Li L, Chang M X, Nie P
College of Fisheries, Huazhong Agricultural University, Wuhan, Hubei Province 430070, China.
Vet Immunol Immunopathol. 2007 Aug 15;118(3-4):270-82. doi: 10.1016/j.vetimm.2007.05.005. Epub 2007 Jun 3.
Complement-mediated killing of pathogens through lytic pathway is an important effector mechanism of innate immune response. C9 is the ninth member of complement components, creating the membrane attack complex (MAC). In the present study, a putative cDNA sequence encoding the 650 amino acids of C9 and its genomic organization were identified in grass carp Ctenopharyngodon idella. The deduced amino acid sequence of grass carp C9 (gcC9) showed 48% and 38.5% identity to Japanese flounder and human C9, respectively. Domain search revealed that gcC9 contains a LDL receptor domain, an EGF precursor domain, a MACPF domain and two TSP domain located in the N-terminal and C-terminal, respectively. Phylogenetic analysis demonstrated that gcC9 is clustered in a same clade with Japanese flounder, pufferfish and rainbow trout C9. The gcC9 gene consists of 11 exons with 10 introns, spacing over approximately 7 kb of genomic sequence. Analysis of gcC9 promoter region revealed the presence of a TATA box and some putative transcription factor such as C/EBP, HSF, NF-AT, CHOP-C, HNF-3B, GATA-2, IK-2, EVI-1, AP-1, CP2 and OCT-1 binding sites. The first intron region contains C/EBPb, HFH-1 and Oct-1 binding sites. RT-PCR and Western blotting analysis demonstrated that the mRNA and protein of gcC9 gene have similar expression patterns, being constitutively expressed in all organs examined of healthy fish, with the highest level in hepatopancreas. By real-time quantitative RT-PCR analysis, gcC9 transcripts were significantly up-regulated in head kidney, spleen, hepatopancreas and down-regulated in intestine from inactivated fish bacterial pathogen Flavobacterium columnare-stimulated fish, demonstrating the role of C9 in immune response.
补体通过溶解途径介导对病原体的杀伤是先天免疫反应的重要效应机制。C9是补体成分中的第九个成员，可形成膜攻击复合物（MAC）。在本研究中，在草鱼（Ctenopharyngodon idella）中鉴定出了一个推定的编码650个氨基酸的C9 cDNA序列及其基因组结构。推导的草鱼C9（gcC9）氨基酸序列与牙鲆和人C9的同一性分别为48%和38.5%。结构域搜索显示，gcC9包含一个低密度脂蛋白受体结构域、一个表皮生长因子前体结构域、一个膜攻击复合物穿孔素（MACPF）结构域以及分别位于N端和C端的两个血小板反应蛋白（TSP）结构域。系统发育分析表明，gcC9与牙鲆、河豚和虹鳟C9聚集在同一个进化枝中。gcC9基因由11个外显子和10个内含子组成，跨越约7 kb的基因组序列。对gcC9启动子区域的分析揭示了一个TATA盒以及一些推定的转录因子结合位点，如C/EBP、HSF、NF-AT、CHOP-C、HNF-3B、GATA-2、IK-2、EVI-1、AP-1、CP2和OCT-1结合位点。第一个内含子区域包含C/EBPb、HFH-1和Oct-1结合位点。逆转录聚合酶链反应（RT-PCR）和蛋白质免疫印迹分析表明，gcC9基因的mRNA和蛋白质具有相似的表达模式，在健康鱼的所有检测器官中组成性表达，在肝胰脏中表达水平最高。通过实时定量RT-PCR分析，gcC9转录本在柱状黄杆菌刺激的失活鱼的头肾、脾脏、肝胰脏中显著上调，在肠道中下调，表明C9在免疫反应中的作用。
