霍乱毒素ctxA基因的克隆及原核表达

The Cloned ctxA Gene of Vibrio cholerae Expresses in E.coli

目的构建霍乱毒素A亚基基因(ctxA)的融合表达载体,并在原核细胞中表达,为霍乱弧菌肠毒素A亚基(CTA)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,PCR扩增获得霍乱弧菌ctxA基因,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),并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Trx-CTA融合蛋白,用SDS-PAGE及Westernblot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,我们扩增出了霍乱弧菌787bp的ctxA基因,成功构建了重组质粒pET-ctxA,经SDS-PAGE及Westernblot分析显示重组质粒pET-ctxA在原核细胞中得到了高效融合表达。结论霍乱弧菌ctxA基因在大肠杆菌中得到了高效表达。

Objective To construct the expression vector of Vibrio cholerae ctxA gene, and realize the Vibrio cholerae ctxA gene to express in E. coli, and lay a basis for future research on the immunogenicity and immunoadjuvant. Methods The ctxA gene, an cholera toxin subunit gene （ctxA） of Vibrio cholerae, was obtained by polymerase chain reaction （PCR） from DNA of Vibrio cholerae, and cloned into prokaryotic expressed vector pET32a（＋） with thioredoxin （Trx） gene. The recombinant plasmid （pET-ctxA） was analyzed to identify with restriction-endonuclease digestion, PCR and DNA sequencing analysis. And then the pET-ctxA was transferred into E. coli strain BL21（DE3） for transformation. The ctxA expression of pET-ctxA was induced with isopropy-β-D-thiogalactoside （IPTG） and the fused protein Trx-CTA was examined by SDS-PAGE and Western blot techniques. Results Restriction endonuclease digestion, PCR and DNA sequencing analyses showed that the ctxA gene of 787 bp was amplified from Vibrio cholerae DNA, and the recombinant plasmid pET-ctxA was successfully constructed, and the ctxA expression in prokaryotlc cell was detected by SDS-PAGE and Western blot techniques. Conculsion The ctxA gene of Vibrio cholerae, in fused protein form with Trx, got a high expression in E. coll.