[杜氏利什曼原虫N端细胞外区域基因的克隆与表达]

[Cloning and expression of extracellular region gene located in N-terminus of Leishmania Donovani].

作者信息

Chen Xian, Chen Jianping, Xu Jia'nan, Wang Xin, Lu Rui, Lu Dianxiang, Ho Xiaosu

机构信息

Department of Parasitology, School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China.

出版信息

Sheng Wu Yi Xue Gong Cheng Xue Za Zhi. 2009 Aug;26(4):820-4.

PMID:19813619

Abstract

The objective of this study was to construct and express recombinant prokaryotic 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(+)-ast1, and then rAST1 was expressed in E. coli BL21(DE3). The results of SDS-PAGE and immunobloting assay showed that a fusion protein rAST1 (relative molecular mass about 27 kDa) was able to express in BL21. The recombinant prokaryotic plasmid pET32a(+)- ast1 was successfully constructed, and noted to be efficiently expressed in E. coli BL21(DE3).

摘要

本研究的目的是构建重组原核质粒pET32a(+)-ast1并在大肠杆菌BL21(DE3)中表达。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；扩增位于无鞭毛体蛋白基因N端的ast1并克隆到原核质粒pET32a(+)中，命名为pET32a(+)-ast1，然后在大肠杆菌BL21(DE3)中表达rAST1。SDS-PAGE和免疫印迹分析结果表明，融合蛋白rAST1（相对分子质量约为27 kDa）能够在BL21中表达。成功构建了重组原核质粒pET32a(+)-ast1，并发现其能在大肠杆菌BL21(DE3)中高效表达。