中文摘要：

【目的】克隆棉铃虫中肠羧酸酯酶基因,了解该酶的分子特性,为研究棉铃虫抗药性机理奠定基础。【方法】制备抗血清,对棉铃虫（Helicoverpa armigera（Hübner））中肠cDNA表达文库进行免疫筛选,得到编码棉铃虫羧酸酯酶基因的cDNA克隆,利用生物信息学软件和网站进行序列分析,构建原核表达载体进行表达,并以α-醋酸萘酯溶液为底物进行活性测定。【结果】测序分析表明,羧酸酯酶基因hc3（GenBank登录号：GU119888）全长2 896 bp,编码795个氨基酸。羧酸酯酶HC3的等电点pI为3.94,活性中心包括3个氨基酸残基（催化三联体）：Ser204,Glu331,His444;第545—773位包含大量高度重复的Gly,Asp、Glu（GDE区域）构成亲水区。该基因在大肠杆菌BL21中成功表达约100 kD的HC3蛋白,检测表达的羧酸酯酶活性为0.32 mmol/100μL酶液。【结论】成功克隆、表达了棉铃虫羧酸酯酶蛋白HC3,并发现GDE亲水结构域。为进一步了解羧酸酯酶的三维结构及其水解作用并设计新型杀虫剂提供了可能。

英文摘要：

【Objective】The objective of the study is to clone carboxylesterase gene and provide a molecular basis for insecticide resistance of organophosphates（OPs）.【Method】 A full-length cDNA clone was identified to code for a carboxylesterase by immuno-screening of the Helicoverpa armigera midgut cDNA expression library and expressed into Escherichia coli.The carboxylesterase activity of HC3 was characterized with the α-NA as a substrate and its enzyme activity of HC3 was analyzed.【Result】 The hc3 is 2 896 bp in length,which encodes 795 amino acids（GenBank accession number is GU119888）.The predicted isoelectric point of HC3 is 3.94.The HC3 possesses a catalytic triad,consisting of a Ser,a His and a Glu residue（Ser204,Glu331 and His444）.The activity for HC3 is 0.32 mmol/100 μL enzyme buffer.【Conclusion】 A cDNA encoding carboxylesterase was cloned and expressed from Helicoverpa armigera.A noval GDE hydrophilic region was found in HC3.All the above understanding will further explore the three-dimensional structure,hydrolysis of carboxylesterase and provide possibility for the design of new types of pesticides.