臺灣博碩士論文加值系統

蕪菁黃化嵌紋病毒(Turnip yellow mosaic virus ；TYMV)主要感染十字花科植物，病毒顆粒本身是一正二十面體球形病毒，直徑大小為28nm；主要由180個鞘蛋白單元包裹一條正股長6.3 kb的核醣核酸基因體。目前的蛋白質檢測方式多採用酵素結合免疫吸附法(Enzyme-linked Immunosobent Assay； ELISA)，但是礙於常用的avidin的結合位有限，使敏感度無法很高，如果我們能夠將修飾過的病毒顆粒來取代avidin，則其將有近百個接位能夠用來增加訊息，其敏感度應該可以被放大。故我們以遺傳工程的技術在鞘蛋白的C-端額外加入一個含有硫根官能基的氨基酸(Cysteine)，進一步我們可以藉由這些額外的硫根官能基，將此病毒發展成一種新的奈米元件，可以用於生物感測器的研發。目前我們已成功的得到突變病毒(TYMV/Cys)，且其感染力與野生型的病毒相似。我們檢測TYMV/Cys以及野生型病毒顆粒表面所含的一些功能性官能基，發現TYMV/Cys病毒顆粒表面至少有100個硫根官能基能夠被用來接各種目標辨識分子，而野生型病毒顆粒表面則沒有硫根官能。TYMV/Cys病毒顆粒表面與野生型一樣都至少有400個氨根官能基能被用來接偵測訊號分子。目前正著手進行的工作包括TYMV/Cys病毒顆粒表面硫根官能基與胡瓜嵌紋病毒鞘蛋白抗體的接合反應，以及TYMV/Cys病毒顆粒表面氨根官能基與螢光訊號分子的接合反應。利用重組病毒顆粒，我們期望可以發展出高效率的生物感測器。

Turnip yellow mosaic virus is an icosahedral plant virus, 28nm in diameter. The viral particle, composed of a 6.3 kb single-strand positive-sense RNA genome encapsidated by 180 copies of capsid protein, offers a uniquely programmable biological nanoscaffold for multiplexed conjugation. Our interest is to create a multivalent display system which would dramatically enhance the sensitivity of conventional ELISA. To achieve this objective, we used molecular techniques to create a recombinant virus that has an extra cysteine residue which chemically is highly active, at the C-terminus of the coat protein. Fluorescein-5-maleimide and Fluorescein N-hydroxysuccinimide were used to determine the number of the exposed thiol-groups and amine-groups, respectively, on the surface of the virus particles. Fluorescein-5-maleimide did not label the wild-type particles suggested that there are no cysteines on the surface, whereas the surface of the mutant particles were strongly labeled at least higher than 100 thiol-groups per particle. Fluorescein N-hydroxysuccinimide labeled the wild-type and the mutant particles with similar efficiency 400 amine-groups on the particle surface were estimated from the labeling intensity. Fluorescence labeling of fluorescein N-hydroxysuccinimide on the virus particle was successful, and the conjugation of an antibody to the virus particle is in progress. The efficiency of this nanobiosensor will be tested in the future.

目錄
頁次

壹、中文摘要 ------------------------------------------- 1
貳、英文摘要 --------------------------------------------2
參、前人研究 --------------------------------------------3
肆、內文 ------------------------------------------------6
簡介 (Introduction) ---------------------------------6
材料與方法 (Materials and Methods) ------------------10
結果 (Results) --------------------------------------13
討論 (Discussion) -----------------------------------17
圖表與說明 (Figure and legend) ----------------------19
參考文獻 (References) -------------------------------31
伍、結論 ------------------------------------------------35

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