The Rescue And Biological Characteristics Of Recombinant Rabies Virus Containing E2 Gene Of Bovine Viral Diarrhea Virus

Bovine viral diarrhea virus(BVDV)causes symptoms such as fever,diarrhea,mucosal erosion and ulcers in cattle,causing serious economic losses to the cattle industry.The development of new vaccines is of great significance.Rabies virus(RABV)as a viral vector can stably express exogenous proteins,and exogenous proteins do not affect viral replication.In this study,the main dominant antigen E2 protein gene sequence of BVDV type 2 was optimized and synthesized.The E2-linker-E2 fragment was obtained by Overlap PCR.The E2-linker-E2 fragment was recombined into the rabies virus vaccine strain LBNSE virus vector by Bsi WI and Nhe I restriction enzyme digestion sites.The recombinant plasmid p LBNSE-2-E2 was constructed by reverse genetics.Then the full-length recombinant plasmid was transfected into BSR cells by liposome transfection to rescue LBNSE-2-E2 with high expression of BVDV E2 protein.The rescued recombinant virus was detected by direct immunofluorescence and test strip technique.At the same time,the growth curve of the recombinant virus was constructed and its pathogenicity was evaluated by mouse challenge test.The expression of recombinant E2-E2 protein was verified by real-time fluorescence quantitative PCR(q PCR)and Western-blot.The immunization test of calves was carried out after the recombinant virus was inactivated,and the BVDV neutralizing antibody was determined.The results showed that the recombinant plasmid p LBNSE-2-E2 was successfully constructed and the recombinant RV was successfully rescued,and the virus titer tended to be stable after F8 generation,the virus titer was higher than that of the parent LBNSE under the same culture conditions.The challenge test showed that the recombinant virus was not lethal to mice,and the pathogenicity was similar to that of the parental virus.The results of q PCR and Western-blot showed that the recombinant E2-E2 protein was successfully expressed in large quantities.After immunizing calves with inactivated recombinant virus,neutralizing antibodies against BVDV were successfully produced.The RV of recombinant BVDV type 2 E2 gene successfully rescued in this study provides a candidate strain for the development of new BVDV vaccines,and provides a new idea for the prevention and control of BVD.