Vip3Aa10与甜菜夜蛾刷状缘膜囊泡的结合特性

The binding properties between Vip3Aa10 and brush border membrane vesicles of Spodoptera exigua

【目的】营养期杀虫蛋白Vip3A是一种由苏云金芽孢杆菌在对数生长期产生的新型杀虫毒素。Vip3A与敏感昆虫中肠上皮细胞的结合是其发挥杀虫活性的关键步骤。为进一步探究Vip3A毒素的杀虫机理,研究Vip3Aa10与甜菜夜蛾刷状缘膜囊泡(BBMVs)的结合特性。【方法】在大肠杆菌中用IPTG诱导表达Vip3Aa10,用亲和层析和离子交换层析的方法纯化蛋白,并用胰蛋白酶活化Vip3Aa10,得到Vip3Aa10-T。然后分析Vip3Aa10前毒素和Vip3Aa10-T对甜菜夜蛾的杀虫活性,用免疫印迹法和配体印迹法分析Vip3Aa10-T与甜菜夜蛾BBMVs的结合特性以及在甜菜夜蛾BBMVs上的结合蛋白。【结果】Vip3Aa10-T与Vip3Aa10前毒素对甜菜夜蛾的杀虫活性很高。Vip3Aa10-T在pH8.0时更容易与甜菜夜蛾BBMVs结合,并且能够与非敏感性昆虫黄粉虫BBMVs结合。在相同pH条件下,Vip3Aa10前毒素比Vip3Aa10-T更容易与甜菜夜蛾BBMVs结合。Vip3Aa10-T在甜菜夜蛾BBMVs上有4条结合蛋白,其分子量分别为80、38、31和22kDa。【结论】活化的Vip3Aa10与甜菜夜蛾BBMVs的结合是pH依赖性的,并且能与BBMVs上的4个蛋白结合。

【Objective】The vegetative insecticidal protein Vip3A,produced by Bacillus thuringiensis during the logarithmic growth phase,is a novel insecticidal toxin.The binding of Vip3Ato midgut epithelial cells of sensitive insect is a key step during the insecticidal process.In order to further investigate the mode of action of Vip3Atoxin,the binding properties of Vip3Aa10to the brush border membrane vesicles(BBMVs)of Spodoptera exigua were studied.【Methods】The expression of Vip3Aa10was induced by IPTG in E.coli.The protein was purified by affinity chromatography and ion exchange chromatography.The purified Vip3Aa10protein was activated by trypsin to produce Vip3Aa10-T.The insecticidal activities of Vip3Aa10 protoxin and Vip3Aa10-T against S.exigua were determined.The binding properties of Vip3Aa10-uc2 T to S.exigua BBMVs and the binding proteins on S.exigua BBMVs were analyzed by Western blot and ligand blot.【Results】The insecticidal activities were high both in Vip3Aa10protoxin and Vip3Aa10-T against S.exigua.The binding of Vip3Aa10-T to S.exigua BBMVs was the highest at pH 8.0.Moreover,Vip3Aa10-T could also bind to BBMVs of Tenebrio molitor,which was a nonsensitive insect of Vip3Aa10.At the same pH,the binding of Vip3Aa10protoxin to S.exigua BBMVs was more easily than that of Vip3Aa10-T.Four proteins on S.exigua BBMVs could bind to Vip3Aa10-T,and their molecular weights were 80,38,31and 22kDa,respectively.【Conclusion】The activated Vip3Aa10can bind to four proteins on S.exigua BBMVs and this binding is dependent on pH.