Isolation And Identification Of Orf Virus And The Expression Of B2L And F1L Gene In Baculovirus System And Its Antigenicity Analysis

Orf virus(ORFV),also known as contagious pustular dermatitis virus(CPDV),induces acute pustular skin lesions in sheep and goats,which also infects human.Aimed to investigate the biological characteristics and epidemiology of the ORFV in White Cashmere goats in the northern region of Shaanxi province,the lip scabs of a lamb with typical orf symptoms from a Cashmere goat farm were collected,and used to isolate ORFV with Madin-Darby bovine kidney(MDBK)cell-line.Meanwhile,the full-length genes of ORFV B2L and F1L were amplified with specific primers,after that,homology and phylogenetic analysis based on B2L and F1L genes were performed.Besides that,B2L,F1L and B2L-F1L were expressed in sf9 cells based on the baculovirus expression system.The expressed product was identified by SDS-PAGE,mass spectrometry and western blot assays.The recombinant protein expressed by the baculovirus were purified,and then been used to establish an indirect ELISA method for detecting ORFV,and the immunogenicity of these recombinant proteins were evaluated in mices.This study revealed novel thoughts for the prevention and control of Orf.1.After inoculation of suspected samples on MDBK cell-line,and five continuous passages,obvious cytopathic effect(CPE)could be observed,subsequently verification has been performed by specific PCR and sequencing results,thus,the isolated ORFV was named as strain Yulin.2.The amplicons of B2L gene and F1L gene were 1 137 bp and 1029bp,respectively.The results of phylogenic analysis based on nucleotides sequences of B2L displayed that ORFV-Yulin was closely related to strain Hub 13,while the phylogenic analysis based on nucleotides sequences of F1L showed that ORFV-Yulin belonged to the same branch as strains isolated in Fujian Province and Shanxi Province.3.pBac-B2L and pBac-cF1L,two vectors for the expression of recombinant protein B2L and F1L,based on Baculovirus expression system,were constructed,subsequently,target proteins were successfully expressed in the sf9 cell-line,although without so much high soluble expression level.4.The recombinant protein GB2LcF1L,based on the B2L and F1L genes without the signal peptide,linked with linker sequences,accomplished with the overlap PCR,was expressed highly in the sf9 cell-line,based on the SDS-PAGE,Mass Spectrometry and Western blot,importantly,almost 40%of the recombinant protein were secreted into the medium of cell cultivation.5.The indirect ELISA method was established based on the recombinant protein rB2LcF1L,which exhibited good specificity?sensibility and repeatability.6.Mice were applied for the immunization of the recombinant protein rB2LcF1L,results of neutralizing antibody titers in mouse were higher than 1:32,the following challenge study revealed that 70%of immunized mice were protected against challenge.