Preparation method of high-purity porcine circovirus type 4 Cap protein

Abstract

The present invention relates to Cap 38～228 The technical field of protein preparation, in particular, relates to a method for preparing high-purity porcine circovirus type 4 Cap protein. Steps include S1. constructing a prokaryotic expression vector pET-28a-His-SUMO-Cap with a double tag based on the nucleotide sequence of PCV4 Cap protein and His-SUMO tag. 38～228 ; S2. using pET‑28a‑His‑SUMO‑Cap 38～228 Transformed into E. coli competent cells BL21, induced expression with IPTG, and optimized expression conditions to produce the target protein His-SUMO-Cap 38～228 ; At the same time, the target protein His-SUMO-Cap 38～228 Purification; S3.His-SUMO-Cap 38～228 After SUMO protease cleavage, the His-SUMO tag was removed again using a Ni-NTA affinity chromatography column to obtain Cap 38～228 The present invention can effectively prepare high-purity PCV4Cap 38～228 The protein has a purity greater than 95%, providing PCV4Cap for serological diagnosis, polyclonal or monoclonal antibodies to Cap protein, and subunit vaccine development. 38～228 Technical basis for protein preparation.

Description

Technical Field

[0001] The present invention relates to Cap 38~228 The technical field of protein preparation, in particular, relates to a method for preparing high-purity porcine circovirus type 4 Cap protein. Background Art

[0002] Porcine circovirus (PCV) is a non-enveloped, single-stranded, circular, negative-sense DNA virus in the genus Circovirus, family Circoviridae. Its virions exhibit icosahedral symmetry and are approximately 17–25 nm in size. Currently, PCV is classified into four subtypes: PCV1, 2, 3, and 4. PCV1 is nonpathogenic to pigs, whereas PCV2, 3, and 4 are known to cause clinical symptoms such as porcine circovirus-associated dikseases (PCVAD), porcine dermatitis and nephropathy syndrome (PNDS), porcine respiratory disease syndrome (PRDC), post-weaning systemic wasting syndrome (PMWS), proliferative necrotizing pneumonia (PNP), neurological disorders, myocarditis, and diarrhea.

[0003] PCV4 is distantly related to the other three subtypes of PCV, with nucleotide similarity ranging from 43.2% to 51.5%. The PCV4 genome is approximately 1770 bp in size and consists of two primary open reading frames (ORFs): ORF1 and ORF2. ORF1 and ORF2 encode the 296-amino acid Rep protein and the 228-amino acid Cap protein, respectively. Similar to PCV1, PCV2, and PCV3, the Rep protein is primarily involved in viral replication, while the Cap protein primarily forms the viral capsid. However, amino acid sequence analysis of the Rep and Cap proteins revealed less than 50% similarity with other porcine circoviruses. Therefore, PCV2 and PCV3 vaccines do not provide cross-protection against PCV4. Since PCV4 is a newly emerging pathogen of ring disease in my country, there is currently no vaccine to prevent and control it. Cap protein is widely used in PCV2 and PCV3 genetically engineered subunit vaccines and diagnostic reagents. Therefore, Cap protein has become the preferred target for the development of PCV4 genetically engineered subunit vaccines and diagnostic kits.

[0004] Currently, research on the PCV4 Cap protein is limited, and there are no existing methods for its high-purity preparation. Consequently, there are no reports on the development of genetically engineered PCV4 subunit vaccines and diagnostic kits based on the Cap protein, further limiting the development of vaccines and diagnostic reagents. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for preparing high-purity porcine circovirus type 4 Cap protein, so as to fill the gap in the prior art.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The method for preparing high-purity porcine circovirus type 4 Cap protein comprises the following steps:

[0008] S1. Construction of a prokaryotic expression vector pET-28a-His-SUMO-Cap with dual tags based on the nucleotide sequences of PCV4 Cap protein and His-SUMO tag 38~228 ;

[0009] S2. Using pET-28a-His-SUMO-Cap 38~228 Transformed into E. coli competent cells BL21, induced expression with IPTG, and optimized the expression conditions to prepare the target protein His-SUMO-Cap 38~228 ; At the same time, the target protein His-SUMO-Cap 38~228 purification;

[0010] S3.His-SUMO-Cap 38~228 After SUMO protease cleavage, the His-SUMO tag was removed again using Ni-NTA affinity chromatography to obtain Cap 38~228 .

[0011] Further preferably, S1 mainly includes:

[0012] Based on the nucleotide sequence of the Cap protein of the PCV4 / GX2020 / FCG49 strain, the codon preference of E. coli was optimized;

[0013] The His-SUMO-Cap protein was constructed by adding a His-SUMO tag nucleotide sequence to the N-terminus of the Cap protein nucleotide. 38~228 target sequence;

[0014] In His-SUMO-Cap 38~228 An RBS sequence and a translation initiation signal were added to the N-terminus of the target sequence. At the same time, XbaI and HindIII restriction sites were added to the 5' and 3' ends of the sequence, respectively.

[0015] The target sequence and pET-28a vector plasmid were double-digested with XbaI and HindIII, respectively, and the double-digested products were recovered by gel electrophoresis and ligated with T4 DNA ligase at 16°C overnight to prepare ligation products;

[0016] The ligation product was transformed into DH5α competent cells, monoclonal colonies were picked and expanded, and the recombinant plasmid pET-28a-His-SUMO-Cap was extracted using a plasmid extraction kit. 38~228 , double enzyme digestion with XbaI and HindⅢ was performed, and sequencing was performed to confirm the correct insertion of the target sequence. Finally, pET-28a-His-SUMO-Cap was constructed. 38~228 Expression vector.

[0017] Further preferably, in S2, pET-28a-His-SUMO-Cap 38~228 Transform E. coli competent cells BL21 and induce expression with IPTG, mainly including:

[0018] Transform E. coli competent cells BL21 with pET-28a-His-SUMO-Cap, pick positive single clones, add 5 mL of liquid LB medium containing 50 μg / mL kanamycin, and culture overnight at 37°C and 220 rpm / min;

[0019] Take out 1 mL of bacterial solution and add it to 50 mL of LB liquid medium containing 50 μg / mL kanamycin;

[0020] Cultivate at 37°C, 220 rpm / min for 4 h. When the OD600 of the bacterial solution reaches 0.6-0.8, add 50 μL of 1 mol / L IPTG at a ratio of 1:1000.

[0021] After culturing at 37°C and 220 rpm / min for 4 h, the cells were collected by centrifugation at 4000 rpm / min.

[0022] Further preferably, in S2, optimizing the expression conditions includes:

[0023] Set the induction time to 10 h; set the induction temperature to 20℃-30℃; set the IPTG concentration to 0.2 mmol / L.

[0024] Further preferably, in S2, the target protein is purified by Ni-NTA affinity chromatography, mainly comprising: first resuspending the bacteria collected by centrifugation in Binding Buffer at a ratio of 1:10 (W / V), and then disrupting the bacteria by ultrasonication;

[0025] Centrifuge at 10,000 rpm / min for 30 min, collect the supernatant, and equilibrate the nickel affinity chromatography column with Binding Buffer;

[0026] Rinse the column with Binding Buffer and then elute the contaminants with Wash Buffer;

[0027] Elute the target protein with Elution Buffer containing 100-500 μL of imidazole.

[0028] Further preferably, S3 mainly includes:

[0029] In His-SUMO-Cap 38~228 Add buffer to the protein solution;

[0030] Add SUMO protease and digest overnight at room temperature;

[0031] After the enzyme digestion effect was analyzed by 12% SDS-PAGE, the PCV4 Cap 38~228 The protein solution flows through the nickel affinity chromatography column, and the flow-through is collected.

[0032] The present invention has at least the following beneficial effects:

[0033] The method of the present invention can effectively prepare high-purity PCV4 Cap 38~228 The protein has a purity greater than 95%, providing PCV4 Cap protein for serological diagnosis, polyclonal or monoclonal antibodies to Cap protein, and subunit vaccine development. 38~228 Technical basis for protein preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0035] Figure 1 For the recombinant expression plasmid pET-28a-His-SUMO-Cap 38~228 Structural diagram;

[0036] Figure 2 Bioinformatics analysis diagram of PCV4 Cap protein;

[0037] Figure 3 Plasmid pET-28a-His-SUMO-Cap 38~228 Double enzyme digestion identification diagram;

[0038] Figure 4 His-SUMO-Cap 38~228 Protein expression identification diagram

[0039] Figure 5 His-SUMO-Cap3 8~228 Protein expression condition optimization diagram

[0040] Figure 6 To purify His-SUMO-Cap 38~228 Protein identification by SDS-PAGE;

[0041] Figure 7 This is the result of Western-blot identification of purified protein;

[0042] Figure 8 Cap 38~228 Protein purification results diagram;

[0043] Figure 9 His-SUMO-Cap 38~228 Protease cleavage identification diagram. DETAILED DESCRIPTION

[0044] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0045] 1 Materials and Methods

[0046] 1.1 Plasmids, strains, and main reagents

[0047] The pET-28a vector was stored in the laboratory; competent Escherichia coli DH5α and BL21 were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; rabbit anti-His-Tag monoclonal antibody and HRP-labeled goat anti-rabbit IgG antibody were purchased from Abcam, UK; HisTrap FF nickel affinity chromatography column was purchased from GE, USA; SUMO protease was purchased from Beyotime Biotechnology Co., Ltd.; and agarose gel DNA recovery kit (enhanced) was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0048] 1.2 Bioinformatics analysis of PCV4 Cap protein

[0049] First, the homology of the PCV4 Cap protein was analyzed using MEGA-X. The primary and secondary structures of the PCV4 Cap protein were analyzed using online software such as ProtParam and PSIPRED4.0, respectively. Furthermore, the transmembrane region, signal peptide, and disordered region of the PCV4 Cap protein were analyzed using online software such as TMHMM2.0, SignalP 5.0, and IUPred 3.0, respectively.

[0050] 1.3 Recombinant expression plasmid pET-28a-His-SUMO-Cap 38~228 Construction and identification

[0051] Based on the nucleotide sequence of the Cap protein of PCV4 / GX2020 / FCG49 strain (112-684 bp), the codon preference of E. coli was optimized, and the nucleotide sequence of the His-SUMO tag was added to the N-terminus of the Cap protein nucleotide to construct the His-SUMO-Cap 38~228 Target sequence and His-SUMO-Cap 38~228 An RBS sequence and a translation initiation signal were added to the N-terminus of the target sequence. At the same time, XbaI and HindⅢ restriction sites (synthesized by Beijing Liuhe BGI Genomics Co., Ltd.) were added to the 5' and 3' ends of the sequence, respectively. The target fragment and pET-28a vector plasmid were double-digested with XbaI and HindⅢ, respectively. The double-digested products were recovered on a gel and ligated overnight at 16°C using T4 DNA ligase. The ligation products were then transformed into DH5α competent cells, single clones were picked and cultured, and the recombinant plasmid pET-28a-His-SUMO-Cap was extracted using a plasmid extraction kit. 38~228 , double enzyme digestion was performed with XbaI and HindⅢ, and the target sequence was sent to Beijing Liuhe BGI Gene Technology Co., Ltd. for sequencing to confirm the correct insertion of the target sequence. Finally, pET-28a-His-SUMO-Cap was constructed. 38~228 Expression vector (see Figure 1 ).

[0052] 1.4 Recombinant His-SUMO-Cap 38~228 Soluble protein expression analysis

[0053] The correctly sequenced recombinant plasmid pET-28a-His-SUMO-Cap was transformed into competent E. coli BL21 cells. Positive single colonies were picked and added to 5 mL of liquid LB medium containing 50 μg / mL kanamycin (Kan). The cells were cultured overnight at 37°C and 220 rpm / min. One mL of the bacterial suspension was removed and added to 50 mL of liquid LB medium containing 50 μg / mL kanamycin (Kan). The cells were cultured at 37°C and 220 rpm / min for 4 h. When the OD600 of the bacterial suspension reached 0.6-0.8, 50 μL of 1 mol / L IPTG was added at a ratio of 1:1000. The cells were cultured at 37°C and 220 rpm / min for 4 h. The cells were then collected by centrifugation at 4000 rpm / min. A control group without IPTG was also prepared. Resuspend the cells in PBS at a 1:10 (w / v) ratio, disrupt the cells by sonication, and centrifuge at 10,000 rpm for 30 min. Collect the supernatant and precipitate from the induced and uninduced cells, respectively. Analyze the solubility of the His-SUMO-Cap protein by 12% SDS-PAGE.

[0054] 1.5 Optimization of expression conditions for recombinant His-SUMO-Cap38~228 proteins

[0055] 1.5.1 Optimal expression temperature optimization

[0056] The stored strain was added to 25 mL of liquid LB medium containing 50 μg / mL kanamycin (Kan) at a ratio of 1:50 and cultured at 37°C, 220 rpm / min for 4 h. When the OD600 reached 0.6-0.8, 25 μL of 1 mol / L IPTG was added at a ratio of 1:1000 to induce expression. The cells were cultured at 15°C, 20°C, 25°C, 30°C, and 37°C for 4 h, and then harvested. After ultrasonic disruption, the cells were centrifuged at 10,000 rpm / min for 30 min, and the supernatant collected for analysis by 12% SDS-PAGE.

[0057] 1.5.2 Optimization of the optimal IPTG concentration

[0058] The stored strain was added to 25 mL of liquid LB medium containing 50 μg / mL kanamycin (Kan) at a ratio of 1:50 and cultured at 37°C, 220 rpm / min for 4 h. When the OD600 reached 0.6–0.8, IPTG was added for induction at final concentrations of 0.2, 0.4, 0.6, 0.8, and 1 mmol / L, respectively. The cells were cultured at 30°C for 4 h and harvested. After sonication, the cells were broken and analyzed by 12% SDS-PAGE.

[0059] 1.5.3 Optimization of optimal induction time

[0060] The stored strain was added to 25 mL of liquid LB medium containing 50 μg / mL kanamycin (Kan) at a ratio of 1:50 and cultured at 37°C, 220 rpm / min for 4 h. When the OD600 reached 0.6–0.8, IPTG was added to induce the strain at a final concentration of 0.2 mmol / L. The strains were cultured at 37°C for 2, 4, 6, 8, and 10 h, and the cells were harvested. The cells were disrupted by ultrasonication and analyzed by 12% SDS-PAGE.

[0061] 1.6 Purification of recombinant His-SUMO-Cap38~228 protein

[0062] Recombinant His-SUMO-Cap was purified by GE's nickel affinity chromatography. 38~228 Protein purification was performed. Resuspend the collected bacterial cells in Binding Buffer (50 mmol / L Tris, 500 mmol / L NaCl, 20 mmol / L imidazole, pH: 8.17) at a 1:10 (W / V) ratio. Disrupt the cells by sonication at 40% power, 3 s on, 3 s off. Centrifuge at 10,000 rpm / min for 30 min, and collect the supernatant. The nickel affinity chromatography column was equilibrated with Binding Buffer, and the supernatant was passed through the nickel affinity chromatography column. The column was then washed with Binding Buffer, and then the impurities were eluted with Wash Buffer (50 mmol / L Tris, 500 mmol / L NaCl, 50 mmol / L imidazole, pH: 8.17). Finally, the target protein was eluted with Elution Buffer (50 mmol / L Tris, 500 mmol / L NaCl, pH: 8.17) containing 100, 150, 200, and 500 imidazole, respectively. Finally, the recombinant His-SUMO-Cap was analyzed by 12% SDS-PAGE. 38~228 Purity of protein.

[0063] 1.7 Western-blot identification

[0064] The purified recombinant His-SUMO-Cap protein was analyzed by Western-blot using rabbit anti-6×His-Tag monoclonal antibody as primary antibody and HRP-labeled goat anti-rabbit IgG antibody as secondary antibody. The results were observed after development with ECL colorimetric solution.

[0065] 1.8 His-SUMO tag removal and capping 38~228 Protein purification

[0066] After purification, recombinant His-SUMO-Cap38~228 The protein solution was prepared with a buffer system of (50 mmol / L Tris, 150 mmol / L NaCl, 3% glycerol, pH: 8.17), and SUMO protease was added and digested overnight at room temperature. After the digestion effect was analyzed by 12% SDS-PAGE, the protein containing PCV4 Cap was 38~228 The protein solution flows through the nickel affinity chromatography column and the flow-through is collected. The His-SUMO fusion tag and His-SUMO enzyme are bound to the nickel affinity chromatography column. 38~228 The protein was present in the flow-through and analyzed by 12% SDS-PAGE, and its concentration was determined using a Nano spectrophotometer.

[0067] 2 Results

[0068] 2.1 Bioinformatics analysis of PCV4 Cap protein

[0069] Conservation analysis of the PCV4 Cap protein by MEGA-X revealed that this protein is highly conserved among PCV4 Cap proteins (results not shown). Analysis of the primary structure of PCV4 Cap using the online software ProtParam (https: / / web.expasy.org / protparam / ) revealed that the protein consists of 228 amino acid residues with a molecular formula of C 1227 H 1868 N 378 O 330 S3 has a molecular weight of 27.29 kDa and a theoretical isoelectric point of 11.05, indicating a positive charge under physiological conditions. Furthermore, the protein has an instability coefficient of 52.96 (>40) and an average hydrophilicity of -0.897 (<0), indicating that it is hydrophilic and potentially unstable.

[0070] The secondary structure of PCV4Cap was analyzed using the online software PSIPRED 4.0 (http: / / bioinf.cs.ucl.ac.uk / psipred / ). Figure 2a), it was found that 3.95% of the PCV4 Cap protein was α-helical (69-71: LKD and 132-137: YDVLAN); 33.33% was β-sheet (39-52: IFHARFMREVTLSV, 61-68: NVGHYDFK, 90-102: YYRIRKVKVEFLP, 116-121: AIQLDG, 142-144: HGF, 150-155: HSRVFT, 181-182: IS, 190-198: HHGLQYSIQ, 204-218: QVWTVRFTLYVQFRE); and 62.72% was random coil (1-38: MPIRSRYSRRRRNRRNQRRRGLWPRASRRYRWRRKNG, 53-60: SSFSTPSW, 71-8 9: FIPKGPGTIVNLYSLPFA, 103~115: LNGINSNRTYSST, 122~131: DYVGEGKNQ, 138~141: HSSR, 145~149: TNIA R, 156~180: PKPQDPSGETHTLHFQPNNKRNQWW, 183~189: MADQDLV, 199~203: NSNFV, 219~228: FDLVNYPKQG).

[0071] The transmembrane region, signal peptide and disordered region of PCV Cap protein were analyzed using online software such as TMHMM 2.0 (https: / / services.healthtech.dtu.dk / service.php?TMHMM-2.0), SignalP 5.0 (https: / / services.healthtech.dtu.dk / service.php?SignalP-5.0) and IUPred 3.0 (https: / / iupred2a.elte.hu / ), respectively. It was found that the protein had no transmembrane region ( Figure 2 b), 1~37 aa is the nucleolar localization signal region ( Figure 2 c), 1~32 aa are N-terminal continuous disorder ( Figure 2 d), indicating that the N-terminal amino acid sequence of 1–37 aa of the PCV4 Cap protein may affect protein expression.

[0072] 2.2 Recombinant expression plasmid pET-28a-His-SUMO-Cap 38~228 Construction and enzyme digestion identification

[0073] Recombinant expression plasmid pET-28a-His-SUMO-Cap 38~228After double digestion with XbaI and HindⅢ, two specific bands were found at approximately 6000 bp and 1000 bp, which were consistent with the expected size ( Figure 3 ). Sequencing results showed that the nucleotide sequence was correct and the recombinant expression plasmid pET-28a-His-SUMO-Cap 38~228 Build successful.

[0074] 2.3 Recombinant His-SUMO-Cap 38~228 Identification of protein expression patterns

[0075] Recombinant expression plasmid pET-28a-His-SUMO-Cap 38~228 After transformation into competent E. coli BL21 cells, the supernatant and precipitate were collected before and after IPTG induction. Analysis by 12% SDS-PAGE revealed a specific band at approximately 34.44 kDa, consistent with the theoretical value. Comparison of the supernatant and precipitate after induction revealed a significant amount of the target protein in the supernatant, indicating that the SUMO tag effectively promoted the soluble expression of the PCV4 Cap protein. Figure 4 ).

[0076] 2.4 pET-28a-His-SUMO-Cap 38~228 Optimization of expression conditions

[0077] The recombinant expression plasmid pET-28a-His-SUMO-Cap 38~228 The BL21 bacterial cultures were cultured in a shaker at 37°C for 4 hours, and the expression conditions such as IPTG induction concentration, induction time, and induction temperature were optimized. After the cells were collected, they were disrupted by ultrasound and the supernatant was collected. The cells were then identified by 12% SDS-PAGE. The results showed that the optimal final IPTG concentration was 0.2 mmol / L ( Figure 5 a) The optimal induction time is 10 h ( Figure 5 b) When the induction temperature is 20℃, 25℃ and 30℃, the soluble expression level is not much different ( Figure 5 c).

[0078] 2.5 Soluble recombinant His-SUMO-Cap 38~228 Protein purification

[0079] The recombinant His-SUMO-Cap 38~228After protein expression, the cells were ultrasonically disrupted and centrifuged at 10,000 rpm for 30 minutes. The supernatant was then collected for purification. The target protein was eluted with buffer solutions containing 50 mmol / L, 100 mmol / L, 150 mmol / L, 200 mmol / L, and 500 mmol / L imidazole, respectively. The purified samples were identified by 12% SDS-PAGE electrophoresis, and a specific protein band was observed at approximately 34.44 kDa ( Figure 6 a) PCV4 His-SUMO-Cap, which is consistent with the expected size and purified 38~228 The purity of the protein can reach more than 95% ( Figure 6 b).

[0080] 2.6 Recombinant His-SUMO-Cap 38~228 Protein Western-blot identification

[0081] The purified His-SUMO-Cap 38~228 Western-blot analysis of the protein showed that the recombinantly expressed His-SUMO-Cap protein could react with rabbit anti-6×His tag monoclonal antibody, and a specific band was observed at approximately 34.44 kDa, which was consistent with the expected result ( Figure 7 ). Therefore, pET-28a-His-SUMO-Cap 38~228 The plasmid can correctly express His-SUMO-Cap 38~228 protein.

[0082] 2.7 His-SUMO tag removal and capping 38~228 Protein purification

[0083] Purified His-SUMO-Cap 38~228 The protein was digested with SUMO protease at 4℃ overnight and then identified by 12% SDS-PAGE. A specific band was found at approximately 22.24 kDa and was closely related to PCV4 Cap 38~228 The size of the protein is consistent with that of the protein, and there is a specific band at approximately 12.2 kDa, which is consistent with the size of the His-SUMO tag ( Figure 8 At the same time, Western-blot showed that the rabbit anti-6×His tag monoclonal clone had a specific reaction with the 12 kDa protein and did not react with the protein at about 22.24 kDa ( Figure 9After the His-SUMO tag was removed by nickel affinity chromatography, 12% SDS-PAGE analysis showed that a band at approximately 22.24 kDa still existed, while the His-SUMO tag at approximately 12.2 kDa disappeared, indicating that the His-SUMO tag was successfully removed ( Figure 8 ).

[0084] 3 Conclusion

[0085] The present invention is to recombinant PCV4 Cap 38~228 The protein was efficiently expressed and purified in a soluble form, with a purity greater than 95%, laying a certain foundation for further serological diagnostic technology, polyclonal or monoclonal antibodies against Cap protein, and subunit vaccine development, especially virus-like particle subunit vaccine development.

[0086] The above shows and describes the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions merely illustrate the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed by the present invention is defined by the appended claims and their equivalents.

Claims

1. High purity porcine circovirus type 4 Cap 38~228 The method for preparing a protein is characterized in that: The following steps are involved: S1. Construction of a prokaryotic expression vector pET-28a-His-SUMO-Cap with dual tags based on the nucleotide sequences of PCV4 Cap protein and His-SUMO tag 38~228 ; Mainly include: Based on the nucleotide sequence of the Cap protein of the PCV4 / GX2020 / FCG49 strain, the codon preference of E. coli was optimized; The His-SUMO-Cap protein was constructed by adding a His-SUMO tag nucleotide sequence to the N-terminus of the Cap protein nucleotide. 38~228 target sequence; In His-SUMO-Cap 38~228 An RBS sequence and a translation initiation signal were added to the N-terminus of the target sequence. At the same time, XbaI and HindIII restriction sites were added to the 5' and 3' ends of the sequence, respectively. The target sequence and pET-28a vector plasmid were double-digested with XbaI and HindIII, respectively, and the double-digested products were recovered by gel electrophoresis and ligated with T4 DNA ligase at 16°C overnight to prepare ligation products; The ligation product was transformed into DH5α competent cells, monoclonal colonies were picked and expanded, and the recombinant plasmid pET-28a-His-SUMO-Cap was extracted using a plasmid extraction kit. 38~228 , double enzyme digestion with XbaI and HindⅢ was performed, and sequencing was performed to confirm the correct insertion of the target sequence. Finally, pET-28a-His-SUMO-Cap was constructed. 38~228 expression vector; S2. Using pET-28a-His-SUMO-Cap 38~228 Transformed into E. coli competent cells BL21, induced expression with IPTG, and optimized the expression conditions to prepare the target protein His-SUMO-Cap 38~228 ; At the same time, the target protein His-SUMO-Cap 38~228 purification; The expression conditions were optimized, including: setting the induction time to 10 h; setting the induction temperature to 20°C-30°C; setting the IPTG concentration to 0.2 mmol / L; S3.His-SUMO-Cap 38~228 After SUMO protease cleavage, the His-SUMO tag was removed again using Ni-NTA affinity chromatography to obtain Cap 38~228 .

2. The high-purity porcine circovirus type 4 Cap according to claim 1 38~228 The method for preparing a protein is characterized in that: In S2, pET-28a-His-SUMO-Cap 38~228 Transform E. coli competent cells BL21 and induce expression with IPTG, mainly including: Transform E. coli competent cells BL21 with pET-28a-His-SUMO-Cap, pick positive single clones, add 5 mL of liquid LB medium containing 50 μg / mL kanamycin, and culture overnight at 37°C and 220 rpm / min; Take out 1 mL of bacterial solution and add it to 50 mL of LB liquid medium containing 50 μg / mL kanamycin; Cultivate at 37°C, 220 rpm / min for 4 h. When the OD600 of the bacterial solution reaches 0.6-0.8, add 50 μL of 1 mol / L IPTG at a ratio of 1:1000. After culturing at 37°C and 220 rpm / min for 4 h, the cells were collected by centrifugation at 4000 rpm / min.

3. The high-purity porcine circovirus type 4 Cap according to claim 1 38~228 The method for preparing a protein is characterized in that: In S2, the target protein was purified by Ni-NTA affinity chromatography, which mainly included: resuspending the centrifuged bacteria in Binding Buffer at a ratio of 1:10 w / v and then disrupting the bacteria by ultrasonication; Centrifuge at 10,000 rpm / min for 30 min, collect the supernatant, and equilibrate the nickel affinity chromatography column with Binding Buffer; Rinse the column with Binding Buffer and then elute the contaminants with Wash Buffer; Elute the target protein with Elution Buffer containing 100-500 μL of imidazole.

4. The high-purity porcine circovirus type 4 Cap according to claim 1 38~228 The method for preparing a protein is characterized in that: S3 mainly includes: In His-SUMO-Cap 38~228 Add buffer to the protein solution; Add SUMO protease and digest overnight at room temperature; After the enzyme digestion effect was analyzed by 12% SDS-PAGE, the PCV4 Cap 38~228 The protein solution flows through the nickel affinity chromatography column, and the flow-through is collected.

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