Isolation And Identification Of Orf Virus And Preparation Of Mabs Against The Viral Envelop Protein B2L

Orf, which can threaten the development of local sheep industry, is one of zoonotic diseasescoused by orf virus worldwide. ORFV not only infects small ruminants and some wild animals,but also transmits directly to humans by contacting with infected animals. At present, traditionalorf virus inactived vaccine and attenuated vaccine play a role in prevention of the disease byinjecting them into the animals, but it seems not effective very much, even the immunizedsheep can be reinfected by ORFV sometimes. As the immunization type of host coused byORFV mainly are cellular immune response. Therefore, multi-epitope genetically engineeredvaccine need combine T-cell epitopes and B-cell epitopes. Increase the path of T-cellimmunization and activating the path of effective B-cell immunization response. it have animportant value on prevention and control of Orf.The suspected cases of Orf was discovered in the region of Daqing of Heilongjiang province.The solated ORFV strain was isolated by series of systemic motheds, and named OV/HLJ/04,the method maily contain antigen isolation, morphologic examination, and indirectimmunofluorescence assay, the alignment of nucleic acid sequences and amino acid sequencesand autologous animal immunity test. OV/HLJ/04had a close relationship with Shanxistrain(HQ221964) by phylogenetic analysis. OV/HLJ/04would additionally belong to genotypeâ…¢ by phylogenetic analysisbased on the B2L gene of orf virus.The gene B2L, which encodes the immunogenic envelope protein, as a target gene wasconstructed into prokaryotic expression vector of B2L gene, then we immunized by injectingthe purified the protein into the4-6week old female BALB/c mice for preparation ofmonoclonal antibody,3hybridoma cell strains can secrete the monoclonal antibodys byhybridoma technology, but only1hybridoma cell line can attenuate the virulence ofOV/HLJ/04, named2E4.2E4could specifically identify the protein B2L by indirect ELISA andWestern Blot; it expressed high antigen-specific antibody responses. The preparation of MAbsagainst protein B2L, biological characteristics analysis and preparation of monoclonal asciteshad been providing a more convenient method for the laboratory diagnosis of ORFV.