霍乱弧菌ctxAB融合基因的克隆及原核表达

Cloning of the Fusion ctxAB of Vibrio Cholerae and Its Induced Expression in E.coli.

目的构建霍乱毒素A亚基基因(ctxA)和B亚基基因(ctxB)的融合表达载体,并在原核系统表达,为霍乱毒素(CT)免疫原性的研究及其作为免疫佐剂的研究提供基础。方法以霍乱弧菌DNA为模板,利用重叠PCR技术,扩增出含有ctxA和ctxB的融合基因片段ctxAB,并与带有硫氧还蛋白(Trx)基因的高效原核表达质粒pET32a(+)定向重组,构建重组质粒,转化大肠杆菌BL21(DE3)。经限制性核酸内切酶酶切鉴定、PCR和核酸序列分析后,以IPTG诱导表达TrxBB-CTAB融合蛋白,用SDS-PAGE及W estern b lot进行鉴定。结果限制性核酸内切酶酶切鉴定、PCR和核酸序列分析表明,扩增出了霍乱弧菌1 158bp的ctxAB融合基因,成功构建了重组质粒pET-ctxAB,SDS-PAGE及W estern b lot分析显示重组质粒pET-ctxAB在原核系统中得到了高效融合表达。结论霍乱弧菌ctxA基因和ctxB基因通过重叠PCR成功地融合在一起,融合基因ctxAB在大肠杆菌中得到了高效表达。

Objective To construct the fused expression vector of ctxA gene and ctxB gene of Vibrio cholerae and to realize the expression of ctxAB gene of Vibrio cholerae in E. coli and to lay basis for future research on the values of immunogenicity and immunoadjuvant. Method In this study, the fusion gene ctxAB containing ctxA and ctxB gene was obtained from DNA of Vibrio cholera by overlap PCR, and to construct recombinant plasmid pET-ctxAB the fusion gene ctxAB was cloned into prokaryotic expressed vector pET32a（ ＋ ） containing thioredoxin gene Trx, and pET-ctxAB was transformed into E. coli strain BL21 （DE3）. pET-ctxAB was analyzed with restriction-endonuclease digestion, PCR and DNA sequencing analysis, and was induced with isopropy-β-D-thiogalactoside （IPTG） to express fusion protein Trx-CTAB, and Trx-CTAB was examined with SDS-PAGE and Western blot techniques. Result Restriction endonuclease digestion, PCR and DNA sequencing a- nalysis showed that the ctxAB gene of 1 158 bp was amplified from Vibrio cholerae DNA, and the recombinant plasmid pET-ctxAB was constructed and detected its expression in prokaryotic cell successfully with SDS-PAGE and Western blot techniques. Conculsion The ctxA and ctxB gene of Vibrio cholerae were fused together by overlap PCR, and the fusion ctxAB gene has highly expressed in E. coli.