Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/36154
標題: 利用蕪菁黃色嵌紋病毒做為螢光顯影劑來增進免疫偵測系統的敏感度
Development of Turnip Yellow Mosaic Virus as Fluoresent Tracer to Improve the Sensitivity of Immunoassay.
作者: 蔡世閔
Tsai, Shih-Ming
關鍵字: Turnip yellow mosaic virus;蕪菁嵌紋病毒
出版社: 生物科技學研究所
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摘要: 
病毒是一個天然的聚合物且具有可自行聚合組裝的特性,所以目前有許多研究都著眼在以病毒做為奈米尺度的材料,利用病毒表現不同的胜肽於病毒外鞘蛋白上,進而衍生出許多新的奈米材料跟應用。蕪菁黃色嵌紋病毒(Turinip yellow mosaic virus; TYMV) 為一正股核酸基因體的植物病毒,是一個病毒外殼由180個外鞘蛋白所構成的正20面體的球形病毒,直徑為28nm。在先前研究中我們已成功的將其外鞘蛋白的C端表現出單一的半胱胺酸,形成一個病毒外鞘表現出180個半胱胺酸的突變病毒(TYMV/Cys),且此病毒在植物中的表現狀況與原生種病毒並無明顯的差異。經由實驗證實此種突變病毒可以同時利用其病毒外鞘半胱胺酸暴露出來的硫基跟離胺酸的氨基來與不同的螢光接合,且病毒表面的硫基可以提供將近110個接位而氨基可以提供約400個接位來進行接合。由於半胱胺酸表現在外鞘蛋白的C端,在病毒表面是處於立體結構的凹陷處,可能會造成空間的立體障礙¬而使病毒的應用有困難。因此我們又進行了新的突變病毒之構築,並且成功獲得在半胱胺酸前面多加入6個丙氨酸的新突變病毒(TYMV/A6C)。我們推測此突變病毒可以降低空間障礙的問題以解決應用上的困難。經過螢光標定的測試推論此新突變株同樣的可以提供90個硫基接位及400個胺基接位。另外一方面,我們也將突變病毒上的硫基與經過修飾可以跟硫基結合直徑1.4nm的奈米金顆粒進行結合,並且以電子顯微鏡觀察病毒表面吸附奈米金顆粒的狀況,進一步證明我們的推論。而利用突變病毒的雙功能基將病毒表面的氨基接上螢光物質,硫基則與抗體進行接合成最終具有螢光病毒標定的抗體。接著再經過陰離子交換管柱純化了期望有螢光病毒結合的抗體。最終希望可以將病毒應用在酵素連結免疫吸附分析(enzyme-linked immunosorbent assay,簡稱ELISA)來取代目前系統中的螢光物質,期望可以增進偵測的靈敏度。

Natural polymers and virus capable of self-replicating and assembly draw many scientists into the development and application of these materials in nanotechnology. Many applications and material development are based on the manipulation of various peptides on the surface of virus particle. Turnip yellow mosaic virus (TYMV) is an icosahedral virus, 28 nm in diameter. It comprises a positive-stranded RNA genome and 180 copies of capsid protein. In a previously study, a mutant virus (TYMV/Cys) which displays one cysteine residue at the C terminus of viral coat protein was constructed showed a similar infectivity to that of wild type TYMV. TYMV/Cys provides 110 and 400 of thiol and amine groups, respectively, on the surface which could conjugate with different kinds of fluorescent markers. Since the C terminus of coat protein on the virus surface is predicted to be in a cleft, the location may be sterically hindered for further applications. A new construct, TYMV/A6C, containing 6 alanine residues with a C-terminal cysteine is predicted to extend the thiol group from the cleft. Characterization of TYMV/A6C indicates that it could provide about 90 and 400 of thiol and amine groups, respectively, on the viral surface. TYMV/A6C was further used to be conjugated with 1.4 nm-diameter nano-gold particles through the thiol groups and the labeling was observed under transmission electron microscope. Purified antibody and fluorescein were conjugated to the thiol and amine groups, respectively, on TYMV/A6C and subsequently purified with an ion-exchange column. This fluorescently labeled viral particle conjugated with antigen specific antibody was tested in a fluorescent immumosorbent assay and compared to the conventialthe sensitivity of conventional ELISA (enzyme-link immunosorbent assay) system.
URI: http://hdl.handle.net/11455/36154
其他識別: U0005-2607200714091700
Appears in Collections:生物科技學研究所

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