Expression and purification of A29L,B6R,and M1R proteins of mpox virus and their application in serological testing of the immunization by vaccinia virus
Objective To express and purify the A29L,B6R,and M1R proteins of mpox virus,and to analyze the application of the three proteins in serological testing of the immunization by vaccinia virus.Methods The codon-optimized A29L,B6R,and M1R antigens of mpox virus were synthesized and inserted into the pVRC vector.After transfection into HEK293T cells,the supernatant was harvested and the protein expression was verified by western blot.The proteins were purified by nickel column and ion exchange chromatography column,and the purities of the proteins were identified by SDS-PAGE and Coomassie Brilliant Blue staining.The purified A29L,B6R,and M1R were used as coating antigens.The antibody titers of IgG in the serum of mice,rhesus monkeys and humans immunized with vaccinia virus were detected by ELISA,and their correlation with the titers of neutralizing antibody in serum against vaccinia virus and mpox virus was analyzed.Results The A29L,B6R,and M1R proteins of mpox virus can all be expressed and secreted into the supernatant of cell culture in large quantity.After purification by nickel columns and ion exchange chromatography columns,the purities may reach over 90％.The specific IgGs against the three proteins can be detected in both animal and human serum immunized with vaccinia virus.The detected titers of IgG against the three antigens were significantly correlated.In the serums after vaccinia virus immunization,the antigen specific IgGs against A29L,B6R,and M1R proteins were also significantly correlated with levels of neutralizing antibody against vaccinia virus,but correlated weakly with neutralizing antibody levels against mpox virus.Conclusions The IgGs that cross reacted with mpox virus A29L,B6R,and M1R proteins can be detected in serums with vaccinia virus immunization,and the titers of IgG correlated significantly with that of anti-vaccinia virus neutralizing antibody,but the correlation with the titer of specific neutralizing antibody against mpox was weak.This study provided references for application of serological detection method and vaccine development for orthopoxviruses infection(including mpox).
Mpox virusA29L,B6R and M1R proteinsEukaryotic expressionProtein purificationELISA
NETL
NSTL
万方数据
