Prokaryotic Expression And Purification Of Chikungunya E2 Protein And Evaluation Of Immunization Effect Of Different Adjuvants

Objective:to achieve prokaryotic expression,purification and renaturation of CHIKV-E2 protein,and then compare the immunopotentiating effects of different adjuvants on subunit vaccines,so as to screen out the synergistic subunit vaccine with good immunogenic adjuvant,which is the subsequent foundation for the development of CHIKV subunit vaccines.Methods:(1)according to the CHIKV-E2 sequence published by Genbank,the E2 gene was synthesized,and the E2 gene was obtained by PCR and gel recovery,and inserted into pEASY bluent Zero cloning vector was sequenced and the E2 gene fragment was digested by restriction enzyme digestion,and the pET-28 a vector was digested to obtain a recombinant plasmid and identified by double enzyme digestion.(2)the recombinant plasmid was transformed into the host strain BL21(DE3)for induction of expression,and the induction conditions(time,temperature,IPTG)were optimized,and the recombinant protein was expressed in a large amount and subjected to Ni column affinity chromatography to obtain a protein of higher purity,and then The purified protein was subjected to dialysis and renaturation to obtain a soluble protein,and WB verification was performed.(3)the obtained soluble E2 protein was supplemented with different adjuvants to co-immunize the mice,the blood of the mice was collected every week and the serum was separated,and then the following five indicators were detected for the serum,respectively,for detecting the proliferation of T lymphocytes.Detection of T lymphocyte subsets,detection of cytokines IL-4,IFN-?,and detection of specific antibodies.Results(1)the recombinant plasmid pET28a-E2 was successfully constructed,and the prokaryotic expression of E2 protein was realized.The optimal induction conditions were as follows: temperature was 37 ?,time was 6 h,and concentration of IPTG was 0.3 mM.the Ni2 affinity chromatography was used to purify the E2 protein,and finally the target protein solution with a purity of 90% and a concentration of 0.54 mg/mL was obtained.The concentration of urea was reduced by dialysis refolding gradient,and finally the soluble protein was obtained.The concentration was 0.23 mg/mL.(2)the subunit vaccine containing adjuvant has better immune effect than the adjuvant-free subunit vaccine,indicating that theadjuvant can improve the immunity of the subunit vaccine,including the E2+ adjuvant(A+D)group and the E2+ adjuvant(The B+E)group has good effects in both cellular and humoral immunity,so the combination of adjuvant(A+D)combination and adjuvant(B+E)can be used as a candidate adjuvant in the development of the CHIKV-E2 subunit vaccine.Conclusion:the prokaryotic expression,purification and renaturation of CHIKV-E2 protein were successfully achieved,and the soluble protein with higher purity was obtained.The adjuvants(A+D)combination and adjuvant(B+E)combination were successfully screened by mouse immunization experiments,which can be used as candidate adjuvants in the development of CHIKV-E2 subunit vaccine.