Prokaryotic Expression Of VP2 Protein And Establishment Of Serological Detection Method Of Seneca Virus A

Seneca Vrius A(SVA)is a small RNA virus of the genus coseneca,which is infectious and pathogenic to pigs.Symptoms are similar to foot-and-mouth disease,swine infectious blister disease and swine vesicular stomatitis,into the early infection of swine appear sleepiness,anorexia and fever,then rhinoscope department,oral epithelium,tongue and coronet of different sizes which appear on the blister,serious cause fester and limp.The mortality rate of piglets within 7 days of age is up to 70%,with occasional diarrhea.In 2014-2015,SVA caused serious economic losses to the Brazilian pig industry.In the same year,SVA first appeared in Guangdong region of China,which is a new exotic disease.Common detection methods include virus isolation and identification,conventional RT-PCR,fluorescent quantitative RT-PCR,virus neutralization test,and the construction of an ELISA by expressing SVA proteins,etc.SVA has only one reading frame,and VP1,VP2,and VP3 constitute the capsid proteins of the virus.Studies have shown that the specificity and sensitivity of ELISA using VP2 protein as the coated antigen is better than that of VP1 or VP3.This experiment used to build the SVA-VP2 protein as antigen,establish SVA determination of serum antibody indirect ELISA and AlphaLISA detection method,This study laid a foundation for the study of immunogenicity of sva-vp2 protein,SVA serological detection kit and genetic engineering vaccine.According to the VP2 gene in SVA isolator GD01/2017,the primers were designed and synthesized based on the carrier characteristics and protein characteristics of pet-30a.The 870bp VP2 gene fragment was successfully amplified by RT-PCR and cloned into a positive plasmid named pet30a-vp2 recombinant plasmid.Subsequently,the recombinant plasmid was transferred into Rosetta(DE3)escherichia coli,and the VP2 protein of 47KDa was successfully expressed at 18? and 12 h after the induction temperature and time were optimized.Meanwhile,the protein was partially soluble,and the non-denatant protein was purified by Ni column affinity chromatography,and the immunoreactivity of VP2 protein was detected by western-blot.Purified VP2 protein was used as an antigen to construct an indirect ELISA method.The optimal dilution ratio of standard positive serum was 1:20.ELISA results showed that the best blocking solution was 2%BSA blocking solution.The optimum dilution of enzyme IgG was 1:5 000.The color rendering solution of TMB substrate reacted at room temperature and away from light for 10 min.The sensitivity of the method was 1:160,and the specific results showed that there was no cross-reaction with the positive serum of porcine reproductive and respiratory syndrome virus(PRRSV),porcine fever virus(CSFV),porcine pseudorabies virus(PRV),porcine foot-and-mouth disease virus(FMDV),porcine epidemic diarrhea virus(PEDV)and porcine infectious pleural pneumonia(APP).The coefficient of variation of inter-and intra-batch repeatability was less than 10%.The results of 200 clinical samples show that the coincidence rate of this method with virus neutralization test(VNT)is 95.5%.The purified VP2 protein was labeled with biotin and the AlphaLISA method was established.Through the exploration and optimization of the reaction conditions,the results show that the total reaction system is 25 ?L and the total reaction time is 2 h.The sensitivity of the test was 1:320,and the specificity was that there was no cross-reaction with FMDV,PRRSV,CSFV,PRV,APP and PEDV positive serum.The coefficient of variation was less than 10%.The soluble VP2 protein was successfully expressed in this experiment.Indirect ELISA and AlphaLISA serological methods for detection of SVA VP2 antibody were initially established,with good specificity,sensitivity and stability.