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题名: 草鱼出血病病毒基因组研究--病毒核酸纯化,全长cDNA文库构建,序列分析,分子杂交,RT-PCR检测,基因表达,免疫检测
作者: 邱涛
答辩日期: 2001
导师: 朱作言 ; 陆仁后
专业: 遗传学
授予单位: 中国科学院水生生物研究所
授予地点: 中国科学院水生生物研究所
学位: 博士
关键词: 草鱼出血病病毒 ; 基因组 ; cDNA文库 ; 序列分析
摘要: 草鱼出血病病毒(Grass Carp Hemorrhage Virus,GCHV)是一种重要的鱼类病原体,它主要引起草鱼发生出血疾病,曾给我国的淡水水产养殖造成巨大损失。该病毒目前为呼肠孤病毒科的一个尝试成员,隶属水生呼肠孤病毒属,具有与其他呼肠孤病毒科成员类似的物理化学属性以及形态学特征。其颗粒由无包膜的双层衣壳蛋白组成,基因组由11条dsRNA节段(segment)组成,可分为三组,即较大节段(L1,L2,L3)、中等节段(M4,M5,M6)和小节段(S7,S8,S9,S10,S11),根据dsRNA的分子量推算总共有大约25000个碱基对。由于过去几十年里草鱼出血病病毒所带来的巨大危害,抗病研究的进一步开展迫切需要对该病毒基因组有一个深入的了解。不象呼肠孤病毒属的其他成员如哺乳动物呼肠孤病毒以及植物呼肠孤病毒,它们的基因组已得到深入广泛的研究,目前水生呼肠孤病毒的研究资料相当缺少。为了能够揭示出草鱼出血病病毒基因组以及为水生呼肠孤病毒提供进一步的数据,在几乎没有任何序列信息可以参照的情况下,本文通过在dsRNA节段两端加上人工接头,再用相关引物进行单引物扩增的方法构建了草鱼出血病病毒基因组的全长cDNA文库。在此过程中,本文还发展了一种新的有效的技术用于完整地克隆大于3.5kb左右的全长eDNA。作为结果,本文获得了9个基因组节段的完整核苷酸序列(长度从820 bp至3877 bp),以此为基础,草鱼出血病病毒基因组的组成以及蛋白编码情况已大致弄清。该病毒颗粒的双层衣壳可能由7个结构蛋白组成,并且其基因组还可能至少编码5个非结构蛋白。除了S7外,每一个基因节段都拥有一个长的开放阅读框(ORF),编码至少一种病毒蛋白。有趣的是S7节段有三个主要的ORF,这一特征在其他呼肠孤病毒的基因组中也存在。S11是另一个特殊的基因节段,它在一个长ORF里还存在另一个框内起始位点,可能通过两个不同的起始位点和同一个终止位点翻译出两个大小不同的非结构病毒蛋白。本文通过Southern印迹,Northern印迹以及DNA序列分析等途径证实了克隆的草鱼出血病病毒全长eDNA的正确性。为了进一步识别该病毒基因组的编码蛋白。本文将其基因组节段M6、S8、S.10的ORF从其克隆载体上扩增出来,插入pET-28a载体上的克隆位点,在大肠杆菌中进行丁表达。通过SDS一聚丙烯酰胺凝胶电泳对提取的大肠杆菌蛋白质进行分析,得到了预期的融合表达蛋白。M6和S8节段的表达产物被转移到硝酸纤维素膜上,与从鸡蛋黄中提取的抗草鱼出血病病毒免疫球蛋白(IgY)进行杂交,其结果显示该免疫球蛋白能够特异地与融合蛋白发生免疫反应,进一步证实了本文所克隆的病毒基因的正确性。通过对基因组各节段的末端序列分析,本文发现了该病毒特有的末端保守序列5'GUUAUU-CAUC 3’,另外,在末端保守序列附近,各个基因节段又拥有节段特异的倒转重复序列。其3'末端的核苷酸序列CAUC 3'与已知的水生呼肠孤病毒SBRV的第10节段的3'末端一致,并且与一些哺乳动物呼肠孤病毒的3'末端也一致。而5’末端的核苷酸序列5 ' GUUAUU则类似于哺乳动物呼肠孤病毒和wound tumor virus(WTV)的5'末端。这些病毒基因组末端结构特征在病毒对其基因组进行分筛以及装配过程中可能起到重要作用。经序列分析,草鱼出血病病毒基因组编码蛋白的氨基酸序列在相当程度上与哺乳动物呼肠孤病毒基因组的编码蛋白存在相似性,以呼肠孤病毒血清类型3为例,基因节段间的相似率最高达到40%左右,显示了草鱼出血病病毒与哺乳动物呼肠孤病毒之间非常近的亲源关系。另外,本文发现了五个草鱼出血病病毒缺损性干扰颗粒(defective interfering,DI)的dsRNA组分,Northern印迹显示它们在基因组中确实存在。这些DI RNA是由于病毒基因组的复制错误而产生的病毒亚基因组成分,它们被错误地包装入病毒颗粒,能够对病毒的生理过程产生影响。经研究分析,在五个DI RNA中,有两个都是包含了母节段的一个内部衰减,这属于典型的DIRNA结构;另外有两个都是缺失了母节段的5'端的区域;而第五个是其两个母节段的3'端序列被剪切后拼接而成。这是目前首例水生呼肠孤病毒缺损性干扰颗粒基因组成分的报道,对它们的研究分析将有助于对病毒基因组的复制与包装信号的识别。DI RNAs的存在也可能是GCHV 873株对草鱼致病能力低的原因。
英文摘要: Grass carp hemorrhage virus (GCHV), an important fish pathogen involved in infecting grass carp Ctenopharyngodon idellus and causing hemorrhagic disease, has caused large losses in fresh water fish culture in China. The virus belongs to the genus Aquareovirus, as a tentative member, and shares the physicochemical properties and morphological characteristics of the family Reoviridae. The GCHV possesses a double-stranded dsRNA genome consisting of 11 segmentspackaged into an non-enveloped icosahedral double capsid. The genome segments are approximately 25,000 nucleotide pairs in total size deduced by the dsRNA molecular weight and separated into three size classes: large (L1, L2, L3), medium (M4, MS, M6) and small (S7, S8, S9, S10, S11). Since the great hazard has been easued by the virus to the aquaculture in our country in the past decades, current antiviral research is crying for the available and detailed genome information on the ViruS. Unlike those of mammalian and plant reoviruses in the family Reoviridae, of which the genomes have been extensively studied, little information is available on the molecular characteristics of the genomes of members of Aquareovirus. In order to obtain the further information on GCHV genome and give available data on Aquareovirus, without any prior sequence information, a full-length eDNA library of GCHV was constructed by ligating adaptors to both ends of the dsRNA genome segments and amplifying with single primer. In the process, an effective approach to completely clone the cDNAs larger than 3.5kb was developed. As a result, nine complete viral genome segments were completely determined (length from 820 bp to 3877 bp). The coding assignments and properties of the proteins encoded in each of the genome segments are now roughly clarified. The virons were predicted to consist of seven structural proteins. The genome encodes at least five nonstructural proteins. Each gene segment of GCHV has one long ORF and codes for the synthesis of at least one protein, except for S7. The S7 segment, interestingly, contains at least three ORFs, of which the feature was also found in other viruses in Reoviridae. The S11, another special segment that has one long ORF but predicted to have another in-frame initiation site, may encode two nonstructural proteins with two distinct initiation sites and the same terminal site. The clones containing full-length cDNAs were confirmed by Southern blotting, Northern blotting, DNA sequencing and RT-PCR assay. In order to clarify the identity of proteins encoded by GCHV genome segments, the ORFs of M6, S8, S 10 were completely amplified from the pGEM-T vector and inserted into the cloning sites of pET-28a vector and expressed in Escherichia coli. The expressed fuse-proteins were identified from the extracted E. coli proteins that resolved by SDS-PAGE as expection. The expressed products of M6 and S8 were transferred to NC membrane, and probed with anti-GCHV immunoglobulin (IgY) that were isolated fiom the yolks of eggs of GCHV-immunized hen. The results exhibited that the anti-GCHV IgY reacted specifically with the protein expressed by these viral genome segments, verifying that the viral genes cloned here are correct. 3'-terminus of each genome segment of GCt-W possesses a conserved terminal sequence 5' GUUAUU CAUC 3', which is special for the virus. In addition, segment-special inverted-repeat sequences were identified adjacent to the conserved terminal nucleotide sequences. The 3' terminal sequence CAUC 3' which is same as that of the striped bass reovirus (SBRV), another member of aquareovirus, and some mammalian reovirus (MRV) species. 5'-terminus 5' GUUAUU is analogous to that of some MRV species and wound tumor virus (WTV). These characters of terminal sequences of dsRNA genome segments may be important in sorting and packing functions of the virus. The deduced amino acid sequences from genome segment sequences of GCHV were found, in some degree, analogous to those of the MRV. Using reovirus serotype III as an example, the identities between deduced proteins of the GCHV and those of MRV is up to 40%, indicating the closer relationship between GCHV and MRV. Furthermore, five defective interfering (DI) dsRNAs of GCHV, which are subgenomic RNAs generated from infectious virus genomes by replicase error, were obtained from the sequence analysis and confirmed by Northern blotting. They are packaged into virus particles capable of interfering the growth of infectious viruses. In sequence analysis, of the five DI RNAs, two that contain a single internal deletion of their parent segments belong to typical DI RNAs; other two lack the 5' terminal regions of the parent segments; The fifth DI RNA consists of 3' termini of its parents: M6 and S10 genome segment. Analysis on DI RNAs will be helpful for idemifying the control signals for replication and packaging of viral genome. The existing of DI RNAs may be the cause of the weakness of pathogenic ability of GCHV-873.
语种: 中文
内容类型: 学位论文
URI标识: http://ir.ihb.ac.cn/handle/342005/12602
Appears in Collections:中科院水生所知识产出(2009年前)_学位论文

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草鱼出血病病毒基因组研究--病毒核酸纯化,全长cDNA文库构建,序列分析,分子杂交,RT-PCR检测,基因表达,免疫检测001.pdf(9365KB)----暂不开放-- 联系获取全文

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草鱼出血病病毒基因组研究--病毒核酸纯化,全长cDNA文库构建,序列分析,分子杂交,RT-PCR检测,基因表达,免疫检测.邱涛[d].中国科学院水生生物研究所,2001.20-25
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