霍乱毒素B亚单位基因（CtxB）的克隆及其表达

从霍乱弧菌中抽提基因组ＤＮＡ，用ＰＣＥＲ方法获取霍乱毒素Ｂ亚单位基因（ＣｔｘＢ）。序列分析结果表明，ＣｔｘＢ基因编码１２４个氨基酸，其中编码６２位Ｔｈｒ的密码子与文献报道有差异。将ＣｔｘＢ基因插入质粒ｐＧＥＸ－４Ｔ－２，构建ｐＧＥＸ－ＣＴＸＢ表达质粒，转化大肠相菌ＢＬ２１（ＤＥ３０，筛选表达菌株ＣＴＸＢ／ＢＬ２１。工程株经ＩＰＴＧ诱导表达，可产生大量的表达蛋白，经ＳＤＳ－ＰＡＧＥ分析，融合蛋白分子

Cloning of the CtxB Gene of Vibrio cholerae and Its Expression in E.coli

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.