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

摘要

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 ELJSA. Results: Vmwas 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.%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.