温度对草鱼出血病影响的初步探讨

将鱼呼肠孤病毒(FRV)感染的草鱼饲养在人工控温的水族箱内,水温在24—30℃恒温时其死亡率无显著差异(P>0.05),而在20℃和33℃恒温时死亡率则明显降低,与24℃—30℃恒温相比死亡率有非常显著差异(P<0.01)。人工感染恒温饲养期间,死亡高峰期随水温降低而推迟,缓慢改变水温能降低死亡率,在低于20℃攻毒并维持一星期左右,即使逐步升温至30℃也不会导致感染鱼的大批死亡。     

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。     

草鱼出血病病毒抗原亚单位的研制

通过比较溶液的离子浓度对病毒形态的结构的影响,发现ＧＣＨＶ在低盐溶液盐低温（－２０℃）保存条件下,外壳蛋白会自动降解,但不十分完全,采用含１％ＮＰ４０（Ｎｏｎｉｄｅｔ－ｐ４０）的稀盐溶液处理感染病毒的细胞,经冻融及差异离心,电镜下观察到纯度较高的病毒双层衣壳,核心衣壳及散在的子粒。纯化的病毒亚单位用核酸酶消化后,与等量的福氏佐剂混合,免疫家兔制备抗体,该抗血清经台和试验测定,显示了较强的中和病毒的     

草鱼出血病病毒多肽的免疫原性

采用中和试验比较了草鱼出血病病毒ＧＣＨＶ８７３的１１个多肽的免疫原性,确定了主要中和抗原。纯化的单个病毒多肽免疫家兔获得抗血清,多太ＶＰ１、ＶＰ５、ＶＰ６和ＶＰ９的抗血清具有中和效价,而ＶＰ２、ＶＰ３、ＶＰ４、ＶＰ８、ＶＰ１０和ＶＰ１１则不能诱生中和抗体。其中ＶＰ５的抗血清中和效价最高,因此,ＶＰ６极可能是病毒多肽中的主要中和抗原。     

几种鱼类细胞对草鱼呼肠孤病毒敏感性的研究

比较研究了鲫鱼异倍体细胞系(CAB-80)、团头鲂尾鳍细胞系(BCC)、大鳞副泥鳅雌核发育单倍体胚胎细胞系(PHC)、草鱼胚胎细胞系(GCE)、草鱼尾鳍细胞系(CCRF-2)、草鱼肾细胞系(CCK—S4)及其四个克隆对草鱼呼肠孤病毒(CCRV)的敏感性。证实了这些细胞(PHG除外)在不同程度上对GCRV敏感,其中以GCK-84的敏感性最强。这表明,在体外培养条件下,GCRV并无严格的种族特异性。用经GCK-84传代的病毒感染草鱼种,能复制出典型的出血病症状。用GCK-84检测了病毒在GCK-84、GCRF-2、CAB-80、BCC和PHG中的滴度(TCID50/mt),其值分别为8．24,7．36,2．90,2．15和1．33。4个克隆与肾细胞系对病毒的敏感程度亦不尽相同,其滴度在6．3到9．32之间变化。上述结果对细胞工程抗病育种预示有较大的潜在意义。在电镜下可见GCRV对被感染的细胞造成了严重的破坏。病毒为平均直径58nm的球形颗粒,具有一个高度电子密度的核心,平均直径约为38nm。病毒在细胞中的分布方式有三种-即散布于细胞质中的、呈晶格状包于一膜状结构中的和整齐或不整齐地聚集在一起但无膜包裹的。     

草鱼出血病病原的研究

草鱼出血病是草鱼种的一种严重疾病,通常在水温25—30℃时发病,其症状表现为全身性组织器官充血。其病原悬液能通过赛氏滤器(Seitz,EKS石棉滤板),能在草鱼种体内及草鱼鳍单层细胞中传代繁殖,在草鱼肌肉单层细胞和性腺单层细胞中感染并出现细胞病变。病原悬液对脂溶剂乙醚敏感,耐热性强,对四环素类药物不敏感。试验结果,我们认为草鱼出血病的病原是病毒。     

草鱼出血病病毒对其它鱼的感染性研究

用草鱼出血病病鱼分离出的草鱼出血病病毒(Grass carp hemorrhage virus,GCHV)感染其它常见鱼并用ELISA方法检查感染鱼组织提取液,结果表明:青鱼、鲢鱼、布氏鳌条对GCHV抗体呈阳性反应;鲤鱼、鳙鱼、鲫鱼、团头鲂、泥鳅则呈阴性反应。综合感染鱼发病症状及死亡特征,初步认为:青鱼对GCHV是易感的,GCHV能在鲢鱼、布氏蟹条体内增值,但毒力较低,鳙鱼、鲫鱼、团头鲂、鲤鱼、泥鳅能抗GCHV感染。     

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。       

草鱼鳃介导草鱼呼肠孤病毒免疫应答（英文）

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。     

草鱼出血病混合感染的嗜水气单胞菌的分离、鉴定与理化特性

从患出血病草鱼的肝脏病灶中分离筛选出2株致病菌。取病鱼样品组织过滤液接种CIK细胞、培养, 电镜下观察到细胞质中含有草鱼呼肠孤病毒样颗粒和包涵体, 病毒颗粒大小65 nm~ 70 nm, 包涵体0.46 μm~1.81 μm。人工回归感染实验显示分离的菌株及细胞毒悬液均能使草鱼致病死亡。对分离菌株进行细胞形态学、理化特性分析及药敏试验, 初步判定所分离的2株菌均为嗜水气单胞菌。进一步对菌株进行DNA分子鉴定, 结果显示2株菌的16S rRNA基因、促旋酶亚单位蛋白(gryB)基因均与GenBank上的嗜水     

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,GCRV S2 片段长为3 877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.     

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     

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.     

草鱼Ⅲ型呼肠孤病毒NS66抗体制备及应用

[背景]草鱼Ⅲ型呼肠孤病毒(grass carp reovirus,GCRV genotypeⅢ) 104株可导致典型性草鱼出血病,对其编码片段的分析有望为临床免疫学检测提供依据。[目的]研究GCRV104株s6基因节段编码蛋白NS66的可能功能,制备良好的GCRV104株NS66蛋白多克隆抗体并分析其特异性。[方法]PCR方法扩增GCRV104株s6基因片段,并克隆至表达载体pGEX-4T-3,转化到大肠杆菌BL21后用IPTG诱导表达,其产物经SDS-PAGE鉴定分析后,通过纯化获得目的蛋白。然后用纯化的pGEX-4T-3-NS66重组蛋白免疫小鼠,获得Anti pGEX-4T-3-NS66多克隆抗体,Western blot测定抗体效价,Western blotting和间接免疫荧光试验(indirect immunofluorescence assay,IFA)鉴定抗体特异性。[结果]SDS-PAGE分析显示表达的重组蛋白约66 kD,大小与预期相符,主要存在于包涵体中;Western blotting测得制备的多克隆抗体效价大于1:50 000,Western blotting和IFA结果表明,制备的多克隆抗体能特异性识别GCRV104病毒。[结论]GCRV104病毒编码的非结构蛋白NS66可能参与了复制和组装过程,形成病毒包涵体,这为建立GCRV104免疫诊断方法及研究GCRV编码的NS66蛋白的功能奠定了前期基础。     

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.     

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 ...