嗜肺军团菌flaA部分基因的克隆及其原核表达  

Expression and Cloning of Partial flaA Gene of Legionella Pneumophila in Prokaryotic Cell

作　　者：刘阳[1] 曹秀琴[2] 杨志伟[1] 

机构地区：[1]宁夏医科大学基础医学院病原生物学与免疫学系,银川750004 [2]宁夏医科大学生育力保持省部共建教育部重点实验室,银川750004

出　　处：《宁夏医科大学学报》2011年第3期206-209,共4页Journal of Ningxia Medical University

基　　金：国家自然科学基金(30860264);宁夏自然科学基金(NZ0992)

摘　　要：目的克隆表达嗜肺军团菌的鞭毛亚单位蛋白flaA部分基因,并纯化重组蛋白。方法以嗜肺军团菌I型DNA为模板,聚合酶链反应(PCR)扩增鞭毛亚单位蛋白flaA部分基因,并将其定向克隆至原核表达载体pET32a(+),构建原核表达重组质粒pET-flaA。经PCR和限制性核酸内切酶鉴定及测序分析后,转化大肠杆菌BL21,IPTG诱导表达,产物进行SDS-PAGE电泳分析,用亲和层析法纯化其表达蛋白。结果扩增出嗜肺军团菌606bp的flaA部分基因,构建的重组质粒pET-flaA表达并纯化出42 kDa融合蛋白质。结论成功构建嗜肺军团菌flaA部分基因的原核表达载体,在原核系统中得到了高效表达。Objective To clone and express the recombinant partial flagellum subunit gene of Legionella pneumophila,and to purify the recombinant protein.Methods The partial flagellum subunit gene of Legionella pneumophila was amplified from the total DNA of Legionella pneumophlia serogroup 1 by polymerse chain reaction and then was cloned into prokaryote expression vector pET32a（＋）.After the recombinant plasmid was identified by PCR,restriction enzyme analysis and sequencing analysis,the recombinant plasmid pET-flaA was constructed and transferred into E.coli strain BL21.The expression of fusion protein was induced with isopropy-β-D-thiogalactoside（IPTG） and examined with SDS-PAGE,and was purified by Affinity chromatography.Results The partial flaA gene of 606 bp in length was amplified.The recombinant plasmid pET-flaA was expressed and purified 42 kDa fusion protein.Conclusion The recombinant plasmid containing Lgeionella pneumophila the partial flaA gene constructed in this study is highly efficient expression in prokaryotic cell.

关 键 词：嗜肺军团菌 鞭毛亚单位蛋白 表达 纯化 

分 类 号：R378[医药卫生—病原生物学]