目的构建结核分枝杆菌rv1837c、rv3803c基因原核表达重组质粒,进行表达、纯化,并分析其免疫原性。方法PCR扩增结核分枝杆菌H37Rv rv1837c、rv3803c基因,并克隆入pTA2质粒。测序正确后,再亚克隆入pET30a(+)质粒,构建pET30a(+):rv1837c、pET30a(+):rv3803c重组体。然后转化入表达宿主大肠杆菌BL21(DE3),经0.4mmol/L异丙基硫代-β-D-半乳糖苷诱导;分别与组氨酸标签单克隆抗体及结核患者血清进行Western blot,鉴定Rv1837c、Rv3803c重组蛋白。经镍离子螯和氮川乙酸-组氨酸标签亲和树脂纯化,将纯化的重组蛋白分别免疫家兔,取兔血清与纯化蛋白通过Western blot方法,检测家兔血清中的抗体。结果pET30a(+):rv1837c、pET30a(+):rv3803c重组体表达相对分子质量为92000及38000的重组蛋白,表达蛋白以包涵体形式存在于胞质中,表达量分别占全菌蛋白质的30%及50%。获得纯度为90%的重组蛋白。纯化蛋白通过Western blot鉴定证实为目的蛋白,有较强的免疫原性。结论成功构建原核表达重组质粒pET30a(+):rv1837c、pET30a(+):rv3803c,并获得Rv1837c及Rv3803c重组蛋白,为血清学诊断活动性结核病奠定了基础。 Objective To construct recombinant plasmid of Mycobacterium tuberculosis rv1837c and rv3803c, express and purify it, and analyze its immunogenicity. Methods The Mycobacterium tuberculosis H37Rv rv1837c and rv3803c genes were amplified by PCR and cloned into pTA2 plasmid. After sequencing, the recombinant plasmid was subcloned into pET30a (+) plasmid to construct pET30a (+): rv1837c and pET30a (+): rv3803c recombinant. The recombinant plasmid was transformed into E. coli BL21 (DE3) and induced by 0.4 mmol / L isopropylthio-β-D-galactoside. Western blot was performed using histidine-tagged monoclonal antibody and the serum of tuberculosis patients, respectively Rv1837c, Rv3803c recombinant protein. The purified recombinant protein was respectively immunized with nickel ion-chelated and nitrocellulose acetate-histidine tag affinity resin, and the rabbit serum and purified protein were detected by Western blot to detect the antibody in rabbit serum. Results The recombinant proteins expressing pET30a (+): rv1837c and pET30a (+): rv3803c had relative molecular mass of 92000 and 38000, respectively. The expressed proteins existed in the cytoplasm as inclusion bodies and accounted for 30% And 50%. A recombinant protein of 90% purity was obtained. The purified protein was verified by Western blot for the purpose of protein, strong immunogenicity. Conclusion The prokaryotic expression plasmid pET30a (+): rv1837c, pET30a (+): rv3803c was successfully constructed, and the recombinant proteins Rv1837c and Rv3803c were obtained, which laid the foundation for serological diagnosis of active tuberculosis.