苏云金杆菌Cry8Ea2蛋白的分离纯化及生物活性测定

摘要

Cry8 Ea2蛋白是一种新型苏云金杆菌杀虫晶体蛋白.通过穿梭载体pSXY422b将cry8Ea2导入Bt无晶体突变菌株中,获得工程菌HD8Ea2.Cry8Ea2蛋白在工程菌中获得表达.通过分子筛层析,成功获得纯化的130 kDa活性毒素蛋白.用改进的生物活性测定方法进行Cry8Ea2蛋白对华北大黑鳃金龟和暗黑鳃金龟一龄幼虫的杀虫活性测定,LC50分别为0.267 9μg/mL和0.071 9μg/mL.研究首次发现Cry8Ea2蛋白对华北大黑鳃金龟具有较高毒力,拓展了该蛋白的杀虫谱,为转基因工程提供了新资源.%Cry8Ea2 protein is a kind of insecticidal crystal protein of Bacillus thuringiensis. In this study, cry8Ea2 was cloned into E. coli- B. thuringiensist shuttle vector pSXY422b,resulting from recombinant pScry8Ea2, and pScry8Ea2 was transformed into the Bt, acrystalliferous mutant generating HD8Ea2 engineering strain. Cry8Ea2 protein was successfully expressed in HD8Ea2 stain. By anion exchange chromatography on molecular sieve chroma-tography,130 kDa of toxin protein Cry8Ea2 was purified. The activities of the Cry8Ea2 toxin were evaluated by improved bioassays, and the LC50 value to neonates of Holotrichia oblita and Holotrichia paralella was 0. 267 9 μg/mL and 0.071 9 μg/mL, respectively. Insecticidal activity of Cry8Ea2 to Holotrichia oblita was firstly reported in this study ,which will broaden the protein insecticidal spectrum and provide new resources for genetically modified project.