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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 賈景山 | |
| dc.contributor.author | Pei-Lin Chen | en |
| dc.contributor.author | 陳珮琳 | zh_TW |
| dc.date.accessioned | 2021-06-13T00:01:57Z | - |
| dc.date.available | 2017-07-27 | |
| dc.date.copyright | 2007-08-08 | |
| dc.date.issued | 2007 | |
| dc.date.submitted | 2007-07-31 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/28164 | - |
| dc.description.abstract | 黏膜免疫系統是身體對抗大多數外來病原體的第一道防線，主要是利用分
泌性的IgA 來防止微生物的附著，也可藉由中和細菌外毒素的作用避免黏膜組織 遭受破壞。藉由黏膜途徑接種能夠引發良好的黏膜及全身性的免疫反應，但是可 溶性抗原本身所能引起的免疫性較弱，因此我們使用一非基因改造且屬GRAS (generally regarded as safe)級的乳酸球菌(Lactococcus lactis)，藉由其低免疫原性 及佐劑功能當作抗原攜帶系統，增強局部的免疫反應。首先我們將抗原建構在帶 有nisA 啟動子和訊息胜肽Usp45 的載體中，經由乳酸鏈球菌素的誘導後產生分 泌型的蛋白質，這些分泌型的蛋白藉由羧基端具有與細胞壁胜肽聚醣結合能力的 protein anchor domain 結合到用酸和加熱處裡過的Lactococcus lactis Gram-positive enhancer matrix (GEM) particles 上。實驗中成功的將轉糖鏈球菌 (Streptococcus mutans)的Immunodominant glycoprotein 60 (IDG-60)建構到上述載 體中，分別使用抗IDG-60、protein anchor 和6×His 的抗體執行西方墨點法，發 現不管是細菌體內或是培養液中的IDG-60 重組蛋白質，都可以被這三種抗體辨 認到。進一步，將培養液中的IDG-60 重組蛋白質或純化出的IDG-60 與GEM particles 混合後發現，兩者都可以黏附到GEM particles 上。採用以鼻腔內免疫途 徑給予純化出的IDG-60 或IDG-60 與GEM particles 的混合物的方式接種BALB/c 小鼠，探討其調節小鼠黏膜及全身特異性免疫反應之影響，接種三次後，發現 GEM particles 可增加小鼠鼻咽相關淋巴組織、頸部淋巴結、脾臟與腸繫膜淋巴 結中對IDG-60 特異性分泌細胞激素IL-2、IL-4、IL-10 及IFN-γ的細胞數，特別 是在鼻咽相關淋巴組織的部位，並可顯著提升小鼠專一性抗IDG-60 唾液及糞便 分泌型IgA 及血清IgG 的產量。 | zh_TW |
| dc.description.abstract | Mucosal immune system is the first line of defense against foreign pathogens that prevents adherence of microorganisms and neutralizes bacterial toxins through secretory IgA to maintain the integrity of the mucosal lining. Mucosal immunization can induce both mucosal and systemic immunity. To improve the immunogenecity of soluble antigens, we used generally regarded as safe (GRAS) bacteria, Lactococcus lactis to generate gram-positive enhancer matrix (GEM) particles with adjuvant properties as antigen display and delivery system. Firstly, we successfully constructed the expression system for immunodominant glycoprotein 60 (IDG-60) of Streptococcus mutans, under the control of a nisin-inducible promoter, with the lactococcal Usp45 signal sequence to drive secretion, and a protein anchor to direct surface localization on the GEM particles. Recombinant IDG-60 could be detected in cell lysates or cell-free culture supernatant and secreted IDG-60 could bind to GEM particles. To investigate the adjuvant effect of GEM particles for mucosal and systemic immune responses, mice were immunized intra-nasally with IDG-60 in soluble or GEM-bound form three times at weekly intervals. GEM particles enhanced IDG-60 specific serum antibody responses, including IgG1, IgG2a, IgG2b and IgG3. GEM particles also increased IDG-60 specific IgA levels in saliva and fecal samples. Although IDG-60 specific Th1- and Th2-type cytokine producing cells could be detected in different lymphoid tissues such as NALT, CLN, spleen or MLN, the incorporation of GEM enhanced significantly an increase in the numbers of cytokine producing cells preferentially in NALT. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-13T00:01:57Z (GMT). No. of bitstreams: 1 ntu-96-R94445126-1.pdf: 1141012 bytes, checksum: 4cb6c74f31fd7853d75a899a4605fda4 (MD5) Previous issue date: 2007 | en |
| dc.description.tableofcontents | 中文摘要............................................... i
英文摘要................................................ii 圖目錄..................................................iii 第一章前言............................................. 1 1 黏膜免疫(Mucosal Immunization) ................................................... 1 2 乳酸球菌黏膜免疫抗原表現及遞送系統(The Antigen Display and Delivery System of L. lactis for Mucosal Immunization)....................................................... 4 2.1 乳酸鏈球菌素調控基因表現系統(Nisin-Controlled Gene Expression System, NICE System) ..................................................................................... 6 2.2 Protein Anchor (PA) ............................................................7 3 免疫優勢蛋白(Immunodominant Glycoprotein-60, IDG-60) .............................. 9 4 實驗目的............................................................................ 10 第二章材料及方法......................................................................11 1 建構IDG-60 表現載體.................................................................11 2 鏈球菌Streptococcus gordonii DL1 轉形法..............................................11 3 鏈球菌質體抽取.......................................................................11 4 L. lactis 勝任細胞之製備............................................................ 12 5 L. lactis 電穿孔法.................................................................. 13 6 L. lactis 質體抽取.................................................................. 13 7 表現IDG-60 重組蛋白質................................................................ 13 8 聚丙烯醯胺膠體電泳法(SDS-PAGE)....................................................... 14 9 西方墨點法(Western blot) ............................................................ 15 10 GEM particles 製備.................................................................. 15 11 IDG-60 重組蛋白質與GEM particles 黏附試驗.......................................... 16 12 IDG-60 蛋白質的純化................................................................ 16 13 純化的IDG-60 蛋白質與GEM particles 黏附試驗....................................... 17 14 動物免疫計畫..................................................................... 18 15 檢體與組織收集.................................................................... 18 16 以ELISA 偵測IDG-60 特異性抗體...................................................... 18 17 以ELISPOT 偵測淋巴組織中IDG-60 特異性分泌細胞激素IL-2、IL-4、IL-10 與IFN-γ 的細胞數...................................................................... 19 第三章實驗結果.................................................................... 21 2 表現分泌型IDG-60 重組蛋白質..................................................... 22 3 分泌型IDG-60 重組蛋白質與GEM particles 黏附試驗.................................. 22 4 純化的IDG-60 蛋白質與GEM particles 黏附試驗...................................... 23 5 鼻腔內途徑免疫小鼠後黏膜及全身性免疫反應........................................... 23 5.1 GEM particles 誘導黏膜抗體反應.................................................... 24 5.2 GEM particles 誘導血清抗體反應.................................................... 24 5.3 淋巴組織中對IDG-60 特異性細胞激素分泌細胞分析.................................... 25 第四章討論............................................................................. 27 1 分泌型重組蛋白質的表現.............................................................. 27 2 純化的IDG-60 蛋白質與GEM particles 黏附試驗.................................... 30 3 鼻腔內途徑免疫小鼠後黏膜及全身性免疫反應......................................... 30 第五章參考文獻.................................................................... 34 | |
| dc.language.iso | zh-TW | |
| dc.subject | 分泌型IgA | zh_TW |
| dc.subject | 黏膜免疫 | zh_TW |
| dc.subject | 佐劑 | zh_TW |
| dc.subject | GEM particles | zh_TW |
| dc.subject | 鼻腔內免疫 | zh_TW |
| dc.subject | mucosal immune | en |
| dc.subject | secretory IgA | en |
| dc.subject | intralnasal immunization | en |
| dc.subject | GEM particles | en |
| dc.subject | adjuvant | en |
| dc.title | 以Lactococcus lactis GEM particles作為鼻腔免疫黏膜佐劑之效果 | zh_TW |
| dc.title | Effects of Lactococcus lactis GEM Particles as Mucosal Adjuvant for Intranasal Immunization | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 95-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 繆希椿,李建國 | |
| dc.subject.keyword | 黏膜免疫,佐劑,GEM particles,鼻腔內免疫,分泌型IgA, | zh_TW |
| dc.subject.keyword | mucosal immune,adjuvant,GEM particles,intralnasal immunization,secretory IgA, | en |
| dc.relation.page | 59 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2007-07-31 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 微生物學研究所 | zh_TW |
| 顯示於系所單位： | 微生物學科所 | |
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