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

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）.