[Cloning, expression and identification of Escherichia coli LTB gene and Vibrio cholerae CTB gene].
To clone the LTB gene of E.coli and the CTB gene of V.cholerae, and to construct expression vectors of these genes.



The LTB gene from E.coli strain 44815 and the CTB gene from V.cholerae strain eastern  74 were amplified by high fidelity PCR. The nucleotide sequences of the two target DNA amplification fragments were sequenced after T-A cloning. pET32a expression vectors with inserted LTB and CTB genes were constructed. The LTB and CTB fusion proteins were expressed in E.coli strain BL21DE3 inducted by IPTG at different dosages. The two expression products were identified by SDS-PAGE and G(M1)-ELISA.



In comparison with the reported LTB and CTB sequences, the nucleotide sequence homologies of the cloned LTB gene and CTB gene were from 99.12% approximate, equals 99.71% and 98.54% approximate, equals 99.42%, while their putative amino acid sequence homologies were as high as 97.58% approximate, equals 99.19% and 96.77% approximate, equals 99.19%. The expression outputs of LTB and CTB fusion proteins in pET32a  LTB  BL21DE3 and pET32a-CTB-BL21DE3 systems were approximately 30% and 10% of the total bacterial proteins, respectively. The LTB and CTB fusion proteins were able to combine with bovine G(M1) confirmed by ELISA.



The expression systems of LTB and CTB genes have been successfully established. Both the expressed LTB and CTB fusion proteins possess mucosal adjuvant immunoactivity.
Xia
Xiao-Ping
XP
Department of Medical Microbiology and Parasitology, College of Medical Sciences, Zhejiang University, Hangzhou 310031, China.
Yan
Jie
J
Zhao
Shou-Feng
SF
D000276:Adjuvants, Immunologic / Q000494:pharmacology*; D000818:Animals; D001427:Bacterial Toxins / Q000235:genetics* / Q000494:pharmacology; D001483:Base Sequence; D002772:Cholera Toxin / Q000235:genetics* / Q000494:pharmacology; D003001:Cloning, Molecular; D004768:Enterotoxins / Q000235:genetics* / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D018928:Immunity, Mucosal; D011817:Rabbits; D014734:Vibrio cholerae / Q000235:genetics*
D000276:Adjuvants, Immunologic; D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D002772:Cholera Toxin; C027538:heat-labile enterotoxin, E coli