Truncated Expression of Cap Protein of Porcine Circovirus Type 3 and Establishment of Indirect ELISA Method for Its Antibody
[Objective]This study was aimed to express truncatly Cap protein(1-197 amino acids)of Porcine circovirus type 3(PCV3)Sichuan strain in prokaryotic expression system,and develop an indirect ELISA(i-ELISA)method for rapid detection of PCV3 antibody,and provide material for serological investigation of PCV3.[Method]The genome of PCV3 Sichuan strain was used as template.The Cap protein coding gene of PCV3 was obtained by PCR amplification and inserted into the prokaryotic expression vector pET-30a(+)to construct the recombinant expression plasmid pET30a-PCV3-Cap.The recombined plasmid pET30a-PCV3-Cap was expressed in E.coli BL21(DE3)competent cells and was induced by IPTG.The recombinant Cap protein with good antigenicity was purified by Ni-NTA,and analyzed by SDS-PAGE and Western blotting.The i-ELISA was developed using the recombinant Cap protein as coating antigen.The optimal concentration of antigen,the optimal dilution ratio of serum to be tested,the dilution ratio of enzyme-labeled antibody and the threshold of negative and positive were determined,and the specificity,sensitivity,repeatability,correlation and coincidence rate of the i-ELISA were analyzed.The established i-ELISA method was used to detect 351 pig serum samples collected from pig farms in Northeast Sichuan from 2018 to 2022 to analyze the prevalence of PCV3 in the region.[Result]The recombinant expression plasmid pET30a-PCV3-Cap was successfully obtained,and the recombinant Cap protein(1-197 amino acids)was successfully expressed in E.coli BL21(DE3)competent cells could react specifically with positive pig serum of PCV3 with a relative molecular weight of 26 ku.The coating concentration of Cap protein was 1 ng/μL,and the detection positive threshold was D450 nm>0.684.The dilution concentration of serum and HRP-rabbit anti-pig IgG were 1∶100 and 1∶60 000,respectively.The i-ELISA was not cross reaction with positive sera of CSFV,PRRSV,PCV2,PRV,JEV and PPV1.The sensitivity of i-ELISA was 1:1 600.The method was of high duplicability with less than 10%variation of intra-and inter-assay coefficients.The result of i-ELISA was positive correlation with that of virus neutralization test,and the coincidence rate with Western blotting method was 95.8%.The positive rate of PCV3 antibody was 7.12%in 351 pig serum samples collected in Northeast Sichuan from 2018 to 2022.[Conclusion]This experiment successfully truncated expression Cap protein(1-197 amino acids)and established i-ELISA of PCV3 with better specificity,sensitivity,repeatability and coincidence rate,which provided material for serological investigation and test kit development of PCV3.
Porcine circovirus type 3(PCV3)Cap proteintruncated expressionELISA