Genomic Analysis Of PCV3 Epidemic Strains In Guizhou And Prokaryotic Expression Of Cap Protein

Porcine circovirus(PCV)is a single-stranded circular DNA virus of the genus Circoviridae.According to the antigenicity and genotype,it is divided into three serotypes: PCV1,PCV2 and PCV3.PCV1 was first discovered in 1974 and is considered to be a pollutant in the cultured cells of PK-15.It has no pathogenic effect on pigs and is prevalent in pigs in many countries without clinical symptoms.PCV2 often causes porcine circovirus-associated disease(PCVAD),which is represented by weaned piglet multisystemic wasting syndrome(PMWS),which brings great harm to the breeding industry.In 2016,Phan T and Palinski R of the United States detected new circovirus(PCV3)in farms in different regions at about the same time.Subsequently,PCV3 was detected and reported in more than 10 countries including China,South Korea and Brazil.It has now become a worldwide distribution,posing a great threat to the pig industry.Guizhou Province’s epidemiological investigation of PCV3 and related gene research have not been reported in detail.In this study,a molecular epidemiological survey was conducted on the prevalence of PCV3 in Guizhou Province.Genetic evolution analysis was carried out based on the genome sequence,and the prokaryotic expression of PCV3,the only structural protein of the main immunogenic protein,was studied.Cap protein can lay the foundation for the research of serological detection method ELISA kit.1.Epidemiological investigation of porcine circovirus type 3 virus in Guizhou Province and genome analysis of epidemic strainsSpecific primers were designed for the PCV3 ORF2 region,and PCR amplification technology was used to specifically amplify the PCV3 ORF2 gene in427 samples from 92 large-scale farms in Guizhou Province collected between 2012 and 2018.The test results were classified and compared.The results showed that among 427 samples,91 were positive for PCV3,and formed double or multiple infections with CSFV,PRRSV,PCV2,PEDV,PRV and other viruses,and the doubleinfection rate with PRRSV was as high as 59.3%.In PCV3 infection,the positive rate increased from 2012 to 2018 year by year;the highest detection rate of PCV3 in Guiyang was 26.1%;the pigs with the highest detection rate of PCV3 was 25% in weaner piglets.At the same time,the paper also cloned and sequenced PCV3 whole genome for some positive samples,and obtained 11 PCV3 genome sequences with a genome size of 2 000 bp.Genetic evolution analysis revealed that among the 11 PCV3 strains,2 belonged to PCV3 b type,9 belonged to PCV3 c type,and no PCV3 a.The nucleotide sequence similarity between the strains is between 98.9% and 100%,and the similarity with foreign strains is between 98.7% and 99.8%.Compared with the domestic PCV3 strain,the nucleotide similarity is between 97.3% and 99.9%,and the similarity with PCV2 and PCV1 is about 40%,and the genetic relationship is far.2.Prokaryotic expression of porcine circovirus type 3 Cap proteinIn order to obtain the PCV3 Cap protein,the Cap gene was amplified by designing specific primers,cloned into pMD19-T vector,and then subcloned into the prokaryotic expression vector pET-32 a by genetic engineering.PCR,double enzyme digestion and sequencing were identified and transformed into the Rosseta E.coli expression system for time,temperature and IPTG optimization induced expression,and subjected to polyacrylamide gelelectrophoresis(SDS-PAGE)and immunoblotting,The expressed PCV3 Cap protein was detected by Western-blotting.The results showed that the prokaryotic expression plasmid of PCV3 Cap protein was successfully constructed.After optimal expression conditions,ie 1.0 mmol/L IPTG,induced by 37 ℃ for 4 h,SDS-PAGE electrophoresis detected the expressed protein at about 43.1 KDa..After transfer of the protein to the PVDF membrane by transfer,the monoclonal-His-tagged antibody was used as the primary antibody,and the fusion-expressed protein was subjected to Western-blotting detection.The result showed that the monoclonal antibodies labeled with His can specifically bind to and colour the fusion expressed target protein.This study laid a foundation for the further study of PCV3 serological differential diagnosis method and ELISA kit.