摘要
目的构建幽门螺杆菌融合基因vacA-hpaA的大肠杆菌-双歧杆菌穿梭表达质粒,并观察其在大肠杆菌(E.coli)和双歧杆菌(B.bifidium)中的表达情况。方法通过PCR得到hpaA-vacA融合基因,将该融合基因定向克隆到大肠埃希菌-双歧杆菌穿梭表达载体pGEX-1λT,构建重组质粒pGEX-hpaA-vacA,电穿孔转化入大肠杆菌BL21和双歧杆菌。转化菌经IPTG诱导,然后用SDS-PAGE和Western blotting方法鉴定表达的重组蛋白。结果构建了重组质粒pGEX-vacA-hpaA,vacA-hpaA融合基因的分子量约为1 548 bp;经SDS-PAGE分析,在大肠杆菌中表达出85 kD的融合蛋白,表达的蛋白约占细菌总蛋白的20.5%;在双岐杆菌中也能得到正确表达,但表达量较大肠杆菌低,占细菌总蛋白约8.5%。Western blotting结果确认了该条带为hpaA-vacA融合基因的产物。结论构建的重组质粒pGEX-hpaA-vacA能够在大肠杆菌及双歧杆菌中获得表达。
[Objective] To construct the E.coil-B.bifidium shuttle plasmid pGEX-vacA-hpaA of Helicobacter pylori and observe its expression in E.coli and B.bifidium. [Methods] VacA-hpaA fusion gene was amplified from plasmid pQE-vacA-hpaA by PCR. Cloned the vacA-hpaA fusion gene into E.coil-B.bifidium shuttle plasmid pGEX-lkT and constructed recombinant plasmid pGEX-vacA-hpaA. 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 blot. [Results] The recombinant plasmid pGEX-vacA-hpaA was successfully constructed with a vacA-hpaA gene fragment of 1 548 bps. A 85 kDa fusion protein was ex- pressed .in E.coli. As demonstrated by SDS-PAGE analyzed, and the amount of the expressed protein was 20.5% 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 8.5% of the total bacterial proteins. This vacA-hpaA gene product was also verified by Western blot analysis. [ Conclusion] That recombinant plasmid pGEX-vacA-hpaA of construction can induce the expression of fusion protein both in E.coli and in B.bifidium.
出处
《中国现代医学杂志》
CAS
CSCD
北大核心
2011年第35期4388-4391,4395,共5页
China Journal of Modern Medicine
基金
云南省自然科学基金(No:2007C147M)
