苏云金芽孢杆菌cry1ac22杀虫蛋白在大肠杆菌中的 - biopublisher.doc

苏云金芽孢杆菌Cry1Ac22杀虫晶体蛋白在大肠杆菌中的表达与纯化 刘卓明1,2　张文飞1,2　谢柳1,2 　叶大维3　柳参奎4*　方宣钧1,2* 1广西大学生命科学与技术学院, 南宁, 530005; 2海南海德热带农业资源研究所, 三亚, 572025; 3南开大学生命科学院, 天津, 300071; 4东北林业大学, 哈尔滨, 150040 *共同通讯作者, xuanjunfang@ 摘要：将苏云金芽孢杆菌W015菌株中的Cry1Ac22基因采用基因重组技术克隆到pQE30高效原核表达载体上，构建了重组质粒pQE30-Cry1Ac22。将构建好的pQE30-Cry1Ac22转化M15宿主菌，IPTG诱导表达出了His-tag-Cry1Ac22融合蛋白,并采用Ni2+-NTA树脂对融合蛋白进行亲和层析纯化。经PCR、BamHⅠ和SalⅠ酶切及DNA测序鉴定表明采用基因重组技术成功构了原核表达载体pQE30-Cry1Ac22。SDS结果表明,转化了pQE30-Cry1Ac22的宿主菌株表达出了分子量为133 kDa的His-tag-Cry1Ac22，表达的融合蛋白在细胞中以包涵体的形式存在。不同的IPTG浓度和温度诱导实验，最佳诱导表达条件1 mmol/L IPTG浓度和28℃的经Ni2+-NTA树脂亲和层析得到了纯化的His-tag-Cry1Ac22融合蛋白，生物活性测定表明工程菌株和蛋白对小菜蛾初孵幼虫具有较高毒力。本研究成功地构建了pQE30-Cry1Ac22原核表达载体,并大量表达和纯化了His-tag-Cry1Ac22,为采用外源性Cry1Ac22蛋白制备抗体和进行定量杀虫活性的测定奠定了基础。 关键词：苏云金芽孢杆菌W015, Cry1Ac22, 融合蛋白, 原核表达, 纯化 Expression and Purification of Bacillus thuringiensis Cry1Ac22 protein in Escherichia coli Liu Zhuoming1,2Zhang Wenfei1,2　Xie Liu1,2　Dawei Yeh3　Liu Shenkui4*　Fang Xuanjun1,2* 1 College of Life and Technology Science, Guangxi University, Nanning, 530004; 2 Hainan Institute of Tropical Agricultural Resources, Sanya, 572025; 3 College of Life Sciences, Nankai University, Tianjin, 300071; 4 Northeast Forestry Uinversity, Harbin, 150040 * Corresponding author, xuanjunfang@ Abstract: By cloning Cry1Ac22 gene from Bacillus thuringiensis W015 to the prokaryotic expression vector pQE30 through gene recombinant technique, we constructed the recombinant vector pQE30-Cry1Ac22. The pQE30-Cry1Ac22 was transformed into E. coli host strain M15 and then induced by IPTG to express His-tag-Cry1Ac22 fusion protein. His-tag-Cry1Ac22 was purified with affinity chromatography on a Ni2+-NTA resin column. PCR, enzyme digestion with BamHⅠ and SalⅠ, and DNA sequencing results showed that pQE30-Cry1Ac22 was successfully constructed. SDSresults demonstrated that the host with pQE30-Cry1Ac22 transformed expressed a 133 kDa His-tag-Cry1Ac22 fusion protein, which was in the form of inclusion body. Inducing experiment with different IPTG concentration an