臺灣博碩士論文加值系統

本研究目的在探討Pseudomonas vesicularis MA103 fucI基因構築至乳酸菌 (Nisin controlled expression system, NICE) 表現系統之 pNZ8149載體上，再以電穿孔法轉殖至 Lactococcus lactis NZ3900 中，以 Nisin 誘導該轉殖株產 Fucosidase 並降解莢托馬尾藻 (Sargassum siliquosum) 之岩藻聚醣 (Fucoidan) ，探討並比較莢托馬尾藻 Fucoidan (SSF) 及其衍生寡醣之抗凝血、抗氧化之生理活性，並評估岩藻寡醣於 Caco-2 細胞吸收之效果。依據菌株 MA103 中 Fucosidase 之 fucI 基因與乳酸菌 pNZ8149限制酶切位，設計引子對分別於 5’ 和 3’ 端加入 SpeI 和 SacI 限制酶切位，另設計一組接上His-tag之引子對。電穿孔轉形至 E. coli DH5α，抽取含 fucI基因之質體與乳酸菌表現載體 pNZ8149 接合，電穿孔轉形至 Lc. lactis NZ3900 之勝任細胞中表現。乳酸菌轉殖株 Lc. lactis NZ3900 rfucI 及 Lc. lactis NZ3900 rfucI-H 以 5 ng/mL Nisin 誘導 3 hr 之胞內酵素活性最佳，分別可得0.056 U/mg protein 及 0.057 U/mg Protein比活性，經 SDS-PAGE 可觀察推估分子量約為 51 kDa之條帶。另外，將 SSF進行高效能液相層析 (High performance liquid chromatograph, HPLC)，與標準品比對，推估其平均分子量約為 25,000 Da，以Lc. lactis NZ3900 rfucI-H胞內粗酵素液 (Fucosidase rfucI-H) 水解後利用超過濾系統區分出小於 3 kDa分子量之寡糖，經過 Sephadex G-10膠體過濾層析 (Gel permeation chromartography, GPC) 後，以HPLC 分析並比對標準品得知，可能是聚合度為 DP3 之寡醣 (Fuc-C3)，硫酸基含量為 0.28 ± 0.14%。在兔血漿在抗凝血實驗中，於活化部分凝血活酶時間 (Activated partial thromboplastin time, APTT) 實驗中，SSF濃度為 5 mg/mL 時，其 APTT 延遲效果等同於 10 g/mL 肝素與空白組相比可延遲 88 sec，而凝集程度則較 10 g/mL 肝素低， Fuc-C3 濃度則至 20 mg/mL 時才具有延遲 APTT 之效果，與空白組相比可延遲 66 sec，無降低凝集程度之能力。而在凝血酶原時間 (Prothrombin time, PT) 實驗中，結果顯示 SSF 及 Fuc-C3 皆無法延遲凝血時間，但濃度為 5 mg/mL 時具有降低凝血程度之效果。測試 SSF及 Fuc-C3 之抗氧化活性，SSF 有較佳的清除 DPPH自由基能力 (27.12 ± 0.01%) 及還原力 (OD700nm = 0.21 ± 0.01)，Fuc-C3 則有較佳的螯合亞鐵能力 (24.29 ± 0.02%)。在人類腸道上皮細胞 Caco-2 吸收實驗上，以 HPLC 分析，可發現隨著吸收時間的增加，上層液 Fuc-C3 之波峰有下降之趨勢，但在下層液部分並未出現其波峰，進一步使用 TLC 確認，當吸收時間達 90 min時，可在下層液中觀察到 Fuc-C3 色斑之產生，測定其全糖量定量，初步推估其吸收率為 0.1%，而其吸收機制與反應條件仍需進一步探討。

The aim of this study is using fucI gene from Pseudomonas vesicularis MA103 construct on LAB nisin controlled expression (NICE) system vector, pNZ8149, then cloned into Lactococcus lactis NZ3900 by electroporating and using nisin induced recombinant crude enzyme to hydrolyzed fucoidan from Sargassum siliquosum. Contrast the effects of Sargassum siliquosum fucoidan (SSF) and fucoidan oligosaccharides on their anticoagulant, antioxidant activities, and its adsorption performance with intestinal cell line Caco-2 are examined. The fucI forward primer encode SpeI restriction site and the reverse primer encode SacI restriction site, and one has his-tag. Then using the gene of Pseudomonas vesicularis MA103 fucosidase fucI gene as a template for PCR reaction, and then transformed into the competent cells of E. coli DH5α. Then pGEM-T vector, which carrying the cloned fucI gene, was digested with SpeI and SacI, and ligated into the LAB vector pNZ8149 and electroporated into the Lc. lactis NZ3900. The clones Lc. lactis NZ3900 rfucI and Lc. lactis NZ3900 rfucI-H induced by 5 ng/mL nisin at 30oC for 3 hr could obtain higher fucosidase activity (0.056 U/mg protein and 0.057 U/mg), which revealed a 51 kDa protein band by SDS-PAGE. Using high performance liquid chromatograph (HPLC) to analysis SSF compare to standard, the average molecular weight was 25,000 Da. The SSF hydrolyzed by Lc. lactis NZ3900 rfucI-H intracellular crude enzyme (Fucosidase rfucI-H) and molecular mass < 3 kDa were collected by ultra filtration (UF) system, then though gel permeation chromartography (GPC) and analysis by HPLC. Compare to standard, the DP of fucoidan oligosaccharides may DP 3 (Fuc-C3). In the experiment of anticoagulant activities of rabbit plasma, activated partial thromboplastin time (APTT) analysis, 5 mg/mL of SSF can delayed 88 sec like 10 g/mL heparin, and also reduction aggregation level better than 10 g/mL heparin. The 20 mg/mL of Fuc-C3 can delayed 66 sec. The anticoagulant activities of rabbit plasma in prothrombin time (PT) analysis were examined, SSF and Fuc-C3 were no obvious delayed in prothrombin time, but there were a tendency for reduction on absorbance while concentration were 5 mg/mL. The experiment on antioxidant activity of SSF and Fuc-C3, SSF has better DPPH scavenging effect (27.12 ± 0.01%) and reducing power (OD700nm = 0.21 ± 0.01), and Fuc-C3 has better Fe2+ chelating effect (24.29 ± 0.02%). In the adsorption performance with Caco-2 cells on transwell device, the HPLC analysis showed that Fuc-C3 did not penetrate to the basolateral liquid but the Fuc-C3 concentration were decreased in apical fluid in a time-dependent fashion. Using thin layer chromatography (TLC) to confirm intestinal absorption, Fuc-C3 was observed in basolateral fluid after 90 min. Further measurement on total suger content and the absorption rate of Fuc-C3 was estimated as 0.1%. The possible absorption mechanism and reaction conditions of Fuc-C3 by intestinal cell need further investigation.

目錄 i
表目錄 vi
圖目錄 vii
附錄目錄 x
中文摘要 xi
英文摘要 (Abstract) xiii
壹、前言 1
貳、文獻整理 3
一、海藻 3
二、海藻多醣 4
2-1. 海藻多醣定義 3
2-2. 海藻多醣之萃取方式 4
2-3. 海藻多醣之生理活性 5
三、海藻寡醣 6
3-1. 海藻寡醣之萃取方法 6
3-2. 海藻寡醣之生理活性 7
四、岩藻聚醣 (Fucoidan) 9
4-1. 岩藻聚醣的組成與結構 9
4-2. 岩藻聚醣之生理活性 10
五、岩藻醣苷酶 (Fucosidase) 12
六、乳酸菌 12
6-1. 定義 12
6-2. 基因轉殖之應用 13
七、Caco-2 細胞 13
7-1. 腸道吸收模式 14
7-2. 寡醣之吸收 15
参、實驗設計 16
肆、材料方法 18
一、實驗材料 18
1-1. 原料 18
1-2. 實驗菌株 18
1-3. 實驗細胞株 18
1-4. 試驗藥品 18
1-4-1. 藥品 18
1-4-2. 細胞培養液 20
1-4-3. 載體 21
1-4-4. 引子對 (Primer pair) 21
1-4-5. 聚合酶連鎖反應試劑 21
1-4-6. 限制酶 21
1-4-7. 蛋白質及DNA萃取純化套裝試劑 21
1-4-8. 電泳標準品 21
1-4-9. 培養基組成 22
1-4-10. 細胞培養液組成 23
1-4-11. Fucosidase 反應基質 24
1-4-12. 電泳溶液 24
1-4-13. 電泳膠片配製 24
1-4-14. 膠過濾緩衝液 24
1-4-15. 無菌過濾 25
1-4-16. 薄層層析矽膠片 25
1-5. 儀器設備 25
二、實驗方法 27
2-1. 菌株保存與活化 27
2-1-1. 菌株之保存 27
2-1-2. 菌株之活化 27
2-2. P. vesicularis MA103 之fucI 基因選殖至乳酸菌 28
2-2-1. 引子設計 28
2-2-2. PCR擴增欲用於TA-cloning 之基因 28
2-2-3. 大腸桿菌之 TA-cloning 轉形作用 29
2-2-3-1. 連接反應 29
2-2-3-2. 大腸桿菌 (E. coli DH5) 勝任細胞製作 30
2-2-3-3. 大腸桿菌 (E. coli DH5) 電穿孔轉形 30
2-2-3-4 利用膠電泳確認插入之基因片段大小 30
2-2-3-5. 利用限制酶剪切純化目標基因 31
2-2-4. 乳酸菌之電穿孔轉形作用 31
2-2-4-1. 連接反應 31
2-2-4-2. 乳酸菌 (Lc. lactis NZ3900) 勝任細胞之製備 32
2-2-4-3. 乳酸菌 (Lc. lactis NZ3900) 電穿孔轉形 33
2-2-4-4 利用膠電泳確認插入之基因片段大小 33
2-2-4-5. 利用限制酶剪切純化目標基因 34
2-2-5. DNA 序列比對 34
2-2-6. Nisin對重組乳酸菌生長之影響 34
2-2-7. 乳酸菌重組 Fucosidase之酵素活性分析 34
2-2-7-1. Fucosidase 活性測定 34
2-2-7-2. Lc. lactis NZ3900 rpfuI 之Nisin 誘導濃度對Fucosidase 活性碳討 35
2-2-7-3. 蛋白質的定量 35
2-2-7-4. 分子量的鑑定 36
2-3. 莢托馬尾藻一般成份分析 36
2-3-1. 水分含量 36
2-3-2. 灰分含量 36
2-3-3. 粗蛋白含量 36
2-3-4. 粗脂肪含量 37
2-3-5. 粗纖維含量 37
2-4.岩藻聚糖之萃取及其經 Fucosidase rfucI-H 水解後寡醣產物之分析 38
2-4-1. 岩藻聚醣之萃取 38
2-4-2. 總醣量測定 38
2-4-3. 還原醣量測定 38
2-4-4. 硫酸酯含量測定 39
2-4-5. 總酚含量測定 39
2-4-6. Fucosidase rfucI-H 之製備 39
2-4-7. 岩藻寡醣之製備 40
2-4-8. 膠體過濾層析 (GPC) 40
2-4-9. 超過濾 (Molecular weight cut off, MWCO) 40
2-4-10. 薄層層析 (TLC) 41
2-4-11. 高效能液相層析 (HPLC) 41
2-4-12. 傅立葉紅外線光譜分析 (FT-IR) 41
2-5. 抗氧化活性測定 41
2-5-1. 清除 DPPH 自由基能力測定 41
2-5-2. 還原能力之測定 42
2-5-3. 亞鐵離子螯合能力之測定 42
2-6. 抗凝血生理活性 42
2-6-1. 凝血酶原時間 (Prothrombin time, PT) 42
2-6-2. 活化部份凝血活酶時間 (Activited
partial thromboplastin time, APTT) 43
2-7. 岩藻寡醣於腸道細胞吸收試驗 43
2-7-1. Caco-2 細胞株之活化 43
2-7-2. Caco-2 細胞株培養方法及繼代方法 44
2-7-3. 細胞數計數 44
2-7-4. 細胞之冷凍保存 44
2-7-5. Caco-2 細胞存活率分析-MTT assay 45
2-7-5-1. 以MTT assay測定不同細胞數對 Caco-2
細胞存活率之影響 45
2-7-5-2. 岩藻寡醣對 Caco-2細胞之毒性試驗 46
2-7-6. Transwell insert 培養法 47
2-7-6-1. Caco-2 細胞於吸收裝置其單層膜完整性測試 47
2-7-7. 岩藻寡醣於腸道細胞吸收率測定 47
三、統計分析 47
伍、結果與討論 48
陸、結論 61
柒、參考文獻 62

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