cry8E启动子指导的非晶体蛋白GabR在苏云金芽胞杆菌HD73菌株中的表达

Expression of GabR protein in Bacillus thuringiensis HD73 strain directed by cry8E promoter

摘要:

【目的】利用cry8E基因启动子指导的高效表达载体pHT315-8E21b，构建一个能够在苏云金芽胞杆菌(Bacillus thuringiensis，简称Bt)中正确表达非晶体蛋白GabR的重组菌株。【方法】将苏云金芽胞杆菌中的功能基因gabR装载到cry8E基因启动子指导的高效表达载体pHT315-8E21b上，转入到HD73?无晶体突变株后获得重组菌株HD-8E-gabR。通过SDS-PAGE和凝胶阻滞等方法对GabR蛋白的表达和功能进行分析。【结果】通过SDS-PAGE及蛋白定量等方法首次证明了在Bt表达体系中cry8E基因启动子指导的高效表达载体能够表达非晶体蛋白GabR，且通过碱裂解的方法可以提高GabR蛋白在Bt系统中的溶解性。进一步凝胶阻滞试验证明GabR能与其调控启动子PgabT结合。【结论】证明了cry8E基因启动子指导的Bt表达系统具有大量表达非晶体类蛋白的能力。

关键词:苏云金芽胞杆菌，cry8E启动子，GabR蛋白，表达载体

Abstract:

[Objective] To express a non-crystal protein GabR in Bacillus thuringiensis, the B. thuringiensis expression system directed by cry8E gene promoter was constructed and verified. [Methods] The gabR gene of B. thuringiensis was cloned into the high-level expression vector pHT315-8E21b initiated by cry8E gene promoter and the resulted vector was introduced into the acrystalliferous mutant HD73–, to obtain HD-8E-gabR. The SDS-PAGE and electrophoretic mobility shift analysis were performed for analysis of expression and function of GabR protein. [Results] SDS-PAGE analysis showed that GabR protein were successfully overexpressed in B. thuringiensis with the high-level expression vector pHT315-8E21b initiated by cry8E gene promoter, which was the first time to express a non-crystal protein in B. thuringiensis. The solubility of GabR protein in B. thuringiensis could be improved in the alkaline buffer. Electrophoretic mobility shift assay showed that GabR could bind with its regulated promoter PgabT. [Conclusion] This study proved that the B. thuringiensis expression system directed by cry8E gene promoter can be utilized to express a large number of non-crystal proteins.

Key words:Bacillus thuringiensis, cry8E promoter, GabR protein, Expression vector