鼠疫F1蛋白原核分泌性表达及鉴定

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。

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

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 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 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.