臺灣博碩士論文加值系統

中文摘要
本研究自樟芝 (Antrodia camphorata) 之菌絲體及人工培養子實體粉末中純化得到三種免疫調節蛋白 acaⅠ、acaⅡ、acaⅢ。樟芝原料依序經均質、超音波破碎、硫酸銨沈澱、透析後，再以 DE-52, Hi-trap Q, Mono Q 離子交換層析純化出三種蛋白質，以 SDS-PAGE 測得分子量約為 29 kDa, 18.4 kDa 及 45 kDa。醣蛋白染色發現 acaⅠ 為醣蛋白而 acaⅡ、acaⅢ 不為醣蛋白。血液凝集試驗發現acaⅠ、acaⅡ、acaⅢ對人及小鼠紅血球無凝集作用;免疫調節活性試驗得知三種樟芝蛋白均可刺激小鼠淋巴細胞之增生，以及細胞激素 IFN-γ 之分泌。此外acaⅠ、acaⅡ、acaⅢ均可直接刺激 RAW 264.7 巨噬細胞分泌 TNF-α 及 NO。這些結果說明新發現之三種樟芝免疫調節蛋白均不為親醣蛋白 (lectin)，且具有顯著之免疫調節活性。這些結果說明了三種新發現的蛋白是樟芝中重要的活性物質之一，且具醫藥及臨床之發展潛力。未來可進一步探討這些樟芝蛋白對不同疾病的影響，及其對免疫細胞的作用機制。
此外我們分別將這些蛋白免疫小鼠後，利用融合瘤技術，製備acaⅠ、acaⅡ、acaⅢ之單株抗體，分析單株抗體之特異性及比較抗體與三種樟芝蛋白的反應性，結果顯示，相同來源之acaⅠ、acaⅡ在結構上是相似的，但不同來源之蛋白acaⅢ在結構上卻明顯不同，所以不同菌種來源的樟芝，是影響免疫活性的原因之一。所得之樟芝單株抗體，可應用於三種蛋白的基因選殖、或是樟芝品種的篩選、開發，可用來鑑定市售樟芝產品之品質。

Abstract
Three new immunomodulatory proteins, acaⅠ, acaⅡ and acaⅢ, were purified from the filtrated mycellium and dried powders of fruiting bodies of Antrodia camphorata. The isolation procedure of acas includes homogenization, sonication, precipitation by ammonium sulfate, and followed by DE-52 ion-exchange chromatography. These proteins from A. camphorata can be further purified using FPLC with Mono Q anion-exchange chromatography. SDS-PAGE analyses showed that the molecular mass of acaⅠ, acaⅡ and acaⅢ was 29 kDa, 18.4 kDa and 45 kDa, respectively. Further staining using the periodic acid/Schiff reagents showed that acaⅠwas an glycoprotein but acaⅡ and acaⅢ were not. Isoelectric focusing electrophoresis reveals that the isoelectric points of these acas were approximately 5.5. Additionally, acaⅠ, acaⅡ and acaⅢ cannot agglutinate mouse red blood cells.
The immunomodulatory activities of these A. camphorata proteins were demonstrated by their potent stimulatory activity toward mouse splenocytes and RAW 264.7 macrophages. All these three proteins increase the proliferation and IFN-γsecretion by mouse spleen cells. Moreover, they can directly activate the cells and enhance the production of nitric oxide and TNF-α by RAW 264.7 macrophages. Therefore, acaⅠ, acaⅡ and acaⅢ are potent immune stimulants that are proposed to strengthen the immune system of its host.
We also produced monoclonal antibodies (MAbs) of these proteins to evaluate the epitopes and structures of those. Six strains of hybridomas, including aca1K1, aca1F1, aca2I1, aca2J1, aca3E1, aca3F1 were obtained. Results of immunoblotting studies indicated that acaⅠand acaⅡ, both purified from the mycellium of A. camphorata, were structurally related. However, acaⅢ isolated from the dried powders of fruiting bodies A. camphorata was structurally not associated with acaⅠand acaⅡ. This result implies that the strains of A. camphorata from various sources may be different.
Further studies should be undertaken to discover the influence of these proteins to various diseases and to study the molecular mechanisms of those toward immune cells. The obtained MAbs can be used in the aids for cloning these proteins, to screen new A. camphorata strains, and to confirm the quality of commercial A. camphorata related products.

目錄
中文摘要 1
英文摘要 2
第一章 研究背景與動機 3
第一節 食藥用菇類的生理活性與活性成分 3
一、食藥用菇類的重要性 3
二、菇類的抗腫瘤與免疫調節活性 3
三、已知的生理活性成分 4
第二節 樟芝簡介 5
一、樟芝的生理與分類 5
二、樟芝的生理機能性 8
第三節 真菌免疫調節蛋白的相關研究與生理作用 9
一、菇類親醣蛋白的生理作用 9
二、真菌免疫調節蛋白的生化特徵與生理活性 11
第四節 單株抗體之生產與應用 14
一、為何要使用單株抗體 14
二、抗體之生產 15
三、單株抗體之特性與應用 20
第五節 研究構想與動機 21
第二章 材料與方法 24
第一節 樟芝免疫調節蛋白純化 24
一、樟芝菌絲體之蛋白純化 25
二、樟芝子實體粉末之蛋白純化 26
三、蛋白質濃度測定 28
第二節 樟芝蛋白生化特性分析 28
一、血球凝集活性試驗 30
二、膠體電泳分析 30
三、醣蛋白染色分析 32
四、等電點電泳分析 33
五、胺基酸組成分析 34
第三節 免疫調節活性試驗 34
一、對巨噬細胞的影響 36
1. 刺激巨噬細胞產生一氧化氮試驗 36
2. 細胞激素TNF-α的測定 37
二、對淋巴球增生的影響 39
1. 細胞激素IFN-γ的測定 39
2. 淋巴球增生試驗 40
(一) 淋巴球的分離與製備 40
(二) 溴尿苷Brd-U攝取之分析 41
(三) 細胞代謝活性分析( MTT / XTT assay) 42
第四節 利用單株抗體評估樟芝免疫調節蛋白結構之相似性 45
一、單株抗體的製作 46
1. 實驗動物與細胞株 47
2. 免疫計畫 47
3. 骨髓瘤細胞培養 47
4. 細胞融合 48
5. 陽性細胞株之篩選 49
6. 融合瘤之單株化 50
7. 融合瘤細胞株之保存 50
二、單株抗體的生產 51
1. 單株抗體的生產 51
2. 腹水之取得 51
三、酵素連結免疫分析法 51
1. 利用非直接非競爭法測定aca抗體特異性 52
2. 菇類蛋白之同源性試驗 53
四、西方轉漬分析 54
第三章 結果 56
第一節 樟芝免疫調節蛋白的純化 56
一、樟芝菌絲體之蛋白純化 56
二、樟芝子實體粉末之蛋白純化 59
三、蛋白質濃度測定
59
第二節 樟芝蛋白生化特性 59
一、血球凝集活性分析 59
二、膠體電泳分析 62
三、醣蛋白染色分析 62
四、等電點電泳分析 62
五、胺基酸組成分析 66
第三節 免疫調節活性試驗 66
一、對巨噬細胞的影響 66
1.acas直接刺激巨噬細胞RAW 264.7產生一氧化氮 66
2.acas可直接刺激巨噬細胞RAW 264.7產生細胞激素TNF-α 76
二、對淋巴球細胞的影響 76
1.樟芝蛋白可提高小鼠脾細胞的細胞激素IFN-γ 76
2.淋巴球增生試驗 76
(一) 溴尿苷Brd-U攝取分析細胞增生活性 76
(二) 細胞代謝活性分析( MTT / XTTassay) 80
第四節 利用單株抗體評估樟芝免疫調節蛋白結構之相似性 83
一、單株抗體的製作 83
二、單株抗體的生產 88
三、酵素連結免疫分析法 89
1. 利用非直接非競爭法測定aca抗體特異性 89
2. 菇類蛋白之同源性試驗 89
四、西方轉漬分析 93
第四章 討論 96
參考文獻 105
圖表目錄
表一、樟芝蛋白與FIP family 之胺基酸組成比較 67
表二、比較FIPs與acaⅠ、Ⅱ、Ⅲ 之生化特性 98
表三、比較樟芝蛋白acaⅠ、Ⅱ、Ⅲ 之生化特性與免疫調節活性
101
表四、單株抗體aca1K1、aca1F1、 aca2I1、 aca2J1、 aca3E1、 aca3F1對不同菇類蛋白的特異性 102
圖一、(A)野生樟芝子實體 (B)樟芝發酵液之菌絲體 (C)樟芝子實體乾燥粉末(D)人工培養子實體 6
圖二、FIP-vvo, FIP-fve, FIP-gts與 LZ-8 胺基酸序列比較圖 13
圖三、單株抗體生產流程圖 16
圖四、細胞融合作用機制 19
圖五、研究架構圖 23
圖六、樟芝蛋白純化流程 27
圖七、樟芝蛋白質acaⅠ, acaⅡ經由DE-52管柱層析純化圖及蛋白質電泳圖 57
圖八、樟芝蛋白質acaⅠ、acaⅡ、acaⅢ經由FPLC MonoQ管柱層析純化圖 58
圖九、樟芝蛋白質acaⅢ經由DE-52管柱層析純化圖及蛋白質電泳分析圖 60
圖十、acaⅠ、acaⅡ、acaⅢ血球凝集活性 61
圖十一、經由SDS/PAGE分析acaⅠ、acaⅡ、acaⅢ分子量 63
圖十二、acaⅠ、acaⅡ、acaⅢ之醣蛋白染色 64
圖十三、acaⅠ、acaⅡ、acaⅢ之等電點分析 65
圖十四、未經LPS處理，acaⅠ、acaⅡ、acaⅢ刺激巨噬細胞活化，NO產生圖 69
圖十五、經LPS處理，acaⅠ、acaⅡ、acaⅢ刺激巨噬細胞活化，NO產生圖 70
圖十六、未經LPS處理，acaⅠ、acaⅡ、acaⅢ刺激巨噬細胞活化， TNF-α產生圖 73
圖十七、經LPS處理，acaⅠ、acaⅡ、acaⅢ刺激巨噬細胞活化， TNF-α產生圖 74
圖十八、acaⅠ、acaⅡ、acaⅢ對小鼠脾臟細胞產生細胞激素IFN-γ之影響 77
圖十九、acaⅠ、acaⅡ、acaⅢ利用Brd-U法測定細胞增生比較
圖 79
圖二十、acaⅠ、acaⅡ、acaⅢ利用MTT法測定細胞增生比較
圖 81
圖二十一、acaⅠ、acaⅡ、acaⅢ利用XTT法測定細胞增生比較
圖 82
圖二十二、單株抗體生產日程表 84
圖二十三、骨髓癌細胞樹狀圖 85
圖二十四、骨髓癌細胞培養及製作融合瘤 87
圖二十五、單株抗體aca1K1、aca1F1對不同菇類蛋白的特異性 90
圖二十六、單株抗體aca2I1、aca2J1對不同菇類蛋白的特異性 91
圖二十七、單株抗體aca3E1、aca3F1對不同菇類蛋白的特異性 92
圖二十八、acaⅠ、acaⅡ、acaⅢ之西方轉漬圖 95

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