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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。
Objective: To prepare a monoclonal antibody (mAb) against Mycobacterium tuberculosis CFP-10 protein. Methods: BALB / c mice were immunized with the His-CFP-10 fusion protein His-CFP-10 expressed in prokaryotic cells BL21 (DE3) -pET-30a (+) - lhp by using lymphocyte hybridoma technique. The purified fusion protein GST Antibody against Mycobacterium tuberculosis CFP-10 mAb was prepared by using -FPA-CFP-10 as a detection antigen. The specificity of mAb was identified by indirect ELISA, Dot-ELISA and Western blot. Results: Two hybridoma cell lines, 6E8 and 2E7, secreting stable anti-Mycobacterium tuberculosis CFP-10 mAb were obtained. The Ig subclasses were IgG1 and IgG2b, respectively. ELISA titers of mAb6E8 and 2E7 ascites were 1: 1000000 and 1: 102424, respectively. In the Dot-ELISA assay, both mAbs reacted only with recombinant E. coli expressing His-CFP-10 and GST-CFP-10 and did not react with the other five strains. In the Western blotting assay, two mAbs reacted only with the CFP-10 protein and a specific band appeared. The results show that mAb6E8, 2E7 are specific mAbs against M. tuberculosis CFP-10. Conclusion: The mAb against Mycobacterium tuberculosis CFP-10 has been successfully prepared and has important application value in the diagnosis and pathogenesis of Mycobacterium tuberculosis.