Soluble Expression Purification And Immunogenicity Analysis Of VP2 Protein Of Bluetongue Virus Type 1

Bluetongue(BT)is a viral infection of ruminants that is caused by insects as the vector of infection caused by Bluetongue virus(BTV).The clinical symptoms are mainly nose,lip mucosal congestion,salivation,dyspnea and tongue cyanosis.BTV is widely spread in the world,which is extremely harmful to the global livestock industry.Vaccination is one of the effective measures to against BT.The vaccines used to prevent and control BT in the market are mainly attenuated live vaccines and inactivated vaccines,but both have certain disadvantages.Currently,there are no safe and high efficiency vaccine in the market.VP2 protein is located in the outermost layer of the virus shell and contains major antigenic determinants.It can induce the body to produce neutralizing antibodies and play an important role in the body's antiviral immunity process.Studies have confirmed that VP2 protein alone is sufficient for the body to produce a protective immune response.Therefore,this study establishes a certain foundation for further research on BTV1 VP2 protein by the soluble recombinant VP2 protein was expressed in prokaryotic expression system and analyze its immunogenicity.In this study,the VP2 target gene of BTV1 was amplified by PCR.Then,the positive recombinant plasmid p ET-28a-VP2 was construced.The plasmid was transformed into BL21(DE3)and Rosetta(DE3)competent cells.Both were induced and identified.The results show that both strains can express the recombinant protein VP2 with a molecular weight of about 109 KDa,the solubility of VP2 protein expressed is better in Rosetta(DE3)strains;At the same time,the expression conditions of Rosetta(DE3)expression strain were optimized.The results showed that the soluble expression of recombinant VP2 protein was the highest at 0.6 mmol/L IPTG and 16? for 16 h;The recombinant VP2 protein was purified by nickel column affinity chromatography and determined by Bradford method at a concentration of 0.4mg/m L.Western blot experiment showed that the recombinant VP2 protein could react with positive sera BTV1.The results showed that the recombinant VP2 protein prepared in this study has good immunoreactivity.Five 6?8 week old Balb/c mice were immunized with purified recombinant VP2protein at a dose of 30?g/head,with 3 weeks interval and 3 immunizations.Dot-ELISA results confirmed that the serum of mice immunized with VP2 could specifically react with BTV1 inactivated virus;Indirect ELISA results showed that the serum titer with VP2 as coating protein was up to 1:2.048×10~5,indicating that the mice immunized with recombinant VP2 protein produced high levels of specific antibodies.Indirect immunofluorescence(IFA)results showed that the antibodies produced after immunization with recombinant VP2 protein can react specifically with VP2 protein expressed by eukaryotic system.The soluble VP2 protein expressed in this study not only provides experimental materials for the research of its structure and function,but also provides a basis for the development of a safe and efficient BT subunit vaccine.