Prokaryotic Expression and Immunogenicity Evaluation of Monkeypox Virus A5L Protein
[Objective]The purpose of this experiment was to obtain high-purity Monkeypox virus(MPXV)A5L protein and prepare mouse anti-A5L highly valent antiserum,which provided materials for the function study of MPXV A5L protein and the research and development of related diagnostic reagents.[Method]The recombinant plasmid pET-28a-A5L was constructed,and the correctly sequenced recombinant plasmid was transformed into Escherichia coli BL21(DE3)competent cells for induction expression.The expression of recombinant protein A5L was analyzed under different IPTG induction concentration,induction temperature and induction time conditions,and the optimal expression conditions were screened.The expression of recombinant protein A5L was identified by Western blotting,and the solubility of recombinant protein was analyzed by SDS-PAGE.A5L protein was purified by His-Bind nickel column affinity chromatography and immunized mice.Antibody titers were detected by ELISA.[Result]The results of double digestion and sequencing showed that the prokaryotic expression vector pET-28a-A5L was successfully constructed.Western blotting results showed that recombinant protein A5L was successfully expressed in Escherichia coli BL21(DE3)competent cells.A5L protein expression was the highest at 42 ℃,IPTG concentration of 1.6 mmol/L and 16 h after induction,with a size of 40 ku.SDS-PAGE analysis showed that A5L protein mainly existed in the form of inclusion bodies,and the optimal imidazole elution concentration was 50 mmol/L.ELISA results showed that the most effective value of the prepared murine anti-A5L protein antibody was 1:205 440.[Conclusion]The recombinant plasmid pET-28a-A5L was successfully constructed and the recombinant protein A5L was successfully expressed in the prokaryotic expression system.The prepared A5L polyclonal antibody had good immunogenicity,and the results laid a foundation for further investigation of the biological function of MPXV A5L protein.
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维普
