摘要
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。
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. biffdium, 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. colf as well as in B. bifidium.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2007年第2期168-171,共4页
Chinese Journal of Zoonoses
