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【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。
【Objective】 The recombinant plasmid pHT315-8E21b, which is under the guidance of cry8E gene promoter, was used to construct a recombinant strain capable of correctly expressing GabR in Bacillus thuringiensis (Bt). 【Method】 The functional gene gabR of Bacillus thuringiensis was loaded into pHT315-8E21b vector, which was directed by promoter of cry8E gene, and transformed into HD73-aphase mutant to obtain recombinant strain HD-8E-gabR. The expression and function of GabR protein were analyzed by SDS-PAGE and gel blocking methods. 【Result】 The results of SDS-PAGE and protein quantification showed that the high efficiency expression vector of cry8E promoter in Bt expression system could express GabR protein, and the increase of GabR protein in Bt system by alkaline lysis Solubility. Further gel retardation test proved that GabR can bind with its regulatory promoter PgabT. 【Conclusion】 The Bt expression system under the guidance of cry8E gene promoter is proved to have the ability to express large amount of non-crystal proteins.