Enhancement of IgG Purification by FPLC for a Serological Study on the Turnip mosaic virus P1 Protein

利用快速流速層析儀發展改良式免疫球蛋白G純化流程進行蕪菁嵌紋病毒P1蛋白血清學之研究

邱敏慈(Ming-Tzu Chiu) ； 林長平(Chan-Pin Lin) ； 林品均(Pin-Chun Lin) ； 林詩舜(Shih-Shun Lin)

《植物病理學會刊》 22卷1期 (2013/03) Pp. 21-30

https://doi.org/10.6649/PPB.201303_22(1).0003

摘要

馬鈴薯Y群病毒（potyvirus）產生的P1/HC-Pro基因是第一個被發現的病毒抑制子，其作用主要是藉由P1蛋白協助HC-Pro針對後轉錄時期基因靜默機制進行抑制作用。然而P1/HC-Pro的真正功能尚未了解十分透徹。P1蛋白在Potyvirus屬中是大小及序列差異最大的蛋白。要有效率的研究P1功能，必須要有好的血清學工具。本研究，我們開發了一個新的利用快速液相層析儀純化蕪菁嵌紋病毒（Turnip mosaic virus, TuMV）的P1免疫球蛋白IgG流程。此外，所純化出的免疫球蛋白IgG配合Centricon濃縮技術，增加免疫球蛋白IgG的濃度。我們結果顯示，新的純化方式可快速地在數小時內有效率地純化出免疫球蛋白IgG。此外，在TuMV所感染的菸草中，病毒產生的P1蛋白難以被未經純化的P1抗血清所偵測；而濃縮及純化的P1免疫球蛋白IgG具高於未純化的P1抗血清的100倍靈敏度，可以順利地偵測出植物體內病毒所產生的P1蛋白。因此，我們推測P1蛋白具有快速代謝降解率，導致所感染之植物體內含量不高，難以偵測。此新的免疫球蛋白IgG純化流程，適合任何血清學研究，有助於提升偵測靈敏度。

關鍵字

蕪菁嵌紋病毒 ； P1蛋白 ； 免疫球蛋白IgG ； FPLC

並列摘要

The P1/HC-Pro of Potyvirus is the first discovered viral gene-silencing suppressor in which helper-component-proteinase (HC-Pro) function in concert with P1 to improve the suppression of post-transcriptional gene silencing (PTGS). However, the mechanism of P1/HC-Pro remains unclear. The P1 protein is the most divergent protein regarding length and the amino acid sequence in potyviruses. An effective serological tool is required to examine the function of P1. To improve the titer of the antiserum to P1 of Turnip mosaic virus (TuMV), we developed a procedure using fast protein liquid chromatography (FPLC) to purify immunoglobulin G (IgG). Moreover, the purified IgGs were further concentrated using appropriate centrifugal filter device to enhance the sensitivity. The results indicated that the new procedure improved the efficiency of IgG purification in a few hours. The low P1 signal was difficult to be detected with unpurified antersum to P1 in the TuMV-infected N. benthamiana plants, whereas the purified IgG to P1 enhanced 100-fold of the sensitivity for detection. These results imply that P1 may have a rapid turnover rate in vivo and the new IgG purification procedure is suitable for any serological study to enhance the detection sensitivity.

並列關鍵字

Turnip mosaic virus ； P1 ； immunoglobulin G ； fast protein liquid chromatography