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 Western blot 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 15.6 kD,等电点为 4.15,N 末端氨基酸序列与理论序列一致。经 Western blot 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。
Abstract:
Objective To construct the recombinant plasmid expressing F1 protein of Yersinia pestis 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 ismainlyexpressed 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 F1 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.
魏东, 王国治. 鼠疫F1蛋白原核分泌性表达及鉴定[J]. 《中国医药生物技术》杂志, 2012, 7(3): 202-205.
WEI Dong, WANG Guo-Zhi. Secreted expression and identification of F1 protein of Yersinia pestis in prokaryocyte. , 2012, 7(3): 202-205.