摘要
目的:制备抗结核分枝杆菌CFP-10蛋白的单克隆抗体(mAb)。方法:应用淋巴细胞杂交瘤技术,以重组菌BL21(DE3)-pET-30a(+)-lhp原核表达的融合蛋白His-CFP-10作为抗原免疫BALB/c小鼠,以纯化的融合蛋白GST-CFP-10作为检测抗原,制备抗结核分枝杆菌CFP-10mAb;采用间接ELISA、Dot-ELISA和Western blot鉴定mAb的特异性。结果:获得2株稳定分泌抗结核分枝杆菌CFP-10mAb的杂交瘤细胞株6E8、2E7,其Ig亚类分别为IgG1、IgG2b。mAb6E8、2E7腹水的ELISA效价分别为1∶1000000,1∶1024000。在Dot-ELISA试验中,这2株mAb均只与表达His-CFP-10及GST-CFP-10的重组大肠杆菌反应,与其他5种菌株均不发生反应。在Westernblot试验中,2株mAb只与CFP-10蛋白发生反应,出现特异性条带。结果表明,mAb6E8、2E7是针对结核分枝杆菌CFP-10的特异性mAb。结论:成功地制备抗结核分枝杆菌CFP-10的mAb,它们在结核分枝杆菌诊断和致病机理研究等方面有重要应用价值。
AIM： To prepare monoclonal antibodies （mAb） against CFP-10 protein of Mycobacterium tuberculosis. METHODS： BALB/c mice were immunized with the purified His-CFP-10 expressed in BL21 （DE3）-pET-30a （ ＋ ）-Ihp. With the purified GST-CFP-10 as detecting antigen, mAb-produced hybridoma cells against CFP-10 were screened by indirect ELISA. The specificity of the mAbs were characterized by indirect ELISA, Dot-ELISA and Western blot. RESULTS： Two hyridoma cell lines secreting mAbs against CFP-10 named 6E8, 2E7 were obtained. The immunoglobulin subclasses of 2 mAbs were IgG1 and IgG2b respectively, and the ELISA titers of 2 mAbs ascitic fluids were 1 ： 1 000 000, 1 ： 1 024 000 respectively. In Dot-ELISA test, the 2 mAbs could only react with BL21 （ DE3）-pET-30a （ ＋ ）-Ihp, BL21-pGEX-6P-1-lhp, which expressed His-CFP- 10, GST-CFP-10, respectively. Western blot analysis confirmed that the 2 mAbs could only react with CFP-10 protein. CONCLUSION： Two mAbs specific to CFP-10 protein of Mycobacterium tuberculosis were obtained, which may have important application value in further studies on diagnosis and pathogenesis.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2009年第12期1143-1145,共3页
Chinese Journal of Cellular and Molecular Immunology
基金
国家重点基础研究发展计划(973)资助项目(CB504404)
国家自然科学基金资助项目(30425031)
国家科技重大专项课题(2008ZX10003-010)
江苏省科技攻关项目(BE2007340)
