|其他题名: ||Expression regulation and antiviral function of two novel interferon-inducible genes in Crucian Carp (Carassius auratus L.)|
|摘要: ||干扰素( Interferon, IFN )系统作为非特异性免疫系统的重要组成部分，是鱼类乃至所有脊椎动物抵抗病毒等病原体微生物入侵的第一道防线。本实验室已经成功建立了一个研究鱼类IFN系统的细胞模型。鲫囊胚细胞（Crucian carp (Carassius auratus L.) blastulae embryonic cells, CAB）在紫外线灭活的草鱼出血病病毒（Grass Carp Hemorrhage Virus, GCHV）诱导下能产生IFN和一系列抗病毒免疫相关的蛋白，并最终建立起抗病毒状态，抵抗病毒的入侵。Gig1（GCHV induced gene 1）和Gig2都是从这样的体系中克隆并鉴定出来的新的IFN诱导基因。GCHV、Poly I:C和粗制IFN（含有IFN的CAB细胞上清）都能诱导它们mRNA水平的上调表达。本文在此基础上，继续对Gig1和Gig2基因的表达调控和抗病毒功能进行了研究。
Gig1是在筛选灭活GCHV诱导CAB细胞的差减cDNA文库时出现频率最高的一个基因。它功能未知并且没有任何已知结构域。在原核表达系统中成功表达并纯化了Gig1蛋白。利用该蛋白免疫家兔成功获得了Gig1的特异抗体，并首次证实Gig1是能编码蛋白的基因。Real-time PCR和Western blot分析揭示灭活GCHV和Poly I:C能诱导Gig1 mRNA和蛋白的上调表达。细胞免疫化学和Western blot分析表明Gig1是一个胞质定位的蛋白。体内研究表明Gig1在草鱼各组织中遍在分布，Poly I:C能诱导各主要免疫相关组织中Gig1蛋白的上调表达。在CAB或鲤上皮瘤细胞系（Epithelioma papulosum cyprinid, EPC）中，瞬时转染过表达Gig1能抑制GCHV复制并保护细胞。通过搜索公共数据库发现目前Gig1是鱼类所特有的基因。所有研究结果表明Gig1是一个鱼类所特有的具有抗病毒功能的新的干扰素诱导基因。
Gig2是在筛选灭活GCHV诱导CAB细胞的差减cDNA文库时获得的又一个功能未知的基因。在原核表达系统中成功表达并纯化了Gig2蛋白。利用该蛋白免疫家兔成功获得了Gig2的特异抗体，并首次证实Gig2是能编码蛋白的基因。Western blot分析揭示灭活GCHV、Poly I:C和原核表达纯化的IFN蛋白都能诱导Gig2蛋白的上调表达。体内研究表明Gig2在草鱼各组织中遍在分布，Poly I:C能诱导各主要免疫相关组织中Gig2蛋白的上调表达。利用Genome walking的方法成功获得了Gig2上游859bp的启动子序列。进一步的序列分析显示Gig2启动子上有3个ISRE（IFN-stimulated response element）序列，5个GAS（gamma IFN activated sequence）序列，9个GAAA/TTTC结构，一个TATA-box和一个Sp1结合位点。利用启动子－报告基因的方法检测Gig2启动子活性，发现Poly I:C、IFN和IRF7都能诱导Gig2启动子活性的上调。这些研究结果进一步证实Gig2是一个新的IFN诱导基因，它在IFN介导的抗病毒免疫中可能起到重要的作用。此外，在Poly I:C和IFN诱导Gig2的表达中，IRF7可能起着重要的调节作用。
|英文摘要: ||As an important component of innate immune system, interferon (IFN) system is the first line of defense protecting fish and other vertebrates against virus and other microbes. An ideal cell model to research fish IFN system has been established in our lab. UV-inactivated grass carp hemorrhage virus (GCHV) is capable to induce an antiviral state in crucian carp (Carassius auratus L.) blastulae embryonic (CAB) cells as evidenced by production of IFN activity and activation of a number of cellular genes involved in IFN antiviral response. GCHV induced gene 1 (Gig1) and Gig2 are two novel IFN inducible genes cloned and characterized from this system. GCHV, Poly I:C and CAB IFN-containing supernatant can induce the expression of Gig1 and Gig2 at mRNA level. In this study, we further analyzed the expression regulation and antiviral function of Gig1 and Gig2.
Gig1 is the most abundant gene found in our subtractive cDNA library. It is still a protein with no recognizable domains and functions. Gig1 protein was successfully produced in prokaryotic expression system. By immunizing rabbit with this prokaryotic expressed and purified Gig1 protein, the specific anti-Gig1 antiserum was generated, and then used to confirm that Gig1 is a structural gene. Real-time PCR and Western blot analyses shown that UV-inactivated GCHV and Poly I:C could up-regulated the expression of Gig1 both at mRNA and protein levels. Indirect immunofluorescence and Western blot analyses shown that Gig1 mainly localized in the cytoplasm. In grass carp, Gig1 was ubiquitously distributed in all tested tissues with a constitutively weak expression both at mRNA and protein levels, and Poly I:C was able to induce an increased expression of Gig1 in several immune tissues. Over-expressed Gig1 protein in CAB cells and Epithelioma papulosum cyprinid (EPC) cells could inhibit virus replication and protect cells. Through searching public databases, we found Gig1 was a fish specific gene. Above all, Gig1 is a fish specific antiviral protein which can be induced by IFN.
Gig2 is another function un-known gene found in our subtractive cDNA library. Gig2 protein was successfully produced in prokaryotic expression system. By immunizing rabbit with this prokaryotic expressed and purified Gig2 protein, the specific anti-Gig2 antiserum was generated, and then used to confirm that Gig2 is a structural gene. Western blot analysis shown that UV-inactivated GCHV, Poly I:C and prokaryotic expressed IFN protein could induce the expression of Gig2 protein. In grass carp, Gig2 was ubiquitously distributed in all tested tissues both at mRNA and protein levels, and Poly I:C was able to induce an increased expression of Gig2 in several immune tissues. An 859 bp upstream region of Gig2 promoter was amplified by gene walking. Gig2 promoter contains three IFN-stimulated response elements (ISREs), five -IFN activated sites (GASs), 9 GAAA/TTTC motifs, a TATA-box and a Sp1 binding site. Promoter-reporter system was used to analyze the activity of Gig2 promoter. We found Poly I:C, IFN and over-expressed IRF7 could induce the activity of Gig2 promoter. In a word, Gig2 is a novel IFN inducible gene, which may have an important function in cellular antiviral immune response. Besides IRF7 might directly upregulate the expression of Gig2 in the context of Poly I:C induction or IFN induction.
Four Gig2 homologues were obtained when further screening our subtractive cDNA library. The full length cDNA sequences of the four Gig2 homologues were then obtained by RACE-PCR. UV-inactivated GCHV could induce the expression of all four Gig2 homologues, which indicated that they might play an important role in antiviral immune response. Gig2 and Gig2 homologues may be members of a gene family which may play an important role in antiviral immune response.
Taken together, the results generated in the present study will be essential for further clarifying fish IFN mediated signal pathway, fish IFN mediated antiviral mechanism in detail, and promoting researches on the antiviral drugs and development of the antiviral breeding technology in fish.|
|Appears in Collections:||中科院水生所知识产出（2009年前）_学位论文|
|File Name/ File Size