权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Molecular cloning and functional analysis of carp complement regulatory factors : Molecular mechanism of C3 fragmentation.

鲤鱼补体调节因子的分子克隆和功能分析：C3片段化的分子机制。

基本信息

批准号：
14560153
负责人：
NAKAO Miki
金额：
$ 2.24万
依托单位：
Kyushu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14560153/
关键词：
fish complement innate immunity factor I regulatory factor cloning inhibotors diversity コイ cDNAクローニング遺伝子多様性

项目摘要

In mammals, biodefensive roles of the complement system depend largely upon several kind& of C3-derived fragments, such as C3b, iC3b and C3d. In fish, little is known how the, complement component C3 is fragmented in physiological conditions. The goal of the present study was to identify a complement regulatory molecule that mediates C3 fragmentation in the common carp, and to analyze its function at the protein level. Two homologous cDNA clones that encode isotypic variants of mammalian factor I orthologue was isolated from a carp hepatopancreas cDNA library. The deduced amino acid sequences are 85% identical. It is interesting to note that one of them contained a novel insertion at its N-terminus and is expressed mainly in the hepatopancrease. Another is expressed predominantly in the ovary, suggesting its function outside of complement-regulation., At the protein level, we have searched factors that inhibit the hemolytic activity of normal carp serum against rabbit erythrocytes. As a result, two distinct factors with molecular masses of 400 kDa and 65 kDa. The 65 kDa putative regulatory factor has been purified by combination of affinity chromatography on heparin-agarose and anion-exchange chromatogarphy on Mono-Q. The partially purified 65 kDa factor was heat-labile. In addition, the 65 kDa factor prevented siginificant C3-deposition on the rabbit erythrocyte membranes, suggesting that the 65 kDa factor inhibits complement activation before the activation cascade proceeds to the reaction step involving C3.

在哺乳动物中，补体系统的生物防御作用在很大程度上取决于几种C3衍生片段，如C3b、IC3b和C3d。在鱼类中，补体成分C3在生理条件下是如何碎片化的，人们知之甚少。本研究的目的是寻找一种补体调节分子，在鲤鱼体内调节C3的断裂，并在蛋白质水平上分析其功能。从鲤鱼肝胰腺cDNA文库中分离到两个编码哺乳动物凝血因子I同源变异体的同源cDNA克隆。推导的氨基酸序列有85%的同源性。有趣的是，其中一个在其N端含有一个新的插入，并且主要在肝胰腺酶中表达。另一种主要在卵巢中表达，提示其在补体调节之外的功能。在蛋白质水平，我们寻找了抑制正常鲤鱼血清对兔红细胞溶血活性的因素。因此，有两个截然不同的因子，其分子质量分别为400 kDa和65 kDa。采用肝素-琼脂糖亲和层析和Mono-Q阴离子交换层析相结合的方法，纯化了65 kDa的调控因子。部分纯化的65 kDa蛋白对热不稳定。此外，65 kDa因子阻止了C3在兔红细胞膜上的显著沉积，这表明65 kDa因子在激活级联到涉及C3的反应步骤之前抑制了补体的激活。