杜氏利什曼原虫无鞭毛体蛋白N端胞外区基因(ast1)的克隆及表达  

Cloning and Expression of Extracellular Region Gene Located in N-terminus of Leishmania Donovani

作　　者：陈宪[1] 陈建平[1] 徐佳楠[1] 王昕[1] 路睿[1] 芦殿香[2] 胡孝素[1] 

机构地区：[1]四川大学华西基础医学与法医学院寄生虫教研室,成都610041 [2]四川大学国家生物材料工程研究中心,成都610041

出　　处：《生物医学工程学杂志》2009年第4期820-824,共5页Journal of Biomedical Engineering

基　　金：国家自然科学基金资助项目(30771883)

摘　　要：构建杜氏利什曼原虫无鞭毛体蛋白N端胞外区基因(ast1)的原核表达重组质粒pET32a(+)-ast1,并在大肠杆菌BL21中进行表达。从杜氏利什曼原虫基因组DNA中扩增无鞭毛体蛋白基因并用SOSUI和Tmpred方法预测其跨膜区结构;根据跨膜区预测的结果,扩增基因ast1并将其连接到原核表达载体pET32a(+)中,重组质粒命名为pET32a(+)-ast1,并在大肠杆菌BL21(DE3)中进行表达。SDS-PAGE和免疫印迹分析表明,在约27kDa处获得重组目的蛋白rAST1的表达,显示成功构建杜氏利什曼原虫原核表达重组质粒pET32a(+)-ast1,并在大肠杆菌BL21中获得有效表达。The objective of this study was to construct and express recombinant prokaryotie plasmid pET32a （＋）- ast1 in E. coli BL21（DE3）. Amastin gene was amplified from genomic DNA of Leishmania Donovani and its transmembran region was predicted by the methods of SOSUI and Tmpred; astl located in N-terminus of amastin gene was amplified and cloned into prokaryotic plasmid pET32a（＋） ,which was named pET32a（＋）-astl ,and then rAST1 was expressed in E. coli BL21（DE3）. The results of SDS-PAGE and immunobloting assay showed that a fusion protein rASTl（relative molecular mass about 27 kDa）was able to express in BL21. The recombinant prokaryotie plasmid pET32a（＋）- astl was successfully constructed, and noted to be efficiently expressed in E. coli BL21（DE3）.

关 键 词：杜氏利什曼原虫 无鞭毛体强白基因 ast1基因 

分 类 号：R531[医药卫生—内科学]