Cloning of the CtxB Gene of Vibrio
cholerae and Its Expression in E.coli
HE Zhi-Yong1,2, LI Ming-Feng1, ZHANG
Wei-Jie2, WU Xiang-Fu1
( 1Shanghai Institute of Biochemistry, Chinese Academy of
Science, Shanghai 200031, China； 2Department of Bioscience and
Biotechnology, Shanghai Jiao-Tong University, Shanghai 200240, China )
Abatract The CtxB
gene encoding cholerae toxin subunit B was amplified from Vibrio cholerae
genomic DNA by PCR. The result of sequencing indicated that CtxB gene
encodes 124 amino acid residues. The sequence of CtxB gene was almost
the same as that of reported except for the codon of Thr 62. The expression
plasmid pGEX-CTXB was constructed by inserting CtxB gene into plasmid
pGEX-4T-2, containing gst gene, immediately downstream of the T7
promoter. The expressed plasmid was introduced into E.coli BL21(DE3)
cells and expression strain CTXB/BL21 was selected. SDS-PAGE analysis revealed
that the GST-CTXB fusion protein was highly expression and accumulated up to
36% of bacterial soluble proteins after the induction by IPTG. A fusion protein
of 40 kD was expressed as inclusion body. The fusion protein was refolded and
purified. The purified fusion protein was cut by thrombin to obtain the
purified CTXB protein.
Key words cholerae toxin subunit B； gene cloning； gene expression； E.coli
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