Prediction, cloning, expression and identification of the functional motif of the recombinant Ricin B

Abstract

Objective To determine the functional motif of the recombinant Ricin B(rRicin B) in Vibrio vulnificus cytolysin (VVC) and understand its molecule pathogenic mechanism.Methods The motif of VVC was predicted through bioinformatics analysis and cloned into a procaryotic expression vector pET28a-rRicin B.The recombinant plasmid was transformed into E.coli BL21 (DE3) and induced by IPTG to express rRicin B.The expressed protein was further analyzed by SDS-PAGE and purified by Ni2+-NTA agarose.Renaturation of the rRicin B were also carried out for further analysis.ELISA assay and confocal microscope was applied to identify the activity of the rRicin B on human Hela cells.Results Ricin B motif located in the 336-465 amino acids of Vibrio vulnificus cytolysin with a relative molecular weight of 20×103.The result of ELISA showed that the antigenicity of rRicin B was 28.71 U/L after renaturation.FITC labeled rRicin B could bind to the cell membrane and enter the cytoplasm of human Hela cells.Conclusion The Ricin B motif in Vibrio vulnificus cytolysin bearing the similar ability with the natural Ricin B can bind to the cell membrane and enter the cytoplasm.This feature may play an important role in the activity of pore-forming and the cytotoxicity of Vibrio vulnificus cytolysin. Key words: Vibrio vulnificus; Cytolysin; Ricin B