Objective: To clone and express the fusion gene encoding Enterohemrrhagic escherichia coli O157 : H7 (EHEC O157 : H7) Shigela toxin 2B subunit (Stx2B) and vibrio cholera toxin B subunit (CTB) as well as to detect the immunogenicity and GM1-binding ability of fusion protein.
Methods: To design a primer to amplify stx2b gene and ctb-stx2b fusion gene encoding Stx2B and CTB-Stx2B respectively and to clone the genes into express plasmid pET30a(+)C in order to construct pET30a-ctb-stx2b after T-A sequencing was varified, then to transform constructed plasmid into E. coli BL21 (DE3) induced by IPTG and purified by a purify kit and to detect molecular weight and immunogenicity by SDS-PAGE and Western-blot.
Results: The amplified ctb-stx2b fragments appeared to be 750 bp and gene sequence was identical to designed sequence. The prokaryotic expression system pET30a-ctb-stx2b/BL21 could express protein weight about M(r) 20 x 10(3) and the expressed protein could react to CTB monoclone anti-body. The fusion protein CTB-Stx2B could bind GM1.
Conclusion: CTB-Stx2B had successfully been expressed in prokaryotic while the expressed protein had good immunogenicity and GM1-Binding ability. This study provided information on further EHEC O157 : H7 vaccine research.