Prokaryotic Expression And Immune Evaluation Of Porcine Circovirus Type 3 Capsid Protein(Cap)

Porcine circovirus type 3（PCV3）is a novel circovirus which is associated with Porcine dermatitis nephropathy syndrome（PDNS）,reproductive failure in sows,multisystem inflammation,heart disease,fetal abortion,and postweaning multisystemic wasting syndrome.Infections have been detected in many countries and regions and reported in many provinces within China.Studies have found that PCV3 can be co-infected with a variety of viruses,seriously endangering the health of pigs and causing huge losses to the breeding industry.The Cap protein is the only structural protein of PCV3,which determines the antigenicity of the virus.The amino acid sequence analysis of Cap shows that PCV3 and PCV2 Cap protein share only 37%of similarities and the Cap of PCV3 and PCV2 have great differences in structure and antigen epitopes,which do not have the characteristics of cross-protection.Thus,the existing commercial PCV2 vaccine cannot effectively protect against PCV3 infection.Therefore,the development of vaccines to prevent PCV3 infection requires urgent attention and efforts.In this study,Cap protein was used as the research object to prepare PCV3 recombinant protein vaccine.First,the prokaryotic expression system of fusion Cap protein was constructed,and the recombinant protein with antigenic activity was obtained.The purified Cap protein was used to immunize mice with different adjuvants,and the immune effects of recombinant protein Cap were analyzed,which laid a solid foundation for the clinical study of PCV3 vaccine.The research contents are as follows.1 Expression and purification of Cap gene of porcine circovirus type 3 in Escherichia coliThe Cap gene sequence of PCV3/CN/Guangdong-CH/2016 strain in Gen Bank（accession number:MFMF589112.1）was genetically optimized and sent to General Biosystems（Anhui）for gene synthesis.The Cap gene fragment of 654 bp was amplified by PCR,and was connected with p ET28a-sumo gene fragment.The expression vector p ET28a-sumo-Cap was constructed and transformed into E.coli Rosetta（DE3）to induce expression.The product was analyzed with SDS-PAGE electrophoresis,and the target protein was about 38.8kda.Western blot（WB）confirmed that the protein was the very Cap protein.Solubility analysis revealed that the fusion protein Cap was mainly expressed in the form of inclusion bodies.Subsequently,the fermentation temperature was optimized,and soluble protein could be obtained by induction at low temperature.Considering many factors affecting protein purification,37°C was finally selected for induction.The inclusion body protein with purity of 68%was obtained by the 8M urea denaturation and dialysis renaturation.The protein purity was further improved by Cut protein glue recovery（>96%）.2 Evaluation of immune effect of recombinant Cap protein of porcine circovirus type 3The immune effects of the fusion Cap protein obtained in the above studies was evaluated at the animal level.First,BALB/c mice were divided into 6 groups with 6 mice in each group.They were negative control PBS group,antigen Cap group,Cap+Cp G,Cap+Al（OH）₃,Cap+ESSAI O/W and Cap+ISA51 adjuvant groups,respectively.The humoral and cellular immunity of BALB/c mice were detected by intramuscular injection of the above solution and protein,and the effects of Cp G,Al（OH）3,ESSAI O/W and ISA51 on Cap immunity were observed in order to select the optimal adjuvant for the development of PCV3 recombinant protein vaccine and to optional the feasibility of PCV3 vaccine development.The results showed that the total antibody level in Cap+Cp G group was the highest compared with that in other groups,which wan reflected in that Ig G2a was higher than that in other groups.The ratio of Ig G1/Ig G2a showed that the Th1 cell response in Cap+Cp G group was dominant,while other groups showed Th2 type immune response tendency.In addition,the proportion of Tem（effect memory T cells,effect memory T lymphocytes）in CD8+T cells in the Cap+Cp G group was slightly higher than that in other groups,indicating that a higher response level of CTL cells（Cytotoxic T lymphocytes）and a higher cell-killing level.In summary,this study realized the full-length expression of Cap protein in E.coli,and the constructed Cap recombinant protein had presented a certain extent of immunogenicity.Compared with the Cap antigen group and other adjuvant groups,the Cap+Cp G group vaccine preparation can induce stronger humoral and cellular immunity in the body.Cp G has the prospect of being used as an adjuvant for PCV3 vaccine adjuvant,providilig a reference for the further development of PCV3 vaccine.