Modification of porcine circovirus type 2 Cap protein, preparation method of virus-like particles and application thereof

Abstract

The present invention obtains PCV2Cap protein with good solubility, immunogenicity and high expression level by modifying the amino acid sequence of porcine circovirus type 2 Cap protein and expressing it in a yeast expression system. The Cap protein constructed by the present invention can be applied to the production of subunit vaccines and VLPs vaccines, and has the characteristics of good immunogenicity and low cost.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to the transformation of porcine circovirus type 2 Cap protein, virus-like particles, and a preparation method and application thereof. Background Art

[0002] Porcine circovirus type 2 (PCV2) is the main pathogen that causes postweaning multisystemic wasting syndrome (PMWS). The virus belongs to the genus Circovirus of the family Circoviridae. It has a single-stranded circular DNA structure. The PCV2 genome is 1767-1768 nt in size and contains two most important reading frames, ORF1 and ORF2. The main function of ORF2 is to encode the viral capsid protein (Cap), which is 27.8 kDa in size. PCV2 Cap is highly conserved and can stimulate the body to produce an immune response. It is currently the most ideal target antigen in the research of PCV2 genetic engineering vaccines and diagnostic technologies.

[0003] Vaccine immunity is one of the most effective ways to prevent and treat PCV2 infection. At present, there are more than 40 PCV2 commercial vaccines approved in China, most of which are inactivated PCV2 vaccines. Only a few have PCV2 genetically engineered vaccines, and these genetically engineered vaccines are PCV2 Cap expressed by baculovirus or E. coli expression systems. At present, research reports have shown that PCV2 Cap protein can also be successfully expressed in yeast systems, and can be assembled into virus-like particles, and can obtain certain immune effects. However, these expression systems are limited by high costs and complex production processes. In the existing technology, the expression of Cap protein still has obvious problems such as low expression level, poor immunogenicity, and poor expression solubility, which is difficult to meet the needs of industrial production.

[0004] Therefore, how to provide a more stable PCV2 Cap protein, obtain better immunogenicity, expression level and solubility, and improve expression efficiency to meet the needs of PCV2 vaccine product production and manufacturing is a technical problem that needs to be urgently solved in this field.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] In order to solve the above problems existing in the prior art, the primary purpose of the present invention is to provide an optimized and modified porcine circovirus type 2 Cap protein.

[0007] To achieve the above purpose, the leucine (Leu, L) at the amino acid position 231 of the porcine circovirus type 2 Cap protein is mutated to any one of isoleucine (Ile, I), cysteine ​​(Cys, C), tryptophan (Trp, W), phenylalanine (Phe, F), threonine (Thr, T), tyrosine (Tyr, Y), valine (Val, V), proline (Pro, P), lysine (Lys, K), methionine (Met, M), serine (Ser, S), glutamine (Gln, Q), histidine (His, H), arginine (Arg, R), glutamic acid (Glu, E), aspartic acid (Asp, D), alanine (Ala, A), asparagine (Asn, N) and glycine (Gly, G).

[0008] As a further technical solution, the leucine (Leu, L) at amino acid position 231 of the porcine circovirus type 2 Cap protein is mutated to any one of isoleucine (Ile, I), cysteine ​​(Cys, C), tryptophan (Trp, W), phenylalanine (Phe, F), threonine (Thr, T), tyrosine (Tyr, Y), valine (Val, V), proline (Pro, P) and lysine (Lys, K), methionine (Met, M), serine (Ser, S) and glutamine (Gln, Q).

[0009] Preferably, the leucine (Leu, L) at amino acid position 231 of the porcine circovirus type 2 Cap protein is mutated to any one of isoleucine (Ile, I), cysteine ​​(Cys, C), tryptophan (Trp, W), phenylalanine (Phe, F), threonine (Thr, T) and tyrosine (Tyr, Y).

[0010] More preferably, the leucine (Leu, L) at amino acid position 231 of the porcine circovirus type 2 Cap protein is mutated to isoleucine (Ile, I).

[0011] As a further technical solution, the leucine (Leu, L) at amino acid position 231 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.2, and its nucleotide sequence is shown in SEQ ID NO.21.

[0012] Preferably, the leucine (Leu, L) at amino acid position 231 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.3, and its nucleotide sequence is shown in SEQ ID NO.22.

[0013] Preferably, the leucine (Leu, L) at amino acid position 231 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.4, and its nucleotide sequence is shown in SEQ ID NO.23.

[0014] Preferably, the leucine (Leu, L) at amino acid position 231 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.5, and its nucleotide sequence is shown in SEQ ID NO.24.

[0015] Preferably, the leucine (Leu, L) at amino acid position 231 of the porcine circovirus type 2 Cap protein is mutated to threonine (Thr, T), and its amino acid sequence is shown in SEQ ID NO.6, and its nucleotide sequence is shown in SEQ ID NO.25.

[0016] Preferably, the leucine (Leu, L) at amino acid position 231 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.7, and its nucleotide sequence is shown in SEQ ID NO.26.

[0017] Preferably, the leucine (Leu, L) at amino acid position 231 of the porcine circovirus type 2 Cap protein is mutated to valine (Val, V), and its amino acid sequence is shown in SEQ ID NO.8, and its nucleotide sequence is shown in SEQ ID NO.27.

[0018] Preferably, the leucine (Leu, L) at amino acid position 231 of the porcine circovirus type 2 Cap protein is mutated to proline (Pro, P), and its amino acid sequence is shown in SEQ ID NO.9, and its nucleotide sequence is shown in SEQ ID NO.28.

[0019] Preferably, the leucine (Leu, L) at amino acid position 231 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.10, and its nucleotide sequence is shown in SEQ ID NO.29.

[0020] Preferably, the leucine (Leu, L) at amino acid position 231 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.11, and its nucleotide sequence is shown in SEQ ID NO.30.

[0021] Preferably, the leucine (Leu, L) at amino acid position 231 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.31.

[0022] Preferably, the leucine (Leu, L) at amino acid position 231 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.13, and its nucleotide sequence is shown in SEQ ID NO.32.

[0023] Preferably, the leucine (Leu, L) at amino acid position 231 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.14, and its nucleotide sequence is shown in SEQ ID NO.33.

[0024] Preferably, the leucine (Leu, L) at amino acid position 231 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.15, and its nucleotide sequence is shown in SEQ ID NO.34.

[0025] Preferably, the leucine (Leu, L) at amino acid position 231 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.16, and its nucleotide sequence is shown in SEQ ID NO.35.

[0026] Preferably, the leucine (Leu, L) at amino acid position 231 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.17, and its nucleotide sequence is shown in SEQ ID NO.36.

[0027] Preferably, the leucine (Leu, L) at amino acid position 231 of the porcine circovirus type 2 Cap protein is mutated to alanine (Ala, A), and its amino acid sequence is shown in SEQ ID NO.18, and its nucleotide sequence is shown in SEQ ID NO.37.

[0028] Preferably, the leucine (Leu, L) at amino acid position 231 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.19, and its nucleotide sequence is shown in SEQ ID NO.38.

[0029] Preferably, the leucine (Leu, L) at amino acid position 231 of the porcine circovirus type 2 Cap protein is mutated to glycine (Gly, G), and its amino acid sequence is shown in SEQ ID NO.20, and its nucleotide sequence is shown in SEQ ID NO.39.

[0030] Another object of the present invention is to provide a method for preparing the porcine circovirus type 2 Cap protein, comprising transferring a recombinant plasmid containing a nucleotide sequence encoding the Cap protein into a yeast strain, and obtaining the porcine circovirus type 2 Cap protein after expression and harvesting.

[0031] As a further technical solution, the preparation method of the Cap protein encoding nucleotide sequence is: obtaining the wild-type porcine circovirus type 2 Cap protein nucleotide sequence by the PCR method, connecting it to a vector, expressing the amino acids, mutating the corresponding positions, codon optimizing and translating to obtain the mutated encoding nucleotide sequence.

[0032] Preferably, in the PCR method, the upstream primer sequence is SEQ ID NO.40, and the downstream primer sequence is SEQ ID NO.41.

[0033] Preferably, the recombinant plasmid is obtained by connecting the nucleotide sequence of the mutant between the BamHI and AgeⅠ restriction enzyme sites of the pPIC3.5K vector through homologous recombination.

[0034] As a further technical solution, the above preparation method also includes a method for detecting yeast strains, and the step is amplification and detection by PCR method.

[0035] Preferably, the upstream primer sequence of the PCR method is SEQ ID NO.42, and the downstream primer sequence is SEQ ID NO.43.

[0036] More preferably, the PCR amplification reaction procedure is: pre-denaturation at 98°C for 30s, denaturation at 98°C for 10s, annealing at 58°C for 30s, extension at 72°C for 20s, 30 cycles, and final extension at 72°C for 2min.

[0037] Another object of the present invention is to provide porcine circovirus virus-like particles assembled from the porcine circovirus type 2 Cap protein.

[0038] The present invention also provides a virus-like particle vaccine against porcine circovirus type 2, comprising the above-mentioned virus-like particles of porcine circovirus.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] (1) The porcine circovirus type 2 Cap protein provided by the present invention has higher immunogenicity, significantly improved expression efficiency, can be expressed in large amounts in a soluble form and at high levels, and is more conducive to further purification and application.

[0041] (2) The porcine circovirus type 2 Cap protein provided by the present invention can self-assemble into virus-like particles. The vaccine prepared using the virus-like particles has a better immune effect and can more effectively prevent porcine circovirus type 2 infection.

[0042] (3) The present invention utilizes 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, and is free of endotoxin contamination, and is suitable for large-scale production applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0044] Figure 1 It is the SEC gel chromatogram of Cap protein;

[0045] Figure 2 Standard curve plotted for BSA standards;

[0046] Figure 3 The results of Western-blot detection of mCap-231-I protein, where M: Protein Marker, 1: mCap-231-I, 2: negative control (PBS), 3: PCV-Ori, 4: original X33 yeast strain;

[0047] Figure 4 This is an electron micrograph of mCap-231-I virus-like particles (VLPs). DETAILED DESCRIPTION

[0048] The embodiments of the present invention will be described in detail below in conjunction with the embodiments and examples, but it will be appreciated by those skilled in the art that the following embodiments and examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. If specific conditions are not specified, proceed according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0049] The main reagents, equipment and their manufacturers used in the following examples are shown in Table 1 and Table 2:

[0050] Table 1 Main reagents and their manufacturers and product numbers

[0051]

[0052]

[0053] Table 2 Main instruments and consumables

[0054]

[0055]

[0056] The present invention is further described below by means of specific examples and comparative examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0057] Example 1 Construction of PCV2 Yeast Expression Vector

[0058] 1. Viruses, cells, vectors

[0059] Porcine circovirus subtype 2b TK / XJ / CHA / 2019 was isolated, identified, and preserved by Tiankang Pharmaceutical Co., Ltd.; Pichia pastoris X-33 strain and vector pPIC3.5K were purchased from Invitrogen; pMD19T vector (T-VectorpMD TM 19 (Simple)), purchased from Takara.

[0060] 2. PCV2b Cap gene acquisition and mutation

[0061] 2.1 Cap gene acquisition

[0062] PCV2b TK / XJ / CHA / 2019 genomic DNA was extracted using a viral DNA / RNA extraction kit (Yaji Biotech) and stored at -20°C. Primers were designed based on the PCV2 SD6 / CHA / 2005 (GenBank: DQ218421) genome sequence and synthesized by Shanghai Shenggong Biotechnology Co., Ltd. The primer sequences are shown in Table 3.

[0063] Table 3 Primer sequences

[0064]

[0065] Using PCV2b TK / XJ / CHA / 2019 genomic DNA as a template, PCR amplified the Cap gene sequence, connected to the pMD19T vector, transformed into DH5α competent cells, screened positive colonies on ampicillin-resistant LB plates, extracted plasmids, and identified by BglⅡ enzyme digestion. The identified positive plasmid was named pMD19T-PCV2-Ori. The plasmid was commissioned to Shanghai Shenggong Biotechnology Co., Ltd. for sequencing. According to the sequencing results, the Cap protein amino acid sequence (SEQ ID NO.1) was obtained and named PCV2-Ori.

[0066] 2.2 Cap amino acid site mutation

[0067] Different mutable sites of the Cap protein were obtained by aggregation tendency analysis combined with alanine scanning. Saturation mutation was used to replace amino acids at the mutable sites, and mutations that could improve antigen stability and reduce aggregation scores without changing the antigen structure and antigenic determinant region were screened out. The mutated protein was named mCap-XY (where X is the mutation position and Y is the amino acid after mutation).

[0068] 2.3 Yeast expression plasmid construction

[0069] The nucleotide sequences of PCV2-Ori and mCap-XY proteins were optimized using the IDT online codon optimization tool. Beijing Qingke Biotechnology Co., Ltd. was commissioned to synthesize the PCV2-Ori and mCap-XY genes, which were connected between the BamHⅠ and AgeⅠ restriction enzyme sites of the pPIC3.5K vector by homologous recombination, respectively. The expression plasmids pPIC3.5K-PCV2-Ori and pPIC3.5K-mCap-XY were constructed and sent for sequencing, confirming that the sequencing results were consistent with the optimized nucleotide sequences.

[0070] Example 2 Construction of PCV2 yeast stable transgenic cells

[0071] The PCV2-Ori and mCap-XY gene plasmids pPIC3.5K-PCV2-Ori and pPIC3.5K-mCap-XY (PureLink TM HiPure Plasmid Filter Maxiprep Kit), then digested with PmeⅠ, and linearized plasmid was recovered by ethanol precipitation nucleic acid extraction method, and the nucleic acid concentration was determined to be 1μg / μL. Plasmid electrotransfer was performed according to the Pichia Expression Kit (Catalog no. K1710-01) operation manual, 1mL YPDS was added, and pPIC3.5K was used as a control. The transfer product was then placed in a 30℃ incubator for 3h, 100μL of the culture was taken and coated on 0.5mg / mL GeneticinYPDS plates, cultured at 30℃ for 3-5 days, and single colonies were picked and inoculated into YPD medium for identification by PCR detection method. The gene amplification primers used for identification are shown in Table 4, and the amplification system was based on High-Fidelity DNA Polymerase was prepared according to the instructions, and the amplification reaction procedure was: pre-denaturation at 98°C for 30 s; denaturation at 98°C for 10 s, annealing at 58°C for 30 s, extension at 72°C for 20 s, 30 cycles; final extension at 72°C for 2 min.

[0072] Table 4 Primer sequences

[0073]

[0074] The PCR product was identified by 1% DNA gel electrophoresis to identify the band position and observe the results. The specific operation was as follows: weigh 1g agarose, put it in a triangular flask, add 100mL of 1xTAE to make the final concentration of agarose 1%, heat it on high heat in a microwave oven, heat it for 20s and then take it out and shake it evenly, heat it 3 times in total, melt the agarose, and the liquid becomes clear. When the gel temperature drops below 55°C, add 1ul of GeneRed, then put the transfer plate into the gel chamber, and insert the comb vertically above the transfer plate, and pour the melted agarose into the gel chamber with the transfer plate. After the gel is completely cooled and solidified and becomes milky white and opaque (20-30min), gently pull out the comb vertically, use the thumb and index finger to gently move the two sides of the gel plate, put the gel into the electrophoresis tank with 1xTAE electrophoresis buffer, and add 1xTAE electrophoresis buffer so that the electrophoresis buffer submerges the gel surface and is 1-2mm higher than the gel surface. Take 1-2ul6xDNA Loading buffer and drop it on PE gloves, mix it with a certain amount of DNA sample, and add it to the gel well. The sample should be precipitated at the bottom of the well. In addition, DNA Marker should be added. Cover the electrophoresis tank lid, select the appropriate electrophoresis voltage (4-10V / cm, the distance is the position of the two electrophoresis tank electrodes, the electrophoresis time is about 30min, and the electrophoresis tank voltage in this experiment is 120V), and the electrophoresis direction, start electrophoresis, and the DNA sample moves from the negative electrode to the positive electrode.

[0075] The correctly identified recombinant strains were named Phica-PCV2-Ori and Phica-mCap-XY.

[0076] Example 3 PCV2 Cap protein expression, purification and identification

[0077] 1. Take several bottles of 50 mL BMGY medium and inoculate them with the recombinant strains Phica-PCV2-Ori and Phica-mCap-XY that were identified as positive by PCR, 3 bottles for each strain.

[0078] 2. Cultivate at 30°C and 250 r / min. After 24 hours, add 0.5 mL of methanol to each bottle and continue culturing.

[0079] 3. When the culture reaches 48h, take out one bottle from the three bottles for each strain, and add 0.5mL methanol to the remaining two bottles and continue to culture. The specific operation of harvesting cells is as follows: 50mL of bacterial solution is centrifuged at 3000g for 10 minutes to collect cells, resuspend the cells with an equal volume of PBS, and crush the yeast cells with a low-temperature high-pressure crusher. The crushing parameters are 1800bar, 4℃, until all cells are crushed. Take 200μL of the crushing mixture and identify the protein expression by SDS-PAGE; take another 200μL of the crushing mixture and centrifuge at 13000g for 20min, transfer the supernatant to a 1.5mL EP tube, resuspend the precipitate with 200μL PBS, and detect the content of the target protein in the supernatant and precipitate by SDS-PAGE electrophoresis.

[0080] The remaining yeast mixture was purified by centrifugation at 13000g for 20 min and discarding the precipitate, precipitating the supernatant with 30% (w / v) ammonium sulfate, centrifuging at 13000g for 20 min and discarding the supernatant, re-dissolving the precipitate with 1 / 2 volume of PBS, and collecting the first elution peak of Superdex 200Increase10 / 300GL column through AKTA Pure25M (the peak position indicates the position of VLPs, see Figure 1 ), the collected samples were quantified by SDS-PAGE and stored at 4°C for future use.

[0081] The SDS-PAGE identification and quantification method in this step is as follows: (1) Take different masses of BSA standards (1 μg, 2 μg, 4 μg, 6 μg, 8 μg and 10 μg) and the sample to be tested, add 6×Protein loading buffer respectively, mix well, and heat at 100°C for 5 min to obtain SDS-PAGE electrophoresis samples; (2) After SDS-PAGE electrophoresis, the gel is stained and destained and the image is collected, and the gray value of each target protein band area is automatically calculated according to the image analysis software; (3) The known concentration and corresponding gray value of each BSA standard are recorded, and the BSA standard curve is drawn with the concentration of the BSA standard as the X-axis and its gray value as the Y-axis; (4) The gray value of the target protein of the sample to be tested is substituted into the standard curve to obtain the concentration of the sample to be tested, that is, the quantitative result.

[0082] 4. After 72 hours of culture, take out one bottle of cells from the remaining two bottles for each strain and harvest them. Follow the same steps as above to detect the Cap protein content and solubility ratio in the 48-hour culture. At the same time, add 0.5 mL of methanol to the remaining bottle and continue culturing.

[0083] 5. After 96 hours of culture, harvest the last bottle of cells and detect the Cap protein content and soluble ratio in the 96-hour culture according to the same steps as above.

[0084] Example 4 Construction and Effect of Mutant Protein

[0085] According to the methods of Examples 1-3, different mutant proteins were constructed, and the specific information is shown in Table 5.

[0086] Table 5 Mutant protein information

[0087] Grouping name Mutation location Amino acid before mutation Amino acid after mutation 1 PCV-Ori / / / 2 mCap-231-I 231 L(Leu) I(Ile) 3 mCap-231-C 231 L(Leu) C(Cys) 4 mCap-231-W 231 L(Leu) W(Trp) 5 mCap-231-F 231 L(Leu) F(Phe) 6 mCap-231-T 231 L(Leu) T(Thr) 7 mCap-231-Y 231 L(Leu) Y(Tyr) 8 mCap-231-V 231 L(Leu) V(Val) 9 mCap-231-P 231 L(Leu) P(Pro) 10 mCap-231-K 231 L(Leu) K(Lys) 11 mCap-231-M 231 L(Leu) M(Met) 12 mCap-231-S 231 L(Leu) S(Ser) 13 mCap-231-Q 231 L(Leu) Q(Gln) 14 mCap-231-H 231 L(Leu) H(HIS) 15 mCap-231-R 231 L(Leu) R(Arg) 16 mCap-231-E 231 L(Leu) E(Glu) 17 mCap-231-D 231 L(Leu) D(Asp) 18 mCap-231-A 231 L(Leu) A(Ala) 19 mCap-231-N 231 L(Leu) N(Asn) 20 mCap-231-G 231 L(Leu) G(Gly) 21 mCap-130-I 130 P(Pro) I(Ile) 22 mCap-167-C 167 L(Leu) C(Cys)

[0088] 1. Expression of each mutant protein

[0089] According to the results of BSA standard (Table 6), a standard curve was drawn ( Figure 2 The expression levels of the Cap protein expressed by the constructed stable yeast cells at different induction times in the shake flask stage are shown in Table 7.

[0090] Table 6 Gray value of standard products

[0091] BSA concentration (g / L) 0.2 0.4 0.6 0.8 1 Gray value 1120.012 1828.447 2688.32 3426.759 4249.625

[0092] Table 7 Expression levels of different mutant proteins

[0093]

[0094] The results showed that when the leucine (L) at amino acid position 231 of the Cap protein was mutated to isoleucine (I), cysteine ​​(C), tryptophan (W), phenylalanine (F), threonine (T), tyrosine (Y), valine (V), proline (P), lysine (K), methionine (M), serine (S), glutamine (Q), histidine (H), arginine (R), glutamic acid (E), aspartic acid (D), alanine (A), asparagine (N), and glycine (G), the expression levels of the Cap protein were higher than those of the Cap protein at 72 h after methanol induction. 0.5g / L, among which mCap-231-I had the highest expression level of 1.792g / L; mCap-231-C, mCap-231-W, mCap-231-F, mCap-231-T, and mCap-231-Y had the second highest expression levels, all higher than 1.55g / L; mCap-231-V, mCap-231-P, mCap-231-K, mCap-231-M, mCap-231-S, and mCap-231-Q had the second highest expression levels, all higher than 1.3g / L.

[0095] 2. Changes in the soluble content of each mutant protein

[0096] The soluble content of the Cap protein expressed in the stably transformed yeast cells constructed after methanol induction for 72 h is shown in Table 8.

[0097] Table 8 Quantitative results of soluble fractions of different mutant proteins

[0098]

[0099] It can be seen that when the leucine (L) at the amino acid position 231 of the Cap protein was mutated to the above-mentioned other amino acids, the content of its soluble part (i.e., the supernatant content) increased. After 72 hours of induction, the supernatant content of the mCap-231-I protein was twice that of PCV-Ori, and the supernatant content of the proteins constructed from groups 3 to 20 was also higher than that of PCV-Ori after the same period of time.

[0100] Example 5 Evaluation of PCV2 virus-like particle vaccine immunization in mice

[0101] 1. Vaccine preparation

[0102] Using the method described in the present application, the PCV2 virus-like particle (VLP) protein antigen content of groups 1 to 20 in Table 9 was diluted to 100 μg / mL, and after passing the sterility test, viscosity test, and stability test required by the appendix of the "Chinese Veterinary Pharmacopoeia" (current edition), it was placed at 4°C for use.

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

[0104] Using the method described in the present application, BALB / c healthy clean mice (PCV2 ELISA antibody titer not higher than 1:50) were immunized, 20 g / mouse, 4-6 weeks old, and the grouping and immunization conditions are shown in Table 9. 28 days after immunization, blood was collected to separate serum, and the PCV2 ELISA antibody titer in the serum was determined. According to the antibody titer of each mouse, the geometric mean (the 10th root of the product of the antibody titers of all mice in the group) and the positive conversion rate (the mouse antibody titer in this group is higher than 1:1600 and is a positive mouse, and the positive conversion rate is the proportion of positive mice to mice in this group) of each group were calculated. The results are shown in Table 10.

[0105] Table 9 Vaccine immunization status

[0106]

[0107]

[0108] Table 10 Antibody titer after mouse immunization

[0109] Group Mouse number Antibody titer (geometric mean) Conversion 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 1:6400 100％ Experimental Group 13 121-130 1:6400 100％ Experimental Group 14 131-140 1:6400 100％ Experimental Group 15 141-150 1:6400 100％ Experimental Group 16 151-160 1:6400 100％ Experimental Group 17 161-170 1:6400 100％ Experimental Group 18 171-180 1:6400 100％ Experimental Group 19 181-190 1:6400 100％ Experimental Group 20 191-200 1:5198 100％ Blank control group 201-210 <1:50 0

[0110] The results showed that the PCV2 ELISA antibody titers of mice in the blank control group were all lower than 1:50, and the PCV2 ELISA antibody titers of mice in the experimental groups were all higher than 1:1600, and the positive conversion rate was 100%; the geometric mean of the antibody titers of experimental groups 1-19 was 1:6400; the geometric mean of the antibody titer of experimental group 20 was 1:5198. It can be seen that the immunogenicity of the Cap protein after mutation by the method of the present invention is still maintained at a better level. The method of the present invention does not destroy the immunogenicity of the Cap protein, but increases the protein expression and the content of the soluble part, saving costs.

[0111] Example 6 Identification of mutant Cap proteins

[0112] 1. Western-blot identification

[0113] Take the mCap-231-I protein, add an appropriate amount of 6×SDS loading buffer and mix, boil at 100℃ for 3min to prepare the sample, and perform SDS-PAGE electrophoresis; after SDS-PAGE electrophoresis of the purified protein, transfer the protein to a polyvinylidene fluoride membrane activated by methanol by wet electrotransfer; wash the membrane three times with PBST, 5min each time; transfer the membrane to 0.5% skim milk PBST, and block at room temperature for 2h; wash the membrane three times with PBST, 5min each time; add 1:2000 diluted pig serum, and act at 37℃ for 1.5h; wash the membrane three times with PBST, 5min each time; add HRP-rabbit anti-pig IgG (1:5000 dilution), and act at 37℃ for 1h; wash the membrane five times with PBST, 5min each time; transfer the membrane to a container containing 5mL PBST, treat the membrane according to the colorimetric reagent kit VectorVIP, take pictures to observe the results, and confirm whether it is Cap protein.

[0114] 2. Identification of Cap protein VLPs

[0115] The proteins detected positive by Western-blot were used to prepare electron microscopy samples. After negative staining with phosphotungstic acid, the morphology of the formed 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 30Kd, indicating that the protein was Cap protein, and the mutation of a single amino acid had no effect on the specificity of PCV2 ( Figure 3 ).

[0118] 3.2 Electron microscopy results show that there are a large number of VLPs with a diameter of about 15 to 20 nm in the field of view of the transmission electron microscope ( Figure 4 ), indicating that the Cap protein expressed by the recombinant strain can aggregate to form VLPs.

[0119] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements 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 porcine circovirus type 2 Cap protein, characterized in that The Cap protein is obtained by mutating the leucine (Leu, L) at amino acid 231 of the wild-type porcine circovirus type 2 Cap protein to isoleucine (Ile, I), wherein the amino acid sequence of the wild-type PCV2 virus Cap protein is shown in SEQ ID NO.

1.

2. A polynucleotide encoding porcine circovirus type 2 Cap protein, characterized in that: The polynucleotide sequence of the Cap protein is shown in SEQ ID NO.

21.

3. The method for preparing the porcine circovirus type 2 Cap protein according to claim 1, characterized in that: The preparation steps are as follows: a recombinant plasmid containing a nucleotide sequence encoding a Cap protein is transferred into a yeast strain, and the yeast is expressed and harvested to obtain the protein.

4. The preparation method according to claim 3, characterized in that: The preparation method of the Cap protein encoding nucleotide sequence is as follows: a wild-type porcine circovirus type 2 Cap protein nucleotide sequence is obtained by a PCR method, a mutation is performed at a corresponding position, a codon is optimized to obtain a mutated encoding nucleotide sequence, and the mutated encoding nucleotide sequence is connected to a vector to express the PCV2 Cap protein.

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

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

7. The preparation method according to claim 6, characterized in that: The upstream primer sequence of the PCR method is SEQ ID NO.42, and the downstream primer sequence is SEQ ID NO.

43.

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

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

10. Use of the porcine circovirus type 2 Cap protein according to claim 1 or the virus-like particle according to claim 9 in preparing a vaccine against porcine circovirus type 2.

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