pEGFP-Cl-MyD88真核表达载体的构建及其在HEK293细胞中的表达

发布时间：2018-01-03 21:49

本文关键词：pEGFP-Cl-MyD88真核表达载体的构建及其在HEK293细胞中的表达　出处：《华中师范大学》2012年硕士论文　论文类型：学位论文

【摘要】：Toll样受体(Toll-like receptors, TLRs)是新近发现的模式识别受体,该家族通过识别病原体相关分子模式pathogen associated molecular patterns, PAMP)激活免疫细胞,在天然免疫防御病原体中起着重要作用。髓样分化因子88(myeloid differentiation factor88, MyD88)作为一种细胞内信号接头蛋白,在TLR家族和白介素1受体(interleukin1receptor, IL-1R)家族信号通路中发挥关键作用。MyD88蛋白具有3个功能结构域：N端的死亡结构域(death domain, DD)、中间区域(intermediate domain, ID)及C端的Toll样受体和白介素1受体相关结构域(TLR/IL-1R related domain, TIR),其中MyD88蛋白的TIR结构域能与TLRs和IL-1Rs的TIR结构域相结合,从而介导信号向下游传导。MyD88是重要的信号转接蛋白,其介导的信号通路与肠炎、动脉粥样硬化、心肌肥大、癌症等在内的多种疾病的发生和发展过程有着密切的联系。MyD88的结构和功能研究将为这些疾病的治疗提供新的方向和思路。本研究以水牛外周血白细胞为材料,提取总RNA,通过RT-PCR的方法扩增MyD88基因全长编码序列,将其连接至pEGFP-Cl质粒,构建(?)EGFP-Cl-MyD88真核重组表达载体,重组载体经菌落PCR、酶切和测序鉴定后,在脂质体的介导下转染进HEK293细胞中,使其发生瞬时表达,并通过G418抗性筛选,构建稳定表达EGFP-MyD88融合蛋白的HEK293细胞系。结果表明：转染48h后,绿色荧光蛋白在转染重组质粒的细胞中呈颗粒状散在分布,而在转染空质粒的细胞中均匀分布；Western Blot结果显示在62kDa处出现一条特异性抗体结合条带；经过G418的持续筛选,获得了稳定表达目的蛋白的阳性克隆,FCM分析结果显示,转染pEGFP-Cl空质粒的细胞系阳性率达93.35%,转染重组质粒的细胞系阳性率达84.33%,表明成功构建了稳定表达MyD88的HEK293细胞系。以上结果为针对MyD88分子的疾病治疗及其功能研究奠定了基础。
[Abstract]:Toll-like receptor (TLRs) is a newly discovered pattern recognition receptor. The family activates immune cells by recognizing the pathogen-associated molecular pattern pathogen associated molecular patterns (PAMPs). Myeloid differentiation factor88, myeloid differentiation factor 88, plays an important role in innate immune defense. MyD88) is a intracellular signaling junction protein in the TLR family and interleukin-1 receptor. IL-1R family signaling pathway plays a key role. MyD88 protein has three functional domains: N-terminal death domain death domain (DDD). Intermediate domain. Toll like receptor and interleukin-1 receptor related domain TLR / IL-1R related domain (TIRs). The TIR domain of MyD88 protein can bind to the TIR domain of TLRs and IL-1Rs, which mediates signal transduction downstream. MyD88 is an important signal transfer protein. It mediates signaling pathways associated with enteritis, atherosclerosis, and myocardial hypertrophy. The structure and function of MyD88 are closely related to the occurrence and development of many diseases, such as cancer, which will provide a new direction and thought for the treatment of these diseases. For material. Total RNAs were extracted, the full-length coding sequence of MyD88 gene was amplified by RT-PCR, and ligated into pEGFP-Cl plasmid. EGFP-Cl-MyD88 eukaryotic expression vector was transfected into HEK293 cells with liposome mediated by restriction endonuclease digestion and sequencing. The HEK293 cell lines expressing EGFP-MyD88 fusion protein stably were constructed by G418 resistance screening. The results showed that the cells were transfected for 48 h. Green fluorescent protein (GFP) was distributed in the cells transfected with recombinant plasmids and distributed uniformly in the cells transfected with empty plasmids. Western Blot showed a specific antibody binding band at 62 kDa. The results of FCM analysis showed that the positive rate of cell lines transfected with empty plasmid of pEGFP-Cl was 93.35%. The positive rate of cell line transfected with recombinant plasmid was 84.33%. The results showed that the HEK293 cell lines stably expressing MyD88 were successfully constructed, which laid a foundation for the study of disease treatment and function of MyD88 molecules.
【学位授予单位】：华中师范大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329;Q78

【引证文献】