Expression Of ORF95and ORF103of Capripoxvirus And Study On Indirect ELISA

Sheeppox and goatpox, called ‘smallpox’of sheep and goats, is a kind of highly contagious diseasecaused by sheeppox virus(SPPV) and goatpox virus(GTPV), respectively, which are included in thegenus of Capripoxvirus. The other member of the genus is LSDV that has major impacts on bovine.Sheeppox and goatpox are endemic throughout Asia, Africa and the Middle East. The disease hasseriously effect on the livestock and livestock products, leading to considerable economic losses offarmers.The genome of Capripoxvirus is dsDNA, and approximately comprised of150kbp. There are143~147ORFs in the genome of SPPV and GTPV. ORF95and ORF103are virion core proteins andplay important roles in virion assembly. Both of them have reaction against positive serum of GTPV andSPPV, so they were considered to be used as candidate antigens to detect the serum from infectedanimals. In view of that, the following work was carried out:1. Cloning and sequence analysis of ORF95and ORF103genesORF95and ORF103genes were cloned from genomic DNA of vaccine strain of GTPV. Sequencecomparison showed that the identities of two genes with homology genes from other isolates were ashigh as96.7%~100%and96.9%~100%at the level of nucleotide, respectively. Phylogenetic analysis oftwo genes showed that the vaccine strain from GTPV was clustered with other isolates of GTPV andseparated from SPPV isolates. Proteins encoded by two genes were predicted that there were no signalpeptide and transmembranes helics.2. Expression and purification of two recombinant proteinsThe two genes were subcloned into pET-30a (+) expression plasmid and expressed in E.coli BL21(DE3) by IPTG (1.0mmol/L) induction at37℃. SDS-PAGE analysis showed that the fusion proteinshad molecular weight of28ku and35ku, respectively, and mainly existed in form of inclusion body. Thefusion proteins were purified readily using affinity chromatography. The results of western-blottingindicated that the recombinant proteins could react with positive serum with good antigencity.3. Development of iELISA for differential diagnosis of capripox infected from vaccinated animalsThe results of optimization of coat antigens (ORF95: ORF103) showed that when the ratio of twoantigens was1:2and the gross of antigens was30ng, the values of OD450nmwere more stable and thevalue of C.V were lower than in other ratio of antigens. The iELISA method, with the characteristic ofreliable, standardized and specific, was established in our laboratory. Moreover, the assay was able todetect antibodies reliably in serum of sheep or goats following vaccination and in animals that werechallenged with a virulent SPPV or GTPV isolate. In summary, this method is suitable forhigh-throughput serosurveillance on a flock or herd.