Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/52054
標題: Secretory production of Enterovirus 71 VP1 partial surface epitopes by Bacillus subtilis and evaluation on the vaccination effect of the secretory epitopes using BALB/c mice.
以枯草桿菌分泌生產腸病毒71型VP1蛋白之部份表位片段及評估此表位片段對BALB/c小鼠之免疫效果
作者: 羅詩晴
Lo, Shih-Ching
關鍵字: Enterovirus 71;腸病毒71型;Bacillus subtilis expression system;VP1;Ganoderma lucidum immunodulatory protein Ling Zhi-8;epitope vaccine;recombinant antigen;枯草桿菌表現系統;腸病毒外鞘蛋白;靈芝免疫調節蛋白質;表位疫苗;基因重組抗原
出版社: 食品暨應用生物科技學系所
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摘要: 
Enterovirus 71 (EV71) is the main causative agent of Hand, Foot and Mouth Disease (HFMD) and is associated with severe neurological diseases resulting in high mortalities. Currently, there is neither vaccine nor therapeutic treatment available. The significant increase in recent EV71 epidemic throughout the Asia-Pacific region was clinically observed recently.
Bacillus subtilis is a Gram-positive, facultative anaerobic rod-shaped endospore-forming bacterium that occurs naturally in soil and water. It has been used in the production of fermented food, extracellular enzymes as well as special chemicals. Some strains of B. subtilis are exploited as probiotics or biological control agents. In the field of genetic engineering, B. subtilis has been developed as a host for the production of homologous and heterologous proteins.
This study developed an effective vaccine by transforming the partial genes of VP1, VP1e and VP1f into Bacillus subtilis. VP1 is a coat protein of EV71 and considered as potent epitope; VP1e is the N-terminal sequence of VP1 (amino acid is 1-81) and VP1f is the hydrophilic sequences fusion (amino acid are 95-109, 162-170, 210-221 and 266-274). In this study, we first generated a Bacillus subtilis secretory production system to produce the VP1e and VP1f epitope protein, high level of recombinant protein was expressed and secreted. Then, vaccination with recombinated protein VP1e, VP1f, or combined VP1e, VP1f and rLZ8 who administered to Balb/c mice. The VP1-specific serum IgG1 and IgG2a were examined. The VP1-specific serum IgG antibody induced by VP1e triggered both IgG subtype responses, indicating that Bacillus subtilis expressing VP1e was successful and high efficiency as vaccine. This study not only demonstrates the feasibility of using Bacillus subtilis secetory system as vaccine producer, but also suggests the probability of enterovirus vaccine development using VP1e as potent epitope.

腸病毒重症屬於台灣第三類法定傳染病,為亞太地區地方性的流行疾病;其中腸病毒71型最易引起手足口病與神經系統等相關嚴重併發症而導致高致死率,且目前並無任何有效的疫苗可供預防和治療。枯草桿菌(Bacillus subtilis)為GRAS(generally recognized as safe)級菌種,此菌之產業應用性極為廣泛,可運用於發酵食品之製造、酵素及特用化學品之生產、作為益生菌(probiotic)及作為植物病害防治試劑等用途;此外亦被開發成為生產同源或異源蛋白質的基因表現宿主。
本實驗以腸病毒71型之外鞘膜上主要抗原決定部位VP1 作為目標,利用枯草桿菌分泌生產其所含之表位蛋白並增進其產量;而疫苗蛋白之一為設計融合經文獻佐證具致免疫性的四段親水性序列片段(VP1 序列胺基酸95-109、162-170、210-221、266-274),以及本實驗室先前已構築出之N端表位蛋白序列VP1e(VP1 序列胺基酸1-81),並將兩者聯合靈芝免疫調節蛋白進行動物實驗檢測其疫苗效果,觀察小鼠的抗體產生情形。結果顯示,VP1e 抗原蛋白可成功誘發腸病毒71型的VP1 特異性抗體,且IgG1 與IgG2a 皆有相同結果;本研究開發出高產量之枯草桿菌分泌生產系統,可用以生產腸病毒71型之抗原表位蛋白VP1e,且該蛋白質極具有疫苗發展之潛力。
URI: http://hdl.handle.net/11455/52054
Appears in Collections:食品暨應用生物科技學系

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