苏云金芽孢杆菌Cry8Ca2蛋白的纯化与活性的研究

A study on purification and activity of Cry8Ca2 protein from Bacillus thuringiensis

cry8 Ca2基因是本实验室自行分离克隆的一种新型苏云金芽孢杆菌(Bacillus thuringiensis,Bt)杀虫晶体蛋白基因,其表达130 kDa蛋白对铜绿异丽金龟(Anomala corpulenta)和黄褐异丽金龟(A.exoleta)幼虫具有较高的活性。本研究对Cry8Ca2蛋白原毒素及胰凝乳蛋白酶消化片断的纯化条件及活性进行研究,最适的消化条件为质量比1∶20,37℃,消化1 h。通过阴离子交换层析得到纯的活性毒素蛋白。用原毒素与纯化的毒素对铜绿丽金龟幼虫进行生测,Cry8Ca2原毒素蛋白的LC50为0.47μg/g土,纯化的毒素的LC50为0.08μg/g土,毒素的杀虫活性可以达到原毒素的6倍。

Cry8Ca2 protein encoded by the gene crySCa2 of Bacillus thuringiensis is a kind of insecticidal crystal protein with molecular mass of 130kDa. It is larvicidal to both Anornala corpulenta and A. exoleta. The solubilized Cry8Ca2 protoxin of 130kDa was activated by chymotrypsin at the enzyme / protoxin ratio of 1：20 （w/w） . It was further purified by anion exchange chromatography on Resource Q column, The activities of the protoxin and the toxin were evaluated by bioassays. The lethal concentration fifty percent （LC50） of the protoxin was 0. 47 μg per gram soil, while that of the toxin was 0. 08μg per gram soil. The activity of the toxins was six folds as that of the protoxin. These results indicate that CrySCa2 toxin may be beneficial for biocontrol of both A. corpulenta and A. exoleta.