腸病毒 71 型鞘蛋白（VP1）於轉殖菸草與番茄之表現與老鼠口服免疫反應

Abstract

腸病毒屬於濾過性病毒之一種，常於夏季、初秋流行；潛伏期約為二至五天，大多數感染者之典型症狀為口腔、手掌、腳掌出現水泡、潰瘍，可能合併發燒；病程約為七至十天；其中腸病毒 71 型歷年來在台灣地區造成不等程度規模之流行，並造成許多致死病例，因此腸病毒已經成為疾病防制的重要課題。 VP1、VP2 以及VP3蛋白質為組成腸病毒外殼鞘蛋白（capsid）之主要成分，而其中 VP1是誘導抗體產生的主要蛋白 ；因此，前人利用含有 VP1 基因的DNA載體（DNA vaccine ) ，或者大腸桿菌的表現系統產生之VP1 蛋白，以肌肉注射的方式進行老鼠的免疫實驗，已經證實了人工表現的 VP1蛋白可以在老鼠體內引起一定程度的免疫反應；然而至今仍未有 VP1 口服疫苗之研究報告發表，本實驗即是研究如何用植物表現腸病毒 VP1蛋白以作為口服疫苗之可能性。 將腸病毒71型VP1蛋白的基因構築於可以在植物中表現的載體，並且在基因序列的前後端，放上植物系統中決定蛋白質運送方向的片段 ( signal peptide ) ；本實驗一共選擇三種，分別是讓蛋白質進入並留在內質網的訊息片段（ER retention signal）、或者進入並儲存於液胞的訊息片段（vacuole signal）、以及分泌至細胞外的訊息片段（secretary signal）。 完成三種載體之後，以農桿菌轉殖方式把載體送入菸草與蕃茄的表現系統之中，並且在確定基因產物是否有表現之後，選擇最佳 VP1蛋白表現之菸草轉殖株進行口服疫苗的定量餵食實驗。實驗結果發現，餵食轉殖菸草蛋白抽出液的實驗組與對照組比較起來，在糞便中IgA 與血液中IgG 的含量的確有升高的趨勢；另外，小鼠脾臟細胞的培養與三種細胞素 (cytokine）的酵素連結免疫分析法（ ELISA ）結果顯示，實驗鼠的體內免疫反應的確因為口服菸草或大腸桿菌表現的VP1蛋白而上升；最後再以小鼠血清進行腸病毒中和試驗，發現實驗組的小鼠血清也能夠中和腸病毒，達到保護細胞的目的。由上述實驗數據推測，以腸病毒71型VP1蛋白的植物口服疫苗進行餵食試驗，的確能引發動物免疫反應的效果。

Enterovirus 71 (EV71) is notable for its etiological role in seasonal epidemics of hand, foot, and mouth disease associated with neurological complications in young children. Moreover, Taiwan had suffered several widespread outbreaks of enterovirus infection in recent years. Since no effective antiviral agent is available, primary prevention, including the development of effective vaccines, is a top priority in terms of control strategies. EV71 is encapsulated in a capsid containing 60 copies of VP1, VP2 and VP3. Neutralization epitopes are densely clustered on VP1, which has been administered as a DNA vaccine or recombinant protein and was able to elicit immunity. The development of transgenic tobacco and tomato expressing the coat protein VP1 from EV 71 was reported here. Fusion protein was designed to improve the expression of VP1 antigen by attempting to mimic the process of VP1 targeting to the endoplasmic reticulum, vacuole, or intercellular space within plant cells. The presence of the transgenes in plants was confirmed by genomic PCR and Western blotting, and then the highest level of VP1 production was observed in plants transformed with VP1 targeting to endoplasmic reticulum. Furthermore, mice fed with the leaf extracts from transgenic tobacco leaf developed VP1-specific IgA from feces and IgG from serum, while positive results were shown in spleen cell proliferation, cytokine assay and EV71 neutralization. These data indicate that the plant-derived VP1 protein is immunogenic when administered orally and that, with further investigation, oral vaccination utilizing transgenic plants may become a viable approach to Enterovirus vaccine development.