Construction Of Bovine Viral Diarrhea Virus E0-E2 Gene Fusion Adenovirus And Immune Evaluation In Mice

In this study,the E0 gene sequence of Si Chuan strain(Gen Bank Accession No.:EU709763)and BVDV V074 strain(Gen Bank Accession No.:A novel bovine viral diarrhea virus vaccine was constructed using adenovirus type 5 as a vector to prevent bovine viral diarrhea virus disease.E0 and E2 genes were amplified by PCR,E0-E2gene was fused by overlapping extended PCR,adenovirus shuttle plasmid PDC316-E0-E2 was constructed,and recombinant adenovirus vaccine Ad5-E0-E2was transfected and packaged,which was verified by Western blotting test,and virus titer was determined.The immune effect of recombinant adenovirus vector vaccine Ad5-E0-E2 constructed by muscle and subcutaneous immunization was studied by ELISA and flow cytometry after animal experiment.The temperature,weight,mental state,appetite and other clinical manifestations of experimental animals were recorded daily,and the trachea,heart,lung,spleen and kidney of experimental animals were collected for pathological tissue sections and observed by HE staining.In order to study the safety of Ad5-E0-E2 of recombinant adenovirus vector vaccine,PCR assay was performed using BVDV E0-E2 primers from aseptic animal tissues.1.Construction of recombinant adenovirus vector vaccine of bovine viral diarrhea E0-E2 geneHEK293T cells were used to proliferate BVDV virus according to the E0 gene sequence of Si Chuan strain(Gen Bank Accession No.:EU709763)and BVDV V074strain(Gen Bank Accession No.:KX170165)E2 gene sequence,Primer5.0 software was used to design and synthesize specific primers for E0 gene and E2 gene,BVDV e0-F/R and BVDV E2-F/R were used as c DNA template to amplify corresponding target fragments.E0-E2 gene fusion was conducted by overlapping extended PCR and the recombinant shuttle vector PDC316-E0-E2 was constructed.The recombinant shuttle vector PDC316-E0-E2 was co-transfected into HEK293T cells and packaged with the Ad Max adenovirus system plasmid.The packaged products were identified by Western-blotting assay.Finally,the titer of the product was measured.The results show that:The target fragments of E0 and E2 genes were successfully amplified,which were 681 bp and 1 122 bp,respectively.The E0-E2 gene was fused by overlapping extended PCR and the recombinant shuttle plasmid PDC316-E0-E2 was constructed.The adenovirus Ad5-E0-E2 was packaged into HEK293T cells after co-transfection with the Ad Max cytoplasmic plasmid.Its titer was 1.1×1010 p FU/m L.Western blotting was used to detect the expression of ad5-E0-E2 exogenous gene in293T cells,and the target band(65k Da)was obtained in accordance with the expectation.2.Immunogenicity of recombinant adenovirus vector vaccine of BOVINE viral diarrhea E0-E2 geneForty SPF mice aged 6-8 weeks(20 males and 20 females)were randomly divided into 4 groups with 10 mice in each group,which were divided into 2experimental groups and 2 control groups(PBS group).Immunization is carried out in accordance with the established immunization methods and procedures.The tail vein blood of experimental animals was collected on the day before immunization,the day before the second immunization and one week after the second immunization,and the serum was separated to detect the humoral immunity level of experimental animals using bovine viral diarrhea ELISA kit.CD4~+and CD8~+T cells were measured by flow cytometry to test the cellular immunity level of experimental animals.The results showed that the recombinant adenovirus vector vaccine Ad5-E0-E2 could induce higher levels of humoral and cellular immunity in mice immunized with muscle and subcutaneous injection.3.Safety evaluation of recombinant adenovirus vector vaccine of bovine viral diarrhea E0-E2 geneForty SPF mice(20 male and 20 female)aged 6 to 8 weeks and weighing 18 to20g were randomly divided into 4 groups with 10 mice in each group.The mice were divided into 2 experimental groups and 2 control groups(PBS group),and were immunized according to the established immunization method and immunization program.Clinical manifestations of mice,HE staining of pathological tissue sections and PCR detection were used to verify whether the constructed recombinant vaccine caused damage to mice,and the safety of recombinant adenovirus Ad5-E0-E2 was systematically evaluated.The results showed that the mental state,body temperature,diet and drinking water,and behavioral activities of mice were normal after inoculation with recombinant adenovirus vector vaccine Ad5-E0-E2 without obvious changes in clinical symptoms and adverse reactions.The visceral organs of mice were collected aseptically for histopathological staining.E0-E2 fusion gene could be detected in the heart,liver,spleen,lung,kidney and intestine of mice by PCR.