草鱼呼肠孤病毒RNA聚合酶基因的表达与产物纯化

草鱼呼肠孤病毒是引起草鱼出血病的主要病原,隶属于呼肠孤病毒科水生呼肠孤病毒属。序列分析表明,GCRVS2片段长为3877核苷酸,编码一个分子量为138kDa的蛋白VP2,具有RNA聚合酶性质。为进一步了解该病毒RNA聚合酶特性,本研究在对GCRV RNA聚合酶基因(GCRV—RdRp)保守区(约1．5kb)重组质粒pR／RRp高效表达的基础上,分别构建了编码GCRV RNA聚合酶保守区N端与C端部分基因的pR／RRpN及pR／RRpC重组表达载体,并在原核细胞中获得成功表达。筛选的重组表达菌株经IPTG诱导培养,得到分子量分别为98kDa、103kDa的目的表达融合蛋白。Western blot分析表明,该表达产物与兔抗GCRV—VP2血清呈阳性反应。通过ProBond柱亲和层析,纯化了融合有6个组氨酸的重组表达产物,并获得约90％纯的目的蛋白。上述结果为GCRV RNA聚合酶特性分析提供了依据。     

草鱼呼肠孤病毒RNA聚合酶基因功能区在原核细胞中的表达

草鱼呼肠孤病毒(grass carp reovirus)为我国分离、鉴定的第一株水生动物病毒.1983年,我国首次报道引起爆发性草鱼出血病的病原为草鱼出血病病毒[1,2],其后相继进行了系统的病毒形态学、生物学、生物化学及分子生物学特性等研究[3-8].自1979年Meyers T R等报道从水生动物中分离出第一株呼肠孤样病毒,迄今国际上已分离鉴定40余种水生呼肠孤病毒(aquareovirus).在这些分离株中,大多数毒株不能引起寄主的病理反应或仅表现出较弱的致病性.然而研究认为,GCRV为水生呼肠孤病毒中致病力最强的毒株[9].可见,以GCRV为模型,研究水生呼肠孤病毒的复制与致病机理具有一定的理论及实际意义.我们在对GCRV反应核心及体外转录研究中,已证实GCRV RNA聚合酶在病毒粒子中的存在及其位置[5];GCRV序列测定及定位结果显示,GCRV-VP2多肽为该病毒RNA聚合酶(RNA dependent RNA polymerase RdRp)[6,7].为了探讨草鱼呼肠孤病毒的侵染与宿主的相关性及复制机制,我们首次进行了该病毒RNA聚合酶基因(GCRV-RdRp)功能区序列在原核细胞中的表达研究,并得到高效表达融合蛋白.这一结果将为该酶的活性及特性分析提供实验依据.下面报道本研究结果.     

草鱼呼肠孤病毒（GCRV）部分基因片段cDNA文库的构建

在随机六聚体引物浓度不变条件下 ,分别采用 0 .5、2、5μg草鱼呼肠孤病毒 (GCRV)单一片段RNA进行反转录cDNA合成及文库构建。结果表明 ,在GCRV RNA模板浓度大于 2 μg时 ,第二链cDNA合成产物可直接通过EB染色的琼脂糖凝胶电泳进行定量及鉴定。选择cDNA与载体连接比率为 5∶1,在高效电转化条件下 ,可获得 10 6 转化子 /μgcDNA的转化效率。阳性克隆子经酶切及PCR鉴定 ,约 4 5%以上的插入片段大于 50 0bp。     

草鱼呼肠孤病毒(GCRV)部分基因片段cDNA文库的构建

在随机六聚体引物浓度不变条件下,分别采用0.5、2、5μg 草鱼呼肠孤病毒(GCRV)单一片段RNA进行反转录cDNA合成及文库构建。结果表明,在GCRV-R NA模板浓度大于2μg时,第二链cDNA合成产物可直接通过EB染色的琼脂糖凝胶电泳进行定量及鉴定。选择cDNA与载体连接比率为5∶1,在高效电转化条件下,可获得10     

水生呼肠孤病毒研究进展

水生呼肠孤病毒为感染水生生物的一类呼肠孤病毒.自Meyers等1979年首次报道水生呼肠孤病毒的分离[1],迄今已分离鉴定出40余株水生呼肠孤病毒[2].     

水生呼肠孤病毒研究进展

水生呼肠孤病毒为感染水生生物的一类呼肠孤病毒。自Meyers等1979年首次报道水生呼肠孤病毒的分离,迄今已分离鉴定出40余株水生呼肠孤病毒。与哺乳动物呼肠孤病毒一样,水生呼肠孤病毒主要通过呼吸肠道感染宿主,导致出血病及肝脏与胰腺疾病,在全球范围内对水产养殖业造成了严     

两株水生呼肠孤病毒部分特性的比较

水生呼肠孤病毒为感染水生动物的一类病原体,隶属于呼肠孤病毒科新建水生呼肠孤病毒属。草鱼呼肠孤病毒(Grass carp reovirus,GCRV)是引起中国南方淡水养殖草鱼暴发性出血病病原,鲅鱼呼肠孤病毒(Threadfin reovirus,TFV)是引起海水养殖鲅鱼病毒病病原。本研究将GCRV与新加坡TFV分离株进行了部分特性比较研究。结果表明,GCRV与TFV均能感染CIK细胞,但对其它鱼类细胞系的敏感性有所差异。此外,凝胶电泳与逆转录聚合酶链式扩增显示,GCRV与TFV核酸属不同的基因型。在多肽特性上,证实了GCRV的5条主要结构多肽具有与。FTV及水生呼肠孤病毒相似的特性。Westem blot检测显示,草鱼呼肠孤病毒与TFV结构蛋白拥有部分相同的抗原决定簇。     

一株中华绒螯蟹呼肠孤病毒RNA1 cDNA文库构建及其RNA聚合酶基因部分序列分析

在中华绒螯蟹体内分离到一株呼肠孤病毒(命名为EsRV905株).采用Trizol试剂提取病毒核酸,经聚丙烯酰胺凝胶电泳,碎胶法回收基因组各节段.随机引物法合成第一节段的cDNA文库,胶回收试剂盒去除小片段,平端连接于载体,化学转化,利用蓝白斑筛选阳性克隆子,酶切鉴定重组质粒.从基因组第一节段的重组质粒中选择2个插入片段约为1.5kb的质粒测序,结果得到包括RNA聚合酶主要特征性结构的一段序列.结果说明,这株蟹呼肠孤病毒的RNA聚合酶定位于基因组第一节段.     

草鱼鳃介导草鱼呼肠孤病毒免疫应答

采用草鱼呼肠孤病毒腹腔注射草鱼, 通过定量RT-PCR检测了12个抗病毒免疫相关基因在鳃中不同时间点的表达模式, 以了解鳃对内源性病毒的免疫应答。模式识别受体基因CiTLR3、CiTLR7、CiTLR22、CiRIG-I、CiMDA5、CiLGP2、CiNOD1和CiNOD2, 以及干扰素基因CiIFN-I的表达在注射病毒后12h、24h、48h及72h基本都上调。IgM基因的表达仅在72h上调。接头分子CiMyD88和CiIPS-1基因的表达在早期下调（6h）, 然后逐渐上升。为了证实病毒感染的可靠性, 通过RT-PCR检测了病毒VP4基因。结果表明草鱼鳃在抗病毒免疫方面发挥着重要作用。       

鱼呼肠孤病毒诱导草鱼肾细胞凋亡

采用荧光显微镜、电子显微镜、琼脂糖凝胶电泳、流式细胞仪分析等技术研究鱼呼肠孤病毒诱导草鱼细胞（ＣＩＫ）调亡。结果显示,鱼呼肠孤病毒感染ＣＩＫ细胞后,光镜下可见空斑形成;荧光染色观察到细胞调亡碎裂核;且电镜下呈现细胞核裂解,核周裂隙增大,细胞膜内陷并出泡形成调亡小体现象;琼脂糖凝胶电泳出现１８０－２００ｂｐ整数倍的ＤＮＡ梯形带;流式细胞仪检测到典型的细胞调亡峰,在病毒感染４８ｈ,细胞调亡百分率达１５     

Suppression of RNA interference pathway <Emphasis Type=Italic>in vitro</Emphasis> by Grass carp reovirus

The means of survival of genomic dsRNA of reoviruses from dsRNA-triggered and Dicer-initiated RNAi pathway remains to be defined.The present study aimed to investigate the effect of Grass carp reovirus...     

High level expression of grass carp reovirus VP7 protein in prokaryotic cells

Sequences analysis revealed Grass carp reovirus (GCRV) s10 was 909 nucleotides coding a 34 kDa protein denoted as VP7, which was determined to be a viral outer capsid protein (OCP). To obtain expressed OCP in vitro, a full length VP7 gene was produced by RT-PCR amplification, and the amplified fragment was cloned into T7 promoted prokaryotic expression vector pRSET. The recombinant plasmid,which was named as pR/GCRV-VP7,was then transformed into E.coli BL21 host cells. The data indicated that the expressed recombinant was in frame with the N-terminal fusion peptide. The over-expressed fusion protein was produced by inducing with IPTG, and its molecular weight was about 37kDa, which was consistent with its predicted size. In addition, the fusion protein was produced in the form of the inclusion body with their yield remaining steady at more than 60% of total bacterial protein. Moreover,the expressed protein was able to bind immunologically to anti-his-tag monoclonal antibody (mouse) and anti-GCRV serum (rabbit). This work provides a research basis for further structure and function studies of GCRV during entry into cells.     

Molecular characterization of nonstructural protein NS38 of grass carp reovirus

Viral nonstructural proteins in both enveloped and non-enveloped viruses play important roles in viral replication. Protein NS38 of Grass carp reovirus (GCRV), has been deduced to be a non-structural protein, and, consistent with other reoviruses, is considered to cooperate with the NS80 protein in viral particle assembly. To investigate the molecular basis of the role of NS38, a complete protein was expressed in E.coli for the first time. It was found that there is a better expression of NS38 induced with IPTG at 28 ℃ rather than 37 ℃. In addition, the antiserum of NS38 prepared with purified fusion protein and injected into rabbit could be used for detecting NS38 protein expression in GCRV infected cell lysate, while there is not any reaction crossed with purified virus particle, confirming NS38 is not a component of the viral structural protein. The result reported in this study will provide evidence for further viral protein-protein and protein-RNA interaction in dsRNA viruses replication.     

Expression and identification of inclusion forming-related domain of NS80 nonstructural protein of grass carp reovirus

Grass carp reovirus (GCRV), a double stranded RNA virus that infects aquatic animals, often with disastrous effects, belongs to the genus Aquareovirus and family Reoviridea. Similar to other reoviruses, genome replication of GCRV in infected cells occurs in cytoplasmic inclusion bodies, also called viral factories. Sequences analysis revealed the nonstructural protein NS80, encoded by GCRV segment 4, has a high similarity with uNS in MRV(Mammalian orthoreoviruses), which may be associated with viral factory formation. To understand the function of the uNS80 protein in virus replication, the initial expression and identification of the immunogenicity of the GCRV NS80 protein inclusion forming-related region (335.742) was investigated in this study. It is shown that the over-expressed fusion protein was produced by inducing with IPTG at 28oC. In addition, serum specific rabbit antibody was obtained by using super purified recombinant NS80(335.742) protein as antigen. Moreover, the expressed protein was able to bind to anti-his-tag monoclonal antibody (mouse) and NS80(335-742) specific rabbit antibody. Further western blot analysis indicates that the antiserum could detect NS80 or NS80C protein expression in GCRV infected cells. This data provides a foundation for further investigation of the role of NS80 in viral inclusion formation and virion assembly.     

High Level Expression of Grass Carp Reovirus VP7 Protein in Prokaryotic Cells

Sequences analysis revealed Grass carp reovirus （GCRV） s10 was 909 nucleotides coding a 34 kDa protein denoted as VP7, which was determined to be a viral outer capsid protein （OCP）. To obtain expressed OCP in vitro, a full length VP7 gene was produced by RT-PCR amplification, and the amplified fragment was cloned into T7 promoted prokaryotic expression vector pRSET. The recombinant plasmid, which was named as pR/GCRV-VP7, was then transformed into E.coli BL21 host cells. The data indicated that the expressed recombinant was in frame with the N-terminal fusion peptide. The over-expressed fusion protein was produced by inducing with IPTG, and its molecular weight was about 37kDa, which was consistent with its predicted size. In addition, the fusion protein was produced in the form of the inclusion body with their yield remaining steady at more than 60% of total bacterial protein. Moreover, the expressed protein was able to bind immunologically to anti-his-tag monoclonal antibody （mouse） and anti-GCRV serum （rabbit）. This work provides a research basis for further structure and function studies of GCRV during entry into cells