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標題: Establishment of Recombinant Orf Virus Expressing Goatpox virus P32 Proteins
作者: 趙譯棋
I-Chi Chao
關鍵字: 羊傳染性化膿性病毒;羊痘病毒;Orf virus;goatpox virus
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Orf virus (ORFV), belonging to parapoxvirus, causes skin lesions in infected sheep and goats. Due to the restricted host range, the skin tropism, the absence of systemic virus spread, and the short-lived ORFV vector-specific immunity allowing repeated immunizations, it has been successfully used as a novel viral vector system for expressing foreign antigens to prevent infectious diseases of swine, rabbit, and avian. Hence, study herein aimed to establish ORFV as a bivalent vaccine platform for goat. In 2008, goatpox (GP) outbreaks caused substantial loss in the production and productivity of goats in Taiwan. Considering that control of goatpox virus (GPV) infection relies on foreign vaccine, the goal of current study is to construct a recombinant ORFV that would express goatpox virus P32, the major immunogenic protein. To do so, coding regions of GPV P32 and eGFP that serves as a selection marker were inserted between the flanking sequences, i.e. open reading frame 127 and 128 of the ORFV genome. By homologous recombination, recombinant ORFV with GPV p32 gene (GPV P32-ORFV) was generated. At the present, the presence and expression of GPV P32 gene were confirmed by polymerase chain reaction and western blot analysis, respectively. The immune characteristics of GPV P32-ORFV will be further tested in animal models.

羊傳染性化膿性病毒(Orf virus,ORFV) 隸屬於痘病毒科的副痘病毒屬,發生於世界各地的山羊與綿羊,其症狀為造成口腔潰瘍和膿皰等,對於成羊來說發生率和死亡率不高,但對於年幼動物ORFV的感染具有致死性,ORFV為具有封套,含雙股DNA的基因體,由於其基因體較大,具有非必要基因可與外源基因置換,基於其限制性的宿主、局部性的皮膚感染、可重複接種等特性,ORFV作為載體之發展具有相當高的潛力,而前人的研究中也有以ORFV為載體表達外源性蛋白,例如:狂犬病的醣蛋白質基因、禽流感的HA、NA、豬瘟病毒E2 蛋白等,台灣於2008年山羊痘疫情爆發,對飼養的經濟動物產業造成嚴重的經濟損失,而為了控制其疫情皆有賴於國外疫苗之進口,台灣目前並無研發和自製之羊痘疫苗,有鑑於此本研究試圖以ORFV為載體,用以架構可以表達羊痘病毒的主要免疫源蛋白(P32)的重組病毒。製備重組病毒所需的transfer vector中含有自ORFV基因體中增幅出的open reading frame 127以及128核酸序列;以此作為flanking sequence,透過homologous recombination將羊痘病毒P32 基因重組入ORFV基因體中。經由PCR、南方墨點法檢測基因型,並以西方墨點法、免疫螢光偵測P32蛋白質的表現,確認已成功製備P32-ORFV重組病毒,未來將藉由動物實驗以證實其所產生之免疫反應。
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