斜带石斑鱼神经坏死病毒CP基因shRNA干扰载体的构建及效果评价

利用Invitrogen公司的在线生物学软件分析斜带石斑鱼神经坏死病毒CP基因,设计针对CP基因不同位置的小发卡RNA( short hairpin RNA,shRNA)干扰序列[其结构特征为正链( 19 nt) -环(4 nt) -负链(19 nt)].化学合成这些序列,并退火连接为双链干扰片段,将双链干扰片段定向克隆到干扰载体pENTRTM/U6中,构建shRNA干扰载体pshRNA -124、pshRNA-896和pshRNA-NNV.然后,用脂质体转染法分别将3种shRNA干扰载体和pEGFP-CP基因共转染导入黑头呆鱼(FHM)肌肉细胞,荧光显微镜观察细胞荧光强度,分析荧光抑制效率,Real-time RT-PCR检测CP基因mRNA的表达水平变化.结果表明,在pEGFP-CP与shRNA干扰载体共转染组,pshRNA-124、pshRNA-896、pshRNA-NNV的荧光抑制效率分别为47％、68％、51％.3种shRNA干扰载体都有干扰效果,均能干扰绿色荧光蛋白的表达,其中pshRNA-896干扰效率最好.Real-time RT-PCR检测表明,干扰质粒pshRNA-124、pshRNA-896、pshRNA-NNV对pEGFP-CP基因的沉默效率分别约为60％、96％和55％,与对照组相比差异显著(P＜0.05).研究表明,靶向斜带石斑鱼神经坏死病毒CP基因的shRNA干扰载体构建成功,为进一步运用RNA干扰技术进行CP基因的功能研究奠定了基础.     

斜带石斑神经坏死病毒外壳蛋白基因克隆与序列分析

从患病毒性神经坏死病的斜带石斑鱼(Epinephelus coioids)的头部提取总RNA，根据已发表的神经坏死病毒外壳蛋白基因设计引物进行RT-PCR扩增，得到预期大小的基因片段。将此基因片段转入pET载体进行序列测定和分析，结果表明：编码斜带石斑神经坏死病毒(Orange-spoued grouper nervous necrosis virus，OGNNV)外壳蛋白基因的阅读框核苷酸数为1017bp，编码338个氨基酸；基因的核苷酸序列与野田村病毒科(Nodaviridae)的几种病毒的外壳蛋白基因序列比较结果显示，该病毒与p野田村病毒属(Betanodavirus)中的赤点石斑神经坏死病毒(red-spotted grouper nervous necrosis virus，RGNNV)的同源性最高(99％)，说明该病毒株是RGNNY血清型的成员。     

逆转录聚合酶链式反应(RT-PCR)检测5种养殖石斑鱼的神经坏死病毒

神经坏死病毒(Nervous necrosis virus)是导致多种海水鱼类神经性病害的致病原.发病及死亡的石斑鱼除了表现神经异常症状外,无明显的临床病症,体表及内脏组织也未发现明显病变及寄生虫感染.2003年4～8月,应用逆转录聚合酶链式反应(RT-PCR)技术从福建南部人工养殖的5种石斑鱼即紫石斑鱼(Epinephelus lanceolatus)、马拉巴石斑鱼(E. malabaricus)、青石斑鱼(E. awoara)、赤点石斑鱼(E. akaara)和云纹石斑鱼(E. moara)中检出5个神经坏死病毒分离株.检测了76份石斑鱼样品,这些石斑鱼NNV病毒的平均感染率约为90%.对这些病毒的RT-PCR产物421 bp核酸进行了测序和序列分析,其相同的序列超过99%.将这些序列与GenBank的石斑鱼(Epinephelus spp.)神经坏死病毒相关基因序列作比较,同源性在97%以上.对神经坏死病毒在石斑鱼体内的分布也进行了分析,在脑和眼组织的检出率最高,部分病鱼的肝、脾和肾组织也能检出病毒.结合流行病学特征,可确认神经坏死病毒为该传染病的主要致病原.RT-PCR方法是检测NNV等病原的一种理想的诊断方法.     

传染性造血器官坏死病毒微型基因组表达干扰素对病毒的抑制效应

为构建传染性造血器官坏死病毒(IHNV HLJ-09)微型基因组并表达虹鳟IFN,采用RT-PCR扩增IHNV HLJ-09株的N、P、L、G和NV蛋白基因并亚克隆入真核表达载体pCI中,构建辅助质粒pCI-N、pCI-P、pCI-L、pCI-G和pCI-NV;将扩增获得的IHNV基因组两末端序列、增强型绿色荧光蛋白(EGFP)报告基因、虹鳟I型干扰素(IFN)基因克隆到真核表达载体pCI中构建出表达EGFP的IHNV微型基因组pCI-LFGT和表达IFN的IHNV微型基因组pCI-LFIT;将pCI-LFIT质粒转染已接种IHNV HLJ-09毒株的EPC细胞,实时荧光定量PCR法测定细胞中IHNV G基因RNA。结果显示:构建的微型基因组不论与辅助病毒还是与5个辅助质粒共转染,外源基因均能正确表达;pCI-LFIT质粒转染已接种病毒的EPC细胞组与对照组相比其中的病毒核酸显著减少。     

RNA干扰技术抑制新加坡石斑鱼虹彩病毒感染细胞多肽ICP46与绿色荧光蛋白融合基因的表达

新加坡石斑鱼虹彩病毒(singapore grouper iridovirus,SGIV)是一种严重的能引起全身性疾病的病原体,对石斑鱼养殖造成了重大的经济损失。将含有SGIV感染细胞多肽ICP46(infected cell polypeptides 46)基因的真核表达载体pEGFP-ICP46转染到胖头鲤细胞(fathead minnow cells,FHM)中进行融合表达,用荧光显微镜观察到ICP46-GFP融合蛋白主要分布于FHM细胞的细胞质中。根据SGIVICP46的序列,设计并体外化学合成了特异性干扰SGIVICP46的siRNA(siRNA-ICP46),与pEGFP-ICP46共转染到FHM细胞中,通过荧光显微镜观察不同时间点荧光强度的变化。转染后24～48h,实验细胞(共转染siRNA-ICP46和pEGFP-ICP46)和阳性对照细胞(共转染siRNA-GFP和pEGFP-ICP46)中的荧光微弱,发荧光的细胞数量较阴性对照(共转染siRNA-Negative和pEGFP-ICP46)少70%左右,但其后实验细胞和阳性对照细胞的荧光强度开始增强,在转染后72h其与阴性对照组已差别不大。说...     

一种大规模制备双链RNA的简单方法及其在斑节对虾中的应用

RNA干扰(RNAi)是由双链RNA(dsRNA)诱发的特异性沉默目的基因表达的过程,一种大规模制备双链RNA的方法可以便利RNAi技术的应用。以斑节对虾kazal型蛋白激酶抑制剂(KPI)基因为例,详细介绍了一种以体内载体表达大量制备dsRNA(>300nt)的方法。使用商业载体pGEMT和pDRIVE,以2步克隆法构建含有发夹环(hairpin loop)dsRNA表达载体,转化RNA酶Ⅲ缺陷的大肠杆菌HT115(DE3)进行体内转录制备dsRNA。构建的发夹RNA表达载体含有494bp的正向靶序列和403bp的反向互补靶序列,其中正向靶序列多出的91bp即可成为loop环,而无需再次克隆加入。培养30mL的细菌,即可得到1mg纯化的dsRNA,而其成本仅为使用商业化体外转录试剂盒的四分之一。为评估RNAi效果,按照每1克虾体肌肉注射2μg dsRNA的剂量,在dsRNA注射后0,6,12和24h采集血淋巴,RT-PCR检测KPI mRNA的基因转录水平。与对照组GFP-dsRNA和NaCl注射组相比,KPI-dsRNA注射组可以在24h内沉默血淋巴中的KPI基因。结果表明该方法是可大规模制备长的dsRN...     

04斜带石斑鱼干扰素调节因子 1 基因的克隆及其原核表达

对斜带石斑鱼(Epinephelus coioides)的干扰素调节因子1(Interferon regulatory factor 1,IRF-1)基因进行了克隆、测序及原核表达.以PBS做对照,利用polvI:C刺激斜带石斑鱼,然后取其肝脏、头肾、脾脏提取总RNA,随机引物反转录获得cDNA.根据相近物种IRF-1的保守序列设计引物,通过PCR得到了保守片段,通过RACE-PCR获得了斜带石斑鱼IRF-1基因的全长cDNA.基因全长为1 730 hp,完整开放阅读框(ORF)906 bp,编码302个氨基酸,5'非编码区153 bp,3'非编码区671 bp.将斜带石斑鱼ORF与其他物种进行比对发现,与海鳜(Siniperca chuatsi)同源性最高,为85%.将根据斜带石斑鱼IRF-1 ORF推测的氨基酸序列与其他物种进行比对,发现斜带石斑鱼与海鳜同源性为90%,与金头鲷(Sparus aurat)为88%,与乌鳢(Channa argus)为86%,与大菱鲆(Scophthalmus maximus)为82%.利用ORF序列,构建原核重组表达质粒.重组质粒经PCR、酶切鉴定及测序验证后转化大肠杆菌BL21(DE3),经IPTG诱导,重组蛋白成功表达,表达蛋白的分子量约55 kD.诱导温度为30℃时,重组蛋白以可溶性蛋白和包涵体2种形式存在.     

闽南地区斜带石斑鱼(Epinephelus coioides)神经坏死病毒的基因型与分子进化研究

本研究以闽南地区具有典型神经坏死病症状的斜带石斑鱼为材料,采用RT-PCR法对其进行病毒检测,对检测到的阳性序列进行双向测序和构建系统发育树,对病毒颗粒进行分离纯化,并通过分子进化模型分析病毒衣壳蛋白基因所受的选择压力。结果显示,采集到的6个石斑鱼样品均呈NNV阳性,系统树分析发现6个样品的PCR扩增片段均为RGNNV基因型序列,表明闽南地区感染石斑鱼的神经坏死病毒主要为RGNNV基因型病毒;通过PEG法对病毒进行分离提纯,获得直径为25~28 nm、呈二十面立体对称结构的无囊膜病毒颗粒;分子进化分析显示NNV外壳蛋白基因经历了纯化选择,表明病毒在进化过程中没有出现遗传变异并以相对恒定保守的速率进化。     

RNA干扰技术抑制新家坡石斑鱼虹彩病毒感染细胞多肽ICP46与绿色荧光蛋白融合基因的表达

新加坡石斑鱼虹彩病毒（singapore grouper iridovirus,SGIV）是一种严重的能引起全身性疾病的病原体,对石斑鱼养殖造成了重大的经济损失。将含有SGIV感染细胞多肽ICP46（infected cell polypeptides 46）基因的真核表达载体pEGFP-ICP46转染到胖头鲤细胞（fathead minnow cells,FHM）中进行融合表达,用荧光显微镜观察到ICP46-GFP融合蛋白主要分布于FHM细胞的细胞质中。根据SGIV-ICP46的序列,设计并体外化学合成了特异性干扰SGIV-ICP46的siRNA（siRNA-ICP46）,与pEGFP-ICP46共转染到FHM细胞中,通过荧光显微镜观察不同时间点荧光强度的变化。转染后24~48 h,实验细胞（共转染siRNA-ICP46和pEGFP-ICP46）和阳性对照细胞（共转染siRNA-GFP和pEGFP-ICP46）中的荧光微弱,发荧光的细胞数量较阴性对照（共转染siRNA-Negative和pEGFP-ICP46）少70%左右,但其后实验细胞和阳性对照细胞的荧光强度开始增强,在转染后72 h其与阴性对照组已差别不大。说明体外化学合成的siRNA-ICP46转染后24~48 h可有效抑制FHM细胞中外源导入SGIV-ICP46基因的表达。     

赤点石斑鱼神经坏死病毒主衣壳重组蛋白多克隆抗体的制备

把赤点石斑鱼（Epinephelus akaara）神经坏死病毒（RGNNV）主衣壳蛋白（MCP）基因的重组表达质粒载体pRSETA-MCP转化至大肠杆菌（Estherichia coli）BL21（DE3）,经IPTG诱导表达,SDS-PAGE显示表达的重组蛋白主要以不可溶的包涵体形式存在,分子量约44.5kD。通过Ni-NTA-Agarose亲和层析柱纯化,经分析纯度达90%,之后免疫新西兰兔制备抗血清,ELISA效价达1：12800以上。Western-blot分析结果显示,该血清与表达的重组蛋白有较强反应,说明通过原核表达的重组蛋白具有良好的免疫原性。     

Enhanced viability of a nervous necrosis virus-infected stable cell line over-expressing a fusion product of the zfBcl-xL and green fluorescent protein genes

Nervous necrosis virus (NNV) infection induces host cell apoptosis by an ill-understood process. We utilized a fusion between enhanced green fluorescent protein (EGFP) and the zfBcl-x(L) gene in GL-av cells to select for zfBcl-x(L) stable cell lines and to assess the effectiveness of the anti-apoptotic protein Bcl-x(L) in circumventing NNV-induced cell death. Stable EGFP and EGFP-Bcl-x(L)-expressing clones were obtained at high purity within 2.5-3 months. In the latter, the EGFP-Bcl-x(L) fusion protein (approximately 58.2 kDa, as ascertained by Western blot) was predominantly targeted to mitochondria. We assayed for apoptosis in red-spotted grouper NNV Tainan no. 1 (RGNNV TN1)-infected cells with terminal deoxynucleotidyl transferase (TdT)-mediated end labelling (TUNEL) of DNA at different virus doses. NNV infection of NNV Bcl-x(L) GL-av cell line revealed a protective effect, with a decrease in TUNEL-positive cells of 7%, 8% and 31.8% at 24, 48 and 72 h, respectively. In addition, RGNNV infection of the Bcl-x(L) GL-av cell line revealed a protective effect, with an enhanced viability of 3%, 40% and 73% at 24, 48, and 72 h, respectively. We conclude that NNV-induced apoptotic cell death can be lessened in transgenic grouper fish cells.     

Establishment and characterization of a new cell line derived from kidney of grouper, Epinephelus akaara (Temminck & Schlegel), susceptible to Singapore grouper iridovirus (SGIV)

A marine fish cell line derived from the kidney of red-spotted grouper, Epinephelus akaara, designated as EAGK was established and characterized. The EAGK cells multiplied well in Leibovitz's L-15 medium containing 10% foetal bovine serum at 25 °C and have been subcultured for more than 90 passages. Karyotyping, chromosomal typing and ribosomal RNA (rRNA) genotyping analysis revealed that EAGK had a modal diploid chromosome number of 82 and was a fibroblast cell line originated from grouper. A severe cytopathic effect was observed in EAGK cells incubated with Singapore grouper iridovirus (SGIV), but not with soft-shelled turtle iridovirus, viral nervous necrosis virus or spring viraemia of carp virus. SGIV replication was further confirmed by immunofluorescence, electron microscopy and virus titre determination. Bright fluorescence was observed after transfection with fluorescent protein reporter plasmids, indicating that EAGK cells can be used to identify gene functions in vitro. In addition, the cell organelles including mitochondria and endoplasm reticulum changed and aggregated around virus factories after SGIV infection, suggested that the EAGK cell line could be an important tool for investigation of iridovirus-host interactions.     

Characterization of grouper nervous necrosis virus (GNNV)

Grouper nervous necrosis virus (GNNV) was isolated from moribund grouper larvae, Epinephelus sp., using a fish cell line GF-1. The present study describes the biochemical and biophysical properties of GNNV and the expression of GNNV in diseased grouper larvae. Viral protein was detectable in most of the GNNV-infected GF-1 cells by the fluorescent antibody technique (FAT) after 12 h post-infection (p.i.), although no cytopathic effect (CPE) appeared at that time. Clear CPE developed on the third day, and complete disintegration of the monolayer occurred over the subsequent two days. The infectivity of GNNV can be blocked following treatment at 60 °C for 1 h. GNNV was sensitive to pH 3 and pH 10–12 with a 4 log₁₀ drop in infectivity. Purified GNNV was analysed by SDS–PAGE, and then stained with periodic acid silver. The positive staining indicated that its two capsid proteins were glycoproteins. Genomic RNAs of GNNV were extracted from purified virions and analysed. The molecular weights of genomic RNAs were 1.02 × 10⁶ and 0.50 × 10⁶ Da. The T2 region of the coat protein gene of GNNV was amplified by polymerase chain reaction (PCR), and the multiple alignment of the T2 sequence of two GNNV isolates with four genotypes of fish nodaviruses revealed that these two isolates (GNNV9410 and GNNV9508) belong to the red-spotted grouper nervous necrosis virus (RGNNV) genotype. The tissue distribution of GNNV in naturally infected grouper larvae was investigated by in situ hybridization using a dig-labelled probe, which showed that GNNV was not only detected in the brain and retina, but also in the gill, skeletal muscle, liver, pyloric gland, intestine and blood cells in the heart.     

Viral encephalopathy and retinopathy is endemic in wild groupers (genus Epinephelus spp.) of the Algerian coast

This work describes betanodavirus infection in two species of groupers (family Serranidae) from the Algerian coast: the dusky grouper Epinephelus marginatus and the golden grouper Epinephelus costae. At necropsy, characteristic clinical signs, external injuries, clouded eyes and brain congestion, generally associated with viral encephalopathy and retinopathy (VER) infection were observed. The partial sequences of RNA1 and RNA2 from two viral strains were obtained, and the phylogenetic analysis revealed the presence of the red-spotted grouper nervous necrosis virus (RGNNV) genotype closely related to strains previously detected in groupers in the same geographic area. Results obtained in this study support the hypothesis that VER disease is endemic in the Algerian grouper population.     

Complete sequencing of Tunisian redspotted grouper nervous necrosis virus betanodavirus capsid gene and RNA‐dependent RNA polymerase gene

Finfish nodaviruses (betanodaviruses) can cause highly destructive infections in numerous species of farmed marine fish larvae and juveniles worldwide. The betanodavirus genome consists of two single‐stranded positive‐sense RNA molecules (RNA1 and RNA2). The virus can be classified into four genotypes based on the partial sequences of the coat protein (CP) gene (T2 and T4 regions). Currently, genomic sequence information for RNA1 regions of RNA2 outside of T2 and T4 is less well documented. This study reports on the characterization of the full RNA2 sequence of a Tunisian betanodavirus with a length of 1433 nt, containing a 339 amino acid open‐reading frame encoding the CP, and typing to the redspotted grouper nervous necrosis virus Ia genotype following phylogenetic analysis. The homology of the capsid protein to other betanodaviruses or alphanodaviruses was compared. In addition, a full length RNA1 sequence of 3104 nt encoding a 982 amino acid RNA‐dependent RNA polymerase was obtained.     

Outbreak of betanodavirus infection in tilapia, Oreochromis niloticus (L.), in fresh water

A betanodavirus associated with a massive mortality was isolated from larvae of tilapia, Oreochromis niloticus , maintained in fresh water at 30 °C . Histopathology revealed vacuolation of the nervous system, suggesting an infection by a betanodavirus. The virus was identified by indirect fluorescent antibody test in the SSN1 cell line and further characterized by sequencing of a PCR product. Sequencing of the T4 region of the coat protein gene indicated a phylogenetic clustering of this isolate within the red-spotted grouper nervous necrosis virus type. However, the tilapia isolate formed a unique branch distinct from other betanodavirus isolates. The disease was experimentally reproduced by bath infection of young tilapia at 30 °C. The reservoir of virus at the origin of the outbreak remains unidentified. To our knowledge, this is the first report of natural nodavirus infection in tilapia reared in fresh water.     

Effects of aromatizable and nonaromatizable androgens on the sex inversion of red-spotted grouper (Epinephelus akaara)

The effects of aromatizable 17α-methyltestosterone (MT) and non-aromatizable 17α-methyldihydrotestosterone (MDHT) on sex inversion in red-spotted grouper, Epinephelus akaara, were investigated. Fish were implanted with MT, MDHT and MT+AI (aromatase inhibitor, AI) respectively for one month. The results showed that the three treated groups turned into transitional stage with intersex gonads, which contained atretic oocytes and spermatogenic germ cells at all stages of spermatogenesis. The controls did not change sex. The gonads of more than half MT-implanted fish were in early transitional stages of sex inversion, whereas those of more than half MDHT and MT+AI-implanted fish were in late transitional stages of sex inversion. No difference in serum estradiol-17β (E₂) levels between the controls and the treated groups were observed, whereas 11-ketotestosterone (11-KT) and testosterone (T) levels increased in all treated groups. Significantly lower gonadosomatic index (GSI) and gonadal aromatase activity were observed in the treated groups, which were in accordance with the lower mRNA expression of P450aromA. However, P450aromB mRNA expression increased in the MT group, while it did not change in the MDHT group. These results suggest that the sex inversion of red-spotted grouper by MT and MDHT implantation might be due to the suppression of P450aromA gene expression, and resulting in both the decrease of the ovarian estrogen –secretion, as well as the increase in the 11-KT levels. Furthermore, the main reason for MT being less effective than MDHT might be due to partial aromatization of MT to estrogen.     

Establishment and characterization of a liver cell line,ALL, derived from yellowfin sea bream,Acanthopagrus latus,and its application to fish virology

Yellowfin sea bream (Acanthopagrus latus) is an important economic fish, which is seriously threatened by various fish viruses. In this study, a cell line designated as ALL derived from the liver of yellowfin sea bream was developed and characterized. The cell line grew well in Dulbecco's modified Eagle's medium containing 10%–20% foetal bovine serum at 28°C. Amplification of the cytochrome B gene indicated that ALL cells originated from yellowfin sea bream. The modal chromosome number of ALL cells was 48. ALL cells were efficiently transfected with pEGFP-N3 plasmids, indicating the potential application of ALL cells in exogenous gene manipulation studies. ALL cells were susceptive to three main fish viruses, including viral haemorrhagic septicaemia virus (VHSV), red-spotted grouper nervous necrosis virus (RGNNV) and largemouth bass virus (LMBV). The replication of VHSV, RGNNV and LMBV in ALL cells was confirmed by quantitative real-time polymerase chain reaction, virus titre and transmission electron microscopy assays. Moreover, ALL cells could respond to VHSV, RGNNV and LMBV infections, as indicated by the differential expression of antiviral genes involving in the innate immune response. In conclusion, the newly established ALL cell line will be an excellent in vitro platform for the study of the virus–yellowfin sea bream interaction.