| Orf is an acute and highly addicted epithelial diseases occured in sheep, goats, peopleand other ruminant animals. The disease is widely prevalent in the sheep industry developedcountries and cause serious economic losses. There is no effective drugs to control the disease.Currently, conventional attenuated and inactivated vaccines exist shortcomings, such asreversion of virulence, insecurity and low immune protection. So it urgently need to develop anew vaccine candidate strains to prevent orf. Goatpox virus as a vector to express foreigngenes was confirmed. Therefore, in order to obtain the vaccine to prevent orf and goat pox,this study selected goatpox virus attenuated vaccine as a carrier to construct the recombinantvirus expressing B2L gene of orf virus. In this study, our major work and the results are asfollows:1.Isolate and identify orf virus.we collected the scabs on the lamb lip in Xinjiang, isolateand culture orf virus in Vero cell and detected by electron microscopy and PCR. The ORFVvirions can be observed and the specific fragment was amplificated. Meanwhile the virus and31isolation virus in the other provinces of Chinese based on B2L gene sequences publishedon GenBank were analyzed in sequence homology and systematic evolution. The results showthat the fragment of B2L is1137bp. The nucleotide sequence homology of96.0%-99.1%issimilarity; The amino acid homology is88.7%-99.2%; Phylogenetic analysis showed that theisolation virus and NX/YC-2010is closest in genetic distance.2.Construct recombinant Goatpox virus and rearch biological characteristics of it. Thisstudy construct recombinant goatpox virus expressing B2L gene of orf virus by homologousrecombination. Firstly, according the sequences published in Genbank, we designed andsynthesized primers for PCR. Secondly, the recombinant goatpox shuttle vector wasconstructed and transfected into the BHK-21cell infected goatpox virus of attenuated vaccineusing lipofectamine. Then the recombinant goatpox virus rGPV-B2L was screened in agarincluding X-gal; And rGPV-B2L had been identified by PCR, indirect immunofluorescenceand Western blot. The results showed that B2L gene can be stably replicating, and expressingin BHK cell. The results of biological characteristics showed that rGPV-B2L was sensitive toacids, alkalis, organic solvents and ultraviolet. The recombinant virus can be inactivated in55℃30min or UV irradiation for60min.3.Evaluate the immune effect of the recombinant virus vaccine. The6-8week old femalemice were injected rGPV-B2L、rGPV-F1L、GPV or PBS by subcutaneous injection. The levelof specific antibodies in serum was detected by indirect ELISA. The cellular and humoralimmune levels were also evaluated by IFN-γ, TNF-α and IgG. The results showed that afterone week injected by recombinant virus, specific antibodies against B2L could be detected.When the recombinant virus infected the mice again, antibody levels reached significantly inserum. This indicates that the recombinant virus can stimulate higher humoral immunity. Thetest of IFN-γ and TNF-α showed that the recombinant virus can activate cellular immuneresponse in the body. Therefore, this study confirmed that the recombinant virus can stimulatemice to produce specific humoral immune responses and cellular immune responses. In addition, recombinant virus can make animals against orf virus.In summary, the recombinant goatpox virus which stably expressed Orf virus B2L gene,was obtained as candidate vaccine. The research suggested that the virus candidate vaccinewith excellent antigenicity and biological activity has excellent antigenicity and biologicalactivity, which provides a new way to prevent Contagious ecthyma as well as Capripox. |
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