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鸡传染性法氏囊病超强毒Gx株感染性分子克隆的构建   总被引:2,自引:0,他引:2  
本研究以鸡传染性法氏囊病超强毒Gx株(野毒株)基因组为模板,用蛋白酶K法提取病毒基因组核酸dsRNA,在cDNA克隆的5'端上游引入了T7启动子序列,采用Long-accurateRT-PCR(LA-PCR)一步法扩增并克隆了病毒基因组A节段与B节段全长cDNA。序列测定结果表明,基因组A节段全长共3267个核苷酸,包括5'及3'端的非编码区和两个部分重叠的开放阅读框,基因组B节段全长共2843个核苷酸,包括5'及3'端的非编码区和一个开放阅读框。将IBDV-Gx株的A节段全长基因组及B节段全长基因组分别克隆入pMD18-T载体,构建成pMD-A、pMD-B两个带有T7启动子的重组质粒。两个重组质粒线性化后,进行体外转录,然后共电转染于鸡胚成纤维细胞37℃培养72h并传代。收获的细胞传代培养物分别用RT-PCR、间接免疫荧光、蚀斑试验等方法进行鉴定,结果用RT-PCR扩增出了VP3及VP5基因片段,间接免疫荧光检测到了特异性的荧光抗体,蚀斑试验结果表明蚀斑形成单位为3×103PFU/mL。  相似文献   

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为研究鸡传染性法氏囊病病毒超强毒株(vvIBDV)细胞嗜性改变的分子基础,本研究通过定点突变、重叠延伸PCR(SOE-PCR)等技术,以vvIBDV HLJ-0504株为骨架构建了两组感染性克隆,并借助已建立的反向遗传操作技术进行病毒拯救。IFA检测、电镜观察及RT-PCR鉴定均显示:Q253H/A284T双点突变的pCAGGHLJ0504A889/980HRT和pCAGGHLJ0504BHRT共转染DF1细胞成功拯救出重组病毒(rHLJ0504HT),而未进行双点突变的pCAGGHLJ0504AHRT和pCAGGHLJ0504BHRT共转染组未获得重组病毒。上述结果表明,双点突变Q253H/A284T能使vvIBDV HLJ-0504适应非允许细胞CEF,但未进行Q253H/A284T双点突变的vvIBDV HLJ-0504不能感染非允许细胞CEF,因此,该研究表明VP2的Q253H/A284T两个氨基酸突变是vvIBDV细胞嗜性改变的分子基础。  相似文献   

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扈留轩  张俊红 《中国家禽》2002,24(11):23-24
1法氏囊 禽类法氏囊是雏鸡免疫系统的中枢免疫器官.它是鸟类特有的免疫器官,所以值得特别关注.许多研究已搞清了其生理学作用.雏鸡前3周是法氏囊快速发展时期,在这个时期法氏囊迅速增殖,3周龄后法氏囊就逐渐退化,研究表明,大约在12周龄,法氏囊就完全退化.  相似文献   

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Clinical outbreaks of severe acute infectious burial disease (IBD) were recorded since the mid- and late 1990s in several countries in the southeastern part of Europe. Epidemiologic data showed that both infectious bursal disease virus (IBDV)-vaccinated and IBDV-nonvaccinated chickens were affected with acute IBD and mortality up to 50% independent of the IBDV vaccination status of the appropriate parent flocks. For investigation of the causative agent of acute IBD, the variable region of VP2 was amplified, cloned, and sequenced. Nucleotide sequence analysis of polymerase chain reaction fragments showed several silent nucleotide exchanges in comparison with the sequence of the very virulent (vv) IBDV strain UK661. Also, restriction enzyme cleavage sites proposed specific for vvIBDV were present in all investigated strains. On the basis of clinical signs in affected flocks, recorded epidemiologic data, and sequence analysis, it is very likely the IBD-causing strains were of the vv phenotype.  相似文献   

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为了解传染性法氏囊病病毒(IBDV)HLJ-0504株的分子生物学特征,本研究利用融合PCR技术获得HLJ-0504株全基因组序列。核苷酸和推导氨基酸序列遗传演化分析发现,HLJ-0504A节段位于IBDV超强毒的分枝上,而B节段则介于超强毒株和减毒株之间,形成一个独立分枝,属于一株新的自然重组病毒。VP2抗原性预测分析表明,HLJ-0504株VP2第Ⅰ亲水区中的氨基酸发生突变(D212N),该突变可能导致了HLJ-0504株抗原性发生漂变。本实验结果为深入研究IBDV的分子特征奠定了基础。  相似文献   

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Gao HL  Wang XM  Gao YL  Fu CY 《Avian diseases》2007,51(4):893-899
The complete genomic sequence of very virulent infectious bursal disease virus (vvIBDV) Gx strain was determined, including the sequences of segment A, encoding the precursor polyprotein, and segment B, encoding the viral RNA polymerase (VP1) and 5'- and 3'-untranslating regions. Alignment of segment A of Gx with the sequences of 12 other vvIBDV strains showed 97.5% to 99.0% amino acid identity, whereas alignment of segment B of Gx with nine other vvIBDV strains revealed high sequence divergence, ranging from 10.3% to 11%. Phylogenetic analysis of segments A and B showed that they were in different branches, indicating that the reassortment occurred in this strain and that segment A and segment B derived from different pathotype strains. The mutant spectrum analysis of quasispecies virus demonstrated that the mean minimum mutation frequency in VP1 was 8.78-fold higher than in the polyprotein. The most frequent mutations were in the first 1986 nucleotides (nonsynonymous mutations) and the last 660 nucleotides (synonymous mutations), indicating that the 219 amino acid residues in the C-terminal of the VP1 form a functional region.  相似文献   

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为研制能够同时预防传染性法氏囊病(IBD)和新城疫(ND)的疫苗,本研究选用表达IBD病毒(IBDV)超强毒株(vvIBDV)VP2基因的重组ND病毒(NDV)LaSota疫苗株(rL-VP2),测定其半数鸡胚感染量、平均鸡胚致死时间、脑内接种指数和静脉内致病指数等指标,按不同剂量接种18胚龄SPF鸡胚,分别于出雏后第9d、第14d和第21d采血,用微量凝集法和ELISA方法测定血清中抗NDV抗体和抗IBDV抗体水平,并于出雏后28d用NDV强毒(F48E9株)和vvIBDVGx株攻毒,评估重组疫苗的胚胎免疫效果。结果显示:按104EID50/枚剂量进行免疫不会影响SPF鸡胚出雏率和出雏后21d存活率,并且该免疫组雏鸡能够对NDV强毒和vvIBDV攻毒提供安全的免疫保护。  相似文献   

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The potential role of the mast cells in the invasion of very virulent infectious bursal disease virus (vvIBDV) is unknown. We evaluated mast cell activity and tryptase production after vvIBDV infection in special pathogen-free (SPF) chickens using cytochemistry and immunohistochemistry analyses. The results were as follows: (1) severe histologic lesions were observed in the thymus, spleen, cloacal bursa, liver, kidney and other tissues. vvIBDV viral antigens were detected and presented extensively in the parenchymatous organs, in particular, the cloacal bursa, liver, kidney, thymus, spleen and pancreas. (2) In the vvIBDV-infected group, the mast cell population increased markedly in the liver, kidney, thymus, glandular stomach, spleen and cloacal bursa on days 1, 2 and 3 after vvIBDV infection (p<0.05). However, very few mast cells were observed in those same tissues in the controls, especially in the bursa of Fabricius. (3) Tryptase, a marker for activated mast cells, has a positive correlation with mast cell distribution. The mast cells identified in the tissues were likely to be activated since they were associated with cell degranulation and the presence of tryptase. Furthermore, the co-localization of mast cells, and presence of vvIBDV antigens suggests that the mast cells were activated by vvIBDV infection. Our results also suggest that tryptase may contribute to the inflammation of acute IBD induced by vvIBDV infection. Our research contributes to the further understanding of inflammatory response mechanisms and the contribution of mast cell activity to this process.  相似文献   

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Banda A  Villegas P 《Avian diseases》2004,48(3):540-549
Very virulent infectious bursal disease viruses (vvIBDVs) were detected in phenol inactivated bursal samples obtained from Brazil, the Dominican Republic, and Venezuela. After nucleotide sequence analysis of the hypervariable region of VP2 gene, the vvIBDVs from Brazil and Venezuela exhibited all of the 14 nucleotide changes that are conserved in the European UK-661 and most other vvIBDV strains. However, the vvIBDV from the Dominican Republic presented 11 nucleotide changes that are conserved in vvIBDV strains. After phylogenetic analysis, the Latin American strains were found to be related to other vvIBDV strains from Europe, Asia, and Africa. However, Brazilian and Dominican vvIBDVs clustered in two separate subgroups, while the vvIBDVs from Venezuela were closely related to other strains from other parts of the world. By deduced amino acid sequence, the three conserved amino acid residues in vvIBDV strains (222 Ala, 256 Ile, and 294 Ile) were confirmed in the Latin American viruses, and one amino acid change (300 Ala) was unique to all vvIBDVs from the Dominican Republic. The occurrence of this change in the Dominican vvIBDVs may have an impact in their antigenic makeup. Results of this study indicate that the vvIBDVs detected in Latin America are genetically similar to IBDV strains from other parts of the world. However, vvIBDVs from Venezuela were more similar to the vvIBDV strains from Europe and Asia. Of all the samples analyzed, vvIBDVs from Brazil and the Dominican Republic exhibited more genetic changes. These changes may have emerged as a result of the different management practices and environmental conditions present in each particular geographic area.  相似文献   

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Comparisons between sequences of very virulent, virulent, and attenuated strains of the infectious bursal disease virus (IBDV) may indicate sites on the genome co-inciding with virulence. In an attempt to detect if such sites exist on the coding region of segment B, viral protein 1 (VP1) (encoded for by segment B) of a very virulent Israeli virus, IL3; its attenuated strain, IL4; and the attenuated Winterfield vaccine 2512 were cloned and sequenced. A comparison was made among them and with six other published sequences of segment B. Six nucleic acids distinguished between IL3 and IL4, three of which were predicted to be expressed as amino acids. A striking similarity between the VP1 sequences of 2512 and P2 (an attenuated German strain) was discovered. Although conclusions could not be drawn concerning attenuation sites on VP1, the analysis performed on the VP1 sequences of the two Israeli strains and the Winterfield 2512 strain sheds light on the phylogeny of IBDV and contributes to the accumulating information that may lead to the identification of virulence-related sites of this virus.  相似文献   

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Recent studies here have demonstrated that increased mast cell populations and tryptase activity contribute to lesion formation in regions of immune organs in special-pathogen-free chickens after infection with very virulent infectious bursal disease virus (vvIBDV). Mast cells and their mediators have been implicated in acute inflammatory injury after vvIBDV infection, but their precise role in this process remains elusive. In this study, the role of mast cells in the vvIBDV infection process was examined using ketotifen, a mast cell membrane stabilizer. On days 1, 2, and 3 postinfection, the bursa of Fabricius (BFs) were collected to quantify mast cells, tryptase and histamine contents by cytochemistry, immunohistochemistry and fluorospectrophotometry analyses, respectively. The results showed that the mast cell populations, tryptase expression, and histamine released increased significantly in the BFs (p < 0.01) of infected birds compared to controls, and acute inflammatory responses were observed in the former. In contrast, in infected chickens pretreated with ketotifen, mast cells, tryptase, and histamine were markedly decreased (p < 0.01) and probably as a result, the BFs remitted significantly. The overall results suggest that mast cells are positively involved in BF injury induced by vvIBDV infection. Inhibition of mast cell degranulation and concurrent mediator release may represent a novel strategy to modulate this process. This study, thus, advances the understanding of the acute inflammatory injury mechanisms triggered by vvIBDV infection and the contribution of mast cell activity in this process.  相似文献   

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本研究从浙江余姚、上虞、海南3个疑似发生传染性囊病的鸡场采集病料共20份,通过观察临床症状、病理变化、RT-PCR检测、SPF鸡胚接种、基因测序等试验,证实鸡场送检样品的鸡群发生了传染性囊病,且分离到5株传染性囊病病毒,对VP2基因进行测序分析后,发现具有超强毒株的特征。并与Gen Bank上的已知的强毒OKYM D49706、HK46 AF092943、D78 AF499929、Harbin-1 AF454945的VP2氨基酸序列的同源性在99.22%。说明此次分离的5株病毒与已知病毒存在一定的差异性。为以后的IBDV病毒的研究提供了一定的依据。  相似文献   

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为检测传染性法氏囊病超强毒株(vvIBDV)重组VP2蛋白的免疫原性,本研究利用RT-PCR方法扩增vvIBDV的结构蛋白VP2基因,并将其克隆到表达载体pGEX-4T-3中,构建重组表达质粒pGEX-VP2。将其转化受体菌E.coli BL21(DE3)plysS,经IPTG诱导后,SDS-PAGE电泳和western blot分析表明,表达的重组蛋白约69 ku,并以包涵体形式存在。表达的重组蛋白经纯化后,免疫6周龄BALB/c小鼠制备免疫血清,ELISA分析表明制备的血清效价在1∶5 120以上,表明vvIBDV VP2具有良好的免疫原性,为建立vvIBDV的ELISA检测方法提供了试验依据。  相似文献   

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