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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 劉?睿 | |
dc.contributor.author | Hsiang-Yun Hsueh | en |
dc.contributor.author | 薛湘雲 | zh_TW |
dc.date.accessioned | 2021-06-15T05:19:37Z | - |
dc.date.available | 2015-07-23 | |
dc.date.copyright | 2010-07-23 | |
dc.date.issued | 2010 | |
dc.date.submitted | 2010-07-21 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/46627 | - |
dc.description.abstract | 益生菌對腸道上皮細胞的吸附能力為菌種篩選要件之一,當其具有高吸附性則更能定殖於腸道中,使腸道上皮形成穩定之正常菌叢,且對於宿主健康有較佳影響。¬而吸附的第一步驟可藉由菌體產生的吸附蛋白與細胞表面接受器進行結合,故吸附蛋白為使菌體具有吸附性的關鍵。
本研究目的即利用基因工程技術,將革蘭氏陽性菌(Gram-positive bacteria)吸附蛋白基因殖入低吸附性的乳酸菌中,讓吸附蛋白表現於乳酸菌菌體表面,增加其吸附能力。首先利用聚合酶鏈鎖反應(polymerase chain reaction, PCR)選殖出Lactobacillus reuteri Pg4膠原吸附蛋白(collagen-binding protein, Cnb)基因與Listeria monocytogenes黏液吸附蛋白(mucus binding protein, Mub)基因,將cnb與mub基因構築於乳酸菌-大腸桿菌穿梭載體pNZ3004後,以電穿孔(electroporation)方式將此重組質體轉殖入Lb. casei ATCC 393中,利用抗生素篩選菌落進行PCR試驗,證實乳酸菌轉殖株Lb. casei pNZ-cnb與Lb. casei pNZ-mub分別帶有外源性吸附蛋白基因cnb與mub。抽取乳酸菌轉形株之RNA並以反轉錄聚酶合鏈鎖反應(reverse transcription-PCR)分析,結果顯示,在乳酸菌轉形株中可觀察到cnb與mub之mRNA表現。西方轉漬分析方法則證實Lb. casei ATCC 393轉形株能表現外源性的29 kDa膠原吸附蛋白或48 kDa黏液吸附蛋白。使用流式細胞儀技術觀察,轉形株菌體表面確實具有膠原吸附蛋白或黏液吸附蛋白存在。進行吸附實驗發現,具有外源性吸附蛋白之乳酸菌轉形株對於腸黏膜Caco-2細胞具有較佳的吸附能力。進一步將乳酸菌與病原菌進行共同競爭(competition)、排斥效應(exclusion)與取代效應(displacement)試驗。結果顯示,Lb. casei pNZ-cnb可藉競爭排斥抑制大腸桿菌O157:H7與Ls. monocytogenes BCRC 15338吸附於Caco-2細胞的能力,Lb. casei pNZ-mub相較於Lb. casei ATCC 393而言,對於Ls. monocytogenes BCRC 15338具有較高的排斥效應以抑制其吸附。 | zh_TW |
dc.description.abstract | The adhesion ability of bacteria to intestinal epithelial cells has been considered as one of the crucial selection criteria for probiotic strains. The superior adhesion is a prerequisite for colonization of bacteria, therefore, it is an importance effect for the establishment of a stable normal flora in the mammalian intestine and host beneficial healthiness. Initial adhesion is followed by firm attachment where adhesins on the bacterial cell surface recognize receptors on the epithelial cell surface.
As the binding proteins play a key role, this study was aimed to clone the Gram-positive bacterial adhesion protein genes and transformed these genes into a poor-adhesive Lactobacillus strain to increase its adhesion ability to intestinal epithelial cells. The DNA sequences encoding collagen-binding protein (Cnb) of Lactobacillus reuteri Pg4 and the mucus-binding protein (Mub) of Listeria monocytogenes were amplified by polymerase chain reaction (PCR). The PCR fragments encoding Cnb and Mub were subcloned into the E. coli-Lactobacillus shuttle vector pNZ3004 to generate pNZ-cnb and pNZ-mub respectively, and then were used to electroporate into Lb. casei ATCC 393. The presence of the cnb and mub gene in the Lb. casei transformants (designated Lb. casei pNZ-cnb and Lb. casei pNZ-mub) was demonstrated by direct colony PCR. The total RNA of the Lb. casei ATCC 393 transformants was extracted for estimation of the transcript levels of cnb and mub by reverse transcription-PCR. The results showed that cnb and mub were successfully expressed by Lb. casei ATCC 393 transformants. The heterologous expression of 29 kDa Cnb and 48 kDa Mub were further confirmed by Western blot analysis. Flow cytometric analysis of the Lb. casei ATCC 393 transformants indicated that Cnb and Mub were display on their cell surface. In addition, the intensity of the fluorescent signals measured on Lb. casei ATCC 393 transformants was higher than that on Lb. casei ATCC 393. In the cell adhesion test, the adhesion ability of the Lb. casei ATCC 393 transformants to Caco-2 cells was increased as compared with Lb. casei ATCC 393. Competition, exclusion and displacement of Listeria monocytogenes BCRC 15338 and Escherichia coli O157:H7 by Lb. casei ATCC 393 transformants from adhesion on Caco-2 cell surfaces were further studied. The results indicated that Lb. casei pNZ-cnb could inhibit the adhesion of Ls. monocytogenes BCRC 15338 and E. coli O157:H7 to Caco-2 cells, while Lb. casei pNZ-mub could only inhibit the adhesive ability of Ls. monocytogenes BCRC 15338. In conclusion, this study demonstrated that Cnb and Mub could be heterologously expressed by Lb. casei ATCC393. The transformed strains Lb. casei pNZ-cnb and Lb. casei pNZ-mub could constitutively express Cnb and Mub on their cell surface, and thus showed a better adhesion ability to Caco-2 cells than Lb. casei ATCC 393 did. Furthermore, Lb. casei pNZ-cnb and Lb. casei pNZ-mub could inhibit the adhesion of pathogens to Caco-2 cells. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T05:19:37Z (GMT). No. of bitstreams: 1 ntu-99-R97626023-1.pdf: 3750847 bytes, checksum: 276a665925b75b536cd9a5554a1c8a03 (MD5) Previous issue date: 2010 | en |
dc.description.tableofcontents | 中文摘要 1
英文摘要 2 第一章、序言 4 第二章、文獻探討 5 一、益生菌簡介 5 (一)益生菌之定義 5 (二)益生菌的特性 5 二、乳酸菌簡介 6 (一)乳酸菌之定義 6 (二)乳酸菌作為益生菌之特性 6 (三)乳酸菌吸附腸道 7 (四)乳酸菌吸附因子 7 三、Listeria monocytogenes簡介 9 (一)Ls. monocytogenes介紹 9 (二)Ls. monocytogenes與腸道 10 四、出血性大腸桿菌O157:H7簡介 10 (一)出血性大腸桿菌O157:H7介紹 10 (二)出血性大腸桿菌O157:H7與腸道 11 五、乳酸菌與病原菌 11 六、乳酸菌吸附蛋白對於病原菌之拮抗作用 12 七、人體腸道模擬系統 12 八、In vitro測量吸附之方法 12 第三章、材料與方法 22 一、Lb. reuteri膠原吸附蛋白之製備 22 (一)膠原吸附蛋白表達質體構築 22 (二)Glutathione-S-transferase(GST)融合蛋白純化 24 二、Ls. monocytogenes黏液吸附蛋白之製備 26 (一)黏液吸附蛋白表達質體構築 26 (二)組胺酸標幟(His-tag)融合蛋白純化 28 三、大腸桿菌-乳酸菌穿梭載體建構 30 (一)Lb. reuteri膠原吸附蛋白穿梭質體構築與選殖 30 (二)Ls. monocytogenes黏液吸附蛋白穿梭質體構築與選殖 30 四、乳酸菌系統表現重組吸附蛋白 31 (一)乳酸菌勝任細胞製備 31 (二)電穿孔(electroporation)反應條件 32 五、吸附蛋白之特性分析 32 (一)吸附蛋白質濃度測定 32 (二)聚丙烯醯胺膠體電泳分析(SDS-polyacrylamide gel electrophoresis) 34 (三)西方轉漬分析(Western blot) 36 六、重組乳酸菌表現吸附蛋白mRNA之檢測 37 (一)抽取菌體RNA 37 (二)反轉錄聚合酶連鎖反應(reverse transcript PCR, RT-PCR) 38 七、間接免疫螢光染色(indirect immunofluorescence stain) 39 八、Caco-2細胞培養 41 九、流式細胞儀分析 42 (一)重組乳酸菌表面蛋白呈現分析 42 (二)重組乳酸菌與Caco-2細胞共同吸附分析 43 (三)重組乳酸菌與病原菌吸附Caco-2細胞分析 44 第四章、結果與討論 50 一、Lb. reuteri膠原吸附蛋白序列分析 50 二、Ls. monocytogenes黏液吸附蛋白序列分析 50 三、以E. coli大量表達Lb. reuteri膠原吸附蛋白及其與GST融合蛋白之純化 51 (一)Lb. reuteri膠原吸附蛋白大量表達 51 (二)Lb. reuteri膠原吸附蛋白純化 52 四、以E. coli大量表達Ls. monocytogenes黏液吸附蛋白及其組胺酸標幟之純化 52 (一)Ls. monocytogenes黏液吸附蛋白大量表達 52 (二)Ls. monocytogenes黏液吸附蛋白純化 53 五、Lb. casei ATCC 393轉形株表達吸附蛋白 54 (一)穿梭質體構築與選殖 54 (二)Lb. casei ATCC 393轉形株mRNA表現測定 54 (三)Lb. casei ATCC 393轉型株蛋白質表現測定 55 六、Lb. casei ATCC 393轉形株菌體表面吸附蛋白分析 55 (一)間接免疫螢光染色分析 55 (二)流式細胞儀分析菌體表面蛋白質表現 56 七、Lb. casei ATCC 393轉形株吸附Caco-2細胞分析 56 八、Lb. casei ATCC 393轉形株與病原菌吸附Caco-2細胞分析 58 (一)病原菌影響Lb. casei ATCC 393轉形株吸附Caco-2細胞 58 (二)Lb. casei ATCC 393轉形株與大腸桿菌O157:H7吸附Caco-2細胞情形 60 (三)Lb. casei ATCC 393轉形株與Ls. monocytogenes吸附Caco-2細胞情形 61 第五章、結論 90 參考文獻 91 作者小傳 98 表目錄 表2-1 常見之益生菌菌種 14 表2-2 乳酸菌吸附因子之特性與功能 15 表2-3 乳酸菌排除病原菌之體外試驗 16 表3-1 PCR之配方 46 表3-2 菌落PCR之配方 47 圖目錄 圖2-1 使用益生菌對健康之益處 18 圖2-2 人體腸道黏膜層與黏膜下層之組成圖 19 圖2-3 革蘭氏陽性菌細胞壁組成示意圖 20 圖2-4 李氏菌感染之生理與病理示意圖 21 圖3-1 實驗流程圖 48 圖3-2 牛血清白蛋白之蛋白質濃度標準曲線圖 49 圖4-1 大腸桿菌表達質體圖譜 64 圖4-2 大腸桿菌-乳酸桿菌穿梭質體圖譜 65 圖4-3 聚丙烯醯胺膠體分析GST-Cnb融合蛋白表現量 66 圖4-4 聚丙烯醯胺膠體分析thioredoxin-His6-S-Tag-Mub融合蛋白表現量 67 圖4-5 RT-PCR分析Lb. casei pNZ-cnb之膠原吸附蛋白cDNA表現量 68 圖4-6 RT-PCR分析Lb. casei pNZ-mub之黏液吸附蛋白cDNA表現量 69 圖4-7 西方轉漬分析Lb. casei pNZ-cnb之膠原吸附蛋白表現量 70 圖4-8 西方轉漬分析Lb. casei pNZ-mub之黏液吸附蛋白表現量 71 圖4-9 共軛焦顯微鏡觀察免疫螢光染色之Lb. reuteri Pg4與Lb. casei pNZ-cnb 72 圖4-10 共軛焦顯微鏡觀察免疫螢光染色之Ls. monocytogenes與Lb. casei pNZ-mub 73 圖4-11 間接免疫螢光染色分析Lb. casei pNZ-cnb菌體表面膠原吸附蛋白表現量 74 圖4-12 間接免疫螢光染色分析Lb. casei pNZ-mub菌體表面黏液吸附蛋白表現量 75 圖4-13 光學顯微鏡觀察Lb. reuteri Pg4與Lb. casei ATCC 393轉形株吸附Caco-2細胞 76 圖4-14 流式細胞儀分析Lb. reuteri Pg4與Lb. casei ATCC 393轉形株吸附Caco-2細胞 77 圖4-15 流式細胞儀分析乳酸菌與大腸桿菌O157:H7吸附Caco-2細胞之紅色螢光改變量 78 圖4-16 流式細胞儀分析乳酸菌與大腸桿菌O157:H7吸附Caco-2細胞之綠色螢光改變量 79 圖4-17 流式細胞儀分析乳酸菌與Ls. monocytogenes BCRC 15338吸附Caco-2細胞之綠色螢光改變量 80 圖4-18 流式細胞儀分析乳酸菌與Ls. monocytogenes BCRC 15338吸附Caco-2細胞之紅色螢光改變量 81 圖4-19 流式細胞儀分析Lb. reuteri Pg4與大腸桿菌O157:H7吸附Caco-2細胞之四象限事件數百分比統計 82 圖4-20 流式細胞儀分析Lb. casei ATCC 393與大腸桿菌O157:H7吸附Caco-2細胞之四象限事件數百分比統計 83 圖4-21 流式細胞儀分析Lb. casei pNZ-cnb與大腸桿菌O157:H7吸附Caco-2細胞之四象限事件數百分比統計 84 圖4-22 流式細胞儀分析Lb. casei pNZ-mub與大腸桿菌O157:H7吸附Caco-2細胞之四象限事件數百分比統計 85 圖4-23 流式細胞儀分析Lb. reuteri Pg4與Ls. monocytogenes BCRC 15338吸附Caco-2細胞之四象限事件數百分比統計 86 圖4-24 流式細胞儀分析Lb. casei ATCC 393與Ls. monocytogenes BCRC 15338吸附Caco-2細胞之四象限事件數百分比統計 87 圖4-25 流式細胞儀分析Lb. casei pNZ-cnb與Ls. monocytogenes BCRC 15338吸附Caco-2細胞之四象限事件數百分比統計 88 圖4-26 流式細胞儀分析Lb. casei pNZ-mub與Ls. monocytogenes BCRC 15338吸附Caco-2細胞之四象限事件數百分比統計 89 | |
dc.language.iso | zh-TW | |
dc.title | 表達外源性吸附蛋白以提高乳酸菌對腸道細胞之吸附性 | zh_TW |
dc.title | Increasing the adhesion ability of Lactobacillus to intestinal epithelial cells by heterologously expression of adhesion proteins | en |
dc.type | Thesis | |
dc.date.schoolyear | 98-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 陳明汝,陳小玲,余碧,劉?德 | |
dc.subject.keyword | 乳酸菌,益生菌,吸附蛋白,競爭排斥, | zh_TW |
dc.subject.keyword | Lactobacillus,probiotic,adhesion protein,competition exclusion, | en |
dc.relation.page | 98 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2010-07-21 | |
dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
dc.contributor.author-dept | 動物科學技術學研究所 | zh_TW |
顯示於系所單位: | 動物科學技術學系 |
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