Porcine circovirus type 2 Cap protein, virus-like particles, their preparation methods and applications

Abstract

This invention obtains the porcine circovirus type 2 (PCV2Cap) protein through mutation, and expresses it using a yeast expression system to obtain PCV2Cap protein with good solubility and immunogenicity, and significantly increased expression levels. The Cap protein constructed in this invention can be used in the production of subunit vaccines and VLPs vaccines, offering advantages such as good immunogenicity and low cost.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to porcine circovirus type 2 Cap protein, virus-like particles, their preparation methods and applications. Background Technology

[0002] Porcine circovirus type 2 (PCV2) is a widespread virus in pigs, belonging to the genus *Circovirus* of the family *Circoviridae*. It is a pathogenic virus circulating globally. Pigs are highly susceptible to PCV2, and infection has caused significant economic losses to the pig farming industry worldwide, making it a highly concerning viral disease. Infected pigs shed the virus through nasal discharge, feces, and other waste products. It infects pigs of all ages via the oral cavity and respiratory tract, causing widespread pathological damage to organs and tissues throughout the body, resulting in stunted growth, depression, anemia, respiratory distress / coughing, swollen lymph nodes, and pale / lesions on the skin. PCV2 severely damages the immune system of pigs, causing immunosuppression and immunodeficiency, making the pigs more susceptible to other pathogens such as PRRSV, PRV, PPV, MhP, PEDV, SIV, and Pasteurella multocida. This can lead to superinfection or triple infection, greatly increasing the mortality rate and causing very serious harm.

[0003] Vaccination is one of the most effective ways to prevent PCV2 infection. Currently used PCV2 vaccines are mainly inactivated vaccines and subunit vaccines. However, inactivated PCV2 vaccines have problems such as difficulty in detecting inactivation efficacy, short immunity period, need for multiple immunizations, and high production costs. Subunit vaccines, such as ordinary subunit vaccines and chimeric epitope vaccines, have problems with immunogenicity and unsatisfactory immunization effects.

[0004] Virus-like particles (VMPs) are a class of structures composed solely of viral structural proteins, structurally similar to parental viral particles. They do not contain viral nucleic acids and can be synthesized genetically and expressed using prokaryotic or eukaryotic expression systems without obtaining active virus. Therefore, VMPs possess immunogenicity similar to the parent virus, exhibiting higher safety. Their production requires less stringent environmental and equipment conditions, which helps reduce production costs and enable large-scale production. PCV2 viral particles contain a unique structural protein or protective antigen, the capsid protein Cap, which contains many protective antigenic determinants. Expression of Cap alone can form VMPs. Current research reports the successful expression and assembly of PCV2 Cap protein into VMPs in E. coli, baculoviruses, and yeast systems, achieving certain immunogenic effects. However, existing Cap protein technologies suffer from problems such as low expression levels, weak immunogenicity, poor expression solubility, high production costs, and complex production processes, making them unsuitable for industrial production.

[0005] Therefore, how to provide a more stable PCV2 Cap protein with better immunogenicity, expression level and solubility, and improved expression efficiency to meet the needs of PCV2 vaccine production and manufacturing is a technical problem that urgently needs to be solved in this field.

[0006] In view of this, the present invention is hereby proposed. Summary of the Invention

[0007] The primary objective of this invention is to provide a porcine circovirus type 2 Cap protein.

[0008] The second objective of this invention is to provide a method for preparing the above-mentioned porcine circovirus type 2 Cap protein.

[0009] A third objective of the present invention is to provide porcine circovirus-like particles assembled from the aforementioned porcine circovirus type 2 Cap protein.

[0010] The fourth objective of this invention is to provide a virus-like particle vaccine against porcine circovirus type 2.

[0011] In a first aspect, the present invention provides a porcine circovirus type 2 Cap protein, wherein the proline (Pro, P) at amino acid position 230 of the porcine circovirus type 2 Cap protein is mutated to any one of cysteine ​​(Cys, C), phenylalanine (Phe, F), tyrosine (Tyr, Y), tryptophan (TRP, W), methionine (Met, M), histidine (His, H), glutamine (Gln, Q), arginine (Arg, R), isoleucine (Ile, I), aspartic acid (Asp, D), serine (Ser, S), asparagine (Asn, N), glutamate (Glu, E), and lysine (Lys, K).

[0012] As a further technical solution, the proline (Pro, P) at amino acid position 230 of the porcine circovirus type 2 Cap protein is mutated to any one of the following: cysteine ​​(Cys, C), phenylalanine (Phe, F), tyrosine (Tyr, Y), tryptophan (TRP, W), methionine (Met, M), histidine (His, H), glutamine (Gln, Q), arginine (Arg, R), isoleucine (Ile, I), aspartic acid (Asp, D), and serine (Ser, S).

[0013] Preferably, the proline (Pro, P) at amino acid position 230 of the porcine circovirus type 2 Cap protein is mutated to cysteine ​​(Cys, C), and its amino acid sequence is shown in SEQ ID NO. 2, and its nucleotide sequence is shown in SEQ ID NO. 16.

[0014] Preferably, the proline (Pro, P) at amino acid position 230 of the porcine circovirus type 2 Cap protein is mutated to phenylalanine (Phe, F), and its amino acid sequence is shown in SEQ ID NO. 3, and its nucleotide sequence is shown in SEQ ID NO. 17.

[0015] Preferably, the proline (Pro, P) at amino acid position 230 of the porcine circovirus type 2 Cap protein is mutated to tyrosine (Tyr, Y), and its amino acid sequence is shown in SEQ ID NO. 4, and its nucleotide sequence is shown in SEQ ID NO. 18.

[0016] Preferably, the proline (Pro, P) at amino acid position 230 of the porcine circovirus type 2 Cap protein is mutated to tryptophan (TRP, W), and its amino acid sequence is shown in SEQ ID NO. 5, and its nucleotide sequence is shown in SEQ ID NO. 19.

[0017] Preferably, the proline (Pro, P) at amino acid position 230 of the porcine circovirus type 2 Cap protein is mutated to methionine (Met, M), and its amino acid sequence is shown in SEQ ID NO. 6, and its nucleotide sequence is shown in SEQ ID NO. 20.

[0018] Preferably, the proline (Pro, P) at amino acid position 230 of the porcine circovirus type 2 Cap protein is mutated to histidine (His, H), and its amino acid sequence is shown in SEQ ID NO. 7, and its nucleotide sequence is shown in SEQ ID NO. 21.

[0019] Preferably, the proline (Pro, P) at amino acid position 230 of the porcine circovirus type 2 Cap protein is mutated to glutamine (Gln, Q), and its amino acid sequence is shown in SEQ ID NO. 8, and its nucleotide sequence is shown in SEQ ID NO. 22.

[0020] Preferably, the proline (Pro, P) at amino acid position 230 of the porcine circovirus type 2 Cap protein is mutated to arginine (Arg, R), and its amino acid sequence is shown in SEQ ID NO. 9, and its nucleotide sequence is shown in SEQ ID NO. 23.

[0021] Preferably, the proline (Pro, P) at amino acid position 230 of the porcine circovirus type 2 Cap protein is mutated to isoleucine (Ile, I), and its amino acid sequence is shown in SEQ ID NO. 10, and its nucleotide sequence is shown in SEQ ID NO. 24.

[0022] Preferably, the proline (Pro, P) at amino acid position 230 of the porcine circovirus type 2 Cap protein is mutated to aspartic acid (Asp, D), and its amino acid sequence is shown in SEQ ID NO. 11, and its nucleotide sequence is shown in SEQ ID NO. 25.

[0023] Preferably, the proline (Pro, P) at amino acid position 230 of the porcine circovirus type 2 Cap protein is mutated to serine (Ser, S), and its amino acid sequence is shown in SEQ ID NO. 12, and its nucleotide sequence is shown in SEQ ID NO. 26.

[0024] Preferably, the proline (Pro, P) at amino acid position 230 of the porcine circovirus type 2 Cap protein is mutated to asparagine (Asn, N), and its amino acid sequence is shown in SEQ ID NO. 13, and its nucleotide sequence is shown in SEQ ID NO. 27.

[0025] Preferably, the proline (Pro, P) at amino acid position 230 of the porcine circovirus type 2 Cap protein is mutated to glutamic acid (Glu, E), and its amino acid sequence is shown in SEQ ID NO. 14, and its nucleotide sequence is shown in SEQ ID NO. 28.

[0026] Preferably, the proline (Pro, P) at amino acid position 230 of the porcine circovirus type 2 Cap protein is mutated to lysine (Lys, K), and its amino acid sequence is shown in SEQ ID NO. 15, and its nucleotide sequence is shown in SEQ ID NO. 29.

[0027] Secondly, the present invention provides a method for preparing the above-mentioned porcine circovirus type 2 Cap protein, comprising transferring a recombinant plasmid containing the nucleotide sequence encoding the Cap protein into a yeast strain, and obtaining the porcine circovirus type 2 Cap protein after expression and harvesting.

[0028] As a further technical solution, the method for preparing the nucleotide sequence encoding the Cap protein is as follows: the wild-type PCV2 virus Cap protein nucleotide sequence is obtained by PCR, ligated into a vector, expressed to obtain amino acids, mutated at the corresponding positions, codon optimized, and translated to obtain the mutated encoding nucleotide sequence.

[0029] Preferably, the upstream primer sequence in the PCR method is SEQ ID NO.30 and the downstream primer sequence is SEQ ID NO.31.

[0030] Preferably, the recombinant plasmid is obtained by homologous recombination, whereby the nucleotide sequence of the mutant is linked between the BamHI and AgeI restriction enzyme sites of the pPIC3.5K vector.

[0031] As a further technical solution, the above preparation method also includes a method for detecting yeast strains, wherein the step is to amplify and detect the strain using a PCR method.

[0032] Preferably, the upstream primer sequence of the PCR method is SEQ ID NO.32, and the downstream primer sequence is SEQ ID NO.33.

[0033] More preferably, the amplification reaction program is as follows: 98℃ pre-denaturation for 30s, 98℃ denaturation for 10s, 58℃ annealing for 30s, 72℃ extension for 20s, 30 cycles, and 72℃ final extension for 2min.

[0034] Thirdly, the present invention provides porcine circovirus-like particles assembled from the porcine circovirus type 2 Cap protein prepared by the above preparation method.

[0035] Fourthly, the present invention provides a virus-like particle vaccine against porcine circovirus type 2, comprising the aforementioned porcine circovirus virus-like particles.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] (1) The porcine circovirus type 2 Cap protein provided by the present invention has higher immunogenicity and can more effectively prevent circovirus infection.

[0038] (2) The porcine circovirus type 2 Cap protein provided by the present invention has significantly improved expression efficiency, can be expressed in large quantities in a soluble manner and has a high expression level.

[0039] (3) The porcine circovirus type 2 Cap protein provided by the present invention can self-assemble into virus-like particles. The vaccine prepared by using the virus-like particles can effectively stimulate the animal body to produce humoral immunity and cellular immunity. Immunological experiments have shown that the immune effect is very good.

[0040] (4) The present invention uses a yeast expression system to express and prepare porcine circovirus type 2 Cap protein, which has the advantages of low production cost, simple production process, and the ability to obtain a large amount of target protein through high-density fermentation. In addition, there is no endotoxin contamination, which can be used for large-scale production and is conducive to protein purification and further research. Attached Figure Description

[0041] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0042] Figure 1 This is a SEC gel chromatography image of the Cap protein;

[0043] Figure 2 Standard curve plotted for BSA standards;

[0044] Figure 3 The results of Western blot analysis of mCap-230-C protein are shown, where M: Protein Marker, 1: mCap-230-C, 2: Negative control (PBS), 3: Original X33 yeast strain, and 4: PCV-Ori.

[0045] Figure 4 Electron micrograph of mCap-230-C virus-like particles (VLPs). Detailed Implementation

[0046] The embodiments and examples of the present invention will be described in detail below. However, those skilled in the art will understand that the following embodiments and examples are for illustrative purposes only and should not be considered as limiting the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0047] The main reagents, equipment, and their manufacturers used in the following examples are shown in Tables 1 and 2:

[0048] Table 1. Main reagents and their manufacturers and product numbers.

[0049]

[0050]

[0051] Table 2 Main Instruments and Consumables

[0052]

[0053]

[0054] The present invention will be further illustrated below with specific embodiments and comparative examples. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0055] Example 1: Construction of PCV2 yeast expression vector

[0056] 1. Viruses, cells, vectors

[0057] Porcine circovirus 2b subtype TK / XJ / CHA / 2019 was isolated, identified, and preserved by Tiankang Pharmaceutical Co., Ltd.; Pichia pastoris strain X-33 and vector pPIC3.5K were purchased from Invitrogen; pMD19T vector (T-Vector pMD) was also used. TM 19 (Simple) was purchased from Takara.

[0058] 2. Acquisition and mutation of the PCV2b Cap gene

[0059] 2.1 Cap gene acquisition

[0060] Genomic DNA of PCV2b TK / XJ / CHA / 2019 was extracted using a viral DNA / RNA extraction kit (Yaji Biotechnology) and stored at -20℃. Primers were designed based on the genomic sequence of PCV2 SD6 / CHA / 2005 (GenBank: DQ218421) and synthesized by Shanghai Sangon Biotech Co., Ltd. The primer sequences are shown in Table 3.

[0061] Table 3 Primer sequences

[0062]

[0063] Using PCV2b TK / XJ / CHA / 2019 genomic DNA as a template, the Cap gene sequence was amplified by PCR, ligated into the pMD19T vector, transformed into DH5α competent cells, and positive colonies were screened on ampicillin-resistant LB agar plates. Plasmids were extracted, digested with BglII, and identified. The correctly identified positive plasmid was named pMD19T-PCV2-Ori. The plasmid was sequenced by Shanghai Sangon Biotech Co., Ltd. Based on the sequencing results, the amino acid sequence of the Cap protein (SEQ ID NO.1) was obtained and named PCV2-Ori.

[0064] 2.2 Cap amino acid site mutation

[0065] Different mutagenic sites of the Cap protein were obtained by aggregation tendency analysis combined with alanine scanning. Amino acids were replaced by saturation mutation at the mutagenic sites, and mutations that could improve antigen stability, reduce aggregation fraction and not change antigen structure and antigen determinant region were screened out. The mutated protein was named mCap-XY (where X is the mutation site and Y is the mutated amino acid).

[0066] 2.3 Construction of yeast expression plasmids

[0067] The nucleotide sequences of PCV2-Ori and mCap-XY proteins were optimized using the IDT online codon optimization tool. The PCV2-Ori and mCap-XY genes were synthesized by Beijing Qingke Biotechnology Co., Ltd., and then ligated between the BamHI and AgeI restriction enzyme sites of the pPIC3.5K vector using homologous recombination. The expression plasmids pPIC3.5K-PCV2-Ori and pPIC3.5K-mCap-XY were constructed and sequenced, and the sequencing results were confirmed to be consistent with the optimized nucleotide sequences.

[0068] Example 2 Construction of PCV2 stable yeast cells

[0069] The PCV2-Ori and mCap-XY gene plasmids pPIC3.5K-PCV2-Ori and pPIC3.5K-mCap-XY (PureLink) were expanded and cultured separately. TMThe linearized plasmid was recovered by PmeI digestion and ethanol precipitation nucleic acid extraction after HiPure Plasmid Filter Maxiprep Kit (HMP5), and the nucleic acid concentration was determined to be 1 μg / μL. Plasmid electrotransfer was performed according to the Pichia Expression Kit (Catalog no. K1710-01) manual, with 1 mL of YPDS added and pPIC3.5K used as a control. The transferred product was then incubated at 30°C for 3 h. 100 μL of the culture was plated onto 0.5 mg / mL Geneticin YPDS plates and incubated at 30°C for 3–5 days. Single colonies were picked and inoculated into YPD medium for identification using PCR. The gene amplification primers used for identification are shown in Table 4. The amplification system was based on… Prepare the High-Fidelity DNA Polymerase according to the instructions. The amplification reaction program is as follows: 98℃ pre-denaturation for 30s; 98℃ denaturation for 10s, 58℃ annealing for 30s, 72℃ extension for 20s, 30 cycles; 72℃ final extension for 2min.

[0070] Table 4 Primer sequences

[0071]

[0072] The PCR products were identified by DNA gel electrophoresis at a concentration of 1%. The specific procedure was as follows: Weigh 1g of agarose and place it in an Erlenmeyer flask. Add 100mL of 1xTAE to bring the final agarose concentration to 1%. Heat the flask on high in a microwave for 20 seconds, then remove and shake well. Repeat this heating process three times until the agarose melts and the liquid becomes clear. When the gel temperature drops below 55℃, add 1ul of GeneRed. Place the transfer plate in the gel chamber and insert the comb vertically above the transfer plate. Pour the melted agarose into the gel chamber containing the transfer plate. After the gel has completely cooled and solidified into a milky white, opaque state (20-30 minutes), gently pull the comb vertically out. Use your thumb and forefinger to gently move the sides of the transfer plate and place the gel into an electrophoresis tank containing 1xTAE electrophoresis buffer. Add 1xTAE electrophoresis buffer until the buffer completely submerges the gel surface, extending 1-2mm above the surface. Place 1-2 μL of 6x DNA Loading buffer on a PE glove, mix it thoroughly with a certain amount of DNA sample, and then add it to the gel wells. The sample should settle at the bottom of the well. Also add DNA marker. Close the electrophoresis tank lid, select an appropriate electrophoresis voltage (4-10 V / cm, distance between the two electrodes, electrophoresis time approximately 30 minutes; in this experiment, the electrophoresis tank voltage is 120 V), and begin electrophoresis. The DNA sample will move from the negative electrode to the positive electrode.

[0073] The correctly identified recombinant strains were named Phica-PCV2-Ori and Phica-mCap-XY.

[0074] Example 3: PCV2 Cap protein expression, purification and identification

[0075] 1. Take several 50mL bottles of BMGY medium and inoculate them with the recombinant strains Phica-PCV2-Ori and Phica-mCap-XY, which were identified as positive by PCR, with 3 bottles for each strain.

[0076] 2. Incubate at 30℃ and 250r / min. After 24 hours of incubation, add 0.5mL of methanol to each bottle and continue incubation.

[0077] 3. After 48 hours of culture, remove one bottle from each strain of the three vials, and add 0.5 mL of methanol to the remaining two bottles for continued culture. Cell harvesting is as follows: Centrifuge 50 mL of bacterial culture at 3000 g for 10 minutes to collect cells. Resuspend the cells in an equal volume of PBS. Disrupt the yeast cells using a low-temperature high-pressure homogenizer at 1800 bar and 4°C until all cells are lysed. Take 200 μL of the disruption mixture and identify protein expression levels using SDS-PAGE. Take another 200 μL of the disruption mixture and centrifuge at 13000 g for 20 minutes. Transfer the supernatant to a 1.5 mL EP tube. Resuspend the precipitate in 200 μL of PBS. Detect the target protein content in the supernatant and precipitate using SDS-PAGE electrophoresis to determine protein solubility. The solubility ratio is calculated as: (Target protein content in supernatant / (Target protein content in supernatant + Target protein content in precipitate)) * 100%.

[0078] The remaining yeast lysate was then purified as follows: centrifuged at 13000g for 20 min and the precipitate was discarded. The supernatant was precipitated with 30% (w / v) ammonium sulfate, centrifuged at 13000g for 20 min and the supernatant was discarded. The precipitate was reconstituted with 1 / 2 volume of PBS. The first elution peak was collected on an AKTA Pure 25M Superdex 200 Increase 10 / 300GL column (the peak position indicates the position of VLPs, see...). Figure 1 The collected samples were quantified by SDS-PAGE and stored at 4℃ for later use.

[0079] The SDS-PAGE identification and quantification method in this step is as follows: (1) Take different masses of BSA standards (1μg, 2μg, 4μg, 6μg, 8μg and 10μg) and the sample to be tested, add 6× Protein loading buffer, mix well, heat at 100℃ for 5min to obtain SDS-PAGE electrophoresis samples; (2) After SDS-PAGE electrophoresis, the gel is stained and destained and the image is acquired. The gray value of each target protein band region is automatically calculated according to the image analysis software; (3) Record the known concentration and corresponding gray value of each BSA standard. Plot the BSA standard curve with the concentration of BSA standard as the X-axis and its gray value as the Y-axis; (4) Substitute the gray value of the target protein of the sample to be tested into the standard curve to obtain the concentration of the sample to be tested, i.e. the quantification result.

[0080] 4. After 72 hours of culture, harvest cells from one of the remaining two bottles for each strain and test the Cap protein content and soluble ratio in the 48-hour culture using the same steps as described above. At the same time, add 0.5 mL of methanol to the remaining bottle and continue culturing.

[0081] 5. After culturing for 96 hours, harvest the last bottle of cells and, following the same steps described above, detect the Cap protein content and soluble ratio in the 96-hour culture.

[0082] Example 4: Construction and Effects of Mutant Protein

[0083] Different mutant proteins were constructed according to the methods in Examples 1-3, and the specific information is shown in Table 5.

[0084] Table 5 Information on mutant proteins

[0085]

[0086]

[0087] 1. Expression levels of each mutant protein

[0088] Based on the results of the BSA standards (Table 6), a standard curve was plotted. Figure 2 The expression levels of Cap protein in the constructed stable yeast cells at different induction times during the shake-flask stage are shown in Table 7.

[0089] Table 6 Gray values ​​of standard samples

[0090] BSA concentration (g / L) 0.2 0.4 0.6 0.8 1 grayscale value 1198.012 1866.447 2906.320 3454.759 4327.625

[0091] Table 7 Expression levels of different mutant proteins

[0092]

[0093]

[0094] The results showed that when the proline (P) at amino acid position 230 of the Cap protein was mutated to C (Cys), F (Phe), Y (Tyr), W (TRP), M (Met), H (HIS), Q (GLN), R (ARG), I (Ile), D (Asp), S (SER), N (ASN), E (GLU), or K (LYS), the expression levels were all higher than 0.5 g / L after 72 hours of methanol induction. Among them, mCap-230-C had the highest expression level at 0.918 g / L, followed by mCap-230-D at 0.915 g / L.

[0095] 2. Solubility of each mutant protein

[0096] The solubility ratio of Cap protein expressed in the constructed stable yeast cells after 72 h of methanol induction is shown in Table 8.

[0097] Table 8. Solubility results of different mutant proteins

[0098]

[0099] The results showed that the constructed proteins from groups 2 to 12 exhibited good solubility after 72 hours of induction, with a solubility rate of ≥94%. Among them, mCap-230-C showed the best solubility, with a solubility rate of 97.83%; followed by mCap-230-F, mCap-230-Y, mCap-230-W, and mCap-230-M.

[0100] Example 5: Evaluation of mice immunized with PCV2 virus-like particle vaccine

[0101] 1. Vaccine preparation

[0102] Using the method described in this application, the PCV2 virus-like particle (VLP) protein antigen content of groups 1 to 12 in the table below is diluted to 100 μg / mL. After passing the sterility test, viscosity test and stability test according to the requirements of the appendix of the Chinese Veterinary Pharmacopoeia (current version), it is placed at 4℃ for later use.

[0103] 2. Antibody evaluation in vaccine-immunized mice

[0104] Using the method described in this application, healthy BALB / c clean-grade mice (PCV2 ELISA antibody titer not higher than 1:50) were immunized, 20g / mouse, 4-6 weeks old. Grouping and immunization details are shown in Table 9. 28 days post-immunization, blood was collected to separate serum, and the PCV2 ELISA antibody titer in the serum was measured. Based on the antibody titer of each mouse, the geometric mean of the antibody titer in each group (the 10th root of the product of the antibody titers of all mice in that group) and the seroconversion rate (mice with an antibody titer higher than 1:1600 were considered positive, and the seroconversion rate was the proportion of positive mice in the group) were calculated. The results are shown in Table 10.

[0105] Table 9 Vaccine Immunization Status

[0106]

[0107]

[0108] Table 10 Antibody titers after mouse immunization

[0109] Group Mouse number Antibody titer (geometric mean) seroconversion rate Experimental group 1 1-10 1:6400 100％ Experimental group 2 11-20 1:6400 100％ Experimental group 3 21-30 1:6400 100％ Experimental group 4 31-40 1:6400 100％ Experimental group 5 41-50 1:6400 100％ Experimental group 6 51-60 1:6400 100％ Experimental group 7 61-70 1:6400 100％ Experimental group 8 71-80 1:6400 100％ Experimental group 9 81-90 1:6400 100％ Experimental group 10 91-100 1:6400 100％ Experimental group 11 101-110 1:6400 100％ Experimental group 12 111-120 1:5971 100％ Blank control group 121-130 <1:50 0

[0110] The results showed that the PCV2 ELISA antibody titers in the blank control group mice were all below 1:50, while the PCV2 ELISA antibody titers in the experimental group mice were all above 1:1600, with a seroconversion rate of 100%. The geometric mean antibody titers of experimental groups 1-11 were 1:6400, and the geometric mean antibody titers of experimental group 12 were 1:5971. This demonstrates that the immunogenicity of the Cap protein remains at a good level after mutation using the method of this invention. The method of this invention does not destroy the immunogenicity of the Cap protein; on the contrary, it improves the protein solubility ratio and expression level, while saving costs.

[0111] Example 6 Identification of Mutant Cap Protein

[0112] 1. Western blot identification

[0113] Take mCap-230-C protein, add an appropriate amount of 6×SDS loading buffer, mix, boil at 100℃ for 3 min to prepare the sample, and perform SDS-PAGE electrophoresis; after SDS-PAGE electrophoresis, the purified protein is transferred to a methanol-activated polyvinylidene fluoride membrane by wet electroporation; wash the membrane three times with PBST, 5 min each time; transfer the membrane to 0.5% skim milk PBST and block at room temperature for 2 h; wash the membrane three times with PBST, 5 min each time; add 1:2000 diluted porcine serum and incubate at 37℃ for 1.5 h; wash the membrane three times with PBST, 5 min each time; add HRP-rabbit anti-porcine IgG (1:5000 diluted) and incubate at 37℃ for 1 h; wash the membrane five times with PBST, 5 min each time; transfer the membrane to a container containing 5 mL of PBST, treat the membrane according to the VectorVIP chromogenic reagent kit, photograph and observe the results to confirm whether it is Cap protein.

[0114] 2. Identification of Cap protein VLPs

[0115] Proteins that tested positive by Western blot were used to prepare electron microscopy samples. After negative staining with phosphotungstic acid, the morphology of the virus-like particles was observed under a JEM-1400 transmission electron microscope.

[0116] 3. Results

[0117] 3.1 Western blot results showed that a protein band of the expected size appeared at 30 kDa, indicating that the protein is a Cap protein, and that mutations in a single amino acid have no effect on the specificity of PCV2. Figure 3 ).

[0118] 3.2 Electron microscopy results showed that a large number of VLPs with diameters of approximately 15–20 nm were present in the field of view of the transmission electron microscope. Figure 4 This indicates that the Cap protein expressed by the recombinant strain can polymerize to form VLP. Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A PCV2 viral Cap protein, characterized in that, The Cap protein is obtained by mutating proline (Pro, P) at position 230 of the wild-type PCV2 virus Cap protein to cysteine ​​(Cys, C), wherein the amino acid sequence of the wild-type PCV2 virus Cap protein is shown in SEQ ID NO.

1.

2. A polynucleotide encoding the PCV2 viral Cap protein, characterized in that, The polynucleotide sequence of the Cap protein is shown in SEQ ID NO.

16.

3. The method for preparing the PCV2 viral Cap protein as described in claim 1, characterized in that, The preparation method involves the following steps: transferring a recombinant plasmid containing a nucleotide sequence encoding the Cap protein into a yeast strain, followed by expression and harvesting.

4. The preparation method according to claim 3, characterized in that, The method for preparing the nucleotide sequence encoding the Cap protein is as follows: the wild-type PCV2 virus Cap protein nucleotide sequence is obtained by PCR, mutations are performed at the corresponding positions, codon optimization is performed to obtain the mutated encoding nucleotide sequence, and after ligation into a vector, the PCV2 virus Cap protein is expressed.

5. The preparation method according to claim 4, characterized in that, The recombinant plasmid was obtained by homologous recombination, which involves linking the nucleotide sequence encoding the Cap protein between the BamHI and AgeI restriction enzyme sites of the pPIC3.5K vector.

6. The preparation method according to claim 4, characterized in that, The method also includes a step of detecting yeast strains using PCR.

7. The preparation method according to claim 6, characterized in that, The upstream primer sequence of the PCR method is SEQ ID NO.32, and the downstream primer sequence is SEQ ID NO.

33.

8. The preparation method according to claim 7, characterized in that, The PCR amplification reaction program is as follows: 98℃ pre-denaturation for 30s, 98℃ denaturation for 10s, 58℃ annealing for 30s, 72℃ extension for 20s, 30 cycles, and 72℃ final extension for 2min.

9. Porcine circovirus virus-like particles assembled from the Cap protein of porcine circovirus type 2 as described in claim 1.

10. The use of the porcine circovirus type 2 Cap protein of claim 1 or the virus-like particles of claim 9 in the preparation of a vaccine against porcine circovirus type 2.

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