【作者】 胡瑜兰；

【导师】 邵健忠； 项黎新； 李亦平；

【作者基本信息】 浙江大学 ， 细胞生物学， 2009， 博士

【摘要】 本论文对斑马鱼中补体经典途径中关键基因C1q以及免疫球蛋白基因进行了鉴定、功能以及进化的研究,共分为两个部分内容。第一部分应用生物信息学方法,利用基因组和EST数据库,在斑马鱼(Danio rerio)中成功预测并克隆了补体经典途径的关键基因C1q的C1qA、C1qB、C1qC三个亚基编码基因,并结合哺乳动物、软骨鱼以及七鳃鳗(Lamprey)发现中报道的C1q基因,对它们进行了基因结构与分子进化等分析。同时,在硬骨鱼中对整个C1q家族成员进行了进化分析,提出了C1q家族和EMU家族存在进化关系。另外,利用原核表达系统表达了斑马鱼C1qA、C1qB和C1qC头部结构域蛋白,研究了它们在补体经典途径中的作用。研究显示,斑马鱼C1qA、C1qB和C1qC并能和体外聚合的斑马鱼IgM重链恒定区结合,但是不能结合体外聚合的IgZ重链恒定区。在以鲫鱼血清为材料进行的补体经典途径研究表明,鱼类中存在补体经典途径,并且C1qA、C1qB和C1qC分子均参与经典途径。此外,鱼类C1qA、C1qB和C1qC还能与人热聚的IgM和IgG结合,鱼类C1qA、C1qB和C1qC的头部结构域能竞争性抑制兔C1q介导的经典途径引起的溶血反应。第二部分克隆了多个斑马鱼免疫球蛋白基因,包括已知的IgM,IgZ基因和另外两个新的免疫球蛋白基因,其中之一与斑马鱼IgZ有一定相似性,称为IgZ-2基因,另一个为由IgM的膜型外显子直接与CH1末端连接的分子,对它们进行了生物信息学分析,在mRNA水平检测了IgZ-2在斑马鱼各个器官以及受精卵各个发育的表达情况,结果表明,IgZ-2主要表达于头肾、肠和皮肤,在受精卵发育的14天开始表达。C1q分子是补体分子经典途径的起始分子,补体经典途径起始于C1q的两个或多个球形头部和免疫复合物中的IgM或IgG分子结合。本文首次克隆了斑马鱼C1q的三个分子C1qA、C1qB和C1qC以及斑马鱼的免疫球蛋白分子,对它们进行了生物信息学分析,并用Elisa分析了C1q和免疫球蛋白之间的关系。通过对硬骨鱼补体经典途径的研究显示,硬骨鱼中存在和哺乳动物类似的补体经典途径,硬骨鱼的C1q在补体经典途径中发挥与人C1q相似的功能。更多还原

【Abstract】 This article focused on the molecular identification,functional characterization, and evolution analysis of complement component C1q genes and immunoglobulin genes in teleost fish.In part one,based on the bioinformatics method such as synteny comparative analysis approach,we successfully identified and cloned the C1qA,B,C chains in Zebrafish(Danio rerio).The gene expression profiling in different tissues were detected.It was found that these genes are globally expressed and the mRNA expressions of Zebrafish C1qA could be detected at all stages during the development. Furthermore,the evolution analysis was performed the elucidate the evolution relationships of C1q family members in teleost fish such as Zebrafish(Danio rerio), Fugu(Fugu rubripe) and Tetraodon(Tetraodon nigroviridis),as well as in other vertebrate species.By phylogenetic analysis,we revealed an evolutionary relationship between C1q and EMu superfamily.Finally,utilizing the soluble recombinant protein which expressed in E.coli,we analyzed the functional characterization of the globular head regions of C1qA(ghA),B(ghB),and C(ghC) chains.The results showed that the ghA,ghB and ghC could bind to heat-aggregated human IgM and IgG and inhibit C1q mediated complement classical pathway.Moreover,it could bind to heat-aggregated and cross linked zebrafish IgM,but it could not bind to cross linked zebrafish IgZ.In part two,we cloned multiple immunoglobulin genes in zebrafish.Two genes, named IgM and IgZ,have been reported previously.And the other two novel immunoglobulin genes,including IgZ-2 and IgMm2,were identified for the first time. According to the structure characterizations,the IgMm2 gene was supposed to be formed by the directly splicing of the transmemebrane region to CH1 of IgM.The IgZ-2 showed high homologies to the newly identified IgZ.It was expressed in the kidney,skin,and intestine and we hypothesis that it may related to mucosal immunity. It was firstly appeared 14 days after fertilization.C1q is the first subcomponent of the classical pathway of complement activation and a major connecting link between classical pathway-driven innate immunity and IgG- or IgM- mediated acquired immunity.It could activate the classical complement pathway via binding of its globular heads to the Fc regions of aggregated IgG or IgM. In the present study,we cloned the C1qA,C1qB,and C1qC chain in zebrafish fish for the first time.Also,we identified several immunoglobulin genes in zebrafish.Thus, we performed an ELISA analysis to find a relationship between C1q and immunoglobulins and studied the classical pathway in teleost fish.In teleost fish,the classical pathway was identified,which showed similar properties as it in the mammals.And the C1q in teleost fish also has the similar functional characterizations as its mammalian counterpart.更多还原