斜纹夜蛾普通气味结合蛋白ⅠcDNA的克隆及在原核细胞中的表达  

Cloning and Prokaryotic Expression of cDNA Encoding General Odorant Binding ProteinⅠfrom Spodoptera litura

作　　者：金丰良[1] 董小林[1] 许小霞[1] 任顺祥[1] 

机构地区：[1]华南农业大学资源环境学院昆虫学系/教育部生物防治工程中心,广州510642

出　　处：《中国农业科学》2009年第3期891-899,共9页Scientia Agricultura Sinica

基　　金：国家基础研究发展计划"973"项目(2006CB102005);国家"十五"攻关项目(2004BA509B0604)

摘　　要：【目的】克隆、序列分析和原核表达编码斜纹夜蛾(Spodoptera litura)GOBPⅠ(SlGOBPⅠ)的cDNA。【方法】采用同源克隆结合RACE的方法克隆SlGOBPⅠ基因的cDNA序列,并在pET-32a/BL21(DE3)系统中进行原核表达。【结果】从斜纹夜蛾触角中克隆了GOBPⅠ的cDNA序列(GenBank登录号为EU086372),序列分析表明,SlGOBPⅠ开放阅读框序列为495bp,编码164个氨基酸,分子量为19.3kD,等电点为5.54。SlGOBPⅠ具有昆虫气味结合蛋白的典型特征,即氨基酸序列中具有6个半胱氨酸残基,呈酸性。SlGOBPⅠ氨基酸序列与鳞翅目昆虫的气味结合蛋白氨基酸序列具有较高的相似性,表明SlGOBPⅠ属于GOBP家族。将SlGOBPⅠ编码序列,克隆到表达载体pET-32a上,构建原核表达载体pET-SlGOBPⅠ,在表达宿主菌BL21(DE3)中,经IPTG诱导成功表达了相对分子质量为32.0kD的可溶性融合蛋白,利用Ni2+-NTA亲和柱一步纯化了SlGOBPⅠ融合蛋白,以此融合蛋白免疫新西兰大白兔制备了SlGOBPⅠ的抗血清,ELISA滴度为1﹕5000,Western印迹检测结果显示,SlGOBPⅠ抗血清与表达的融合蛋白质呈阳性反应,表明所表达的融合蛋白保持原有的免疫原性。【结论】克隆、分析和表达了编码斜纹夜蛾气味结合蛋白GOBPⅠcDNA序列,为今后深入研究斜纹夜蛾GOBPⅠ基因的结构和功能奠定了基础。[ Objective] The objective of this study aims at cloning, sequence analysis and prokaryotic expression of a new cDNA encoding the general odorant binding protein Ⅰ from Spodoptera litura （named as SIGOBP Ⅰ ）. [Method] The cDNA encoding S1GOBP I was isolated from the antennae of S. litura （S1GOBP I, GenBank Accession No. EU086372） by homology cloning and rapid amplification of cDNA ends （RACE）, and then the open reading frame （ORF） of S1GOBP Ⅰ was cloned into prokaryotic cells to test its expression. [Result] Results of sequencing and structural analysis showed that the ORF of S1GOBP I was 495 bp, encoding 164 amino acid residues, with the predicted MW of 19.3 kD and pI of 5.54, respectively. It shared the typical structural features of odorant binding proteins from other insects, including the six conservative Cys lotus in the sequence. The deduced amino acid sequence showed a high identity to the reported sequences of GOBP Ⅰ from other lepidopteran insects, and showed that SIGOBP Ⅰ may belong to the family of GOBPJ The SIGOBP I was constructed into pET-32a vector and expressed in Escherichia coli BL21 （DE3） after induction with IPTG. The analysis of SDS polyacrylamide gel electrophoresis and Western blot indicated that the molecular weight of fusion protein was about 32.0 kD. The fusion protein was purified by one step Ni-NTA affinity chromatograph column and injected into New Zealand White rabbit to raise antiserum successfully. The analysis of ELISA showed the titer of antiserum was 1 ： 5 000. Western blot analysis showed that the expressed S1GOBP I protein was crossreactive with the anti-S1GOBP Ⅰantiserum, which indicated the expressed fusion protein belonged to GOBP I of the insect. [Conclusion] The successful cloning and expression of the coding sequence of SIGOBP Ⅰ have laid a basis for further study on the structure and function of SIGOBP Ⅰ.

关 键 词：斜纹夜蛾 触角 气味结合蛋白 基因克隆 原核表达 

分 类 号：S433[农业科学—农业昆虫与害虫防治]