无标签鼠疫耶尔森菌Ⅴ抗原突变体在大肠杆菌中的表达、纯化及免疫原性分析  

Expression and Purification of Non-Tagged Yersinia pestis LcrⅤ Antigen Mutant in Escherichia coli and its Immunogenicity in BALB/c Mice

作　　者：王猛[1] 任军[1] 张金龙[1] 李浩[1] 蔡晨光[1] 李建民[1] 陈薇[1] 

机构地区：[1]军事医学科学院微生物流行病研究所病原微生物与生物安全国家重点实验室,北京100071

出　　处：《生物技术通讯》2012年第1期26-29,共4页Letters in Biotechnology

基　　金：国家自然科学基金(81000710)

摘　　要：目的:在大肠杆菌中表达、纯化无标签鼠疫耶尔森菌低钙反应V抗原突变体,并对其免疫原性进行研究。方法:采用融合PCR方法将Ⅴ抗原基因中编码半胱氨酸的碱基缺失突变,将获得得Ⅴ抗原突变体基因克隆到原核表达载体pET32a(+)后转化大肠杆菌BL21(DE3),用IPTG诱导表达并进行柱层析纯化,纯化产物以Western印迹进行鉴定并免疫BALB/c小鼠,通过ELISA检测免疫血清效价。结果:目的蛋白在大肠杆菌中获得了可溶性高表达,经三步柱层析后纯度高于95%,经Western印迹检测可与野生型V抗原单克隆抗体特异性结合,免疫小鼠后获得高效价免疫血清。结论:获得了无标签的鼠疫耶尔森菌Ⅴ抗原突变体蛋白,并证实其具有免疫原性,将对V抗原结构和功能的研究提供帮助。Objective： To express and purify non-tagged Yersinia pestis LcrV antigen mutant in Escherichia coli and investigate its immunogenicity.Methods： Bases encoding Cys273 of LcrⅤ were deletion mutated by using fusion PCR techniques.This LcrV mutant gene was cloned into expression vector pET-32a（＋） and then transformed into E.coli BL21（DE3）.The mutant protein Vm was induced by IPTG,purified with chromatographic column,identified by Western blotting,and used to immunize BALB/c mice.Serum antibody was detected by ELISA.Results： Vm was expressed in soluble form at high levels.After purified with three steps,the purity was more than 95%.Anti-Ⅴ antibody could specifically react with Vm by Western blotting.BALB/c mice vaccinated with Vm induced a strong and specific antibody.Conclusion： The non-tagged mutant protein Vm with strong immunogenicity was obtained,which could be helpful to study the structure and function of LcrⅤ.

关 键 词：鼠疫耶尔森菌 Ⅴ抗原突变体 表达纯化 免疫原性 

分 类 号：Q78[生物学—分子生物学] R392.1[医药卫生—免疫学]