【作者】 陈家艳；

【导师】 梁宇君； 张士璀；

【作者基本信息】 中国海洋大学 ， 生物工程， 2011， 硕士

【摘要】 Properdin蛋白即P因子,是补体替代激活途径中的正向调节因子,起着稳定替代途径中C3转化酶(C3bBb)的作用。为了进一步阐明Properdin的结构和功能之间的关系,探索Properdin与C3b及B因子的结合位点,我们从斑马鱼中克隆并表达了该基因,以鉴定其功能特性。Properdin基因的开放阅读框全长为1314bp,编码一条含有437个氨基酸的多肽,其前20位氨基酸为信号肽,预测分子量大小为48.2KDa,也是由6个Ⅰ型凝血酶敏感蛋白重复序列构成。Properdin成熟蛋白中有40个半胱氨酸,预测有15个二硫键形成,31和58位处氨基酸可能为二个潜在的糖基化位点。对不同物种间Properdin的蛋白序列建进化树分析,结果显示斑马鱼Properdin蛋白和其它硬骨鱼Properdin聚成一簇。不同物种的Properdin蛋白序列及结构域进行多序列比对,发现斑马鱼properdin蛋白中存在保守的氨基酸基序WXXWXXW和RXR。与人、鼠、爪蟾、虹鳟(cfp1、cfp2、cfp3)的比较发现,同源性分别为42%、41%、43%、48%、48%和49%。实时荧光定量PCR结果显示:斑马鱼Properdin直到胚胎发育到30%下包时有母源性表达,且表达量随发育进行逐渐降低,但从20体节开始,合子基因开始表达。不同组织的实时荧光定量PCR结果表明,肝脏、肠、心脏、脑、眼睛、精巢、卵巢等组织中均有表达,在肠、鳍、肌肉的表达量相对较低,精巢、肝脏、眼睛、心脏、鳍的表达量较高,其中精巢中表达最多。综上所述,斑马鱼Properdin的结构域特征与哺乳动物中该蛋白的结构域特征一样,与其在系统中的发育地位是相符的。预测的一个糖基化位点位于Cys45和Cys63之间,而这两个半胱氨酸预测会形成二硫键,因此推测此二硫键为与糖类的结合位点。Properdin有母源性表达,可能提示其在发育早期起作用。不同物种间Properdin表达部位不同是否提示其功能有差异,还有待于深入研究。这是关于斑马鱼中Properdin基因表达及其表达模式的首次报道,我们已经纯化得到了Properdin重组蛋白,为以后利用免疫结合等生物学技术探索Properdin蛋白的结构、功能及作用机制奠定了基础。深入了解其功能,有助于我们阐明人类补体缺乏病理机制,也促进我们对鱼类补体系统的功能有更深入的理解。更多还原

【Abstract】 Properdin, also known as P factor, is an upregulator of the alternative complement pathway and acts by stabilizing the alternative pathway convertase (C3bBb). To elucidate the relationship between structure and function of properdin, and find the binding sites for C3b and factor B, we cloned and expressed the properdin gene in zebrafish to identify its functional properties.The open reading frame cDNA of properdin is 1314bp, coding a polypeptide of 437 amino acids with a calculated molecular weight of about 48.2 kDa, composed of six thrombospondin repeat typeⅠdomains(TSR-1–TSR-6) too. There were forty Cys in the mature Properdin, predicted fifteen possible disulfide bonds, and two potential N-linked glycosylation site located at the residual position 31 and 58. Phylogenetic tree analysis of different Properdin protein sequences showed that zebrafish Properdin clustered in bony fish clade. On aligning the zebrafish properdin and TSR with other species, zebrafish properdins protein were found to have consensus sequence motifs WXXWXXW and RXR, The amino acid identity between zebrafish and that of humans, mouse, xenopus and trout pfc1, pfc2 and pfc3 was 42%、41%、43%、48%、48% and 49%, respectively. Zebrafish Properdin gene expression pattern at different developmental stages and in different tissues was analyzed by qRT-PCR. Results show Properdin to have maternal expression with a tendency to decrease gradually as the development continues. No expression was recorded at 30%-epiboly stage. However, from the 20-somite stage, the zygotic expression of Properdin commenced again. In adult zebrafish, Properdin was found to be expressed in all the tissues. Relatively lower expression was found in the muscles, fins, intestine, ovaries and brain; higher expression was found in the heart, testis, liver, eyes and heart.In Summary, zebrafish properdin resemble its mammalian counterpart, which coincides with the phylogenetic status of zebrafish. One of the predicted glycosylation sites located between Cys45 and Cys63, which were predicted to form a disulfide bond, possibly serving to bind saccharide. Zebrafish Properdin has maternal expression, indicating its role in the early development. This is the first report of properdin in zebrafish. We have purified the recombinant Properdin protein thus making it possible to conduct immunoassays and other biological investigations in order to study the structure, function and mechanism of the action of Properdin. Further characterization of Properdin will help us clarify the pathological mechanism of the human complement defect and help us understand the functioning of the complement system.更多还原