暗黑鳃金龟几丁质酶基因的克隆、表达及活性分析  

Gene cloning,expression and activity analysis of chitinase Hpchi from Holotrichia parallela(Coleoptera:Motschulsky)

作　　者：邹爽[1] 赵丹[2] 郭巍[2,3] 徐大庆[1] 张雅昆[1] 

机构地区：[1]河北农业大学生命科学学院,河北保定071001 [2]河北农业大学植物保护学院/河北省农作物病虫害生物防治工程技术中心,河北保定071001 [3]北京农学院植物科学技术学院,北京102206

出　　处：《河北农业大学学报》2014年第6期91-94,105,共5页Journal of Hebei Agricultural University

基　　金：国家重点基础研究发展规划("973"计划)项目(2009CB118902);国家自然科学基金项目(30971910);现代农业产业技术体系建设项目;河北省教育厅项目

摘　　要：本研究通过免疫筛选暗黑鳃金龟幼虫中肠cDNA表达文库,获得其几丁质酶基因Hpchi。序列分析表明Hpchi开放阅读框长1 443bp,编码480个氨基酸,预测分子量为51.4kDa。Hpchi蛋白N-端带有18个氨基酸的信号肽,C-端存在有一个几丁质结合结构域,催化区位置显示有特异活性位点,判定Hpchi蛋白属于Ⅰ型的几丁质酶。分别构建原核和真核表达载体,转化到大肠杆菌BL21和昆虫细胞系sf9,BTI-Tn-5BI-4(Highfive)中进行表达。SDS-PAGE和Western Bolt杂交试验显示,Hpchi重组蛋白在大肠杆菌及昆虫细胞系中都获得了成功表达。酶活分析表明真核表达的Hpchi重组蛋白具有几丁质酶活性。Insect chJtinases are a kind of active proteins that are involved in life activities, and are also potential biological pesticides. Identification of new chitinase gene is of importance. In this study, by screening Holotrichia parallela larva midgut eDNA expression library, using the polyclonal antiserum against H. armigera, a full-length cDNA Hpchi clone encoding chiti- nase was obtained. The Hpchi cDNA was 1 583 bp in length （GenBank Accession No. KJ009563）. The open reading frame（ORF） encoded 480 amino acids, with the predicted MW of 51.4 kDa and pI of 5.08. Hpehi protein shares the typical character of chitinase protein, in- cluding a signal peptide, a chitinase active site, a C-terminal threonine-rich region, a chitin- binding domain, belonging to the chitinases type I. The Hpchi was transformed into E. coli BL21 and HighFive cell line to express by two different pairs of primer. SDS-PAGE and Western Blot analysis revealed that recombinant Hpchi had been successfully expressed in E.coli BL21 and HighFive cell line, respectively. Enzyme activity test showed that Hpchi had chitinase activity.

关 键 词：暗黑鳃金龟 CDNA表达文库 几丁质酶 真核表达系统 活性分析 

分 类 号：Q786[生物学—分子生物学]