Construction And Immunogenicity Of Recombinant Vaccinia Virus With BTV VP2 Gene

Bluetongue(BT)is an animal disease that must be reported by OIE,which is transmitted by arthropods such as cousin transmitted by blood and all ruminants can be infected.BT has been detected in 22 provinces,Bluetongue virus is the pathogen of BT.The general methods for diagnosing BT include clinical diagnosis and laboratory diagnosis such as AGID and ELISA.One of the most effective way to prevent BT is vaccination.Currently,inactivated vaccines for commercial purposes and live attenuated vaccines have their shortcomings.Live carrier vaccine method can effectively avoid the spread of live attenuated vaccines to the environment and lead to nucleic acid recombination.The biosafety hazard of virulence is also effective in preventing insufficiency of multiple high-dose immunizations.Therefore,it is important to study and construct a recombinant vaccinia virus vaccine which can stably express VP2 protein and has perfect immunogenicity and safety.In this study,the BTV 16 VP2 gene on plasmid p SK-VP2 was amplified by PCR amplification and its physicochemical properties such as hydrophobicity.Moreover,secondary structure and genetic evolution analysis were used to recombine BTV 16 VP2 gene.VP2 was optimized based on the BTV16 type VP2 gene sequence,three terminators recognized by poxviruses were mutated,and a Kozak sequence was added to the upstream of the BTV-VP2 start codon in order to enhance its expression.The VP2 gene was ligated into the shuttle vector by restriction enzyme ligation to obtain pSTKE-VP2.Then pSTKE-VP2 and vaccinia virus VTT were co-transfected into 2*106 monolayer BHK-21 cells,and the recombinant vaccinia virus rVTT-VP2 was obtained by screening the diseased plaque with reporter gene EGFP.The rVTT was detected by PCR,RT-PCR and Western blot for-identification of VP2.The successfully identified recombinant vaccinia rVTT-VP2 was immunized with 6 weeks old BALB/C mice by intramuscular injection at a final concentration of 105 PFU.The 6-week-old BALB/c mice were randomly divided into 10 groups,with rvtt-vp2,VTT and DMEM,respectively,for each group.The vaccine was administered by intramuscular injection.The mice were weighed every two days to assess the safety of the recombinant virus.Spleen lymphocytes were taken from mice in accordance with the experimental procedures.The T cell subtype,proliferation,and secretion of IFN-were detected.The levels of il-2,il-4,and serum specific antibodies were detected after peripheral blood serum isolation.RESULTS:The BTV 16 VP2 gene was 2880 bp long,the VP2 protein was 112 k Da,and the PI was 6.74.According to DNAstar analysis,VP2 has a good antigenic structure.Compared with the 27 serotype genes downloaded from NCBI,the results showed that the VP2 gene was type 16 in this study.It was found that the BTV 16 VP2 gene used in this study was likely to be derived from Japanese isolates,and Chinese popular type 1 The homology of the type at the nucleic acid level and the amino acid level were low,only 46.9%.After 10 times of plaque purification,the results showed that the target gene VP2 was successfully integrated into the recombinant vaccinia virus genome.RT-PCR and Western blot showed that BTV-16 VP2 was successfully expressed in infected cells.After 20 consecutive passages,PCR and RT-PCR could detect the integration and transcription of the foreign gene,and failed to amplify the TK gene,indicating that the recombinant vaccinia virus rVTT-VP2 has been selected pure and has good genetic stability.The virus was identified by Western blot and showed a band of capsid protein VP2,which proved that rVTT-VP2 virus can be expressed.After immunization according to the immunization protocol,the body weight of the rVTT-VP2 and VTT mice was not significantly different from that of the DMEM group,and the overall trend was upward.The surface recombinant virus rVTT-VP2 and VTT were safer.The T lymphocyte subset analysis showed that the gated rate of CD4~+and CD8~+cells was significantly increased compared with the DMEM group,indicating that rVTT-VP2 and VTT effectively stimulated the differentiation of murine T lymphocytes,and the immune response was good.Lymphocyte transformation experiments demonstrated that the specific antigens of rVTT-VP2 and VTT could significantly activate spleen lymphocytes and multiply proliferatively relative to the DMEM group.The serum IL-2 level in the rVTT-VP2 group was significantly higher than that in the DMEM group,and the IFN-?content induced by the inactivated rVTT-VP2 antigen was significantly higher than that in the DMEM group and lower than the PHA group.The combination of the two indicated that rVTT-VP2 induced BALB./C mice produce a cellular immune response.There was no significant difference in serum IL-4levels between the groups.CONCLUSION:The homology of VP2 gene with Japanese BTV16KSB-7/C/08 VP5 and BTVINDG53/ABT/HSR is high,and VP2 antigenicity is good.The recombinant VP2 shuttle vector pSTKE-VP2 was successfully constructed.The recombinant BTV16 VP2 vaccinia virus rVTT-VP2 was successfully obtained.The recombinant vaccinia virus rVTT-VP2 has good immune effect in vivo,high safety and effective stimulation of specific response.