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羧酸酯酶是昆虫体内重要的解毒酶系之一,与昆虫抗药性产生相关。利用粉纹夜蛾Trichoplusia ni(Hübner)肠粘蛋白多克隆抗体免疫筛选甜菜夜蛾Spodoptera exigua(Hübner)中肠cDNA表达文库,得到编码羧酸酯酶的全长cDNA克隆。该cDNA克隆全长1812bp(GenBank登录号EF580101),开放阅读框长1605bp,编码535个氨基酸。该蛋白活性中心包括3个氨基酸残基(催化三联体):Ser186,Glu319和His443,3个N-联糖基化位点,具备羧酸酯酶的结构特征,属羧酸酯酶家族(EC:3.1.1.-)。将该基因与pQE30载体重组,经IPTG诱导,spot-blot鉴定,蛋白获得了表达;以α-醋酸萘酯为底物,检测表达的羧酸酯酶活性为1.3nmol/100μL酶液。
Carboxylesterase is one of the most important detoxification enzymes in insects and is related to the development of insect resistance. The midgut cDNA expression library of Spodoptera exigua (Hübner) was immunoprecipitated with Trichoplusia ni (Hübner) polyclonal antibody against intestinal mucosa to obtain the full-length cDNA clone encoding carboxylesterase. The cDNA clone was 1812bp in length (GenBank accession number EF580101). The open reading frame was 1605bp and encoded 535 amino acids. The active site of this protein consists of three amino acid residues (catalytic triad): Ser186, Glu319 and His443, three N-linked glycosylation sites with the structural features of carboxylesterase, belonging to the carboxylesterase family (EC : 3.1.1.-). The gene was recombined with pQE30 vector, induced by IPTG and identified by spot-blot. The expressed protein was obtained. The activity of carboxylesterase was detected as 1.3nmol / 100μL enzyme solution using naphthyl acetate as substrate.