Isolation And Identification Of Orf Virus Strain And Establishtion Of ELISA Method

Orf, also know as contagious ecthyma, which is caused by orf virus, is a common epitheliotrophic viral disease of sheep,goats, and wild ruminants. Orf virus is the prototype species of the Para-poxvirus genus of the Poxviridae family that includes Pseudocowpoxvirus, Bovine papular sto-matitis virus and the Parapoxviruses of red deer,squirrels and seals. Orf virus has a worldwide distribution, meantime, Orf virus is a zoonotic pathogen as humans are also susceptible to orf, which people concern a lot.The ORFV genome consists of linear double-stranded DNA, it is 138 kbp and contains 132 putative genes. The envelope gene (B2L) of the ORFV encodes for a highly immunogenic major envelope protein of about 42 kDa, which is a homologue of vaccinia virus major envelope antigen p37K. The B2L gene has been used for the detection, molecular characterization and phylogenetic analysis of ORFV. With the virus host expanding, a standard diagnostic test will be useful to assess the prevalence of ORFV and other parapoxvirus-saaociated disease, helping to overcome the difficulty in quantifying the impact of ORFV on livestock production.Based on them, we carried out the following study.1 Isolation and Identification of orf virus Gansu strainIn this test, orf virus was isolated from samples of sick sheep crusta with HeLa cell. According to the nucleotide sequence of ORFV B2L gene published in NCBI, a specific pair of primers were designed using Oligo6.0, and PCR technique was used to amplify the conseved B2L gene. B2L nucleotide sequences of parapoxvirus were aligned, meantime, phylogenetic tree was constructed in maximum likehood method. Results indicated that Orf virus can produce a cytopathic effect after 1~2 blind passages in HeLa cell. The most common feature of the CPE were balling, rouding and degeneration of cells with detachment from the surface. The expected PCR fragments, approximately 1137bp in length, were obtained from genomic DNA. The sequencing results showed that it shared 99% smilarities with the reference sequence and submitted to NCBI GenBank(accession number is HQ694772). Phylogenetic relationship indicated that the isolated orf virus which belongedⅠbranch, had high homology with the Jilin strain(GU903501). Above all, one orf virus srain had been successfully isolated and named Orfv-GS strain.2 Prokaryotic expression and purification of ORFV B2L fusion proteinB2L fusion protein was expressed in E.coli Rosetta(DE3)plyS successfully. Expriments were designed to qualify the induction condition, and we finally choosed 28℃,0.4mmol/L IPTG and inducing for 4h as the optimized induction condition. The inclusion body was disposed of DOC and SKL, and then purified through Ni column. Results of SDS-PAGE showed that the puried expected protein, approximately 42 kDa ,was obtained. Meantime, the result of western-blotting indicated that the combinant protein had good immunogenic,which was consistant with the reference.3 Establishtion of orf ELISA detection methodIn this study, we choosed combinant B2L protein as incubation antigenic and made sure a series of condition in indirect ELISA. Trough matrix test, the optimized dense of antigenic is 5μg/mL, and the optimized dilution of serum is 1:100. In addtion, other expriments showed that this ELISA method had good specificity,sensibility and repeation.