【作者】 李莉；

【导师】 聂品；

【作者基本信息】 华中农业大学 ， 水产养殖， 2008， 博士

【摘要】 C9是补体溶解途径膜攻击复合体的一个成员。穿孔素是主要存在于自然杀伤细胞和细胞毒性T淋巴细胞细胞毒颗粒中的一种糖蛋白,两个分子都可以聚合形成环状结构插入靶细胞膜导致细胞溶解,具有结构和功能的相似性。本研究对草鱼Ctenopharyngodon idellus补体C9(grass carp C,gcC9)和穿孔素(grass carpperforin,gcPFP)基因进行了克隆鉴定和特征分析。使用RACE方法扩增得到gcC9的cDNA全长2123 bp,开放阅读框包含1953bp,编码650个氨基酸,理论分子量71.045 kDa。结构域搜索和多序列比对显示gcC9含有血小板反应素(thrombospondin domain,TSP)、低密度脂蛋白受体(lowdensity lipoprotein receptor class A,LDL-R)、表皮生长因子前体(epidermal growthfactor precursor,EGFP)和与穿孔素相关的MACPF结构域。使用PCR和染色体步移法获得gcC9基因全长7003 bp,包含11个外显子和10个内含子,启动子区域822 bp,含有一个典型的TATA框及C/EBP,HSF,NF-AT,CHOP-C,HNF-3B,GATA-2,IK-2,EVI-1,AP-1,CP2和Oct-1等潜在的转录因子结合位点。RT-PCR和实时荧光定量PCR显示gcC9在草鱼未受精卵中已表达,受精后48 h gcC9表达量达到峰值。使用pQE-30载体对gcC9进行原核表达,获得重组蛋白免疫兔子制备多克隆抗体,Western免疫印迹和RT-PCR显示gcC9在健康草鱼肝脏、肠道、脾脏、头肾、中肾、胸腺、皮肤、肌肉、鳃、心脏、脑和血液中表达,其中肝脏中表达量最高。实时荧光定量PCR分析显示经灭活的柱状黄杆菌Flavobacterium columnare诱导后1 d和7 d gcC9转录分别在肝脏和脾脏中有显著上调;Poly I:C诱导后1 d和3 d,在脾脏、肝脏和头肾中显著上调。使用简并引物扩增得到gcPFP-1和gcPFP-2的两个片段,使用RACE扩增获得gcPFP-1 cDNA全长2514 bp,开放阅读框包含1767 bp,编码588个氨基酸;gcPFP-2 cDNA全长2254 bp,其中开放阅读框1740 bp,编码579个氨基酸。gcPFP-1和gcPFP-2氨基酸相同率为58%,结构预测显示都存在MACPF和C2的PFP的特征结构域。gcPFP-1和gcPFP-2基因全长分别是4090 bp和3059 bp,均由4个外显子和3个内含子组成。RT-PCR显示gcPFP-1和gcPFP-2转录本在健康草鱼被检组织中都有分布,在草鱼早期发育阶段两个基因都在孵化后6 h开始表达,48 h表达量达到峰值,之后表达下降,呈现相同的表达规律。蛋白免疫印迹显示gcPFP-1和gcPFP-2可以交叉识别兔抗gcPFP抗体,草鱼组织中gcPFP-1和gcPFP-2均为68 kDa左右的蛋白,在健康草鱼组织中呈组成型表达。灭活的柱状黄杆菌诱导后7 d,在脾脏和头肾中gcPFP-1和gcPFP-2表达上调;Poly I:C诱导后1 d在脾脏,诱导后3 d在头肾和肠道中检测到gcPFP-1和gcPFP-2表达上调。两种诱导剂作用后3 d,肝脏中gcPFP-1表达上调而gcPFP-2表达下调。GcC9和gcPFP结构相关,氨基酸序列同源性为22%,共同含有MACPF和富含半胱氨酸的EGFP结构域,这些区域具有使蛋白构成跨膜通道和单体多聚化的功能。本研究首次提出鱼类穿孔素存在多种亚型,斑马鱼Danio rerio、黑青斑河豚Tetraodon nigroviridis和三棘刺鱼Gasterosteus aculeatus等鱼类模式生物基因组数据库信息支持该论点,推测穿孔素多态性是硬骨鱼类进化过程中基因组复制的结果。更多还原

【Abstract】 C9 is the ninth member of complement components,creating the membrane attack complex(MAC) in lytic pathway.Perforin is a soluble,pore forming cytolytic glycoprotein synthesized in CTL and NK cells and sequestered into secretory cytotoxic granules.Both molecules have been shown to undergo polymerization to form circular lesions,and they share some structural and functional similarities.In the present study,the complement C9(gcC9) and perforin(gcPFP) genes were cloned in grass carp Ctenopharyngodon idella.GcC9 gene with a full cDNA length of 2123 bp has been cloned using RACE-PCR,which contains an open reading frame(ORF) of 1950 bp coding for 650 aa.Domain search revealed that gcC9 contains a LDL receptor domain,an EGF precursor domain,a MACPF domain and two TSP domain.GcC9 gene consists of 11 exons with 10 introns,spacing over 7003 bp of genomic sequence.Analysis of gcC9 promoter region-of 822 bp revealed the presence of a TATA box and some putative transcription factors such as C/EBP,HSF,NF-AT,CHOP-C,HNF-3B,GATA-2, IK-2,EVI-1,AP-1,CP2 and OCT-1 binding site.By RT-PCR and quantitative real-time PCR analysis,the transcription of gcC9 was detected in eggs,and the peak value occurred at 48 h post fertilization(hpf).Using pQE-30 expression vector, recombinant gcC9 protein was obtained and used to immunize rabbits for generating polyclonal antibody.The distribution of gcC9 transcripts and protein in different organs of healthy grass carp were examined by RT-PCR and Western blotting analysis,respectively. The gcC9 had a constitutive expression in all examined organs,and had a significantly high expression in liver,about 72 kDa was detected in liver,head kidney, renal kidney,spleen,brain,heart,gill,blood,muscle,thymus,skin and intestine.In response to F.columnare injection,the gcC9 mRNA increase was observed in liver at day 1 and in spleen at day 7 post treatment;and in response to Poly I:C treatment,the gcC9 transcription increase was observed in spleen at day 1,in liver.and head kidney at day 3 post treatment.The gcPFP-1 and gcPFP-2 were cloned by using degeneracy primers,the gcPFP-1 with the full cDNA length of 2514 bp which contains an ORF of 1764 bp coding for 588 aa,and the gcPFP-2 full cDNA length is 2254 bp,including a 1737 bp ORF which encodes 579 aa.The amino acid sequence of gcPFP-1 is 58%identical to that of gcPFP-2,and the two gcPFPs all contain MACPF and C2 domain which are found in perforin.The gcPFP-1 and gcPFP-2 all consist of 4 exons with 3 introns, spacing over 4090 bp and 3059bp of genomic sequence,respectively.The distribution of gcPFP-1 and gcPFP-2 transcripts had a similar pattern in all detected organs,the transcription of gcPFPs were detected at 6 hpf during development,with the peak value detected at 48 hpf,followed by decline.The polyclonal antibodies to gcPFP-1 and gcPFP-2 show immunological cross-reactivity.GcPFP-1 and gcPFP-2 are all about 68 kDa,and had a constitutive expression in all examined organs.In response to F.columnare injection,gcPFP-1 and gcPFP-2 mRNA increase was observed in spleen and head kidney at day 7 post treatment;in response to PolyI:C injection,gcPFP-1 and gcPFP-2 transcription increase was observed in spleen at day 1,in intestine and head kidney at day 3,post treatment,gcPFP-1 transcription increase but gcPFP-2 decline was observed in liver at day 3 post two treatment.The gcC9 and gcPFP are structurally related;gcPFP has approximately 20% amino acid identity with gcC9,and they all have the MACPF and EGFP domains. PFP and C9 share two domains that fulfill roles essential to their overall functions, namely those providing for lipid insertion and polymerization.The present study suggested that fish possess multiple isoforms of PFP,and gene duplication events may have given rise to diverse gene isotypes in the PFP family.This suggestion was supported by the genomic information of zebrafish,pufferfish and stickleback.更多还原