检索历史 清除

目的 对两种结核分枝杆菌(Mycobacterium tuberculosis)蛋白Dnak(Rv0350)和MPT83(Rv2873)进行抗原性评价,为结核病免疫诊断和疫苗研究提供实验依据.方法 通过基因重组和蛋白质纯化技术,克隆表达和纯化结核分枝杆菌蛋白Dnak(Rv0350)和MPT83(Rv2873).收集人群包括结核病患者、非结核病其他肺部疾病患者和健康志愿者的血液标本,以基因重组Dnak(Rv0350)和MPT83(Rv2873)蛋白作抗原,采用酶联免疫吸附试验(ELISA)和效应T细胞酶联免疫斑点试验(ELISPOT)分别进行人群体液和细胞免疫学检测,分析其免疫学性能.结果 成功获得基因重组表达和纯化的结核分枝杆菌蛋白Dnak(Rv0350)和MPT83(Rv2873),以这两种蛋白质作抗原进行人群体液和细胞免疫学检测.体液免疫检测,用ELISA检测135名结核病患者、56名非结核病其他肺部疾病患者和94名健康人的血清特异性IgG抗体,结果显示,Dnak(Rv0350)检测的灵敏度、特异度和准确率分别为77.80％(105/135)、56.70％(85/150)和66.67％(190/285);MPT83(Rv2873)检测的灵敏度、特异度和准确率分别为76.30％(103/135)、43.30％(65/150)和58.95％(168/285).细胞免疫检测,用ELISPOT检测59名结核病患者、65名非结核病其他肺部疾病患者和64名健康志愿者的外周血单核细胞被抗原刺激后效应T淋巴细胞产生γ干扰素(IFN-γ)的水平,结果显示,Dnak(Rv0350)检测的灵敏度、特异度和准确率分别为66.10％(39/59)、62.79％(81/129)和63.83％(120/188);MPT83(Rv2873)检测的灵敏度、特异度和准确率分别为47.46％(28/59)、79.84％(103/129)和69.68％(131/188).结论 结核分枝杆菌Dnak(Rv0350)和MPT83(Rv2873)蛋白均具有较好的抗原性,并且刺激T细胞产生免疫应答的能力较强,两者联合可能对于结核病免疫诊断和新型抗结核疫苗研究有更好的应用价值.

Objective To evaluate the antigenicity of two proteins of Mycobacteium tuberculosis (M. tuberculosis), Dnak(Rv0350) and MPT83(Rv2873), in order to provide a scientific basis for immuno-logical diagnosis of tuberculosis and research on vaccines. Methods The two antigen proteins, Dnak (Rv0350) and MPT83(Rv2873), were cloned, expressed and purified using the methods of genetic recom-bination and protein purification technology. Blood samples were collected from subjects including tuberculo-sis patients ( TB) , non-tuberculosis patients with other pulmonary diseases ( non-TB) and healthy volunteers (HV). To analyze the immunological properties of the recombinant Dnak (Rv0350) and MPT83 (Rv2873) proteins, they were used as antigens to detect humoral and cellular immunity in the subjects with enzyme linked immunosorbent assay ( ELISA ) and effector T cell enzyme-linked immunospot assay ( ELISPOT ) . Results The recombinant and purified Dnak (Rv0350) and MPT83 (Rv2873) proteins of M. tuberculosis were successfully obtained and used as antigens in the detection of humoral and cellular immunity in the sub-jects. Specific antibodies ( IgG) in the serum samples of 135 TB, 56 non-TB and 94 HV were tested with ELISA. The results showed that the sensitivity, specificity and accuracy of Dnak ( Rv0350 ) protein were 77. 80％ (105/135), 56. 70％ (85/150) and 66. 67％ (190/285). Similarly, the sensitivity, specificity and accuracy of MPT83 (Rv2873) protein were 76. 30％ (103/135), 43. 30％ (65/150) and 58. 95％(168/285). Cellular immunity was tested with the levels of IFN-γproduced by effector T lymphocytes after stimulating peripheral blood monouclear cells ( PBMC) collected form subjects of 59 TB, 65 non-TB and 64 HV with Dnak (Rv0350) and MPT83 (Rv2873) protein antigens. The results showed that the sensitivity, specificity and accuracy of Dnak (Rv0350) and MPT83 (Rv2873) proteins were 66. 10％ (39/59), 62. 79％ (81/129) and 63. 83％ (120/188), and 47. 46％ (28/59), 79. 84％ (103/129) and 69. 68％(131/188), respectively. Conclusions M. tuberculosis Dnak (Rv0350) and MPT83 (Rv2873) proteins have good antigenicity and could stimulate T cells to produce stronger immune responses. The two proteins used in combination might have promising potential in the research of immunodiagnosis of tuberculosis and the development of new anti-tuberculosis vaccines.