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.