Toll样受体3过表达与抑制表达对草鱼呼肠孤病毒致病的影响及机理研究

项目名称： Toll样受体3过表达与抑制表达对草鱼呼肠孤病毒致病的影响及机理研究

项目编号： No.30871917

项目类型： 面上项目

立项/批准年度： 2009

项目学科： 轻工业、手工业

项目作者： 苏建国

作者单位： 中国科学院水生生物研究所

项目金额： 35万元

中文摘要： GCRV为双链RNA病毒。TLR3基因作为识别进入内体中双链RNA的模式识别受体。本研究通过同源克隆及RACE技术获得了草鱼TLR3基因cDNA全长序列。筛选BAC文库获得gDNA序列。草鱼TLR3在肝胰脏中的表达在GCRV感染后显著上调，而对GCRV不感染的鲤鱼TLR3在肝胰脏中的表达被显著抑制。构建过表达载体，转染草鱼肾细胞系(CIK)，GCRV感染，抗病毒能力高于对照，抗病毒效应分子表达量升高；用化学合成的siRNA转染CIK，抗病毒能力低于对照。通过DNA池测序发现了13个多态位点，关联分析发现2个多态位点及1个单体型与草鱼抗GCRV显著相关。通过TLR3基因甲基化检测，发现启动子区甲基化修饰水平与GCRV感染显著相关。TLR22基因是识别胞外双链RNA的模式识别受体。本研究获得了草鱼TLR22的cDNA和gDNA序列及在GCRV刺激下体内外的表达模式，发现了5个多态位点，其中1个多态位点与GCRV抗性显著相关。本研究还发现了识别病毒单链RNA的草鱼TLR7和TLR8对GCRV体内外感染也表现出显著的变化，说明草鱼机体通过TLR基因家族多个成员共同识别GCRV并激发免疫反应。

中文关键词： 草鱼；Toll样受体3；草鱼呼肠孤病毒；基因功能；先天性免疫

英文摘要： GCRV is dsRNA virus. TLR3 recognizes dsRNA as a pattern recognition receptor. Full length cDNA sequence of grass carp TLR3 was obtained by homologous cloning and RACE techniques. gDNA sequence was got by screening BAC library. The mRNA expression of TLR3 was significantly elevated post GCRV infection in hepatopancreas in grass carp, however, the transcripts were significantly down-regulated post challenge in common carp. Over-expression plasmid was transfected into CIK cell line, infected by GCRV. The antiviral ability is stronger than that in control. The expressions of antiviral effector molecules were up-regulated. CIK cells was tranfected by chemical synthetic siRNA. The antiviral ability decreases. 13 SNPs were discovered by DNA pool sequencing. Two SNPs and one haplotype were associated with resistance of grass carp to GCRV by association analysis. Methylation level in the promoter region was significant association with resistance of grass carp to GCRV. TLR22 recognizes extracellular dsRNA as a pattern recognition receptor. cDNA and gDNA sequences of grass carp TLR22 were obtained. The expression profiles were studied post GCRV infection in vitro and in vivo. Five SNPs were detected and one SNP associated with the resistance of grass carp to GCRV. In the present studies, the expressions of grass carp TLR7 and TLR8 recognizing ssRNA were induced after GCRV infection. All together, grass carp recognizes GCRV and triggers immune response through several members of TLR family.

英文关键词： grass carp; Toll-like receptor 3; grass carp reovirus; gene function; innate immunity