Preparation Of Porcine Circovirus Type 3 Virus-like Particles And Establishment Of Diagnostic Method

Since porcine circovirus type 3(PCV3)was first found in the United States in 2016,many countries in the world have reported that PCV3 has become widespread.There is no doubt that this has caused serious losses to the pig industry and related industries around the world.Therefore,it is of great significance to establish effective PCV3 prevention and control measures.At present,it is difficult to isolate the virus from cell lines.However,virus like particles(VLPs)based on genetic engineering technology can reveal the characteristics of the virus,and this purpose can be achieved without isolation of the virus.The research based on PCV3 VLPs may provide new ideas,making people have a better understanding of PCV3,and have potential therapeutic and diagnostic value.The capsid protein(Cap)is the sole structural protein of the virus,which is also an important antigen for diagnosis and vaccine development.The main purpose of this research is to efficiently express recombinant PCV3 Cap protein in E.coli and self-assemble into VLPs,and use it for the development of subsequent diagnostic methods.Therefore,5 codon-optimized Cap genes were designed and synthesized,including the Cap gene(wt-eCap)of the wild-type PCV3 strain that was amplified from clinical samples in our laboratory,3 sets were based on E.coli codon preferences,and the truncated cap gene(opti-dCap)which is based on the deletion of NLS.Then these gene fragments were inserted into pet30 a expression vector,and the recombinant plasmid was transformed into E.coli.Through SDS-PAGE analysis,the recombinant protein expressing opti-eCap-3 can be efficiently expressed in E.coli and soluble form,and then the purified recombinant protein is identified by transmission electron microscopy(TEM),and a large number of diameters can be clearly observed round hollow particles of uniform size around 10 nm.Therefore,it can be determined that part of the codon-optimized opti-eCap-3 can be stably expressed in E.coli and can self-assemble into VLPs,which can be used for the development of subsequent diagnostic methods.After obtaining high-purity PCV3 VLPs,they are used to coat antigens to establish a serological diagnostic method based on VLPs.The optimal concentration of antigen coating was 5 ?g/ml,the dilution of serum was 1:200,the dilution of enzyme standard second antibody was 1:5000,the incubation time of serum was 60 min,the incubation time of second antibody was 45 min,and the color development time of substrate was 15 min.The indirect ELISA(I-ELISA)method was highly specific and sensitive,and PCV3 antibody was successfully detected in 373 clinical pig serum.The final results show that the I-ELISA based on PCV3 VLPs will be a valuable tool to detect the presence of PCV3 antibody in serum samples,which will help to screen a large number of pig serum in clinical practice and provide a simple and rapid diagnostic method.