[Clone and express ctb-stx2b fusion gene in Enterohemrrhagic escherichia coli O157:H7 Shigeal toxin 2B subunit and V cholera toxin B subunit and the detection of their immunogenicity].
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.


CONCLUSIONS
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.
State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China.
Zhen-Jun
ZJ
Li
Qiang-Zheng
QZ
Sun
Huai-Qi
HQ
Jing
Jian-Guo
JG
Xu
D015153:Blotting, Western; D002772:Cholera Toxin / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D019453:Escherichia coli O157 / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D022641:Shiga Toxin 2 / Q000235:genetics*; D014734:Vibrio cholerae / Q000235:genetics*
D011993:Recombinant Fusion Proteins; D022641:Shiga Toxin 2; C450841:shiga toxin 2 B subunit; D002772:Cholera Toxin