【作者】 潘萍萍；

【导师】 邵健忠； 项黎新；

【作者基本信息】 浙江大学 ， 细胞生物学， 2010， 硕士

【摘要】 论文以模式生物斑马鱼作为研究对象,开展了鱼类白介素-4(interleukin-4,IL-4)及其受体IL-4Rα的分子克隆与功能研究。论文第一部分为绪论部分。主要分两个部分介绍了IL-4及其特异性受体IL-4Rα的研究近况。第一部分介绍了IL-4的发现、基因克隆、基因结构、表达调控机制、受体的组成、生物学功能以及相关信号传导途径等方面的研究结果和进展;第二部分主要介绍了IL-4Rα的基因克隆、表达调控、信号传导、选择剪接形式及生物学功能等方面的研究进展。IL-4Rα和IL-4是高度亲和的一对特异性相互作用的分子,两者的亲和作用对发挥IL-4的生物学功能起到了非常重要的作用。论文第二部分为实验部分。主要分为三个小节:第一小节,为斑马鱼IL-4基因的克隆、生物信息学分析以及组织分布的鉴定,同时利用原核表达系统得到可溶型的重组斑马鱼IL-4蛋白质。利用生物信息学软件,分析了斑马鱼IL-4分子在进化上的规律,并且分析了其在进化上与高等脊椎动物及其他物种的关系。第二部分,在斑马鱼中克隆得到了IL-4特异性受体IL-4Rα的基因,同时还克隆到一个IL-4Rα样(IL-4Rα-L)的基因,与IL-4Rα基因座位相邻,它与IL-4Rα基因在序列和结构上存在高度的同源性,最大的区别是IL-4Rα-L缺失大部分的胞内区,同时两者通过选择性剪接均得到一个可溶形式的蛋白。我们对这四个分子进行了其在斑马鱼中组织分布的规律的研究,同时在原核表达系统中表达并纯化得到可溶性的IL-4Rα蛋白质,并制备得到多克隆抗体。利用生物信息学软件,对IL-4Rα及其IL-4Rα-L与其他物种中的IL-4Rα进行分析比对,发现IL-4Rα在基因结构,基因共线性,蛋白质的空间构象,蛋白质信号转导的结合位点等各方面都是相当保守的。第三部分,我们对IL-4/IL-4Rα进行了功能分析,提供了IL-4和IL-4Rα作为一对特异性互作分子的证据,同时发现IL-4能刺激B细胞增殖,对抗体产生起上调作用,并且鉴定了IL-4提高Th2类细胞因子及转录因子的表达,证实了IL-4/IL-4Rα作为Th2类免疫调节中一个重要的信号调节途径在鱼类中的发生。更多还原

【Abstract】 This article focused on the molecular cloning, functional characterization of Th2 immunity related molecules and preliminary identification of Th2 immunity in Danio rerio.Chapter 1 is an introduction of the recent researches on IL-4 and its specific receptor IL-4Rα. It was described in two parts. In the first part, we mainly introduce interleukin-4 (IL-4) and IL-4Ra (IL-4 receptor alpha chain) gene including its discovery, molecular cloning, gene structure, regulatory mechanisms of them expression, its receptors and the corresponding signal pathways, the biology functions of IL-4, as well as various types of alternative splicing of IL-4Rα. IL-4 and IL-4Ra was two reciprocal molecules with high affinity, they play the important function in Th2 immunity.Chapter 2 is the description of the main researches. It was described in three parts. In first part, we performed the molecular cloning and characterization of IL-4 from zebrafish, and its prokaryotic expression in E.coli and purification of soluble recombinant protein. By bioinformatics methods, we carried out the study on the evolution of vertebrates IL-4 genes. In the second part, we cloned IL-4Rα, and we also get a IL-4Rαlike (IL-4Rα-L) gene from zebrafish. IL-4Ra-L adjacent to IL-4Ra, they had high identity in cDNA sequences and gene structures, the most difference is that IL-4Rα-L laking the most cytoplasmic region. Both of them have its solubles from alternative splicing. By bioinformatics methods, we found that the gene structure, gene locus, protein structure and the main motifs for signaling were conserved across all species aligned. In the third part, we performed the function analysis on IL-4/IL-4Rα, provided the evidence on the two reciprocal molecules. And we found that, IL-4 could stimulate the proliferation of B cell, up-regulate the production of antibody, and promote the expression of Th2 cytokines and transcription factors. All the evidence showed that a possible Th2-mediated regulatory mechanism had been developed in the adaptive immunity during early vertebrate evolution.更多还原