转人乳铁蛋白基因草鱼抗GCHV的初步研究

国家自然科学基金重点项目(39823003)

The innate immune response to grass carp hemorrhagic virus (GCHV) in cultured Carassius auratus blastulae (CAB) cells

Virus infection of mammalian cells activates an innate antiviral immune response characterized by production of interferon (IFN) and the subsequent transcriptional upregulation of IFN-stimulated genes (ISGs) by the JAK-STAT signaling pathway. Here, we report that a fish cell line, crucian carp (Carassius auratus L.) blastulae embryonic (CAB) cells, can produce IFN activity and then form an antiviral state after infection with UV-inactivated grass carp hemorrhagic virus (GCHV), a double-stranded (ds) RNA virus. From UV-inactivated GCHV-infected CAB cells, 15 pivotal genes were cloned and sequenced, and all of them were shown to be involved in IFN antiviral innate immune response. These IFN system genes include the dsRNA signal sensing factor TLR3, IFN, IFN signal transduction factor STAT1, IFN regulatory factor IRF7, putative IFN antiviral effectors Mx1, Mx2, PKR-like, Viperin, IFI56, and other IFN stimulated genes (ISGs) IFI58, ISG15-1, ISG15-2, USP18, Gig1 and Gig2. The identified fish IFN system genes were highly induced by active GCHV, UV-inactivated GCHV, CAB IFN or poly(I).poly(C), and showed similar expression patterns to mammals. The data indicate that an IFN antiviral innate immune response similar to that in mammals exists in the UV-inactivated GCHV-infected CAB cells, and the IFN response contributes to the formation of an antiviral state probably through JAK-STAT signaling pathway. This study provides strong evidence for existence of IFN antiviral innate immune response in fish, and will assist in elucidating the origin and evolution of vertebrate IFN system. (c) 2006 Elsevier Ltd. All rights reserved.

The molecular characterization of RNA segment S9 of grass carp hemorrhage virus (GCHV), an aquareovirus

Although reovirus infection is one of the major virus diseases of grass carp in China, the available knowledge on the structure and function of genes and proteins of the virus is limited. The complete sequence of the S9 genome segment of grass carp hemorrhage virus (GCHV) was determined. The segment consists of 1130 nucleotides and has a large open reading frame (ORF) encoding a protein of 352 amino acids with predicted molecular mass of 37.7 kDa. Amino acid sequence comparison revealed that the deduced protein encoded by GCHV S9 is closely related to the sigma NS proteins of mammalian reovirus (MRV) and avian reovirus (ARV). Secondary structure analysis displayed that the form of alpha -helices (40.1%) and beta -sheets (49.4%) are the richest two contents in the protein encoded by S9, and this protein is predicted to be a nonstructural protein. (C) 2001 Elsevier Science B.V. All rights reserved.

Molecular characterization and expression of the M6 gene of grass carp hemorrhage virus (GCHV), an aquareovirus - Brief report

7The complete nucleotide sequence of M6 gene of grass carp hemorrhage virus (GCHV) was determined. It is 2039 nucleotides in length and contains a single large open reading frame that could encode a protein of 648 amino acids with predicted molecular mass of 68.7 kDa. Amino acid sequence comparison revealed that the protein encoded by GCHV M6 is closely related to the protein mul of mammalian reovirus. The M6 gene, encoding the major outer-capsid protein, was expressed using the pET fusion protein vector in Escherichia coli and detected by Western blotting using chicken anti-GCHV immunoglobulin (IgY). The result indicates that the protein encoded by M6 may share a putative Asn-42-Pro-43 proteolytic cleavage site with mul.

A detection method for grass carp hemorrhagic virus (GCHV) based on a reverse transcription polymerase chain reaction

A rapid, sensitive and highly specific detection method for grass carp hemorrhagic virus (GCHV) based on a reverse transcription-polymerase chain reaction (RT-PCR) has been developed. Two pairs of PCR primers were synthesized according to the cloned cDNA sequences of the GCHV-861 strain. For each primer combination, only one specific major product was obtained when amplification was performed by using the genomic dsRNA of GCHV-861 strain. The lengths of their expected products were 320 and 223 bp, respectively. No products were obtained when nucleic acids other than GCHV-861 genomic RNA were used as RT-PCR templates. To assess the sensitivity of the method, dilutions of purified GCHV-861 dsRNA total genome (0.01 pg up to 1000 pg) were amplified and quantities of as little as 0.1 pg of purified dsRNA were detectable when the amplification product was analyzed by 1.5% agarose gel electrophoresis. This technique could detect GCHV-861 not only in infected cell culture fluids, but also in infected grass carp Ctenopharyngodon idellus and rare minnow Gobiocypris rarus with or without hemorrhagic symptoms. The results show that the RT-PCR amplification method is useful for the direct detection of GCHV.

病毒感染草鱼胸腺的EST分析和免疫相关基因的鉴定

以感染草鱼(Ctenopharyngodon idellus)出血病病毒(GCHV)的草鱼胸腺为材料,构建了草鱼胸腺的SMARTcDNA文库。筛选文库获得到1933条有效EST序列。BLASTX分析显示,583条序列在公共数据库中能找到同源基因(E-value≤1.00E10-3,Identities≥30%),另外1350条序列则找不到显著同源性。已知基因按具体功能可划分为6类,大部分与细胞内的各种生理过程、细胞结构以及免疫防御相关。研究结果从分子水平上表明鱼类的胸腺在机体感染病毒的免疫反应中发挥重要作

双链RNA病毒诱导的牙鲆胚胎细胞差减cDNA文库的构建

以紫外线灭活的dsRNA病毒草鱼出血病病毒(GCHV)诱导和模拟诱导的牙鲆胚胎细胞为材料,利用抑制性差减杂交(SSH)技术,成功构建了双链RNA病毒诱导的牙鲆胚胎细胞(FEC)差减cDNA文库。以管家基因αtub lin作为差减指标,经检测,该文库差减效率达210倍,表明经病毒诱导后某些差异表达基因也得到了相应倍数的富集。将获得的cDNA片段连接到pGEM T载体,PCR检测显示差减片段在250bp~2 000bp之间。该差减cDNA文库的构建为从分子水平研究牙鲆培养细胞对dsRNA病毒的免疫反应、以及进

鱼类干扰素系统基因的克隆鉴定及其特征分析(英文)

干扰素 (IFN)系统是脊椎动物抵抗病毒入侵的第一道防御系统 .除了哺乳类 ,有关低等脊椎动物的IFN系统基因知之甚少 .在鱼类 ,近 40年来能证明IFN系统存在的证据主要有两个 :一是检测经病毒诱导后的多种鱼类机体和细胞 ,证明能产生类似哺乳类IFN的抗病毒活性物质 ;二是近年来 ,已经证明在少数几种鱼类中存在与哺乳类IFN系统基因Mx同源的基因 .以前的研究结果表明 ,紫外线灭活的草鱼出血病病毒 (GCHV)能够诱导鲤科鱼类培养细胞 ,如鲫鱼囊胚细胞 (CAB)产生类IFN活性物质 ,并建立宿主细胞

鱼类培养细胞抗病毒基因差减cDNA文库的构建

紫外线灭活的草鱼出血病病毒 (GCHV)能诱导鲫囊胚培养细胞 (CAB)产生高滴度的干扰素 ,从而诱导宿主细胞基因表达的改变并处于抗病毒状态。提取灭活病毒诱导未经病毒诱导的CAB细胞mRNA ,利用抑制性差减杂交技术 ,成功构建了鱼类培养细胞抗病毒基因差减cDNA文库。以鲫管家基因α tubulin和 β actin作为差减指标 ,检测差减cDNA文库的差减效率分别高达 2 15和 2 7倍 ,表明经过病毒诱导后的细胞中 ,某些差异表达基因的富集效率也接近 2 15倍。鱼类抗病毒基因差减cDNA文库的建立

从灭活病毒诱导的培养细胞中鉴定鱼类抗病毒相关基因

紫外线灭活的草鱼出血病病毒(GCHV)能诱导鲫鱼囊胚培养细胞(CAB)产生高滴度的干扰素并抵抗病毒入侵,其机制可能是通过诱导一组抗病毒相关基因的表达,而使寄主细胞建立起抗病毒状态.利用抑制性差减杂交技术,构建了灭活病毒诱导鱼类培养细胞基因差异表达的差减cDNA文库.从差减文库中筛选鉴定出272个差异cDNA片段,测序分析发现它们为69个基因,其中46个为已知基因的同源物,23个为未知的假定基因.已知基因包括I型干扰素信号通路的基因Statl和JakI,抗病毒基因Mx和Viperin,以及一系列干扰素激活基

应用逆转录聚合酶链反应直接检测草鱼出血病病鱼组织的研究

人工感染GCHV－861后，对处于潜伏期、发病期和恢复期等不同时期的草鱼内脏组织匀浆上清液进行逆转录聚合酶链式反应（RT－PCR）扩增，除恢复期的1条草鱼外，其余样品均得到特异扩增带，而对照组都没有，预示着RT－RCR技术对于草鱼出血病的早期诊断、防治及抗病有种具有重要意义。另外，对于显症出血病草鱼的肝、肾、脾、鳃、肌肉和肠道等组织器官进行检测，结果都为阳性，首次证实了GCHV存在于肝脏中，并对此作了进一步的讨论。

用逆转录聚合酶链式反应检测草鱼出血病病毒的研究

于1994年5月-1995年7月，根据已克隆的草鱼出血病病毒GCHV-861株cDNA的部分序列，设计合成了两对PCR引物，采用RT-PCR技术对GCHV-861及GCHV-873两病毒株的dsRNA进行扩增。结果表明，两对引物仅能特异地检测出GCHV-861病毒株核酸的存在，而不能对GCHV-873病毒株的核酸进行特异扩增，该方法最小可检测出0.1Pg纯化的GCHV-861病毒dsRNA;采用该方法对GCHV-861人工感染的草鱼和稀有鲫组织进行RT-PCR检测，不仅能检测到发病期显症病鱼中GCHV-8

草鱼出血病病毒人工感染稀有(鱼句)鲫出血病鱼主要器官组织的超薄切片观察

用电子显微镜观察了稀有(鱼句)鲫出血病(Hemorrhage of Gobiocypris rarus)鱼的病理切片及对照鱼的超薄切片。在对照鱼的鳃、肠道、肾脏、脾脏、肌肉和肝脏中没有见到任何病毒颗粒。在病鱼鳃血管内皮细胞质中观察到聚集的直径70nm左右的GCHV颗粒,说明鳃是GCHV侵袭的主要器官之一,并讨论了鳃对GCHV敏感在GCHV传播中的意义。还在病鱼肠道,肾脏中观察到成片的病毒颗粒,它们也是GCHV侵袭的主要组织。在病鱼脾脏的电子密度低的细胞质中观察到散在的病毒颗粒。很少见到病毒聚集体,脾脏中的

Expression regulation and functional characterization of a novel interferon inducible gene Gig2 and its promoter

Grass carp hemorrhagic virus (GCHV)-induced gene 2 (Gig2) is a novel gene previously identified from UV-inactivated GCHV-treated Carassius auratus blastulae embryonic (CAB) cells, suggesting that it should play a pivotal role in the interferon (IFN) antiviral response. In this study, a polyclonal anti-Gig2 antiserum was generated and used to study the inductive expression pattern by Western blot analysis, showing no basal expression in normal CAB cells but a significant up-regulation upon UV-inactivated GCHV, polyinosinic:polycytidylic acid (Poly I:Q and recombinant IFN (rIFN). However, constitutive expression of Gig2 is observed in all tested tissues from grass carp (Ctenopharyngodon idellus), and Poly I:C injection increases the relative amount of Gig2 protein in skin, spleen, trunk kidney, gill, hindgut and thymus. Moreover, the genomic sequence covering the whole Gig2 ORF and the upstream promoter region were amplified by genomic walking. Significantly, the Gig2 promoter contains three IFN-stimulated response elements (ISREs), nine GAAA/TfTC motifs and five gamma-IFN activating sites (GAS), which are the characteristics of genes responsive to both type I IFN and type 11 IFN. Subsequently, the complete Gig2 promoter sequence was cloned into pGL3-Basic vector, and its activity was measured by luciferase assays in the transfected CAB cells. The Gig2 promoter-driven construct is highly induced in CAB cells after treatment with Poly I:C or rIFN, and the functional capability is dependent on IFN regulatory factor 7 (IRF7), because its activity can be stimulated by IRF7. Collectively, the data provide strong evidence that Gig2 is indeed a novel IFN inducible gene and its expression is likely dependent on IRF7 upon Poly I:C or IFN. (C) 2009 Elsevier Ltd. All rights reserved.

Effect of beta-glucan on activity of antioxidant enzymes and Mx gene expression in virus infected grass carp

The effects of beta-glucan, an immunostimulatory agent, on the superoxide dismutase (SOD) and catalase (CAT) activities of erythrocytes and Mx gene expression were studied from grass carp that were challenged with grass carp hemorrhage virus (GCHV). The SOD and CAT activities in erythrocytes and Mx gene expression in spleen from the fish were detected by spectrophotometry and RT-PCR, respectively. Negative control fish were injected with PBS; positive control groups were injected with either P-glucan or GCHV only; and the experimental groups were pre-injected with beta-glucan 15 days prior to injection with GCHV. The results show that the SOD and CAT activities were higher in fish injected with beta-glucan for 15 days than the negative control group injected with PBS. The SOD and CATactivities significantly decreased when the fish were challenged with GCHV, but it was higher in the group pre-treated with beta-glucan than in infected fish not pre-treated, 15 days after GCHV infection. Mx gene expression levels increased during the early stages (at 12 h and 36 h) of GCHV infection, and it remained at higher levels from the 6th till the 10th day in the beta-glucan pre-treated group, but it was failing from the 6th day in the beta-glucan untreated group. The GCHV-infected group pre-treated with P-glucan had a higher survival rate (60%) than the group not pre-treated with P-glucan (20%), suggesting that beta-glucan possesses or enhances anti-viral responses. (C) 2009 Elsevier Ltd. All rights reserved.