利用噬菌體展現技術製造蘭花病毒鞘蛋白抗原之雞抗體片段重組基因庫

蘭花是開花植物中最大的一科，估計約有1萬5千至3萬種，遍及全世界。由於極具觀賞價值以及商業性而被大量栽種。相對的在蘭花病蟲害的防治上極其重要，蕙蘭嵌紋病毒(Cymbidium mosaic virus, CyMV)是目前最為普遍造成蘭花傷害的其中一屬。受到病毒感染的蘭花可產生嚴重的系統性黃化病徵，造成蘭花外觀有極大變化與傷害，相關產業也遭受損失，嚴重影響國內經濟。感染途徑主要是經由機械性傷口感染蘭花本身，而本篇的研究目標是利用重組表現CyMV蘭花病毒鞘蛋白片段來免疫雞隻，藉由嗜菌體展現技術(Phage display technique)來建立重組抗體基因庫(antibody library)篩選對於CyMV蘭花病鞘蛋白具有特異性結合力的單株抗體片段(scFv, single chain variable fragment)。重組的CyMV蘭花病毒鞘蛋白基因經酵素作用確認，將重組基因送入大腸桿菌表現並純化重組鞘蛋白，藉由SDS-PAGE染色證實。將純化後的鞘蛋白片段與佐劑混合均勻注射雞隻大腿肌肉部位，連續四周免疫雞隻，收集每次免疫雞蛋，萃取蛋中抗體Poly-IgY，藉由西方墨點法確認。利用西方墨點法(western blot)以及酵素連結免疫分析實驗(enzyme-linked immunosorbent assay；ELISA)偵測經四次免疫後雞蛋中的抗體IgY效價，證實在免疫四次後雞隻體內的確誘發出高劑量的抗體反應並可辨認His-CyMV融合蛋白。將雞隻犧牲，取出脾臟，萃取RNA反轉成cDNA並利用多鏈聚合酶反應(PCR)做出抗體重鏈和輕鏈基因片段，將做出的重鏈與輕鏈接到pComb3X載體中，建構出CyMV為1.1× 103蘭花病毒外鞘蛋白的雞隻抗體片段基因庫，經隨機挑取15個clone，萃取其DNA證實。再利用嗜菌體感染並使其表現出抗體片段基因庫上的scFv。經四次篩選(panning)，萃取四次scFv DNA確認。同時表現與重組CyMV鞘蛋白最具有特異性結合力的scFv抗體片段，最後共篩選出14個有表現的抗體片段，將抗體片段基因定序分析其中的相似性，最後共分成三群具有不同序列的片段。將三群抗體片段從中各挑出一個作為代表大量純化，利用純化後可溶性抗體片段以western blot與ELISA方法證實抗體與抗原間結合的能力，發現不同抗體片段具有與抗原間不同的結合能力，對於往後在蘭花病毒的防治及偵測上，期待更有良好的效果。

關鍵字

噬菌體；蘭花病毒:鞘蛋白；抗體；基因庫

並列摘要

The Orchidaceae is the largest and most diverse plant family, consisting of more than 25,000 species in approximately 900 genera. It is important to control virus diseases in orchid collections. Orchids are affected by more virus disease problems than others. CyMV (CyMV, family Flexiviridae) has been reported in many cultivated orchid species in many countries, and than is now considered to be spread worldwide. The symptoms of infected orchids were such as system yellowing of leaves, necrotic spot. In this study, we must develop CyMV scFv (single-chain variable fragment) antibody library to produce specific antibody by phage display, and screen the binding ability of CyMV coat protein antibodies. First, we constructed CyMV coat protein genes into pET32a vector and expression the fusion protein; second, we used fusion protein to immunize chicken for IgY production. After four times of boost, the titers of anti CyMV coat protein antibody were increased. And than, identified antibodies with western blot and ELISA analysis. Additionally, we have to analysis the binding ability with total IgY antibody and CyMV coat protein. Finally, We successfully used phage display to generate CyMV scFv library and the size was 1.1× 103. After four times panning amplified the phage, we found 14 scFv clones were expressed antibody protein.After all, we alignmented these 14 scFv clone sequences and evaluated specificity of scFv antibodies with CyMV coat protein for western blot and ELISA analysis. We hope they could use to prevent and detect the orchids virus in the future.

並列關鍵字

single chain ； phage display ； orchid virus ； library

參考文獻

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2. Goodman, R.M. and A.F. Ross, Enhancement of potato virus X synthesis in doubly infected tobacco occurs in doubly infected cells. Virology, 1974. 58(1): p. 16-24.

3. Ajjikuttira, P., C.S. Loh, and S.M. Wong, Reciprocal function of movement proteins and complementation of long-distance movement of Cymbidium mosaic virus RNA by Odontoglossum ringspot virus coat protein. J Gen Virol, 2005. 86(Pt 5): p. 1543-53.

4. Seoh, M.L., S.M. Wong, and L. Zhang, Simultaneous TD/RT-PCR detection of cymbidium mosaic potexvirus and odontoglossum ringspot tobamovirus with a single pair of primers. J Virol Methods, 1998. 72(2): p. 197-204.

5. Verchot-Lubicz, J., A new cell-to-cell transport model for Potexviruses. Mol Plant Microbe Interact, 2005. 18(4): p. 283-90.

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利用噬菌體展現技術製造蘭花病毒鞘蛋白抗原之雞抗體片段重組基因庫

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