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题名: 虹彩病毒三个免疫逃避基因的克隆与特征分析
作者: 黄友华
答辩日期: 2007-06-15
导师: 张奇亚
授予单位: 中国科学院水生生物研究所
授予地点: 水生生物研究所
学位: 博士
关键词: 虹彩病毒 ; 免疫逃避 ; G蛋白耦联受体 ; Bcl-2 ; 肿瘤坏死因子受体 ; 凋亡
其他题名: Cloning and characterization of three immune evasion genes from iridovirus
摘要: 淋巴囊肿病毒中国分离株(Lymphocystis disease virus isolated from China, LCDV-C)和蛙虹彩病毒(Rana grylio virus, RGV)是我国分离到的水产动物重要病毒病原,分别来自患病牙鲆和美国青蛙。在实验室对LCDV-C全基因组进行测定和序列注释以及对RGV理化性质鉴定的基础上,本文对LCDV-C 三个免疫逃避蛋白基因进行了克隆和功能检测,并对RGV诱导凋亡的机制进行了研究,具体结果如下: 1.通过同源性搜索表明:LCDV-C 编码三个免疫逃避蛋白基因同源框:G蛋白耦联受体(G protein coupled receptor,GPCR)(ORF124R)、肿瘤坏死因子受体(tumor necrosis factor receptor,TNFR)(ORF095R)以及一个Bcl-2基因(ORF189R)。并对以上三个基因进行特征分析。 (1)利用跨膜分析显示,LCDV-C GPCR编码一个具有典型七次跨膜结构域的受体蛋白,并有一个高度保守的DRY模体。通过氨基酸同源性搜索显示LCDV-C GPCR和牙鲆编码的趋化因子受体CCR9具有高达37%的同源性,比哺乳动物以及哺乳动物病毒编码的趋化因子受体同源性略高。以LCDV-C基因组为模板,设计特异引物扩增得到LCDV-C GPCR全长,构建融合蛋白GPCR-GFP和GPCR-pcDNA3.1,在细胞水平进行了亚细胞定位和功能的检测。结果表明:LCDV-C GPCR在鱼类细胞EPC和FHM中表达具有泛细胞分布的特征。瞬时过量表达对鱼类细胞没有明显的毒性影响。稳定表达LCDV-C GPCR的FHM细胞在正常的细胞培养条件下,能够促进细胞的增殖;在软琼脂培养条件下,LCDV-C GPCR表达的细胞具有失去定着依赖性生长的典型特征。对LCDV-C GPCR表达的FHM细胞进行不同浓度的放线菌酮(CHX)和放线菌素D(ActD)诱导,结果显示LCDV-C GPCR表达细胞在ActD诱导之后的凋亡小体数目比对照细胞要少;且流式细胞仪检测证实LCDV-C GPCR的表达能够明显抑制ActD和CHX诱导的凋亡。LCDV是一种能够长期潜伏感染鱼类、引起肿瘤发生的病毒,因此可推测,GPCR抑制凋亡、转化细胞是LCDV-C致肿瘤的一种重要途径。 (2)经计算机辅助分析表明,LCDV-C Bcl-2和斑马鱼Mcl的同源性达到34%,并且C端具有一个跨膜区域。通过构建LCDV-C Bcl-2的GFP 融合蛋白,进行显微观察表明,LCDV-C Bcl-2和内质网完全共定位,而其跨膜缺失突变体的定位则发生了变化,揭示跨膜区对于LCDV-C Bcl-2的内质网定位是必需的。细胞毒性检测试验表明,LCDV-C Bcl-2的瞬时表达不诱导FHM细胞出现凋亡,但是其稳定表达能促进ActD诱导的凋亡,而跨膜区的缺失对促进凋亡作用有一定的影响。此外,LCDV-C Bcl-2过量表达还能抑制ActD诱导凋亡过程中核转录因子NF-κB的激活。 (3)对LCDV-C TNFR的结构域分析表明,其N端是肿瘤坏死因子家族所含有的半胱氨酸富集区。LCDV-C TNFR和淋巴囊肿病毒欧洲株LCDV-1、LCDV-2编码TNFR类似物的同源性都低于20%,显示虹彩病毒不同分离株之间基因结构存在差异。通过GFP融合蛋白的亚细胞定位结果显示,LCDV-C TNFR主要分布在内质网,与已知病毒编码TNFR类似物的表达定位明显不同。体外细胞毒性试验表明,LCDV-C TNFR的过量表达不诱导鱼类细胞出现凋亡,但是能促进ActD和CHX诱导的凋亡,并能抑制ActD诱导凋亡过程中转录因子NF-κB的激活。 2.利用电镜及荧光显微镜观察、报告基因检测、细胞内钙离子浓度变化以及caspase活性测定等方法,研究RGV感染引起细胞凋亡的机制。结果表明RGV感染能够引起FHM细胞出现线粒体的片断化、caspase-9的激活、核转录因子NF-κB和AP-1的激活以及细胞质内钙离子浓度升高等变化。结合对RGV感染能够引起线粒体的细胞分布发生变化以及线粒体膜电势降低的认识,提出RGV诱导的凋亡是线粒体介导的观点,并绘制出RGV诱导细胞凋亡的模式图。 本文的研究结果不仅为在分子水平上研究虹彩病毒的免疫逃避以及病毒与宿主细胞的相互作用积累重要信息,而且为进一步了解虹彩病毒的致病机理奠定坚实的基础。
英文摘要: Lymphocystis disease virus isolated from China (LCDV-C) and Rana grylio virus (RGV) were important virus agents isolated from the diseased flounder and Rana grylio in China, respectively. Based on the sequencing and annotation of the LCDV-C genome, and the studies on the morphogenesis and physicochemical characterizations of RGV, we cloned and detected the functions of three immune evasion genes from LCDV-C, and investigated the mechanism of RGV induced apoptosis in fish cells. The results are given as follows: 1. The reannotation of open reading frame (ORF) from LCDV-C indicated that at least three immune evasion gene homologs were present in LCDV-C, including G protein coupled receptor (GPCR) (ORF124R), tumor necrosis factor receptor (TNFR) (ORF095R) and Bcl-2 (ORF189R). The characterization of these genes was described as following. (1). The transmembrane prediction of LCDV-C GPCR indicated that it had a central core domain constituted of seven transmembrane regions and a highly conserved DRY motif. The homology search showed that LCDV-C GPCR shared 37% identity to flounder chemokine receptor 9 (CCR9), following by rainbow trout CXCR, zebrafish CXCR4 and other mammal virus. Full length of LCDV-C GPCR was obtained by PCR amplification using LCDV-C DNA as template. The recombinant plasmid GPCR-GFP and pcDNA-GPCR were constructed and confirmed by sequencing, and the subcellular localizatization and function in fish cells were detected. The results showed that: LCDV-C GPCR was expressed in both the cytoplasm and nucleoplasm of fish cells and independent of the cell type; No obvious cytotoxic effect was examined after transient overexpression of LCDV-C GPCR in fish cells; Stabled expressed LCDV-C GPCR not only increased the cell proliferation, but also had the transformation potential in FHM cells. In addition, LCDV-C GPCR overexpression also inhibited apoptosis under external apoptotic stimuli, such as CHX and ActD. Considering Lymphocystis is a chronic disease characterized by wart-like lesions, the transforming potential of LCDV-C GPCR, which maybe functioned via inhibiting apoptosis, was purposed to contribute to the massive enlargement of epidermal cells and viral persistent infection. (2). The computer assisted analysis on LCDV-C Bcl-2 showed that it shared 34% identity to zebrafish Mcl and had a transmembrane domain at C terminal. The detection of subcellular localization of LCDV-C Bcl-2 by using GFP fusion protein showed that LCDV-C colocalised to endoplasmic reticulum (ER), while its transmembrane mutant had a changed localization. These data indicated that the transmembrane domain is important for LCDV-C Bcl-2 localization. The transient expression of LCDV-C Bcl-2 had no obvious cytotoxic effect on fish cells, but the overexpression of LCDV-C Bcl-2 increased the apoptosis induced by ActD and the deletion of transmembrane domain influenced the ability of enhancing apoptosis. In addition, LCDV-C Bcl-2 expression inhibited the NF-κB activation induced by ActD. (3). The domain analysis of LCDV-C TNFR showed that it had the conserved cysteine rich domain which is a typical chacterization of TNFR family members. Although it shared 38% identity to mouse TNFR2, the identity between LCDV-C TNFR and LCDV-1 or LCDV-2 TNFR was less than 20%. It is indicated that the difference of gene structure existed between various iridovirus isolates. The examination of LCDV-C TNFR localization showed that LCDV-C TNFR was also colocalized to ER, this data is different from the subcellular localization of other viral TNFRs. Transient expression LCDV-TNFR could not induce apoptosis in fish cells, but the stably overexpression of LCDV-C TNFR enhanced the apoptosis induced by ActD and CHX, and inhibited the ActD activated NF-κB. 2. Using electron micrscopy and fluorescent microscopy observation, reporter gene assay, examination on the intracellular Ca2+ and caspase activity, we investigate the mechanism of RGV induced apoptosis in fish cells. The data showed that RGV infection induced a series of changes, such as mitochondrial fragmentation, caspase activation, NF-κB and AP-1 activation and intracellular Ca2+ increase. Combining the previous results that RGV infection altered the mitochondrial distribution and the mitochondrial membrane potential (Δψm) collapse, we purposed that RGV infection should trigger mitochondrion mediated apoptosis. Taken together, the results not only provided important clues for the studies on iridovirus immune evasion and the virus-host interaction at molecular level, but also contributed to understanding the iridovirus pathogenesis.
语种: 中文
内容类型: 学位论文
URI标识: http://ir.ihb.ac.cn/handle/342005/12138
Appears in Collections:中科院水生所知识产出(2009年前)_学位论文

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Recommended Citation:
虹彩病毒三个免疫逃避基因的克隆与特征分析.黄友华[d].中国科学院水生生物研究所,2007.20-25
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