Isolation And Identification Of Bovine Rotavirus HB01 Strain And Preliminary Study On VP7 Subunit Vaccine

Bovine rotavirus(Bovine rotavirus,BRV)belongs to the Reoviridae family and belongs to the genus Rotavirus.It is a segmented double-stranded RNA virus and is a group A rotavirus(Rotavirus,RV).BRV is most susceptible to infection of calves within 7 days of age,with an incidence rate of about 90%to 100%,and a fatality rate of 50%;for most adult cattle,it is a latent infection,which will continue to excrete to the outside world,causing serious damage to the cattle industry all over the world.significant economic loss.The 11 gene segments of RV are prone to gene reassortment,and the emergence of reassortant strains makes prevention and control more difficult.Therefore,understanding the variation law of BRV is beneficial to the development of vaccines and the prevention and control of epidemic diseases.In this experiment,a strain of BRV was isolated and identified from a calf diarrhea disease material in a cattle farm in Hebei Province,and the whole genome was amplified and sequenced,and the genetic evolution analysis of the VP7 and VP4 gene sequences was carried out.The eukaryotic expression plasmid of BRV VP7 was constructed,the VP7 protein was obtained by expression and purification,and the VP7 subunit vaccine was prepared.The immunogenicity was determined by immunizing BALB/c mice,which provided a reference for the prevention and control of BRV.1.Isolation,identification and genetic evolution analysis of BRVIn this study,the feces of calves with diarrhea in a cattle farm in Hebei Province,which were positive by RT-PCR,had cytopathic changes in MA-104 cells after continuous blind passage for 5 generations.A strain of BRV was isolated and identified by PCR and indirect immunofluorescence techniques.It was later named RVA/Cow-wt/HB01/China/2021/G6P[1](referred to as HB01),and its complete serotype was G6-P[1]-I2-R2-C2-M2-A3-N2-T6-E2-H3,and focus on the genetic evolution analysis of the VP7 and VP4 genes of this strain,through the nucleotide sequence comparison with the domestic and foreign classic strains and amino acid The homology analysis showed that the HB01 strain had the highest homology to the VP7 and VP4 genes of the classic strain NCDV strain,and they were in the same branch.The research results provide reference for the genetic evolution and vaccine development of BRV.2.Eukaryotic expression and purification of BRV VP7 protein and study on animal level of subunit vaccine immunizationIn this study,the BRV VP7 gene was codon-optimized,and the pCAGGS-BRV-VP7 eukaryotic expression plasmid was constructed.The VP7 protein was expressed by the HEK-293F eukaryotic cell expression system,and the expressed protein was subjected to protein affinity chromatography and gel layer.The VP7protein with higher purity was obtained after analytical purification.The eukaryotic expression of BRV VP7 protein was used to immunize BALB/c mice to study the immunogenicity of the subunit vaccine.The results showed that the proportion of CD4~+and CD3~+subtypes of spleen lymphocytes in the immunized mice with VP7protein increased by flow cytometry;the proliferation rate of spleen lymphocytes in the experimental group was increased by MTT method;The contents of IFN-?and IL-4 cytokines in the experimental group increased;the titer of BRV neutralizing antibody in the serum of mice in the experimental group was 1:46.The results provide reference for the development of BRV VP7 subunit vaccine.