Construction And Immunogencitiy Evaluation Of VLP Vaccine And DNA Vaccine Of Porcine Circovirus 4

Porcine circovirus type 4（PCV4）is a newly discovered DNA virus with no envelope structure and single strand circular positive strand.Since 2019,PCV4 positive samples have been detected in some cities in South Korea and 17 provinces in China.It is reported that PCV4 can be detected in healthy pigs and sick pigs with severe respiratory diseases,diarrhea,porcine dermatitis and nephrotic syndrome at all ages.However,the infectivity,pathogenicity and host range of PCV4 and its economic losses to the global pig breeding industry are unknown.Therefore,at present,the most urgent thing is to develop a safe and effective PCV4 virus vaccine with immune efficacy,so as to control the occurrence and prevalence of PCV4.Cap protein encoded by PCV4 is the only structural protein containing multiple antigenic determinants.Therefore,in this experiment,Cap protein was selected as the target protein for the following research:（1）This study also used the prokaryotic expression of PCV4 Cap protein to establish an indirect ELISA method for the detection of anti PCV4 Cap serum,which is suitable for the immunogenicity of clinical and candidate vaccines.The critical values for clinical and candidate vaccine detection methods established in this study can reach 0.262 and 0.157 respectively;The specificity results showed that only PCV4 showed specific reaction,and there was no cross reaction to TGEV,PEDV,PDCo V,SVV,Ni V,PCV2 and PCV3;The sensitivity can reach 1:102400.（2）According to the sequence characteristics of PCV4 Cap gene,the gene sequence suitable for intracellular expression is designed,the codon of the corresponding expression system is optimized.The codon optimized cap gene is amplified and connected to the baculovirus expression vector,p Fast Bac1 and to construct the recombinant shuttle plasmid and the recombinant rod to obtain the infectious recombinant baculovirus r BV-PCV4.After infecting Sf9adherent cells,PCV4 Cap VLP with biological activity was successfully obtained to construct PCV4 VLP vaccine with immune efficacy;The amplified and optimized Cap gene was connected to eukaryotic expression vector that named as p VAX1 and transfected into 293 cells.Western blot verified that Cap protein was successfully expressed.Consequently,p VAX1-PCV4 was constructed PCV4 DNA vaccine.After the mice were immunized with PCV4 VLP vaccine,DNA vaccine and corresponding control groups,the body weight,the expression level of specific antibody,the clustering status of CD4⁺T lymphocytes,CD8⁺T lymphocytes,the proliferation of lymphocytes and the expression level of cytokines were detected.The results showed that PCV4VLP vaccine and DNA vaccine could make the host produce obvious humoral immune response and cellular immunity response against PCV4.PCV4 VLP vaccine is mainly secreted by IFN-γto stimulate the proliferation and differentiation of T lymphocytes to participate in and assist cellular immunity.PCV4 DNA vaccine mainly secreted IL-2 to stimulate CD8⁺T lymphocytes to differentiate into CTL cells,causing and enhancing cellular immune response.In conclusion,in this study,PCV4 VLP vaccine and DNA vaccine were successfully constructed based on PCV4 Cap protein,and a specific antibody detection method was established.Immune experiments show that both vaccines can effectively induce humoral and cellular immune responses.This study has laid a foundation and provided new ideas for the further development of pcv4 clinical diagnosis and new vaccine research and development.