negative PCV2 ELISA antibodies and negative PCV2 antigen were divided into 3 groups, with 5 pigs in each group. Group 1 was used as the vaccine immunization group, which was injected with 1.0 ml of vaccine into the neck muscle for one immunization. Groups 2 and 3 were not vaccinated, and served as the attack control group and blank control group, respectively.

[0119] The results showed that 5 / 5 of the vaccine-immunized group were protected after challenge with the virus, 5 / 5 of the control challenge group became ill, and all of the blank control groups were normal.

[0120] The present invention is illustrated by the above examples, but it should be understood that the present invention is not limited to the specific examples and embodiments described herein. The purpose of including these specific examples and embodiments here is to help those skilled in the art to practice the present invention. Any person skilled in the art is easy to make further improvements and perfections without departing from the spirit and scope of the present invention, so the present invention is only limited by the content and scope of the claims of the present invention, and it is intended to cover all alternatives and equivalents included in the spirit and scope of the present invention defined by the appendix claims.

[0121]

Claims

1. A method for preparing porcine circovirus type 2 Cap protein virus-like particles, characterized in that: The preparation method comprises the following steps: 1) preparing a porcine circovirus type 2 Cap protein solution; and 2) Autonomous assembly to form VLPs in slightly acidic buffer; Wherein, the porcine circovirus type 2 Cap protein solution in step 1) is a monomer and / or oligomer solution of the Cap protein, and the amino acid sequence of the Cap protein is shown in SEQ ID NO.

2.

2. The preparation method according to claim 1, characterized in that: In step 1), the purity of the porcine circovirus type 2 Cap protein solution is not less than 80%, and the Cap protein concentration is not higher than 37 mg / ml.

3. The preparation method according to any one of claims 1 or 2, characterized in that: The porcine circovirus type 2 Cap protein has a tag selected from the group consisting of poly-His, FLAG, c-myc and HA connected to the amino terminus or carboxyl terminus of the amino acid sequence shown in SEQ ID NO.

2.

4. The preparation method according to claim 3, characterized in that: The method for preparing the porcine circovirus type 2 Cap protein solution comprises the following steps: 4-1) cloning the coding gene sequence of porcine circovirus type 2 Cap protein as shown in SEQ ID NO.1 after codon optimization into a prokaryotic expression vector to obtain a recombinant plasmid containing the porcine circovirus type 2 Cap protein coding gene; 4-2) transforming the recombinant plasmid containing the porcine circovirus type 2 Cap protein encoding gene into competent Escherichia coli cells to obtain a recombinant expression strain; 4-3) culturing and screening the recombinant expression strains in step 2) to obtain highly expressing strains; 4-4) fermenting and culturing the highly expressing strain described in step 3), and inducing with IPTG to obtain bacteria that highly express Cap protein; and 4-5) Take the bacterial cells in step 4), resuspend them in a lysis buffer, break them by high-pressure homogenization, collect the supernatant by centrifugation, and purify them by affinity chromatography to obtain a high-purity Cap protein eluate.

5. The preparation method according to claim 4, characterized in that: In step 4-1), the prokaryotic expression vector is pET28a.

6. The preparation method according to claim 4, characterized in that: In step 4-2), the Escherichia coli strain is selected from one of BL21 (DE3), BL21 star (DE3), and Arctic Express (DE3).

7. The preparation method according to claim 4, characterized in that: In step 4-5), the affinity chromatography is one of nickel affinity chromatography, Flag affinity chromatography, and Strep column affinity chromatography.

8. The preparation method according to claim 4, characterized in that: The method also includes adding saturated ammonium sulfate to the purified Cap protein eluate in step 4-5) to precipitate the Cap protein; and then adding a slightly acidic buffer to the Cap protein precipitate to redissolve it to obtain a Cap protein solution; wherein the ammonium sulfate saturation is 30-50%.

9. The preparation method according to claim 8, characterized in that: The slightly acidic buffer is selected from phosphate-citrate buffer and phosphate-carbonate buffer.

10. The preparation method according to claim 1, characterized in that: In step 2), the slightly acidic buffer solution is a buffer solution with a pH value of 3.5-6.

5.

11. Use of porcine circovirus type 2 virus-like particles obtained by the method for preparing porcine circovirus type 2 virus-like particles according to any one of claims 1 to 10 in preparing vaccines for diagnosing, preventing and treating porcine circovirus.

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