绿色木霉TvALP-linker-TvRBL融合基因的构建与原核表达  

Construction and Prokaryotic Expression of Trichoderma viride TvALP-linker-TvRBL Fusion Gene

作　　者：徐杨玉 温世杰[1] 李海芬[1] 李玲[2] 梁炫强[1] 

机构地区：[1]广东省农业科学院作物研究所,广州510640 [2]华南师范大学生命科学学院,广州510631

出　　处：《生物技术通报》2015年第1期131-137,共7页Biotechnology Bulletin

基　　金：国家"863"计划项目(2006AA10Z156);广东省自然科学基金重点项目(07117967)

摘　　要：在Tv ALP基因和Tv RBL基因间加入一段柔性链接头(linker)基因序列,构建融合基因。生物信息学分析表明该融合基因含有一段信号肽序列。利用RT-PCR技术从绿色木霉菌丝体总RNA中扩增出Tv RBL基因、Tv ALP基因和去除信号肽的ΔTvALP基因,分别克隆到原核表达载体p ET30a,构建重组质粒p ET30a-Tv ALP-linker-Tv RBL和p ET30a-ΔTv ALP-linker-Tv RBL,转化大肠杆菌BL21(DE3)p Lys S,经异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达。SDS-PAGE电泳分析结果表明,去除信号肽的表达质粒p ET30a-ΔTv ALP-linker-Tv RBL在大肠杆菌BL21(DE3)p Lys S中获得了表达,在60 k D处有一条蛋白质特异条带,与预测的目的产物蛋白条带大小一致。Fusion gene were constructed by linking theTvALP andTvRBL genes with a flexible chain（linker）. Bioinformatics analysis showed that the fusion gene contained a signal peptide. TheTvRBL、TvALPand△TvALPgenes were cloned by RT-PCR from the total RNA of Trichoderma viride mycelium, which in turn were cloned into expression plasmid pET30a to construct prokaryotic expression plasmids pET30a-TvALP-linker-TvRBL and pET30a-△TvALP-linker-TvRBL, then these two expression plasmids were transformed intoE.coliBL21（DE3） pLysS,and protein expression were induced by IPTG. SDS-PAGE was used to analyze the expression of the fusion protein. The result showed that expression plasmid pET30a-△TvALP-linker-TvRBL was obtained expression inE.coliBL21（DE3）pLysS, which was a specific band at about 60 kD in size and identical with the expected molecular weight of the fusion protein.

关 键 词：绿色木霉 TvALP-linker-TvRBL融合基因 信号肽 原核表达 

分 类 号：Q78[生物学—分子生物学]