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目的表达和纯化结核分枝杆菌早期分泌蛋白ESAT-6重组二聚体(rdESAT-6),并探讨其在结核病血清学诊断中的价值。方法以HG856A核酸疫苗质粒为模板,经PCR扩增获得2×esat-6基因,克隆至pET-28a质粒,构建原核表达质粒pET2E6;转化大肠杆菌BL21(DE3),IPTG诱导表达;用Ni2+柱亲和层析纯化重组蛋白,并用Western blot鉴定其反应原性,ELISA检测其敏感性和特异性。结果ESAT-6重组二聚体以包涵体形式表达,表达量约占菌体总蛋白的35%;纯化的rdESAT-6纯度可达95%,可与ESAT-6小鼠抗血清发生特异性反应;用于结核病血清学诊断的敏感性为30%,特异性为95.8%。结论已成功表达并纯化了rdESAT-6,可作为结核病血清学诊断或皮试检测用的抗原之一。
Objective To express and purify the early secreted protein ESAT-6 recombinant dimer (rdESAT-6) of Mycobacterium tuberculosis and explore its value in serodiagnosis of tuberculosis. Methods The 2 × esat-6 gene was amplified by PCR using plasmid HG856A as a template. The recombinant plasmid was cloned into pET-28a plasmid and transformed into E.coli BL21 (DE3) The recombinant protein was purified by chromatography and identified by Western blot. The sensitivity and specificity were determined by ELISA. Results Recombinant dimers of ESAT-6 were expressed as inclusion bodies, accounting for about 35% of the total bacterial proteins. Purified rdESAT-6 was up to 95% pure and could specifically react with ESAT-6 mouse antiserum ; For serological diagnosis of tuberculosis, the sensitivity was 30% and the specificity was 95.8%. Conclusion rdESAT-6 has been successfully expressed and purified, which can be used as one of the antigens for serological diagnosis or skin test of tuberculosis.