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1.
鳜传染性脾肾坏死病毒p31基因结构及序列分析   总被引:3,自引:0,他引:3  
邓敏 《水产学报》2001,25(6):542-546
报道了鳜传染性脾肾坏死病毒(ISKNV)的p31基因结构及其序列分析。对ISKNV DNA HindⅢE酶切片段的序列分析结果发现该序列中含有完整的p31基因,ISKNV p31基因完整读码框为675bp,GC含量为49.78%,等电点为7.61,编码一个长为225aa、分子量为25.3kD的推定蛋白。结果分析发现该基因具有启动动子TATAbox和CAATmotif,下游有反向重复序列可形成茎环,另外还有一段直接重复序列和二联体结构。ISKNV与其它3种虹彩病毒(包括FV3、LCDV-1和EHNV)的p31基因氨基酸序列具有一定的同源性,但ISKNV与它们的同源性不高,序列比较和系统树分析发现ISKNV与蛙病毒属和淋巴囊肿病毒属的病毒都不尽相同。  相似文献   

2.
虎纹蛙病毒甲基转移酶基因的克隆和分析   总被引:1,自引:0,他引:1  
苗素英 《水产学报》2002,26(2):157-160
经PCR扩增得到虎纹蛙病毒DNA甲基转移酶完整基因片段,并将其克隆到pUCm-T载体,测序可知该基因读码框大小为642bp,编码一由214个氨基酸组成的,预期分子量为24.8kD的多肽。RTV的MTase基因与蛙病毒属的蛙病毒-3型、叉尾Hui病毒和Regina病毒的一致性为96%~97%,与淋巴囊肿病毒属的比目鱼病毒的一致性为56%,从而进一步证明RTV蛙病毒的分类地位;与其它脊椎动物的虹彩病毒一样,该基因包含原核生物5′甲基转移酶的前四个高度保守区而缺少第五个区域,可能只是构成甲基转移酶的一个亚基,RTV、裂唇鱼病毒和大口黑鲈病毒之间MTase基因的一致性与衣壳蛋白基因的差异较大,说明同一种类的不同基因甚至同一基因的不同区域间演化速率不同,因此在虹彩病毒的演化研究中选择合适的基因或基因区域极为重要。  相似文献   

3.
应用同源PCR技术,从被一种球状病毒感染的患病大菱鲆(Scophthalmus maximus)脾脏和肾脏组织中扩增出了一段长度为620bp的DNA片断。序列测定和Blast分析表明,该DNA片断与鱼类虹彩病毒主要衣壳蛋白(MCP)C末端编码区的DNA序列高度相似,由此证实感染养殖大菱鲆的这种球状病毒为一种鱼类虹彩病毒,暂命名为大菱鲆红体病虹彩病毒(TRBIV)。多序列比对和分析发现,TRBIV MCP C末端的205个氨基酸序列与GenBank中20种虹彩病毒相应序列的相似性分别为99.47%(韩国大菱鲆虹彩病毒)、97%~98%(待指定病毒属的7种病毒),以及50%以下(蛙病毒属、淋巴囊肿病毒属、虹彩病毒属的12种病毒),由此绘制出了包含TRBIV在内的21种虹彩病毒的系统发育树。研究结果表明,感染中国养殖大菱鲆的TRBIV属于虹彩病毒科待指定病毒属,位于该属ISKNV亚群和RSIV亚群之间,是该病毒属的一个新成员。  相似文献   

4.
鳜传染性脾肾坏死病毒核糖核酸酶Ⅲ基因结构及序列分析   总被引:2,自引:0,他引:2  
测定了鳜传染性脾肾坏病毒(ISKNV)核糖核酸酶Ⅲ(RNaseⅢ)基因的核苷酸全序列。该基因完整读码框为771bp,GC含量为52.53%,编码一个长为256个氨基酸、分子量为28.9kD的推定蛋白。其上下游序列各有一个TATA box,终止子下游有一段回文序列。与其它物种相比,ISKNV与虹彩病毒(包括LCDV-1和CIV)的核糖核酸酶Ⅲ基因的氨基酸序列同源性较高,与酵母、线虫等物种的相应基因的同源性较低。  相似文献   

5.
李亚男  刘春  林华剑  秦真东  林蠡 《水产学报》2023,47(8):089415-089415
为了进一步丰富蛙虹彩病毒检测方法,实验针对蛙病毒3型(frog virus 3,FV3)核衣壳蛋白(major capsid protein,MCP)基因保守区设计一对特异性引物,并选取另外2种蛙病毒PCR检测方法进行对比实验,通过反应体系的优化、反应特异性和敏感性实验,建立了一种针对FV3的PCR检测方法。结果显示,该方法最低检测限可达1.2个拷贝数的病毒粒子,与神经坏死病毒、虾血细胞虹彩病毒、大口黑鲈虹彩病毒、锦鲤疱疹病毒、鲤浮肿病毒、传染性脾肾坏死病毒、加州鲈弹状病毒等常见水产动物致病毒株无交叉反应。临床样品检测表明,该方法所获得的检测结果与另外2种方法一致,结果可靠。本研究所建立的FV3 PCR检测方法,具有简便、快速、敏感度好、特异性高、低成本等特点,可用于FV3蛙病毒的快速诊断和分子流行病学调研。  相似文献   

6.
大口黑鲈病毒性溃疡病病原的分离和鉴定   总被引:8,自引:1,他引:7       下载免费PDF全文
对广东省佛山地区2008年夏季高温时期暴发的大口黑鲈溃疡病的病原进行分离,从患病鱼的病灶肌肉组织中分离到5株嗜水气单胞菌,人工感染健康大口黑鲈,均未出现溃疡暴发病的典型症状病鱼体表大片溃烂,裸露肌肉坏死并有出血,尾鳍、胸鳍和背鳍基部红肿溃烂。制备病灶肌肉组织的除菌过滤液,背部肌肉注射感染健康大口黑鲈,7 d后出现典型的溃疡病症状。取自然发病鱼和人工感染的患病鱼病灶肌肉组织制作超薄切片,经电镜观察,均发现组织中有大量病毒颗粒,病毒粒子有囊膜,呈六角形,为正20面体对称结构,大小约为145.5 nm。根据已知虹彩病毒主要衣壳蛋白(MCP)基因序列设计特异引物,提取人工感染发病鱼病灶肌肉组织的DNA进行PCR扩增,将扩增片段进行序列测定与分析,结果表明该序列与已报道的虹彩病毒MCP基因有着较高同源性(39.5%~100%)。从电镜观察和MCP基因测序分析结果确认该病毒为虹彩病毒科蛙病毒属中的一种病毒,与国外报道的大口黑鲈病毒(LMBV)在分子特性和引起的疾病特征上有一定差异。研究结果表明引起大口黑鲈溃疡性暴发病的病原是虹彩病毒,将该病暂命名为大口黑鲈病毒性溃疡病。  相似文献   

7.
根据Gen Bank中大鲵虹彩病毒主衣壳蛋白MCP(major capsid protein,MCP)基因序列(序列号:KF512820),设计一对特异性引物,以大鲵虹彩病毒贵州分离株基因组DNA为模板,PCR扩增大鲵虹彩病毒MCP基因并测序,与Gen Bank中大鲵虹彩病毒MCP基因进行比对,然后将其亚克隆到原核表达载体p ET-32a(+)中,转化大肠杆菌BL21(DE3)感受态细胞,经IPTG诱导后进行Western blot分析。结果显示:PCR扩增出长度为1 392 bp的片段,与Gen Bank中大鲵虹彩病毒MCP基因核苷酸序列相似性为99.7%~99.9%,SDSPAGE电泳显示该重组蛋白的相对分子质量约为67×103。免疫原性检测结果表明,该重组蛋白可与兔抗大鲵虹彩病毒阳性血清特异性反应,具有免疫原性。  相似文献   

8.
斜带石斑神经坏死病毒外壳蛋白基因克隆与序列分析   总被引:2,自引:3,他引:2  
陈晓艳 《水产学报》2004,28(2):183-188
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。  相似文献   

9.
为了查明2009年10月广东省佛山地区养殖大口黑鲈(Micropterus salmoides)中暴发的传染性疾病的病原,对病鱼的肝脏、脾脏和腹隔膜进行切片电镜观察,发现细胞质中有大量病毒颗粒,切面为六角形,直径约145~150 nm,病毒为二十面体对称结构、无囊膜、似虹彩病毒的病毒粒子.用除菌的病鱼组织滤液感染健康大口黑鲈,被感染鱼死亡率达90%以上.根据已知虹彩病毒主要衣壳蛋白(MCP)基因序列设计特异引物,提取人工感染发病鱼的肝脏、脾脏、肾脏组织的DNA进行PCR扩增,将扩增片段进行序列测定与分析,结果表明该序列与已报道的鳜(Siniperca chuatsi)传染性脾肾坏死病毒(Infectious Spleen and Kidney Necrosis Virus,ISKNV)MCP基因同源性为98%.电镜观察和MCP基因测序分析结果显示,该病毒的分类地位为虹彩病毒科(Iridoviridae)细胞肿大病毒属(Megalocytivirus).  相似文献   

10.
2021年7月,浙江省象山某养殖场养殖的大黄鱼(Larimichthys crocea)出现类似大黄鱼虹彩病毒引起的疾病。采用鲤上皮瘤细胞培养和病毒主要衣壳蛋白测序分析的方法,从患病的大黄鱼中分离到一株病毒。该病毒接种到鲤上皮瘤细胞(EPC)后出现空斑、脱落的细胞病变症状。根据虹彩病毒MCP和ATPase基因保守序列设计特异性引物对病毒组织样本进行PCR扩增,得到分别为1 367 bp和740 bp的目的基因片段。将MCP基因扩增片段测序,经BLAST对比及系统发育树聚类分析,确定该分离的病毒属虹彩病毒科细胞肿大病毒属。通过蔗糖密度梯度离心纯化,用透射电镜观察该病毒粒子呈正六边形,直径为120~150 nm。用纯化病毒作为抗原免疫小鼠获得抗大黄鱼虹彩病毒的多克隆抗体,效价为1∶7 000;通过SDS-PAGE和Western blotting初步确定3个免疫蛋白。本研究为大黄鱼虹彩病毒纯化提供一种新方法,并初步分离出免疫蛋白,为该病毒相关分子生物学研究、蛋白研究以及疫苗制备等提供理论依据。  相似文献   

11.
Molecular characterization was carried out on an iridovirus isolated from yellow grouper, Epinephelus awoara . The major capsid protein (MCP) gene was located, sequenced and compared with homologous genes from other iridoviruses. The nucleotide sequence is 1392 bases long and contains a single open reading frame beginning at an ATG codon from the 5' end and terminating at a TAA codon at the 3' end. The open reading frame encodes a protein of 463 amino acids with a predicted molecular weight of 50 272 Da. Pairwise amino acid alignments detected a high degree of sequence identity between grouper iridovirus (GIV) MCP and the homologous genes of other iridoviruses. The MCP gene of GIV was most similar to the MCP gene from frog virus 3 (FV3) with 70% nucleotide and 73% amino acid sequence identity. The predicted molecular weight of the protein of this gene is comparable with the apparent weight obtained by SDS–PAGE. Pathogenicity of the GIV was investigated in yellow grouper by intraperitoneal injection of 107 and 104 TCID50 virus. Cumulative mortalities reached 100% within 11 and 25 days post-infection, respectively, while no grouper died in the control group. The molecular studies demonstrated that GIV is a member of the genus Ranavirus .  相似文献   

12.
Grouper Epinephelus spp. is one of the most important mariculture fish species in China and South-East Asian countries. The emerging viral diseases, evoked by iridovirus which belongs to genus Megalocytivirus and Ranavirus, have been well characterized in recent years. To date, few data on lymphocystis disease in grouper which caused by lymphocystis disease virus (LCDV) were described. Here, a novel LCDV isolate was identified and characterized. Based on the sequence of LCDV major capsid protein (MCP) and DNA polymerase gene, we found that the causative agents from different species of diseased groupers were the same one and herein were uniformly defined as grouper LCDV (GLCDV). Furthermore, H&E staining revealed that the nodules on the skin were composed of giant cells that contained inclusion bodies in the cytoplasm. Numerous virus particles with >210 nm in diameter and with hexagonal profiles were observed in the cytoplasm. In addition, phylogenetic analysis based on four iridovirus core genes, MCP, DNA polymerase, myristoylated membrane protein (MMP) and ribonucleotide reductase (RNR), consistently showed that GLCDV was mostly related to LCDV-C, followed by LCDV-1. Taken together, our data firstly provided the molecular evidence that GLCDV was a novel emerging iridovirus pathogen in grouper culture.  相似文献   

13.
‘Gold standard’ OIE reference PCR assay was utilized to detect the presence of infectious spleen and kidney necrosis virus (ISKNV) in freshwater ornamental fish from Malaysia. From total of 210 ornamental fish samples representing 14 species, ISKNV was detected in 36 samples representing 5 fish species. All positive cases did not show any clinical signs of ISKNV. Three restriction enzymes analyses showed that the fish were infected by identical strains of the same virus species within Megalocytivirus genus. Major capsid protein (MCP) genes of 10 ISKNV strains were sequenced and compared with 9 other reference nucleotide sequences acquired from GenBank. Sequence analysis of MCP gene showed that all strains detected in this study were closely related to the reference ISKNV with nucleotide sequence identity that was ranging from 99.8% to 100%. In addition, phylogenetic analysis of MCP gene revealed that viruses from genus Megalocytivirus can be divided into three genotypes: genotype 1 include reference ISKNV and all other strains that were detected in this study, genotype 2 include viruses closely related to red sea bream iridovirus (RSIV), and genotype 3 include viruses closely related turbot reddish body iridovirus (TRBIV).  相似文献   

14.
Iridoviruses infect a wide variety of wild and cultured fish. Those iridoviruses belonging to the genus Ranavirus, in the Iridoviridae family, cause systemic disease in infected animals with a high morbidity and mortality. This paper reports the cloning, sequencing, and expression of the rock bream iridovirus (RBIV) major capsid protein (MCP) in an Escherichia coli expression system for subsequent immunological studies. The completeness of the expressed protein was confirmed by peptide mass fingerprinting (PMF) analysis using MALDI-TOF MS. The recombinant MCP (rMCP)-specific mouse polyclonal antibody reacted with the viral 52 kDa protein, indicating that this rMCP induces an immunological response. Fish antibodies induced against iridovirus infection were also detected using ELISA when rMCP was used as an antigen. As a result, it was found that many cultured rock bream (92.5%) were naturally infected with iridovirus and that the rMCP might be useful for serological tests.  相似文献   

15.
为了早期快速诊断近年来流行于广东省养殖大口黑鲈(Micropterussalmoides)中的病毒性溃疡综合征,本研究用基因组步移的方法获得了大口黑鲈溃疡综合征病毒(Largemouthbassulcerativesyndromevirus,LBUSV)主要衣壳蛋白(MCP)基因,该基因编码区全长1392bp。通过序列比较分析,在MCP基因内确定了一段241bp的特异性较强的片段作为靶序列,设计并合成引物,经过优化PCR反应条件,建立了可以快速检测大口黑鲈溃疡综合征病毒的PCR方法。实验表明,在PCR进行到30个循环反应时可以检测到的质粒最小浓度是104拷贝数/μL,相当于104个病毒粒子。利用该方法,从天然感染LBUSV的大口黑鲈脾脏组织DNA可扩增出241bp的片段,而健康大口黑鲈和感染了传染性脾肾坏死病毒样病毒的大口黑鲈脾脏组织则没有扩增条带。本研究建立的PCR检测方法具有检测快速、成本低、准确性高的特点,适用于大范围早期病害诊断的推广应用。  相似文献   

16.
2008年11月~2010年11月,采集山东海域大菱鲆、石鲽、鲈鱼各20批,按照世界动物卫生组织推荐的PCR检测方法对真鲷虹彩病毒病(Red Sea Bream Iridoviral Disease,RSIVD)进行初步调查.结果显示,共检出4例RSIVD感染样品.以真鲷虹彩病毒(Red Sea Bream Iridovirus,RSIV)和传染性脾肾坏死病毒(Infectious Spleen and Kidney Necrosis Virus,ISKNV)主要衣壳蛋白基因为基础,设计简并引物,PCR扩增本次检出阳性样品的RSIV/ISKNV MCP基因.将MCP基因PCR扩增产物测序,提交GenBank,并以MCP基因为基础,对被检出的阳性样品进行虹彩病毒属系统分类,绘制进化树.由进化树得出,4例阳性病毒株均属于虹彩病毒科细胞肿大病毒属.  相似文献   

17.
Epizootic haematopoietic necrosis virus: purification and classification   总被引:1,自引:0,他引:1  
Abstract. Epizootic haematopoietic necrosis virus (EHNV) has been isolated from redfin perch, Perca fluviatilis L., and cultured rainbow trout, Oncorhynchus mykiss (Walbaum), in Victoria and New South Wales, Australia. On the basis of virion size, icosahedral morphology and ultrastructural characteristics of virus-infected cells, EHNV was designated as an iridovirus. The following observations outlined in this paper confirm the designation: (1) The number and complexity of proteins in purified EHNV and the ordered appearance of virus-specific proteins in infected cells are similar to that described for the iridovirus, frog virus 3 (FV3). (2) EHNV isolated from the cytoplasm of infected cells contains a lipid membrane associated with 7–9 nm capsomeric subunits containing a 52-kD protein. (3) The morphogenesis of EHNV within cytoplasmic inclusion bodies resembles that of FV3 and a piscine iridovirus isolated from goldfish, Carassius auratus (L.) (4) EHNV also resembles FV3 in its association with the cytoskeleton of infected cells. (5) EHNV DNA, like that of other iridoviruses, is resistant to digestion with HpaII, a restriction endonuclease which does not cleave DNA at CCGG sequences where the internal cytosine is methylated.  相似文献   

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