结核分枝杆菌CFP10、ESAT6蛋白的原核表达、纯化及ELISPOT检测方法的建立与应用  

Evalution of an ELISPOT method developed based on the recombinant expression and purification of CFP10 and ESAT6 proteins from mycobacterium tuberculosis

作　　者：王晓玮[1] 程筱雯[1] 张焰[1] 徐东芳 王东萍 韦薇 王庆 徐元宏[1] 

机构地区：[1]安徽医科大学第一附属医院检验科,合肥230022 [2]安徽省胸科医院检验科,合肥230022

出　　处：《安徽医科大学学报》2015年第9期1347-1351,共5页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金(编号:81171606);安徽省临床检测技术公共服务平台(编号:PT20081011)

摘　　要：以结核分枝杆菌CFP10、ESAT6蛋白原核表达、纯化体系为基础,建立稳定的ELISPOT方法;检测164份疑似结核患者标本的外周血ELISPOT,与临床诊断及T-SPOT·TB结果对比。表达所得重组蛋白浓度和纯度如下:CFP10为0.5 mg/ml,84%;ESAT6为4 mg/ml,98%。ELISPOT方法最佳实验条件为每孔细胞密度2×105/孔,CFP10、ESAT6蛋白抗原浓度分别为10μg/孔,细胞孵育时间20 h。ELISPOT的灵敏度与特异度分别为88.50%、82.35%,检测结果与TSPOT·TB方法结果一致(χ2=0.57)。Building a stable method of ELISPOT by the expression and purification of the protein CFPIO and ESAT6 from mycobacterium tuberculosis. 164 suspected tuberculosis cases were checked by ELISPOT and T-SPOT TB with peripheral blood samples, compared with the clinical diagnosis. It was found that the concentration and purity of the recombinant ESAT6 were 0. 5 mg/ml,84% ;the concentration and purity of recombinant of the CFP10 were 4 mg/ml, 98%. The most appropriate conditions of ELISPOT were 2 × 105/well as cell concentration, 10μg/ well ESAT6/CFP10 recombinant protein as stimulus and 20 hours of incubation period. There was no statistically significant difference between ELISPOT and T-SPOT · TB （ χ2 = 0. 57）.

关 键 词：结核病诊断 Γ-干扰素 ELISPOT CFP10 ESAT6 

分 类 号：R446-61[医药卫生—诊断学]