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標題: 鴨環狀病毒感染性選殖株之構築與核酸疫苗 之研發
Construction of infectious clones and development of DNA vaccines for duck circovirus
作者: 陳巧津
Chen, Ciao-Jin
關鍵字: duck circovirus
infectious clones
DNA vaccine
出版社: 獸醫微生物學研究所
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摘要: 鴨環狀病毒(Duck Circovirus,DuCV)為新興的水禽感染性病原體,主要侵犯鴨隻淋巴器官而引起免疫抑制,進而增加其它病原體感染機率。本研究有兩個目標,一為構築DuCV感染性選殖株(Infectious clone),並藉以研究DuCV基因表現之調控機制,二為研發DuCV核酸疫苗,以協助DuCV疫情之控制。在感染性選殖株之構築方面,本研究以PCR增幅DuCV之全長DNA,再進行接合反應,以形成DuCV之感染性DNA。構築方法是依據DuCV病毒株之序列所設計,經定點突變含有Eco RI切位,可與野生型區分。在動物實驗方面,每隻正番鴨採肌肉注射施打1.4 μg劑量之感染性DNA,攻毒後犧牲鴨隻取胸腺、華氏囊抽取病毒DNA並分析其序列,證實所構築之感染性DNA可成功感染鴨隻,並於其體內複製。DNA疫苗之製備則是將全長外殼蛋白(Capsid protein)基因選殖到載體pcDNA3.1/ TOPO,並以肌肉注射施打400 μg劑量,追加一次劑量後以DuCV野外株進行攻毒之後與對照組比較,定量PCR分析發現有施打DNA疫苗的鴨隻中,其華氏囊中之病毒量明顯較未施打疫苗的對照組少,顯示DNA疫苗亦有效。此外本研究也將全長之DuCV Capsid protein基因選殖到表現載體pET32a,再轉型到E. coli BL21 codon plus RIPL 中,大量表現重組蛋白,再以這些重組蛋白為抗原,利用western blot技術,偵測攻毒後不同週數鴨隻中血清抗體力價的變化。
Duck circovirus (DuCV) infection is an emerging disease of waterfowls. This disease invades the lymphoid organ of ducks and subsequently leads to immune suppression and increased incidence of other infections. The goal of this study is divided into two parts. The first is to construct infectious DNA of DuCV for investigating mechanisms involved in regulation of gene expression of DuCV, whereas the second is to develop DNA vaccine for the control of DuCV. Toward the first goal, we used PCR to amplify the full-length genome of DuCV. After ligation, the amplified DNA could serves as infectious DNA of DuCV. The infectious DNA of DuCV amplified by PCR contained an Eco RI cutting site and thus could be discriminated from wild-type viruses. For animal experiments, each Muscovy duck was injected intramuscularly with 1.4 μg of infectious DNA. After the injection, the DuCV DNA could be detected in the thymus and bursa of Fabricius of ducks. This result suggests that the infectious DNA could replicate in ducks. The DNA vaccine was constructed by cloning the gene encoding the capsid protein of DuCV into the vector pcDNA3.1/TOPO. Each Muscovy duck was injected with 400 μg of DNA and after a booster immunization, the ducks were challenged with a field strain of DuCV. The result of quantitative PCR tests showed that the virus titers in vaccinated ducks was lower than those in the control ducks. This result indicates that the DNA vaccine is effective. This study also clones the gene encoding the capsid protein of DuCV into the expression vector pET32a. After transformation into E. coli strain BL21 codon plus RIPL, the recombinant capsid protein was highly expressed. This recombinant was used as the antigen for Western blot analyses to determine the antibody tires against DuCV in ducks challenged with the virus.
其他識別: U0005-2008200801180100
Appears in Collections:微生物暨公共衛生學研究所



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