人IL-17基因克

发布时间：2018-03-10 03:05

本文选题：hIL-17　切入点：单克隆抗体　出处：《汕头大学》2010年硕士论文　论文类型：学位论文

【摘要】： 背景与目的:IL17-A是IL17家族(包括IL-17A, IL-17B, IL-17C, IL-17D, IL-17E和IL-17F)的成员,作为Th17细胞亚型因主要分泌的蛋白分子近年来受到广泛关注。Th17细胞与同属于CD4T淋巴细胞的Th1、Th2亚型在刺激条件和转录因子等方面有显著不同。在类风湿性关节炎(RA)、系统性红斑狼疮(SLE)、银屑病等病患中发现IL17-A表达升高,但并不清楚IL17-A在疾病发病过程中的具体作用。因此,制备经济有效的IL17-A以及抗hIL-17单克隆抗体(mAb)为今后相关实验研究具有重要意义。方法及结果:克隆并原核表达hIL-17蛋白,以之为免疫原免疫BALB/c小鼠,取免疫小鼠脾细胞和小鼠骨髓瘤NS-1细胞融合。通过间接ELISA法筛选可特异性分泌抗hIL-17 mAb的杂交瘤细胞。采用Ig类与亚类鉴定试剂盒鉴定mAb的Ig亚类;通过ELISA、Western blot、免疫组织化学染色法鉴定mAb的特异性。获得7株可稳定分泌抗hIL17-A mAb的杂交瘤细胞,其分泌的mAb六株为IgM,一株为IgG2a。Western blot分析表明,此mAb所识别的靶分子的相对分子质量(Mr)约为15400。免疫组织化学染色表明,mAb与食管癌、胃癌反应呈阳性。结论:成功地制备IL17-A以及抗hIL-17 mAb,为今后相关实验研究奠定了基础。
[Abstract]:Background and objective: IL17-A is a member of the IL17 family (including IL-17A, IL-17B, IL-17C, IL-17D, IL-17E and IL-17F). In recent years, the protein molecules secreted by Th17 cells have attracted extensive attention. Th17 cells are significantly different from Th1T 2 subtypes, which belong to CD4T lymphocytes, in stimulation conditions and transcription factors, and in rheumatoid arthritis. Increased expression of IL17-A was found in patients with systemic lupus erythematosus and psoriasis. However, the role of IL17-A in the pathogenesis of the disease is not clear. Therefore, the preparation of economical and effective IL17-A and anti hIL-17 monoclonal antibody (mAb) is of great significance for the related experimental research in the future. Methods and results: hIL-17 protein was cloned and expressed in prokaryotic cells and immunized BALB/c mice. The hybridoma cells secreting specific anti-#en2# mAb were selected by indirect ELISA method. The immunized spleen cells were fused with mouse myeloma NS-1 cells. The Ig subclasses of mAb were identified by Ig and subclass identification kit. The specificity of mAb was identified by Elisa Western blot and immunohistochemical staining. Seven hybridoma cells secreting hIL17-A mAb stably were obtained, six of which secreted mAb and one was identified by IgG2a.Western blot analysis. The relative molecular weight of the target molecule recognized by this mAb was about 15400.Immunohistochemical staining showed that mAb was positive to esophageal carcinoma and gastric cancer. Conclusion: IL17-A and anti-hIL-17 mAbwere successfully prepared, which laid a foundation for related experimental research in the future.
【学位授予单位】：汕头大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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