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1、斑马鱼补体调节因子CF!基因的鉴定、功能初探及CFH家族成员的急相反应补体调节因子J(CFI)是哺乳动物补体系统的重要组成成分，发挥着免疫调节的作用，在辅因子C4bp、MCP和CFH的作用下通过酶解C3b和C4b分子调节补体系统的过度激活。已有研究表明哺乳动物中CFI有重要作用且与疾病发生相关，但CFI在其它物种中研究较少。斑马鱼作为一种重要的模式动物，其基因组测序已经完成，与人类基因的保守性高达95，这使得斑马鱼适于研究基因的表达与功能。本论文成功从斑马鱼中克隆了补体因子CFI并分析了其特征、表达，对功能进行了初步探索，同时对CFI的辅因子CFH在胚胎发育中的表达模式及其对LPS和LTA感染
2、的应答进行了分析。斑马鱼CFI mRNA全K为2384bp，其中编码区为2193bp，编码含731个氨基酸的蛋白质，多肽链的分子量为81 4kDa，等电点pl为6 17。与其它脊椎动物CFI蛋白的同源比对结果显示序列相似性高达35 567 1，且序列中存在大量保守的半胱氨酸。CFI蛋白具有段由18个氨基酸组成的信号肤和5个保守域，即FIMAC、SRCR、LDLrl、LDLr2和sP结构域。在信号肽与FIMAC结构域之间还有一段特异性的序列，这段序列可能是鱼类所特有的。成熟的CFI是由重链和轻链组成的异质二聚体，FIMAC、SRCR、LDLrl和LDLr2组成重链，sP结构域组成轻链，在轻链中存
3、在保守的催化三联体HisAsp-Set和一个位子催化部位底部的Asp残基。作为一种分泌型糖蛋白，斑马鱼的CFI序列中舍有O糖基化位点和N糖基化位点，O糖基化位点主要位于信号肽和F1MAC结构域之间的特异性序列区域N-糖基化位点则分散存在于整个CFI序列之中。蛋白结构预测显示斑马鱼CFI蛋白同人CFI蛋白的空问结构具有很高的相似性，这预示它们功能上的相似性。进化分析显示，CFI蛋白在进化上高度保守，哺乳动物CFI基嗣聚为一枝斑马鱼与硬骨鱼的CFI蛋白聚为一枝。LDLrl结构域和LDLr2结构域的进化树中两个结构域各裳为。簇，在每一个结构域的分枝中表现出与CFI蛋白全瞄赴化树相同的聚类趋势。实时定
4、量PCR检测结果显示斑马鱼CFI基因在并组织t J，J“泛表达，其中在卵巢、精巢、肝脏、脑、肠和眼中的表达量很高。当受到LPS蟮染时，甜7在肝脏中的表达最r调。CFI堆嗤l和CFH家族基冈在不同发育时期胚胎中具有不J司的表达模式，(蜒因在胚胎发肯的体节期表达量揖多，而大部分(仰家族成员在】dpf腓胎巾表达量开始显著增多，其中CFHL4在胚胎发育整个过程中部具有很高的表达量。在体节期，当胚胎受到LPS和LTA感染时CFI基因的表达量显著F喘，而(1刖家族基蹦在胚胎发育不同对期则呈现不同的应苔趋势，且表达量变化显著。推测CFH家族基因在斑马鱼胚胎发育过程巾，通过多种形式对朴体选径进行若精确的调控。
5、胚胎整体原位杂交结果显示，CFl基因在胚胎发育的过程中在卵黄合胞体上直具有特异性表达，在体节期时信号最强。当胚胎发育至1天对CFI基因在松果体中也出现特异性表达。这提示CFI除了参与朴体调节外，可能在早期发育过程中也起重要作用。初步实验表明过表达CFlmRNA并没有可见的对胚胎发育的影响。同时，还尝试了原核表达C兀，体外诱导表达重组蛋白CFI的大小为64 2kDa这为进步在蛋白水平研究斑马鱼CFI的功能奠定了基础。关键词：斑马鱼：补体调节；CFI；CFHCharacterization and functional analysis ofthe complementfactor 1 and a
6、cute phase response ofCFH family members inzebrafishAbstractThe complement system in vertebrates plays a crucial role in the elimination ofpathogens To regulate complement on self-tissue and to prevent spontaneousactivation and systemic depletion，complement is controlled by both fluidphase andmembra
7、nebound inhibitors One such inhibitor,complement factor I(CFI)regulatescomplement by proteolyfic cleavage of components C3b and C4b in the presence ofspecific cofactors Complement factor H(CFH)，the main cofactor for CFI，regulatesthe alternative pathway ofcomplement activation by acting in the breakd
8、own of C3bto iC3b To gain further insight inlo the regulators of complement activation in bonyfish，zebrafish CFl was cloned and its chariacterizatfon and fonction was studied Theexpression ofCFHfamily genes and CFandtheir regulation by LPS and LTA duringthe ontogeny ofzebrafish embryoslarvae were al
9、so studiedThe full-length of zebrafish CFcDNA sequence is 2384bp and encodes apredicted protein with 731 amino acids The molecular weight of CFI protein is81 4kDa and the pI is 6 17 II shares as high as 35 5-67 jamino acid identities withother CFI of ve rfebrates There are lots of conserved Cys resi
10、dues in the peptideSimilar to mammals CFI，zebraflsh CFI has a leader peptide，FIMAC，SRCR，LDLrl，LDLr2 and SP domains，except that there is a teleost-specific sequence between theleader peptide and the FIMAC domain The mat_Llre CFI protein is a heterodimer witha heavy chain and a light chain The heavy c
11、hain contains FIMAC，SRCR，LDLrl andLDLr2 domains and the light chain only contains the SP domain In the SP domain，His Asp-Set residues comprise the catalytic triad and the conserved Asp residue in thebottom of the catalytic pocket As a solube glycosylated protein，the zebrafish CFIpeptide has 5 Nglyco
12、sylated sites which scatter on the whole peptide and 8O-glycosylated sites which locate on the region between the leader peptide and theFIMAC domain fhe 3D structure ol zebrafish CFl Is quite similar with thai ofhumanindicating that it may perform the same function The phylogenetic trees show thatCF
13、I proteins are highly conserved during c、+olution and the zebrafish CFI is clusteredwith other fish CFI The CFI gene ol zebrafish expressed widely in various tissuesand mainly in ovary lestiz1ive r1 brain intestines and eyes CFHs and CFI havediflerent expression patterns during the ontogeny of zebra
14、fish embryoslarvae andthey respond differently to LPS and LTA challenge We speculate that CFH familygenes play important roles through diflhrenl ways and regulate the complementactivation pathways accurately Whole mount in situ hybridization shows that CFI hasa dynamic expression pattern in early st
15、age,l!expresses mainly抽YSL in early smge,and later on，it specifically expresses in pineal gland and YSL Overexpression ofCFlmRNA shows no visible phenotype，but whether CFI is involved in early developmentstill needs further investigation In vitro expression of the recombinant proteinCFI。28a has been
16、 performed in E coli and the protein we obtained is 64 2 kDa(MW)，this layes a basis for further study ofzebmfish CFI functionKeywords：zebrafish，complement regulation，CFI，CFH缩略语英文全称AcrylamideBisacDlamideAmpcillinalternate pathwayAmmonium persulfate5-Bromo-4-chloro-3一indolyl-phosphateC4 binding proteincomplement factor Hcomplement factor H relatedcomplement factor 1classical pathwayDiethyl pyrocarbonateDimethylformamide；NNDimethylformamideDeoxv曲
