ESAT-6/CFP-10融合蛋白诊断结核性感染和结核病的研究

Research of ESAT-6/CFP-10 fusion protein on diagnosis of tuberculosis and mycobacterium tuberculosis infections

目的::探讨ESAT-6/CFP-10融合蛋白刺激外周血单个核细胞(PBMC)释放IFN-γ反应及其对结核性感染和结核病的诊断价值。方法:选取健康对照组(34例),密切接触组(19例)和结核病组(39例)3组对象,按其有无卡介苗(BCG)接种各分成两个2组共6小组:健康对照组BCG未接种者(15例)、健康对照组BCG接种者(19例)、密切接触组BCG未接种者(6例)、密切接触组BCG接种者(13例)、结核病组BCG未接种者(7例)和结核病组BCG接种者(32例)。分离PBMC并分别与PPD和ESAT-6/CFP-10融合蛋白共同培养5d,以酶联免疫吸附测定法(ELISA)检测培养上清液中IFN-γ,观察6组对象IFN-γ释放反应。结果:PPD刺激后,健康对照组BCG未接种者与密切接触组BCG未接种者结果之间差异有统计学意义(P<0.01),而3组BCG接种者之间结果差异无统计学意义(P>0.05)。ESAT-6/CFP-10融合蛋白刺激后,健康对照组、密切接触组、和结核病组3组总阳性率分别为0%,21.1%,87.2%,阳性率明显增加。健康对照组BCG未接种者与密切接触组BCG未接种者结果之间差异有统计学意义(P<0.05);健康对照组BCG接种者与密切接触组BCG接种者结果之间差异有统计学意义(P<0.01)。结论:ESAT-6/CFP-10融合蛋白为结核杆菌特异性抗原,能够区分BCG接种和结核病,在结核性感染和结核病的诊断中有应用价值。

Objective：To investigate the role of in-vitro interferon-gamma （IFN-γ） release assay based on ESAT-6/CFP 10 fusion protein stimulating PBMC in the diagnosis of tuberculosis and Mycobacterium tuberculosis infection. Method：There were three group objects： healthy controls（n=34）, closed contacts （n= 19） and tuberculosis cases （n= 39）. According to the history of BCG vaccination, they were separated into healthy controls without BCG vaccination （n= 15）, healthy controls with BCG vaccination （n= 19）, closed contacts without BCG vaccination （n = 6）, closed contacts with BCG vaccination （n = 13）, tuberculosis cases without BCG vaccination （n =7） and tuberculosis cases with BCG vaccination （n=32）. PBMCs were collected from six groups as previously described and were co-cultured for 5 days with PPD or ESAT-6/CFP-10 fusion protein. IFN-γ release quantity were detected by ELISA. Result：The positive rates of six groups were 0%（0/15） ,100% （19/19） ,16.7%（1/6）, 100%（13/13） ,85.7%（6/7）,100%（32/32）,0% （0/15）,0% （0/19）,33.3% （2/6）,15.4% （2/13）, 71.4% （5/7）, and 90.6%（29/32）respectively. After PBMC were co-cultured with PPD, there was a significant difference of IFN-γ release assay between healthy controls without BCG vaccination and closed contacts without BCG vaccination （P〈0.01）, but there were no significant differences among three groups with BCG vaccination （P〈0.05）. After PBMC were co cultured with ESAT-6/CFP-10 fusion protein, the total positive rates of healthy controls, closed contacts and tuberculosis cases were 0%, 21.1% and 87.2%, respectively. There was a significant difference of IFN-γ release assay between healthy controls without BCG vaccination and closed contacts without BCG vaccination （P〈0.05）. There was a significant difference between healthy with BCG vaccination and closed contacts with BCG vaccination （P〈0.01）. Conclusion： ESAT-6/CFP-10 fusion protein is the specific antigen of myeobacterium tuberculosis. It can distinguish BCG vaccination from tuberculosis, and is valuable in diagnosis of tuberculosis and Mycobacterium tuberculosis infections.