嗜肺军团菌Ⅳ型菌毛蛋白在大肠杆菌中的表达及纯化

目的扩增编码Ⅳ型菌毛蛋白的嗜肺军团菌pilE基因，构建重组质粒pET32a（＋）-pilE并在原核系统中表达．纯化Ⅳ型菌毛蛋白PILE．为进一步探讨Ⅳ型菌毛蛋白的作用及其作为军团病的诊断抗原提供实验基础。方法采用聚合酶链式反应（PCR）从嗜肺军团菌扩增得到pilE基因，构建重组质粒pET32a（＋）-pilE，转化大肠杆菌BL21（DE3）并用聚合酶链式反应、限制性酶切分析、序列分析鉴定后，IPTG诱导表达PIIJE蛋白，用硫酸十二烷酸钠-聚丙烯酰胺凝胶电泳（SDS—PAGE）、Western印迹鉴定。使用HisTrap^TM HP亲和层析柱纯化Ⅳ型菌毛蛋白PILE。结果扩增出430bp的pilE基因；构建了重组质粒pET32a（＋）-pilE；表达并纯化出35700Mr的目标蛋白。结论成功构建了嗜肺军团菌pilE基因的原核重组质粒，并在原核系统中得到了高效表达。成功纯化Ⅳ型菌毛蛋白PILE。

Expression and Purification of Type IV Pilin Protein of Legionella pneumophila in Escherichia coli

Objective To construct the recombinant plasmid pET32a（＋）-pilE and to detect its expression in the prokaryotic cells BL21 （DE3） and to purify type Ⅳ pilin protein of the Legionella pneumophila encoded by pilE gene so as to set the basis for future research on the type Ⅳ pilin protein function and as the diagnostic antigen on Legionnaires disease. Method The pilE gene was amplified by PCR from a template of sero group 10 of the LegioneUa pneumophila and the recombinant plasmid pET32a（＋）-pilE was constructed and transferred into E.coli strain BL21 （DE3）. The recombinant plasmid was identified by PCR, restriction enzyme analysis and sequence analysis. The recombinant protein was examined by Western-blot. The type Ⅳ pilin protein PILE was purified by HisTrapTM HP affinity columns. Result The pilE gene of 430bp in length was cloned and the recombinant plasmid pET32a（＋）-pilE was constructed.The PILE protein of approximately 35700 Mr in size was expressed in E.coli and purified. Conclusion The pilE gene of 430bp in length was successfully cloned and the recombinant plasmid pET32a（＋）-pilE was successfully constructed and expressed efficiently. The PILE protein was successfully purified.