[Preparation and biological activity analysis of chimeric antibody against capsular F1 antigen of Yersinia pestis].

OBJECTIVE

To express human-mouse chimeric antibody against Yersinia pestis F1 capsular antigen (F1 antigen) and analyze its biological activities.

METHODS

The heavy chain gene of the chimeric antibody was obtained by fusing the variable region gene of the mouse mAb heavy chain with human IgG1 constant region gene. The light chain gene of the chimeric antibody was obtained by fusing the variable region gene of the mouse mAb light chain with the human kappa constant region gene. Both the heavy and light chain genes of the chimeric antibody were further verified by sequencing. The chimeric antibody heavy and light chain genes were inserted into EcoR I/Not I of pcDNA3.1 (+) to construct expression plasmids termed pcDNA3.1-L and pcDNA3.1-H, respectively. Then, two plasmids were mixed and transfected into CHO-S cells. Finally, the stable cell clone secreting chimeric antibody was obtained by G418 selection. The culture supernatants of serum-free medium were collected and the chimeric antibody was purified by MabSelect SuRe affinity chromatography. The purified chimeric antibody was analyzed by SDS-PAGE, Western blotting, ELISA and evaluated in the protective effect in vivo.

RESULTS

PCR and sequencing analysis proved that plasmids pcDNA3.1-H and pcDNA3.1-L were correctly constructed. Dot blot showed that a cell line with high-level expression of chimeric antibody was obtained. SDS-PAGE and western blot showed that the chimeric antibody was successfully purified. ELISA showed that the chimeric antibody could specifically bind to F1 antigen. In vivo activity assay showed that 80% BALB/c mice treated with the chimeric antibody survived from 36 MLD virulent Yersinia pestis.

CONCLUSION

The chimeric antibody against F1 antigen with neutralizing activity was successfully expressed in CHO-S cells, which laid a foundation for the preparation of anti-plague passive immunity agents.