霍乱弧菌肠毒素B亚单位基因在大肠杆菌和双歧杆菌的表达

目的构建霍乱弧菌肠毒素B亚单位(Cholera toxin B subunit,CTB)基因的大肠杆菌表达重组质粒,并观察其在大肠杆菌和双歧杆菌中的表达。方法从pBI121质粒PCR扩增获得CTB基因片断,克隆到大肠杆菌载体pGEX-4T-1上,构建重组质粒,然后转化大肠杆菌DH5α和双歧杆菌。转化菌经IPTG诱导,然后用SDS-PAGE和Western blot方法鉴定表达的重组蛋白。结果构建了重组质粒pGEX-4T-CTB,CTB基因片段分子量约为376bp;在大肠杆菌中表达出35kD的霍乱弧菌B亚单位融合蛋白,经SDS-PAGE分析,相对分子量与文献相符,表达的蛋白约占细菌总蛋白的10%;在双岐杆菌中也能得到正确表达,表达量较大肠杆菌低,占细菌总蛋白约5%。Western blotting结果确认了该条带为CTB基因的产物。结论构建的重组质粒pGEX-4T-CTB能够在大肠杆菌及双歧杆菌中获得表达。

Cloning and expression of cholera toxin B subunit gene in E.coli and Bifidobacterium bifidium

To construct a recombinant plasmid carrying the cholera toxin B subunit(CTB) gene and observe its expression in E.coli and Bifidobacterium bifidium,CTB was amplified from plasmid pBII21 by PCR and a recombinant plasmid pGEX-4T-CTB was constructed.This recombinant plasmid was then transformed into E.coli and B.bifidium.After induction with IPTG, the expression of the recombinant protein was verified by SDS-PAGE and Western blotting.Finally,the recombinant plasmid pGEX-4T-CTB was successfully constructed with a CTB gene fragment of 376 bps.A 35 kDa cholera toxin B subunit protein was expressed in E.coli.As demonstrated by SDS-PAGE analysis,its relative molecular weight was just the same as reported in the literature,and the amount of the expressed protein was 10% of the total bacterial proteins.Also,this protein could be expressed in B.bifidium,but the expression amount was lower than that expressed in E.coli,only accounting for 5% of the total bacterial proteins.This CTB gene product was also verified by Western blot analysis.It is concluded that the recombinant plasmid pGEX-4T-CTB can induce the expression of CTB fusion protein in E.coli as well as in B.bifidium.