1. NCHU Institution Repository
  2. 農業暨自然資源學院
  3. 食品暨應用生物科技學系
Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/52054
DC FieldValueLanguage
dc.contributor蔡英傑zh_TW
dc.contributor林金源zh_TW
dc.contributor.advisor葉娟美zh_TW
dc.contributor.author羅詩晴zh_TW
dc.contributor.authorLo, Shih-Chingen_US
dc.contributor.other中興大學zh_TW
dc.date2010zh_TW
dc.date.accessioned2014-06-06T08:55:39Z-
dc.date.available2014-06-06T08:55:39Z-
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dc.identifier.urihttp://hdl.handle.net/11455/52054-
dc.description.abstractEnterovirus 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.en_US
dc.description.abstract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。zh_TW
dc.description.tableofcontents中文摘要i 英文摘要ii 壹、 前言1 一、 枯草桿菌之簡介1 二、 免疫系統之簡介2 (一) 免疫系統概述2 三、 疫苗之簡介3 (一) 預防接種的定義3 (二) 疫苗分類3 四、 腸病毒簡介4 (一) 疾病概述4 (二) 分類與致病性5 (三) 流行病學6 (四) 傳染方式8 (五) 潛伏期8 五、 腸病毒71型分子構型與疫苗設計原理8 (一) 病毒分子學8 (二) 疫苗設計9 六、 靈芝免疫調節蛋白11 貳、 實驗緣起與目的13 參、 實驗策略14 肆、 材料與方法15 一、 菌種與質體15 (一) 菌種15 (二) 質體16 二、 藥品和試劑17 (一) 藥品相關資訊17 (二) 培養基18 三、 質體DNA 抽取方法18 (一) 大腸桿菌質體抽取19 (二) 枯草桿菌質體抽取19 四、 染色體DNA 抽取方法19 (一) 枯草桿菌染色體抽取19 五、 DNA 分子生物操作技術20 (一) DNA 分子電泳20 (二) DNA 分子剪切20 (三) DNA 分子回收20 (四) PCR 產物回收21 (五) DNA 分子黏合21 六、 聚合酶連鎖反應21 (一) VP1 全長序列之增幅22 (二) VP1e N端序列之增幅22 七、 電勝任細胞（electrocompetent cell）製備及電轉形條件22 (一) 大腸桿菌22 (二) 枯草桿菌23 八、 轉形株篩選與重組質體確認23 (一) 菌落聚合酶鏈鎖反應23 (二) 重組質體確認24 九、 表現載體構築流程24 (一) VP1全長序列表現質體pNW33NVP1之構築24 (二) VP1全長序列表現質體pSECS7VP1之構築24 (三) VP1全長序列表現質體pSECS7VP1h之構築25 (四) VP1全長序列表現質體pOAVP1之構築25 (五) VP1e N端序列表現質體pSLYSPVP1e之構築25 (六) VP1f 親水性序列表現質體pSLYSPVP1f之構築26 十、 重組蛋白之表現、偵測與純化濃縮26 (一) 胞內重組蛋白VP1 之處理26 (二) 胞外分泌重組蛋白之處理28 十一、 質譜分析（Mass Spectrometer）與蛋白質身份鑑定31 (一) 質譜分析樣品製備31 (二) 蛋白質身份鑑定樣品製備31 十二、 實驗動物致免疫模式32 (一) 小鼠分組與飼32 (二) 介入方式與血液樣本取得32 (三) 特異性抗體偵測33 (四) 統計分析34 伍、 結果與討論35 一、 大腸桿菌誘導型系統之VP1全長序列生產35 二、 枯草桿菌分泌表現系統載體之構築35 (一) VP1 全長序列質體35 (二) 部份表位片段質體37 三、 VP1 部份表位片段蛋白之分泌表現38 (一) N端序列蛋白VP1e 分泌表現系統38 (二) 親水性序列蛋白VP1e 分泌表現系統39- 四、 質譜分析與蛋白質身份鑑定結果39 (一) VP1e 蛋白質譜分析39 (二) VP1e 蛋白質身份鑑定40 五、 連續式純化系統建立40 六、 疫苗效能分析與探討41 (一) 介入期間小鼠體重變化41 (二) 測定特異性抗體誘發結果41 陸、 結論43 柒、 參考文獻93 捌、 附錄102zh_TW
dc.language.isoen_USzh_TW
dc.publisher食品暨應用生物科技學系所zh_TW
dc.subjectEnterovirus 71en_US
dc.subject腸病毒71型zh_TW
dc.subjectBacillus subtilis expression systemen_US
dc.subjectVP1en_US
dc.subjectGanoderma lucidum immunodulatory protein Ling Zhi-8en_US
dc.subjectepitope vaccineen_US
dc.subjectrecombinant antigenen_US
dc.subject枯草桿菌表現系統zh_TW
dc.subject腸病毒外鞘蛋白zh_TW
dc.subject靈芝免疫調節蛋白質zh_TW
dc.subject表位疫苗zh_TW
dc.subject基因重組抗原zh_TW
dc.titleSecretory 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.en_US
dc.title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zh_TW
dc.typeThesis and Dissertationzh_TW
item.openairetypeThesis and Dissertation-
item.openairecristypehttp://purl.org/coar/resource_type/c_18cf-
item.languageiso639-1en_US-
item.grantfulltextnone-
item.fulltextno fulltext-
item.cerifentitytypePublications-
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