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標題: 鴨環狀病毒感染性選殖株之構築與病毒ORF3基因功能分析
Construction of infectious clone and functional study of the ORF3 gene from duck circovirus
作者: 藍順瀚
Lan, Shun-Han
Contributors: 張伯俊
Poa-Chun Chang
微生物暨公共衛生學研究所
關鍵字: 鴨環狀病毒;感染性選殖株
duck circovirus;DuCV;infectious clone;open reading frame 3;ORF3
日期: 2012
Issue Date: 2013-11-19 12:51:30 (UTC+8)
Publisher: 微生物暨公共衛生學研究所
摘要: 鴨環狀病毒(Duck Circovirus, DuCV)為新興的水禽感染性病原,會造成鴨隻出現生長遲緩、體重下降等症狀。病毒主要侵犯鴨隻淋巴器官,感染鴨隻後會引發免疫抑制及可能之二次感染,對養殖業所衍生之經濟損失不容小覷。由於DuCV目前尚無任何活體外(in vitro)培養系統可應用,病毒基因功能與致病機轉尚未完全釐清。感染性選殖株(infectious DNA clone)具有可修飾病毒基因體之優點,為研究環狀病毒致病機轉與疫苗開發之重要工具,且質體(plasmid)的保存比病毒容易,大量製備也非常便利。因此,本研究有兩個主要目標,一為構築DuCV毒株TC4/2002之感染性選殖株,另一目標則是分析DuCV ORF3基因之功能。在感染性選殖株方面,首先設計一組兩端含Pst I切位之引子,以PCR增幅DuCV之全長基因體(DNA genome),將其選殖至載體pCRII-TOPO,即可利用E. coli進行增殖並抽取大量質體,之後使用限制酵素Pst I進行反應,將目標DNA切下,進行電泳分離和膠體DNA純化,回收DuCV之全長雙股DNA,再進行自體接合(self-ligation)反應,即形成鴨環狀病毒感染性DNA (infectious DNA)。動物實驗方面,於土番鴨(Mule duck)腿部接種35 μg之鴨環狀病毒感染性DNA,接種方式採肌肉內注射(I.M.),施打後每周分離周邊血液單核細胞(PBMC)並萃取核酸,利用PCR進行檢測病毒DNA並分析其序列,證實所構築之感染性DNA可成功感染鴨隻,並於其體內增殖。鴨環狀病毒ORF3基因功能之研究方面,首先設計一組含限制酵素切位和Kozak sequence之引子,以PCR增幅完整的ORF3基因,將其構築至載體pEGFP-N1並利用E. coli進行增殖質體,之後將重組質體轉染至鴨胚纖維母細胞(duck embryo fibroblast, DEF),培養24小時後,以倒立式螢光顯微鏡進行分析。顯示毒株TC4/2002之ORF3表現後,蛋白主要位於細胞核內,此結果指出毒株TC4/2002之ORF3具有核定位(nuclear localization)之功能,此現象與豬環狀病毒第二型之ORF3相似,可能與病毒誘發淋巴細胞凋亡之能力相關。另一方面,本研究利用定位點突變(site-directed mutagenesis)技術,剔除(knock out)鴨環狀病毒感染性選殖株的ORF3基因,成功構築DuCV ORF3 null-infectious clone並進行土番鴨動物實驗,結果顯示ORF3基因缺損之感染性選殖株於鴨隻體內之複製能力與正常之感染性選殖株並無顯著差異,顯示ORF3基因之缺損,並不影響病毒之複製,此結果與PCV2 ORF3基因之有無並不影響病毒複製的報導相符。本研究已成功建立鴨環狀病毒感染性選殖株,未來可配合分子技術進行基因體修飾,以探討DuCV之致病機轉與基因功能,並協助鴨環狀病毒疫苗之開發,對鴨環狀病毒之防疫工作將有所助益。
Infections with duck circovirus (DuCV) are associated with growth retardation and developmental problems in farmed ducks. DuCV invades lymphoid tissues and lead to immunosuppression and an increased probability of secondary infections. This disease has been the cause of considerable economic losses to the duck industry worldwide. Because no in vitro culture system is yet available for propagation of DuCV, the pathogenesis of this virus remains unclear. The availability of infectious clones offers an opportunity for analysis and modification of viral genomes at the molecular level and has greatly aided research on virus replication, pathogenesis and vaccine development. The objective of this study is divided into two parts. The first is to construct infectious DNA clone of TC4/2002 strain of DuCV, whereas the second is to investigate the function of the ORF3 gene. Toward the first objective, the complete DuCV DNA genome was cloned into the vector pCRII-TOPO. After the DuCV genome was excised from the plasmid and circularized by ligation to produce the DuCV infectious DNA, the DNA mixture was inoculated in Mule ducks by intramuscular route. The DuCV DNA could be detected in PBMC, thymus and bursa of ducks by PCR weekly thereafter. This result suggests that the infectious DNA could initiate active DuCV infection. Secondly, the ORF3 gene of DuCV including complete open reading frame was amplified by PCR, and it was cloned into the vector pEGFP-N1. The recombinant plasmid was transfected into duck embryo fibroblast (DEF). After incubation for 24 hours, expression of ORF3 was analyzed by using fluorescence microscope. The ORF3 protein was dominantly localized in the nucleus. This result indicate that there is nuclear localization signal on the ORF3 of TC4/2002 strain of DuCV. It is possible that ORF3 of DuCV might induce apoptosis similar to PCV2. On the other hand, site-directed mutagenesis was performed in the DuCV infectious clone to knock out the ORF3 gene and the effect of null of ORF3 on the ability of replication of the muDuCV and the wildtype-DuCV in duck was investigated. The result shows that DuCV ORF3 protein is not essential for replication similar to PCV2.
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