嗜肺军团菌pip基因原核质粒的构建及其表达  

Construction and Expression of Pip Gene of Legionella Pneumophila in Prokaryotic Cell

作　　者：刘胜泉[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》2012年第1期10-13,21,共5页Journal of Ningxia Medical University

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

摘　　要：目的扩增嗜肺军团菌基因pip,构建重组质粒pET-pip并在原核系统中表达。方法采用PCR,从嗜肺军团菌基因组DNA中扩增出pip基因,将其定向克隆至原核表达载体pET-32a(+),构建重组质粒pET-pip,经限制性内切酶、PCR鉴定及测序分析后,转化BL21,IPTG诱导表达,并用SDS-PAGE进行鉴定。使用His-tag纯化重组蛋白PIP。结果扩增出了嗜肺军团菌726bp的pip基因,构建的原核表达重组质粒pET-pip表达并纯化出了约46kD Trx-PIP的融合蛋白。结论成功构建了嗜肺军团菌pip基因的原核重组质粒,并在大肠杆菌中得到了高效表达。Objective To construct the recombinant plasmid pET-pip and to detect its expression in the prokaryotic cells BL21（DE3）.Methods The pip gene was amplified by PCR from DNA of Legionella pneumophila,then inserted it into the prokaryotic expression vector pET-32a（＋）.The recombinant plasmid pET-pip was constructed.The recombinant plasmid was identified by restriction-endonuclease digestion,PCR and DNA sequencing analysis and transferred into BL21.The expression of pET-pip was induced with IPTG and the fusion protein was analyzed with SDS-PAGE.It was purified by His-tag.Results The pip gene of 726bp in length was amplified and the recombinant plasmid pET-pip was constructed.The PIP protein of approximately 46KD in size was expressed in E.coli and purified.Conclusion The pip gene of 726bp in length was successfully cloned and the recombinant plasmid pET-pip was constructed successfully and expressed efficiently.

关 键 词：嗜肺军团菌 pip基因 表达 纯化 

分 类 号：R378[医药卫生—病原生物学]