Research On Molecular Epidemiology Survery And ELISA Based On An Expressed S1 Antigen Of PDCoV

PDCo V is a single-stranded positive-stranded RNA virus with envelope structure.It was first discovered and reported by Hong Kong scholars in 2012.PDCo V broke out in the United States as early as possible,showing that the characteristics of pigs susceptible to infection in all ages,especially for piglets.The sick pigs are characterized by severe vomiting,diarrhea,dehydration and drowsiness.A pig can be found to be filled with undigested liquid in the stomach and intestines,and the intestinal wall becomes thin and brittle.Histopathological observation shows villous atrophic enteritis of the jejunum and cecum,and the epithelium of the cecum and colon can be observed at the same time.In this study,314 samples were collected from the dead pigs in parts of Guangdong province which were suspected of PDCo V.After RT-PCR test,a total of 42 samples were tested positive,with a positive rate of 13.4%.In this study,we designed and synthesized a set of primers for the whole genome sequencing,by amplifying 4 samples from different sources,two full-gene sequences and four partial S-gene sequences were obtained.The sequences were compared with other strains by genetic evolutionary tree,homology and predicted B cell antigen epitopes.The results showed that the homology of the two strains was 88.0%～99.4% with that of other strains,which was closest to that of Chinese strains.There were some amino acid mutations in B cell antigen epitopes of S gene.In this study,a primer containing a digestion site was designed based on the S1 gene sequence of PDCo V/CHGD/2016 strain,which was preserved in the laboratory.After amplification,the target fragment was linked to the p MAL-c5 x vector,and the recombinant plasmid PDCo V-S1-p MAL was successfully constructed.After induction,SDS-PAGE electrophoresis showed that the protein was about 100 k D.In order to induce more soluble proteins,the best inducing concentration,inducing time and inducing conditions were continuously screened.Finally,soluble proteins were obtained.The recombinant protein was prepared in large quantities under the above optimum conditions.After concentration,the recombinant protein was identified by Western blot assay.The results showed that the recombinant protein had good antigenicity.The concentrated protein was purified through affinity chromatography column,and the purified recombinant protein was finally obtained,which laid a foundation for further scientific research.An indirect ELISA method was established to detect the recombinant protein.The optimal concentration of antigen was 5.5 ug/m L,the dilution of serum was 1:200,the best sealing solution was 1% BSA,the best coating time was 2 h,the best incubation time of primary antibody was 1 h,the best incubation time of secondary antibody was 1.5 h,and the best development time was 20 min.The threshold value of negative and positive is 0.237,and the repeatability and specificity test results are good.The serum of 200 diseased animals was detected on the spot.32 of them were positive,and the positive rate was 16%.This study initially explored a set of indirect ELISA methods.