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標題: 利用反向遺傳學技術構築重組傳染性華氏囊病毒載體
Construction of recombinant infectious bursal disease viruses expressing foreign proteins by reverse genetics technology
作者: 汪語心
Yu-Hsin Wang
關鍵字: 傳染性華氏囊病;反向遺傳學技術;病毒載體;Infectious bursal disease virus;Reverse gentics;viral vector
反向遺傳學技術已經成功地開發了許多RNA病毒作為遞送外來抗原的病毒載體。傳染性華氏囊病毒(Infectious bursal disease virus, IBDV)是雞傳染性華氏囊病(Infectious bursal disease, IBD)的病原,然而最近因其作為外來抗原遞送系統的潛力而持續的被研究中。本研究的目的是利用反向遺傳學技術構築重組IBDV作為外來抗原遞送系統。通過使用反向遺傳學技術,以強毒型IBDV (vvIBDV)毒株P3009為骨架,插入或置換部分基因序列來產生重組IBDVs,將含有新城病毒(Newcastle disease virus, NDV)融合(Fusion protein, F)蛋白的病毒中和抗體決定位的基因插入IBDV的非結構VP5的起始密碼子後端,形成新的重組病毒命名為rP3009/VP5-F。NDV的全長F基因或增強型綠色螢光蛋白(EGFP)基因被用來替代IBDV VP3的部分序列。分別命名為rP3009/VP3-F和rP3009/VP3-EGFP。以間接免疫螢光分析法分析表達的外來蛋白,證實成功產生含有NDV之VN抗原決定位或EGFP的重組IBDVs。重組病毒rP3009 / VP5-F和rP3009 / VP3-EGFP之複製效率與未插入外來基因之IBDV重組病毒 (rP3009)相似,但在感染後36, 48, 60小時則其病毒複製效率較rP3009低。然而,實驗中並無法有效的增殖出含有F蛋白的重組IBDV (rP3009/VP3-F)。這些結果證實於VP5 N端插入或VP3基因部分置換適當大小的蛋白,皆能形成重組病毒。因此,IBDV可以成為一個病毒載體系統於VP5或VP3中攜帶外來基因以開發雙價疫苗。

Reverse genetics technology has been successfully developed for numerous RNA viruses as virial vectors for foreign antigen delivery. Recently, infectious bursal disease virus (IBDV), a causative agent of infectious bursal disease (IBD) in chicken, has been explored for its potential to serve as a deliver system. The purpose of this study was to construct a recombinant IBDV as a foreign antigen delivery system by reverse genetics technology. By using the reverse genetics technology, recombinant IBDVs containing insertions or substitutions of sequences encoding the foreign proteins were engineered base on the backbone of very virulent IBDV strain, P3009. The sequences containing virus-neutralizing (VN) epitopes of Newcastle disease virus (NDV) fusion (F) protein was inserted into the N-terminus of the nonstructural VP5 of IBDV to rescue recombinant IBDV designated as rP3009/VP5-F. The F gene of NDV or enhanced green fluorescent protein (EGFP) gene was used to substitute partial sequences of IBDV VP3 to rescue recombinant IBDV designated as rP3009/VP3-F and rP3009/VP3-EGFP, respectively. The recombinant IBDVs containing VN epitopes or EGFP were successfully rescued and the expressed foreign proteins were recognized by indirect immunoflurecence assay and western blotting. However, the rescue of the recombinant IBDV containing F protein had failed. When compared to the recombinant rP3009 (without containing ant foreign gene), the growth kinetic of recombinant rP3009/VP5-F and rP3009/VP3-EGFP showed similar pattern with significant lower virus at 36, 48 and 60 hours after infection in DF-1 cells. These results indicated that VP3 and the N-terminus of VP5 of IBDV has shown to be capable of tolerating the substitution and insertion of small proteins, respectively. Thus, IBDV is potentially employed as a viral vector to carry foreign proteins fused with VP5 or VP3 to develop bivalent vaccines.
Rights: 同意授權瀏覽/列印電子全文服務,2020-08-28起公開。
Appears in Collections:微生物暨公共衛生學研究所

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