鼠疫耶尔森氏菌rV270抗原的克隆、表达及其免疫保护作用评价

目的：为研制鼠疫亚单位疫苗，克隆、表达并纯化去除产生免疫抑制作用序列后的鼠疫耶尔森氏菌LcrV抗原（rV270）。方法：依据已知的LcrV的核苷酸序列，避开其产生免疫抑制作用的区段设计引物，扩增rV270基因并克隆到pET24a载体中，在大肠杆菌BL21中表达His-rV270融合蛋白：表达产物先后经Co^2＋亲和层析和Sephacryl S-200HR凝胶柱纯化，并在纯化过程中应用凝血酶切除His标塔；氢氧化铝佐剂吸附重组抗原后免疫BALB／c小鼠，初次免疫后第21天加强免疫1次，第5周使用104CFU鼠疫菌141强毒株攻毒，测定其免疫保护作用。结果：rV270以可溶性方式表达；应用Co^2＋亲和层析柱和Sephacryl S-200HR凝胶柱结合凝血蛋白酶切除His标签的方法可得到不含标签的较高纯度的重组蛋白；攻毒实验中实验组小鼠全部存活，而对照组全部死亡。结论：获得了具有良好免疫保护作用的rV270蛋白，可用于鼠疫亚单位疫苗的研究。

Cloning and Expression of Yersinia pestis rV270 Antigen and its Efficacy Against Y.pestis Virulent Strain Challenge

Objective： To reduce immunosuppressive properties of LcrV antigen of Yersinia pestis and retain its immuno-protection activities. Methods： rV270, a variant lacking amino acids 271 to 326 of LcrV, was cloned into pET24a vector, expressed as a His fusion protein in Escherichia coli BL21, digested with thrombin to cut the peptide of His-Tag and purified by a combination of Co^2＋ affinity chromatography and Sephaeryl S-200HR gel filtration. Groups of mice were immunized with rV270 antigen adsorbed to 25% alhydrogel in PBS. After primary immunization, on day 21 the animals were boosted with an identical dose at the same injection site. The animals from the immunized and control groups were challenged s.c. with 104 CFU of Y.pestis strain 141 at five weeks after the final immunization, and then closely observed for 14 days. Results： The rV270 was expressed in E.coli BI21. All the control animals succumbed to Y.pestis strain 141 at a dose of 104 CFU. In contrast, all the immunized animals survived exposure to Y.pestis at 104 CFU without the development of symptoms in the 14 days post-challenge observation period. Conclusion： It was indicated that rV270 antigen is highly immunogenic, and can render significant protection in BALB/c mice against challenge with virulent Y.pestis. It was suggested that rV270 may serve as an improved plague vaccine.