Journal of Pure and Applied MicrobiologyVol. 8 No. Special Edition Nov. 2014

Prokaryotic Expression and Purification of Heat-labile Toxin B Subunit Gene and EtpA Adhesion Gene from Enterotoxigenic Escherichia coli

Wanzhe Yuan1,2,3*, Xiuyuan Zhang1,2,3, Qingan Han4 and Jiguo Sun1,2,3

1College of Animal Medicine, Agricultural University of Hebei, Baoding, Hebei 071001, China. 2Hebei Engineering and Technology Research Center of Veterinary Biological Products, Baoding, Hebei 071001, China. 3North China Research Center of Animal Epidemic Pathogen Biology, China Agriculture Ministry, Baoding, Hebei 071001, China. 4Hebei Center for Animal Disease Prevention and Control, Baoding, Hebei 071001, China.

Received on 10 August 2014 and accepted on 30 October 2014

 

ABSTRACT

Enterotoxigenic Escherichia coli (ETEC) is an important pathogen causing diarrheal disease in humans and animals in developing countries. EtpA adhesin and heat-labile enterotoxin B subunit (LT B) are the main virulence factors of ETEC. The purpose of this study is to construct a prokaryotic expression vector for EtpA and LT B and to purify these two  proteins. The cloning of the EtpA and LT B genes from ETEC using polymerase chain reaction (PCR) was performed. The fragments were then identified and cloned into the prokaryotic expression vector pET-32a. The recombinant plasmids were transformed into E. coli BL21(DE3) and induced to express with IPTG. The products of expression were analyzed by SDS-PAGE and western-blot. We obtained the optimum expression conditions of pET-EtpA and pET-LT B. The maximal expression quantity of EtpA was observed at 5 h of induction by 1 mM IPTG at 37 °C, and, whereas that of LT B was observed at 6 h of induction by 1 mM IPTG at 37 °C. The expression of EtpA and LT B provides the material basis for the further development of novel ETEC vaccine.

Keywords : Enterotoxigenic Escherichia coli (ETEC); EtpA; LT B; Prokaryotic Expression; Purification.