绿木霉ZY-01中纤维素酶EGⅣ基因的克隆及其在E.coli中的原核表达  

Cloning of cellulase EGⅣ gene fromTrichoderma viride ZY-01 and its prokaryotic expression in E.coli

作　　者：胡侨 罗伟[1] 阮涛[1] 侯亚利[1] 黄皓[1] 杨忠华[1] 

机构地区：[1]武汉科技大学化学工程与技术学院,湖北武汉430081

出　　处：《武汉科技大学学报》2016年第2期156-160,共5页Journal of Wuhan University of Science and Technology

基　　金：国家自然科学基金资助项目(21376184)

摘　　要：以前期筛选到的纤维素酶高产菌株绿木霉T.viride ZY-01的总RNA为模板,利用RT-PCR技术克隆出内切葡聚糖酶EGⅣ基因,连接至pET-28a构建出重组质粒,并转化至E.coli BL21(DE3)中表达。该EGⅣ基因约有1089bp,在启动子T7lac的控制和IPTG的诱导下,目的基因成功地在原核细胞中表达。通过SDSPAGE检测得出目标蛋白分子量为39kDa,重组酶的比酶活为1.05U/mg,该酶最适pH值为6、最适反应温度为50℃。Endoglucanase Ⅳ (EGⅣ)gene was cloned by using RT-PCR with the total RNA of Tri-choderma viride ZY-0 1 ,a strain with high cellulase productivity and screened in our previous work,as template.Then the gene was ligated into pET-28a to construct the recombinant plasmid,and trans-formed into E.coli BL21(DE3)to be expressed.The cloned EGⅣ gene fragment is about 1089 bp. Controlled by promoter T7lac and induced by IPTG,the target gene is successfully expressed in pro-karyote.Molecular weight of the target protein is 3 9 kDa according to SDS-PAGE analysis.Specific enzyme activity of the recombinase is 1.05 U/mg.The optimum pH value and reaction temperature of this enzyme is 6 and 50 ℃,respectively.

关 键 词：绿色木霉 纤维素酶 内切葡聚糖酶 EGⅣ基因 基因重组 原核表达 

分 类 号：Q786[生物学—分子生物学]