一种新的Bt杀虫基因HJC原核表达蛋白活性的鉴定

Activity of a New Recombinant Insecticidal Crystal Protein Encoded by HJC Gene from Bacillus thuringiensis

HJC基因是由2个Bt基因(cry1Ab和vip3)经过人工融合而成,具有更广谱的杀虫活性,可延缓害虫产生交互抗性的时间。将已构建好的携带HJC基因的重组质粒pET28a-HJC转化到大肠杆菌BL21中诱导表达。该HJC融合蛋白主要以包涵体形式存在,变性条件下使用镍亲和层析柱对其进行纯化,并经尿素梯度透析复性后,进行免疫反应活性及美国白蛾杀虫活性测定。Western blot结果显示,该原核表达蛋白与转HJC基因水稻中的HJC蛋白有相同的免疫反应性,对美国白蛾也有一定的杀虫活性,可以替代植物外源蛋白进行转HJC基因产品的食用安全性评价。

For a better management of insect cross-resistance against Bt toxins, a fusion gene derived from crylAb and rip3 genes was created and named HJC. The gene was expressed in E. coli strain BL21 upon transformation with plasmid pET28a-HJC. The his-tagged HJC protein mainly existed as inclusion body in E. coli, and was purified by Ni-NTA resin affinity chromatography under denatured condition. After refolding upon gradient urea dialysis, recombinant HJC protein was analyzed for its immune recognition and insecticidal activity. The results showed that the HJC protein expressed from E. coli was substantially equivalent to that of a transgenic rice carrying an HJC gene in terms of immune recognition. The refolded HJC protein exhibited high insecticidal activity against Hyphantria cunea. To the end, this protein can be used to substitute extrinsic plant protein in food-safty assessment of genetically modified products with HJC gene.