新型凝集素LSECtin相互作用蛋白研究及基因打靶载体构建
发布时间：2018-07-28 12:34
【摘要】：对于自己和非己的识别是机体免疫反应的基础,而完成这一功能的分子被称作模式识别受体(pattern recognition receptor, PRRs)。C型凝集素(C-type lectins)作为一类新型的模式识别分子,近年来逐渐显示出在病原体识别中的重要作用。同时,某些C型凝集素与配体结合后可以产生免疫抑制信号,因而许多病原微生物包括艾滋病毒、丙肝病毒等借此以逃避机体的免疫监视,但迄今对其胞内的信号转导途径仍不清楚,此方面的研究引起了人们的高度关注。LSECtin(liver and lymph node sinusoidal endothelial cells C-type lectin)是本室鉴定的一个新的C型凝集素,特异表达在肝脏和淋巴结窦内皮细胞上,具有糖基结合能力,但是其胞内的信号转导途径尚未揭示,其生理功能和分子机制了解甚少,我们对此开展了研究。 本文首先通过酵母双杂交方法筛选LSECtin的胞内区结合蛋白,并选择了四对可能存在的相互作用在酵母中进行了初步验证,证明均真实存在。继而在综合比较结合力、功能相关性、亚细胞定位的基础上,选取了其中两个蛋白IFP35(interferon-induced protein 35)和TRIP-1(TGF-β type Ⅱ receptor interacting protein-1)在哺乳动物细胞中进行进一步验证。荧光定位实验表明,当单独表达时,LSECtin为质膜上的不均匀分布和细胞质中的少量点状分布,IFP35为细胞质中的点状或弥散状分布,TRIP-1在质膜和细胞质中均有分布。而当IFP35或TRIP-1分别与LSECtin共表达时,二者均被募集到LSECtin高表达的区域,并且存在明显的共定位。同时,免疫共沉淀实验也证明二者确与LSECtin存在相互作用。此外,我们对LSECtin与TRIP-1的相互作用的生物学意义进行了探索。报告基因实验显示,LSECtin对TGF-β诱导的靶基因PAI-1表达具有抑制作用,而同家族的DC-SIGNR(DC-SIGN related gene)没有此功能,可能与其特有的生理功能相关。这些结果提示,LSECtin的胞内信号转导可能与TGF-β和干扰素途径有关。同时,为阐明LSECtin的生理功能,我们还开展了小鼠LSECtin的基因打靶工作,完成了mLSECtin基因组序列的筛选。
[Abstract]:The recognition of self and non-self is the basis of the body's immune response, and the molecule that completes this function is called the pattern recognition receptor (pattern recognition receptor, PRRs). C lectin (C-type lectins) as a new type of pattern recognition molecule. In recent years, it has gradually shown the important role in pathogen recognition. At the same time, some type C lectin binding with ligands can produce immunosuppressive signals, so many pathogenic microorganisms, including HIV, hepatitis C virus and so on, can escape the body's immune surveillance. However, the intracellular signal transduction pathway is still unclear, and this study has aroused great concern that. LSECtin (liver and lymph node sinusoidal endothelial cells C-type lectin is a new type C lectin identified in our laboratory. Specifically expressed in liver and lymph node sinusoidal endothelial cells, it has glycosyl binding ability, but its intracellular signal transduction pathway has not been revealed, and its physiological function and molecular mechanism are not well understood. In this paper, the intracellular binding proteins of LSECtin were screened by yeast two-hybrid method, and four pairs of possible interactions were selected to verify the existence of these proteins in yeast. On the basis of comprehensive comparison of binding ability, functional correlation and subcellular localization, two proteins IFP35 (interferon-induced protein 35) and TRIP-1 (TGF- 尾 type 鈪，
本文编号：2150153
[Abstract]:The recognition of self and non-self is the basis of the body's immune response, and the molecule that completes this function is called the pattern recognition receptor (pattern recognition receptor, PRRs). C lectin (C-type lectins) as a new type of pattern recognition molecule. In recent years, it has gradually shown the important role in pathogen recognition. At the same time, some type C lectin binding with ligands can produce immunosuppressive signals, so many pathogenic microorganisms, including HIV, hepatitis C virus and so on, can escape the body's immune surveillance. However, the intracellular signal transduction pathway is still unclear, and this study has aroused great concern that. LSECtin (liver and lymph node sinusoidal endothelial cells C-type lectin is a new type C lectin identified in our laboratory. Specifically expressed in liver and lymph node sinusoidal endothelial cells, it has glycosyl binding ability, but its intracellular signal transduction pathway has not been revealed, and its physiological function and molecular mechanism are not well understood. In this paper, the intracellular binding proteins of LSECtin were screened by yeast two-hybrid method, and four pairs of possible interactions were selected to verify the existence of these proteins in yeast. On the basis of comprehensive comparison of binding ability, functional correlation and subcellular localization, two proteins IFP35 (interferon-induced protein 35) and TRIP-1 (TGF- 尾 type 鈪，
本文编号：2150153
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