[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.
Li
Zhen-Jun
ZJ
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.
Sun
Qiang-Zheng
QZ
Jing
Huai-Qi
HQ
Xu
Jian-Guo
JG
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