Expression of the Truncated vip3A Gene at the 3'-terminus from Bacillus thuringiensis in Escherichia coli

将苏云金芽孢杆菌(Bacillus thuringiensis)缺失C端154个氨基酸编码区的vip3A基因(vip3T)插入原核表达载体pQE30，构建了重组表达载体pQEvip3T，并转化大肠杆菌(Escherichia coli ) M15进行IPTG诱导表达，比较了完整的Vip3A蛋白和C端缺失的蛋白Vip3T的可溶性和杀虫活性。与Vip3A不同，融合蛋白Vip3T以不可溶的包含体形式存在，诱导表达的菌液中没有检测到可溶性Vip3T蛋白。生物测定结果表明，M15(pOTP)诱导表达的Vip3A蛋白对初孵斜纹夜蛾(Spodoptera litua)和甜菜夜蛾(S. exigua)幼虫具有较高的杀虫活性，其提纯的包含体无毒，但包含体的碱性裂解液却又恢复了对夜蛾科害虫的活性；M15(pQEvip3T)菌液、包含体及其碱性裂解液对这两种昆虫幼虫则完全无毒，说明在大肠杆菌中，Vip3A蛋白C端氨基酸可能对Vip3A蛋白的可溶性和杀虫活性具有重要的影响。

Expression of the Truncated vip3A Gene at the 3'-terminus from Bacillus thuringiensis in Escherichia coli

vip3T gene from Bacillus thuringiensis, which deleted 154 amino-acid encoding sequences at the carbon terminus of Vip3A, was cloned into the expression vector pQE30. The constructed recombinant plasmid pQEvip3T was transformed into Escherichia coli M15 and induced with 1mmol/L IPTG. Protein solubility and insecticidal activity of Vip3T were compared with those of Vip3A. Different from Vip3A, Vip3T existed only in the insoluble inclusion body of induced M15(pQEvip3T). Bioassay showed that M15(pOTP) expressing Vip3A protein performed high toxicity against Spodoptera litura and S. exigua larvae. The purified Vip3A inclusion body lost toxicity against the insects, however, the solubilized inclusion body with Na2CO3 regenerated the insecticidal toxicity. In contrast, the insecticidal activity of induced M15(pQEvip3T), Vip3T inclusion body and its solubilized form against the insect larvae were completely abolished. This suggests that C-terminal amino acids of Vip3A may have an effect on its solubility and insecticidal activity.