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Title: | 貓傳染性腹膜炎病毒單株抗體之生產與定性 Generation and Characterization of Monoclonal Antibodies against Feline Infectious Peritonitis Virus |
Authors: | Ya-Yun Lu 呂亞芸 |
Advisor: | 張晏禎(Yen-Chen Chang) |
Keyword: | 貓傳染性腹膜炎病毒,單株抗體,中和抗體表位,交叉反應, feline infectious peritonitis virus,monoclonal antibody,neutralizing epitope,cross-reactivity, |
Publication Year : | 2022 |
Degree: | 碩士 |
Abstract: | 貓傳染性腹膜炎病毒 (FIPV) 屬於冠狀病毒科,是具有封套的正鏈單股核糖核酸病毒。由於目前仍缺乏有效的疫苗和治療方法,FIPV至今仍是高致死性的疾病,尤其在幼貓。FIPV基因可轉譯出11種蛋白質,包括四種結構蛋白,即棘蛋白(spike, S)、封套蛋白(envelop, E)、膜蛋白(membrane, M)和核殼蛋白(nucleocapsid, N),以及數種非結構蛋白。S蛋白與病毒進入細胞有關且具有中和表位(epitope)。根據S蛋白的序列,FIPV可再被細分為第一血清型(FIPV I)和第二血清型(FIPV II)。雖然在世界各地FIPV I是主要流行的血清型,但其在體外分離和培養十分困難,故有關於FIPV I的相關研究仍相當缺乏。此外,FIPV具有抗體依賴性增強(antibody dependent enhancement, ADE)效應,補體和抗體Fc受體會促進病毒進入巨噬細胞與複製,進而限制了FIPV疫苗與治療藥物的開發。在本研究中,為了研究FIPV I 和FIPV II 的S蛋白上的中和表位,以作為未來設計FIPV治療藥物之基礎,我們分別以FIPV I病毒株UU4的三聚體S蛋白及FIPV II 病毒株NTU156製造單株抗體,並且進行定性試驗與中和試驗。本研究總共篩選出11株單株抗體,其中5株來自FIPV I免疫之小鼠,6株來自FIPV II免疫之小鼠。所有來自FIPV I的單株抗體皆對FIPV II病毒株NTU156之感染細胞具有不等程度的交叉反應能力,並且其中4株能夠中和NTU156毒株。而所有來自FIPV II的單株抗體皆對表現FIPV I病毒株UU4之S蛋白之沒有交叉反應,也無法中和NTU156毒株。本研究篩選出的具有交叉反應之中和單株抗體不僅將有助於研究兩種血清型之共同中和表位,未來亦可作為研發廣效治療藥物之依據。 Feline infectious peritonitis virus (FIPV) belongs to Coronaviridae, which are a group of positive-sense single-stranded enveloped RNA viruses, and causes a fatal disease in cats, especially in young cats, due to the lack of effective vaccines and therapeutics so far. The FIPV genome encodes 11 proteins, including four structural proteins, namely spike (S), envelope (E), matrix (M), and nucleocapsid (N), and several non-structural proteins. The S protein is considered to be responsible for cell entry and harbors neutralizing epitopes. Based on the sequence of S protein, FIPV is subdivided into serotype I and II. Since the difficulty of viral isolation and propagation in vitro, most information about serotype I FIPV, which is the predominant serotype worldwide, is still undetermined. In addition, antibody dependent enhancement (ADE) is reported in FIPV infection, in which complements and Fc receptors facilitate the viral entry and replication in macrophages, and limits the development of vaccine. To study the neutralizing epitope on the S protein of serotype I and II FIPV, and to design novel therapeutics, we generated serotype I UU4 strain trimeric S protein- and serotype II FIPV NTU156 strain-specific monoclonal antibodies (mAbs) and characterized the property of the selected monoclonal antibodies. A total of eleven clones were generated in this study, including five clones from serotype I FIPV S protein immunized mice and six clones from FIPV II NTU156 strain immunized mice. All of the five FIPV I S-specific mAbs showed variable levels of cross-reactivity against FIPV II-infected cells, and four of them demonstrated neutralizing ability against serotype II FIPV NTU156 strain. None of the six FIPV II-specific mAbs demonstrated cross-reactivity against serotype I FIPV UU4 strain S protein-expressing HEK cells, and nor did these clones show neutralizing ability against serotype II FIPV NTU156 strain. These mAbs could be useful tools for investigating the common neutralizing epitopes and provide insight in developing broad therapeutic drugs. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/84418 |
DOI: | 10.6342/NTU202203842 |
Fulltext Rights: | 同意授權(限校園內公開) |
metadata.dc.date.embargo-lift: | 2024-09-23 |
Appears in Collections: | 分子暨比較病理生物學研究所 |
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U0001-2209202218154600.pdf Access limited in NTU ip range | 3.1 MB | Adobe PDF | View/Open |
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