臺灣博碩士論文加值系統

β-多醣體 (β-glucan) 為葡萄糖聚合物，具有免疫調節之生物活性。Dectin-1為C型醣類辨識受體，其位於細胞外之醣類辨識功能部位(carbohydrate recognition domain; CRD) 可以辨識真菌細胞壁中β-glucan，並會誘發活性氧原子與促發炎細胞激素之產生。冰核蛋白 (ice nucleation protein; INP) 是由Pseudomonas syringae 而來的一種外膜蛋白，可將外源性蛋白質表現於細胞表面。本實驗已成功選殖出人類與小鼠Dectin-1基因之hCRD與mCRD 核酸片段，分別將hCRD 與mCRD錨定在大腸桿菌的細胞表面上。經誘導表現之人與小鼠重組INPN-CRD融合蛋白，其分子量分別為38 kDa、37 kDa。利用西方墨點法分析確認INPN-CRD融合蛋白，並以酵素聯結免疫吸附分析法 (enzyme-linked immunosorbent assay; ELISA) 與免疫螢光顯微鏡證明hCRD 與mCRD確實表現於大腸桿菌細胞表面，且具結合水溶性β-glucan之活性。本系統建立之親和力結合分析法，將應用於競爭型親和力分析，以篩選不同來源與結構中與Dectin-1具有高親和力之β-glucan。

β-glucan, a glucose polymer, has been demonstrated to have immunomodulatory activities. Dectin-1 is a C-type lectin receptor that recognizes fungal β-glucan through a single extracellular C-type lectin-like carbohydrate recognition domain (CRD), and mediates the production of reactive oxygen species and inflammatory cytokines. The ice nucleation protein (INP) is a membrane-associated protein from Pseudomonas syringae. It has been demonstrated that truncated INP derivatives containing the N-terminal domain can display targeted foreign proteins on the cell surface. In this experiment, hCRD and mCRD were cloned and expressed with INPN as an anchor motif for surface expression on Escherichia coli. Finally, hCRD and mCRD anchored on the cell surface were confirmed using Western blotting and immunofluorescence microscopy. The whole-cell enzyme-linked immunosorbent assay further validated the displayed hCRD and mCRD with binding activity to souble β-glucan. Thus, the expressed hCRD and mCRD can be applied as an in vitro screening system for biological forms of β-glucan with high affinity for Dectin-1.

目錄
中文摘要 I
Abstract III
謝誌 V
目錄 VI
圖表目錄 XI
第1章 前言 1
第2章 文獻回顧 3
2.1 免疫系統 3
2.1.1先天性免疫反應 3
2.2　Dectin-1受體 4
2.2.1　Dectin-1受體之表現 4
2.2.2 Dectin-1之結構特性 5
2.2.3 Dectin-1受體之重要性 6
2.2.4　Dectin-1及其他PRRs之交互作用 7
2.3 β-glucan之結構特性 9
2.3.1 β-glucan 之化學構造與活性之關係 10
2.3.2 β-glucan 之專一性受體 10
2.3.3 具免疫活性之β-glucan 11
2.3.4 β-glucan之免疫活性與臨床應用 11
2.4 菌體表面表現系統 13
2.4.1 冰核蛋白 13
2.4.1.1 冰核蛋白之結構及功能 13
2.4.2 冰核蛋白於菌體表面呈現系統之應用 14
第3章 材料與方法 16
3. 1 本實驗使用菌株及細胞株種類 16
3.1.1 菌株種類 16
3.1.2 細胞株種類 16
3.1.3 載體 16
3.1.4 儀器設備 17
3.1.5 試劑 18

3.2 人類與小鼠CRD表面呈現系統之建立 21
3.2.1 pGEMT-INPN載體之構築 21
3.2.1.1 萃取P. syringae染色體DNA 21
3.2.1.2 以PCR技術增幅INPN基因 21
3.2.1.3 DNA電泳 21
3.2.1.4 膠體純化 23
3.2.1.5 TA cloning接合反應 23
3.2.1.6 勝任細胞之製備 23
3.2.1.7 轉型作用 24
3.2.1.8 藍白篩選 24
3.2.1.9 以PCR方式確定是否含有所選殖片段INPN 24
3.2.2 INPN於表現載體之構築 24
3.2.2.1 質體的抽取 24
3.2.2.2 重組質體pET21b-INPN之構築 25
3.2.2.3 INPN及pET21b以電泳法回收DNA片段 25
3.2.2.4 膠體純化 25
3.2.2.5 接合反應 26
3.2.2.6 轉型作用 26
3.2.2.7 以PCR方式確定是否含有所選殖片段INPN 26
3.2.2.8 核酸定序 26
3.2.3　RAW264.7細胞株之培養條件 26
3.2.3.1 細胞繼代培養 27
3.2.3.2 細胞冷凍保存 27
3.2.3.3　RAW 264.7總量RNA之萃取及cDNA之合成 27
3.2.3.3.1 總量RNA之萃取 27
3.2.3.3.2 RNA電泳 28
3.2.3.3.3 核糖核酸濃度的定量與計算 28
3.2.3.3.4 反轉錄作用 28
3.2.4 pGEMT-hCRD與pGEMT-mCRD質體之構築 28
3.2.4.1 引子之設計 29
3.2.4.2 以PCR技術增幅hCRD與mCRD基因 29
3.2.4.3 DNA電泳 29
3.2.4.4　TA cloning接合反應 29
3.2.4.5 勝任細胞之製備 29
3.2.4.6 轉型 30
3.2.4.7 藍白篩選 30
3.2.4.8 以PCR方式確定是否含有hCRD與mCRD片段 30
3.2.5 INPN-hCRD與INPN-mCRD於表現載體之構築 30
3.2.5.1 質體的抽取 30
3.2.5.2 pET21bINPN-hCRD與pET21bINPN-mCRD之構築 30
3.2.5.3 以PCR方式確定是否含有所選殖片段CRD 31
3.2.5.4 利用限制酶切割檢查是否有重組成功 31
3.2.5.5 核酸定序 31
3.2.5.6 菌種保存 31
3.2.6 INPN-hCRD與INPN-mCRD融合蛋白於菌體表面之表現 31
3.2.6.1 聚丙烯醯胺膠體電泳 32
3.2.7 西方墨點法 33
3.2.8 INPN-hCRD與INPN-mCRD融合蛋白之免疫螢光鏡檢分 析 33
3.2.8.1 INPN-hCRD與INPN-mCRD融合蛋白之免疫螢光鏡 檢分析 34
3.2.9 E. coli BL21 (DE3)/pET21bINPN-hCRD與E. coli BL21 (DE3)/pET21bINPN-mCRD之生長曲線測定 34
3.2.10 INPN-hCRD與INPN-mCRD融合蛋白之親和力分析法 34
3.2.10.1 海藻多醣之生物素化 35
3.2.10.2 親和力結合分析法 35
3.3　哺乳動物細胞表現系統之建立 35
3.3.1 細胞株培養 36
3.3.1.1 HEK293T細胞株之培養條件 36
3.3.1.2 細胞繼代培養 36
3.3.1.3 細胞冷凍保存 36
3.3.2 RAW 264.7總量RNA之萃取及cDNA之合成 36
3.3.3　hCRD與mCRD於表現載體之構築 36
3.3.3.1以PCR技術增幅CRD基因 36
3.3.3.2 DNA電泳 36
3.3.3.3 CRD及pcDNA3.1限制酶切割 37
3.3.3.4 CRD及pcDNA3.1以電泳法回收DNA片段 37
3.3.3.5 膠體純化 37
3.3.3.6 接合反應 37
3.3.3.7 勝任細胞之製備 37
3.3.3.8 轉型作用 37
3.3.3.9 藍白篩選 37
3.3.3.10 以PCR方式確定是否含有所選殖片段CRD 37
3.3.3.11 核酸定序 37
3.3.4　哺乳動物細胞之轉染作用 38
3.3.4.1 大量質體之萃取 38
3.3.4.2 細胞之轉染作用 39
3.3.4.3 穩定表現株之篩選 39
3.3.5　穩定表現細胞株之蛋白分析 40
3.3.5.1 西方墨點法 40
3.3.5.2.1細胞蛋白質萃取 40
3.3.5.2.2 蛋白質定量 40
3.3.5.2.3 蛋白質轉漬 40
3.3.5.2.4 西方墨點法呈色分析 40
第4章 結果 41
4.1 表面呈現系統之建立與表現分析 41
4.1.1 pET21b-INPN載體之構築 41
4.1.2人類與小鼠mCRD基因之選殖 41
4.1.2.1人類醣類辨識功能部位hCRD之增幅 41
4.1.2.2小鼠巨噬細胞株RAW264.7總量RNA之萃取及小鼠醣類辨識功能部位mCRD之選殖 46
4.1.2.3 pET21bINPN-hCRD與pET21bINPN-mCRD質體之構築 46
4.2 CRD基因之序列比對 46
4.3重組蛋白之SDS-PAGE蛋白分析 54
4.4重組蛋白之西方墨點分析 54
4.4.1 E. coli BL21 (DE3)/pET21bINPN-hCRD與之西方墨點分析 54
4.4.2 E. coli BL21 (DE3)/pET21bINPN-mCRD與之西方墨點分析 54
4.5重組蛋白之免疫螢光顯微鏡檢分析 57
4.5.1 E. coli BL21 (DE3)/pET21bINPN-hCRD之免疫螢光顯微鏡檢分析 57
4.5.2 E. coli BL21 (DE3)/pET21bINPN-mCRD之免疫螢光顯微鏡檢分析 57
4.6重組蛋白之生長曲線測定 57
4.6.1 E. coli BL21 (DE3)/pET21bINPN-hCRD之生長曲線測定 57
4.6.2 E. coli BL21 (DE3)/pET21bINPN-mCRD之生長曲線測定 58
4.7 菌體表面融合蛋白之表現及其親和力分析法 64
4.7.1 菌體表面INPN-hCRD融合蛋白之表現及其親和力分析法 64
4.7.2 菌體表面INPN-mCRD融合蛋白之表現及其親和力分析法 64
4.8　人類與小鼠CRD基因於哺乳動物細胞表現載體之構築 64
4.8.1 人類與小鼠CRD基因於哺乳動物細胞表現載體之構築 64
4.9　hCRD與mCRD於HEK293T細胞株之表現 70
4.9.1 人類與小鼠CRD之西方墨點分析 70
第5章 討論 72
第6章 結論 75
參考文獻 76
作者簡介 82




圖表目錄
圖1、 Dectn-1之結構圖 6
圖2、 Dectin-1居間之訊息傳遞 8
圖3、 β-多醣體之結構 9
圖4、 P. syrimgae之染色體DNA萃取之電泳分析圖 42
圖5、 以1%瓊膠電泳分析INPN之PCR產物 43
圖6、 pET21b-INPN經限制酶NdeI和HindIII作用之電泳分析圖 44
圖7、 以1%瓊膠電泳分析hCRD之PCR產物 45
圖8、 小鼠巨噬細胞總量RNA萃取之電泳分析圖 47
圖 9、 以1%瓊膠電泳分析mCRD之PCR產物 48
圖10、 pET21bINPN-hCRD經限制酶HindIII和XhoI作用之電泳分析圖 49
圖11、 pET21bINPN-mCRD經限制酶NotI和XhoI作用之電泳分析圖 50
圖12、 INPN-hCRD之重組基因核苷酸序列及推衍之胺基酸序列 51
圖13、 INPN-mCRD之重組基因核苷酸序列及推衍之胺基酸序列 52
圖14、 CRD胺基酸序列比對分析 53
圖15、 IPTG誘導E. coli BL21 (DE3) 表現INPN-hCRD與INPN-mCRD蛋白質之SDS-PAGE分析 55
圖16、 西方墨點法分析INPN-hCRD與INPN-mCRD融合蛋白 56
圖17、 E. coli BL21 (DE3)/pET21bINPN-hCRD 免疫螢光顯微鏡檢 分析 (600× 放大倍率) 59
圖18、 E. coli BL21 (DE3)/pET21bINPN-mCRD 免疫螢光顯微鏡檢分析 (600× 放大倍率) 60
圖19、 E. coli BL21 (DE3)/pET21bINPN-hCRD 免疫螢光顯微鏡檢 分析 (600× 放大倍率) 61
圖20、 E. coli BL21 (DE3)/pET21bINPN-mCRD免疫螢光顯微鏡檢分析 (600× 放大倍率) 62
圖21、 E. coli BL21 (DE3)/pET21b-hCRD、E. coli BL21 (DE3)/ pET21b-mCRD、E. coli BL21 DE3)/pET21bINPN-hCRD 與E. coli BL21 DE3)/ pET21bINPN-mCRD之生長曲線 63
圖22、 定量E. coli BL21 (DE3)/pET21bINPN-hCRD (pET21bINPN -mCRD) 與生物素化之laminarin標準液親和性結合 66
圖23、 pcDNA3.1-hCRD經限制酶BamHI和AgeI作用之電泳分析圖 67
圖24、 pcDNA3.1-mCRD經限制酶BamHI和AgeI作用之電泳分析圖 68
圖25、 西方墨點法分析hCRD與mCRD游離蛋白 71
表1、 本研究所使用之引子序列與限制酶 22
表2、 比較人與小鼠CRD胺基酸序列之相似度 69

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