结核分枝杆菌模拟抗原的筛选及初步应用

目的 通过对甲期分泌抗原-6(ESAT-6)及培养基滤过蛋白-10(CFP-10)肽库的结核分枝杆菌模拟抗原的筛选,旨在建立一个临床鉴别诊断结核分枝杆菌感染的新方法.方法 基于蛋白ESAT-6和CFP 10的序列,随机设计合成一组1 9肽库,通过γ-干扰素释放试验筛选特异性结核分枝杆菌抗原模拟多肽,并研究确立其工作浓度.结果 经过多轮筛选获得3条特异性模拟多肽,分别是P1MG、P8NV、P11LD,灵敏度分别为93.3％、90.0％、80.0％,特异性均＞90.0％;工作浓度均为2 μg/ml;将3种模拟多肽等浓度混合作为刺激原,初步临床验证试验表明,其灵敏度为95.3％,特异性为96.2％.结论 基于蛋白ESAT-6和CFP-10的序列设计、筛选、制备的混合型模拟多肽抗原,有望建立一种临床结核分枝杆菌鉴别诊断的新方法.

Simulation antigens screening from Mycobacterium tuberculosis and its preliminary clinical study

OBJECTIVE To establish a new clinical diagnosis method of Mycobacterium tuberculosis infection by ESAT-6 and CFP-10 peptide library screening of M.tuberculosis antigen simulation.METHODS A random peptide library was designed and synthesized based on the sequences of ESAT-6 and CFP-10 protein.M.tuberculosis antigen-specific analog peptides were screened by γ-interferon release tests,the working concentration was also determined.RESULTS Three specific analog peptides were obtained after several rounds of screening,including P1MG,P8NV,and P11LD,with the sensitivity of 93.3%,90.0%,80.0% respectively;all the specificity was greater than 90%.The concentration of the working concentration is 2 μg/ml.The three analog peptides worked as the original stimulus in detecting γ-interferon release when they were mixed in the equal levels.The preliminary clinical validation study showed that the sensitivity was 95.3%,and specificity was 96.2%.CONCLUSION By using the mixed analog peptide antigen which were designed,selected,prepared based on protein ESAT-6 and CFP-10 sequence,it is expected to establish a new method of clinical diagnosis of M.tuberculosis.