鱼类干扰素系统基因的克隆鉴定及其特征分析(英文)

Cloning, Identification and Characterization of Interferon System Genes in Crucian Carp (Carassius auratus L.)

干扰素 (IFN)系统是脊椎动物抵抗病毒入侵的第一道防御系统 .除了哺乳类 ,有关低等脊椎动物的IFN系统基因知之甚少 .在鱼类 ,近 40年来能证明IFN系统存在的证据主要有两个 :一是检测经病毒诱导后的多种鱼类机体和细胞 ,证明能产生类似哺乳类IFN的抗病毒活性物质 ;二是近年来 ,已经证明在少数几种鱼类中存在与哺乳类IFN系统基因Mx同源的基因 .以前的研究结果表明 ,紫外线灭活的草鱼出血病病毒 (GCHV)能够诱导鲤科鱼类培养细胞 ,如鲫鱼囊胚细胞 (CAB)产生类IFN活性物质 ,并建立宿主细胞的抗病毒状态 .为了揭示鱼类培养细胞抗病毒免疫的分子机制 ,首先建立了一个研究鱼类抗病毒免疫相关基因的细胞模型系统 ,通过用灭活GCHV诱导CABIFN并进行理化、生物学特性鉴定的基础上 ,成功建立了一个囊括鱼类细胞抗病毒基因在内的差减cDNA文库 .其次 ,筛选文库揭示了一批与哺乳类IFN系统基因同源以及找不到同源性的EST ,表达分析证实它们也是IFN刺激基因 .再次 ,根据哺乳类IFN系统研究的最新进展 ,从该细胞模型系统中克隆、鉴定了 1 9个IFN系统基因的全长cDNA序列 ,包括鲫鱼IFN基因 ,IFN信号传导通路基因STAT1 ,IFN诱导表达调控基因IRF7,IFN行使抗病毒作用的效应基因Mx1、Mx2、PKR、Viperin、IFI5 6。

Interferon (IFN) system is the first line of defense against virus invasion in vertebrate. So far relative little was known concerning IFN system genes other than in mammals. In fish, nearly 40 year evidence available for existence of IFN system mainly lies in the observation that fish and fish cells are much earlier known to produce molecules with IFN like activity as measured by a cell protection test, and that IFN inducible gene, Mx homologue, has been identified in a limited number of fish species in the latest years. Previously we reported that UV inactivated GCHV was able to induce high level of IFN like molecule for establishment of antiviral state in cyprinid fish cell lines, such as crucian carp (Carassius auratus L.) blastulae embryonic (CAB) cells. In the current study, in order to better understand innate immune mechanisms of fish cells responsive to virus infection, an ideal cell model system for identifying fish antiviral relevant genes was firstly established, and on the basis of induction and characterization of CAB IFN, a subtractive cDNA library accumulating an enhanced mRNA level of fish antiviral relevant genes was also constructed. Secondly, a subset of fish IFN system genes and some unidentified ESTs that were found no similarity by BLASTX were retrieved by screening of the library, and expression analysis demonstrated that they were IFN stimulated genes (ISGs) as well. Thirdly, according to the latest research of mammals, 19 full length cDNAs of fish IFN system genes were cloned, including crucian carp IFN, IFN signal transduction factor STAT1, IFN regulatory factor IRF7, putative IFN antiviral effectors Mx1, Mx2, PKR, Viperin, IFI56, and some ISGs with function unknown, including IFI58, ISG15 1, ISG15 2, Gig1, Gig2 and so on. Finally, further studies on the characterization of structure, expression, induction and evolution of some important relevant genes reveal that several mechanisms in fish innate immune following virus infection, including signal recognition, signal transduction and regulation, antiviral pathways, were similar to those in mammals.