鼠疫F1蛋白原核分泌性表达及鉴定  

Secreted expression and identification of F1 protein of Yersinia pestis in prokaryocyte

作　　者：魏东[1] 王国治[1] 

机构地区：[1]中国食品药品检定研究院细菌一室,北京100050

出　　处：《中国医药生物技术》2012年第3期202-205,共4页Chinese Medicinal Biotechnology

基　　金：国家高技术研究发展计划(863计划)(2006AA02Z461)

摘　　要：目的构建重组质粒,在大肠杆菌中表达鼠疫菌F1抗原。方法用PCR方法扩增出带有信号肽的F1基因,将其克隆到表达载体pET30a(+)上,转化大肠杆菌BL21(DE3);用IPTG诱导目的基因表达,层析方法纯化F1蛋白,测定其分子量、等电点、N末端氨基酸序列,用Westernblot法检测其抗原性。结果根据双酶切和DNA测序结果显示,F1基因成功连接到表达载体pET30a(+)中,F1蛋白主要为分泌性可溶表达。测定纯化后F1蛋白的相对分子量约为15.6kD,等电点为4.15,N末端氨基酸序列与理论序列一致。经Westernblot鉴定,能被兔抗鼠疫菌EV株血清识别。结论成功克隆并构建了F1蛋白分泌性原核表达系统,所表达的重组F1蛋白具有较好的抗原性,为新型鼠疫疫苗研制提供基础。Objective To construct the recombinant plasmid expressing F1 protein of Yersiniapestis in E.coli BL21（DE3）. Methods The F1 gene was amplified by PCR, cloned into prokaryotic expression plasmid pET30a（＋） and then transformed into E.coli BL21（DE3）. The recombinant E.coli BL21（DE3） was induced by IPTG. The protein was purified, and its molecular weight, isoelectric point and N-terminal amino acid sequence was identified. The antigenicity of the protein was measured by Western blot. Results The F1 protein is mainly expressed in a secreted form by the recombinant E.coli BL21（DE3） strain. Its molecular weight is 15.6 kD identified by SDS-PAGE, and isoelectric point is 4.15. The N-terminal amino acid sequence of the protein is completely the same as expected. As Western blot result shows, the FI protein could react with plague anti-sera from rabbit. Conclusion pET-30a/F1 expressing secretive F1 protein is successfully constructed. The F1 protein developed by this study has good immunoreactivity with plague anti-serum.

关 键 词：耶尔森菌 鼠疫 克隆 分子 基因表达 

分 类 号：R392[医药卫生—免疫学]