结核分枝杆菌Rv3425-Rv1168c蛋白融合表达与血清学评价

目的 原核表达结核分枝杆菌Rv3425-Rv1168c融合蛋白并进行纯化,通过酶联免疫吸附试验(ELISA)方法评价重组蛋白在结核病血清学诊断中的价值。方法 以结核分枝杆菌H37Rv基因组为模板,通过重叠PCR扩增得到Rv3425-Rv1168c全核酸序列克隆至表达载体pET-24b中,转入大肠杆菌BL21(DE3)进行诱导、表达和纯化,通过ELISA检测100份确诊结核病人、20例非结核呼吸疾病患者、100份健康人血清,评价融合蛋白进行临床血清学诊断的可行性。结果 Rv3425-Rv1168c融合蛋白在原核系统内获得高表达,纯化的融合蛋白经ELISA方法测定,在血清学实验中敏感性为54%,特异性为95%。结论 Rv3425-Rv1168c融合蛋白具有较高的抗原特异性和免疫原性,在结核病血清学诊断方面具有很大的应用价值。

Fusion expression in prokaryotic system and serological evaluation of Mycobacterium tuberculosis Rv3425-Rv1168c protein

In order to obtain fusion protein Rv3425-Rv1168c of Mycobacterium tuberculosis expression, purification in prokaryotic system and to evaluate its potential value for tuberculosis serological diagnosis, we amplified Rv3425-Rv1168c gene by overlap PCR from genome of Mycobacterium tuberculosis H37Rv. The fragments were cloned into pET-24b vectors. After induction, expression and purification from E. coli BL21(DE3), the feasibility was evaluated of fusion protein for clinical serological diagnosis by ELISA assay with 100 TB patients, 20 respiratory patients with a non-tuberculous and 100 healthy people sera. Rv3425-Rv1168c fusion protein was highly expressed, sensitivity and specificity were about 54% and 95% with tuberculosis serological diagnosis. Recombinant protein showed a strong antigenic ability. Results suggested Rv3425-Rv1168c protein had an important value in diagnosis of tuberculosis, and might be selected as one of diagnostic antigens of tuberculosis.