结核分枝杆菌特异性抗原Rv3117的克隆表达和诱导小鼠免疫应答的实验研究

Cloning,expression and immune response in mice of specific antigen Rv3117 from M. tuberculosis

目的克隆表达结核分枝杆菌Rv3117蛋白,并进行诱导小鼠免疫应答的初步研究。方法通过聚合酶链反应(PCR)扩增结核分枝杆菌Rv3117基因,克隆入pET32a载体,构建重组质粒pET32a-Rv3117,转化入大肠埃希菌BL21(E.coli)plysS(DE3),异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达,镍柱亲和纯化,通过SDS-PAGE鉴定其表达情况;以目的蛋白免疫C57BL/6小鼠血清并行Western blotting分析。结果成功构建原核表达载体pET32a-Rv3117,获得纯化的目的蛋白,其在E.coli BL21 plysS(DE3)中主要以可溶性形式表达,相对分子质量(48 100)与预期相符。Western blotting分析结果显示在目标位置有阳性条带。结论成功克隆结核分枝杆菌重组蛋白Rv3117,其能够诱导C57BL/6小鼠的免疫应答。

Objective To clone recombinant protein Rv3117 from M. tuberculosis, and investigate its immune response in mice. Methods The gene encoding Rv3117 protein was amplified by PCR from genome DNA of M. tuberculosis, and was then cloned into corresponding site of the expression vector pET-32a. The recombinant plasmid pET32a-Rv3117 was transformed into E. coli BL21 plysS （DE3）, induced with isopropyl-β-D-thiogalaetopyranoside （IPTG）, and purified by Ni- NTA purification system. The expression of recombinant protein was identified by SDS-PAGE analysis. Western blotting was performed on sera from Rv3117-immunlzed C57BL/6 mice. Results The prokaryotic expression vector pET32a-Rv3117 was successfully constructed. The target protein was expressed in soluble form in E. coli BL21 plysS （ DE3）, with the relative molecular weight of 48 100, which was in line with the expectations. Western blotting revealed a positive stripe at the destination. Conclusion The recombinant protein Rv3117 has been successfully cloned from M. tuberculosis, which could induce immune response in mice.