结核分枝杆菌早期分泌抗原ESAT-6的高效表达及其在结核分枝杆菌特异性细胞免疫检测中的应用  

Expression of Mycobacterium tuberculosis early secretory antigentic target-6 protein and its application in detection of Mycobacterium tuberculosis antigen-specific interferon-γ response

作　　者：张晶[1] 刘威龙 周伯平 张明霞 杨倩婷 朱秀云 陆坚 邓群益[2] 陈心春 

机构地区：[1]深圳市东湖医院呼吸内科,518020 [2]深圳市东湖医院结核科 [3]深港传染病研究中心

出　　处：《中华结核和呼吸杂志》2009年第1期55-59,共5页Chinese Journal of Tuberculosis and Respiratory Diseases

基　　金：国家自然科学基金资助项目（30771900）;广东省自然科学基金资助项目（7008514）;深圳市科技局重点资助项目（200701008）

摘　　要：目的获得纯化的MTB早期分泌抗原ESAT-6，并对其抗原性及在MTB特异性细胞免疫检测中的应用价值进行评价。方法采用基因工程技术表达MTBESAT-6蛋白，通过Western blot法分析ESAT-6与特异性抗体的反应性。以纯化ESAT-6为抗原，用酶联免疫斑点技术（Elispot）检测肺结核患者、健康医务工作者、自然村居民外周血γ-干扰素的应答水平，同时与用于隐性结核感染及结核病诊断的QFT-G检测法进行平行比较，对其在MTB感染细胞免疫检测中的应用价值进行评价。结果成功表达和纯化了ESAT-6重组蛋白，所表达的蛋白能被特异性抗ESAT-6抗体识别，同时与肺结核外周血单核细胞反应，刺激后者产生γ-干扰素。Elispot技术检测肺结核患者、健康医务工作者、自然村居民的阳性率分别为36／49（73．5％）、11／62（17．7％）、17／194（8．8％），同时用QFT-G法对肺结核患者和健康医务工作者进行检测阳性率分别为38／49（77．6％）、14／58（24．1％），两种检测方法比较敏感度（73．5％，77．6％；X^2=0．381，P〉0．05）和特异度（82．3％，75．9％；X^2=0．406，P〉0．05），差异无统计学意义。结论利用pET原核表达系统可成功地表达和纯化MTBESAT-6重组蛋白，以ESAT-6为抗原建立的Elispot外周血IFN-γ检测技术与QFT-G的特异性和敏感性相当，可用于结核病的辅助诊断。Objective To obtain the purified early secretory antigentic target-6 （ESAT-6） protein and to evaluate its application in detection of Mycobacterium tuberculosis antigen-specific interferon-γ（IFN-γ） response. Methods ESAT-6 protein was expressed by genetic engineering. The antigen specificity and reactivity of ESAT-6 were evaluated by Western blot. Using ESAT-6 as the antigen, the antigen-specific IFN-γ response in patients with tuberculosis, healthy medical workers, and village residents was detected by the Elispot method. The results were also compared with those obtained by a commercial kit （ QuantiFERON- TB-GOLD, QFT-G）. Results ESAT-6 protein was successfully expressed and purified, and the antigen specificity of ESAT-6 was confirmed by its recognition by the antigen-specific antibody （anti-ESAT-6）. The specificity and sensitivity of the Elispot assay using ESAT-6 as the antigen in detecting the IFN-γ response was comparable with those of the commercial kit （QFT-G）. The positive rates of the Elispot assay for patients with tuberculosis, healthy medical workers and villagers were 36/49 （73.5%）, 11/62 （17.7%）, and 17/194（8.8% ）, respectively, while the rates of the OFT-G method for patients with tuberculosis and healthy medical workers were 38/49 （77.6%） and 14/58 （24. 1% ）, respectively. The sensitivity （73.5%, 77.65%;X^2= 0. 381, P 〉 0. 05 ） and specificity （ 82. 3%, 75.9% ; = 0. 406, P 〉 0. 05） of these two methods did not differ significantly. Conclusions Recombinant ESAT-6 protein was expressed and purified. Elispot using recombinant ESAT-6 protein as antigen showed high sensitivity and specificity for detection of Mycobacterium tuberculosis antigen-specific IFN-γ response. The purified ESAT-6 can be used for diagnosis of Mycobacterium tuberculosis infection.

关 键 词：分枝杆菌 结核 抗原 酶联免疫吸附测定 

分 类 号：R686[医药卫生—骨科学]