一株猪A群轮状病毒的分离及VP6多克隆抗体的制备OA

The aim of this study was to isolate and identify the prevalent strains of porcine group A rotavirus(PoRVA)in a pig farm and to prepare polyclonal antibodies for the VP6 protein.The positive fecal samples of PoRVA from diarrheic piglets were collected,selected,pro-cessed,and inoculated into Marc145 cells for virus isolation.The strains were identified through RT-PCR and indirect immunofluorescence(IFA)assays.The full-length sequence of VP6 was amplified and cloned into the prokaryotic expression vector pET-28a.The correctly re-combinant expression plasmid was transformed into the Escherichia coli BL21(DE3)competent cells.The soluble analysis and nickel column purification of the recombinant protein induced by IPTG were performed,which were identified by SDS-PAGE and Western blot.Finally,BALB/c mice were immunized with the purified VP6 protein,and the serum was collected from the mice for Western blot and IFA identifica-tion.The results showed that a PoRVA strain GDYT2024 was successfully isolated,which belonged to G12 genotype(VP7).The recombi-nant proteins were expressed mainly in the form of inclusion bodies.Purification determined the band to be of 43 kDa,consistent with the ex-pected result.The titer of the prepared polyclonal antibody measured by indirect ELISA was more than 1∶64 000,and the Western blot(the dilution ratio of polyclonal antibody ranged from 1∶500 to 1∶3 000)and IFA(the dilution ratio of polyclonal antibody ranged from 1∶500 to 1∶1 500)test results of the samples infected with PoRVA all presented positive signals,indicating that the prepared polyclonal antibody produced a good antigen-antibody reaction with the viral protein.This study laid a foundation for subsequent research on isolation and identi-fication of PoRVA,and on the infection and pathogenic molecular mechanisms of the strain.