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研究生:張文瀠
研究生(外文):Wen-Ying Chang
論文名稱:台灣金線連免疫調節蛋白之純化與活性分析
論文名稱(外文):Purification and Function Analysis of the Immunomodulatory Protein from Anoectochilus formosanus
指導教授:許輔許輔引用關係
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:園藝學研究所
學門:農業科學學門
學類:園藝學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:138
中文關鍵詞:免疫調節台灣金線連
外文關鍵詞:Anoectochilus formosanusimmunomodulatory
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台灣金線連 (Anoectochilus formosanus Hayata) 經硫酸銨沈澱以及DE-52 / Hi-Trap Q離子交換層析,可以純化出一個66 kDa及一個14 kDa之蛋白以及一種未知紅色化合物,稱為台灣金線連免疫調節區分物(immunomodulatory fraction from A. formosanus Hayata, IFAF)。IFAF並非醣蛋白,亦不具有凝集小鼠紅血球之活性。IFAF可活化巨噬細胞,促進該細胞分泌NO及TNF-alpha,並提高iNOS, TNF-alpha, IL-1 beta�n以及IL-18的mRNA表現。此外,IFAF也可以活化小鼠脾臟細胞,刺激其增生以及細胞激素IFN-gamma分泌量,並可增加IL-2, IL-5及IFN-gamma的mRNA表現量。另一方面,利用細胞融合技術製作得到3株融合瘤細胞,其分泌之單株抗體可分別辨識不同IFAF之蛋白質結構。為進一步瞭解IFAF的活性成分,將蛋白質以丙酮沈澱法來純化,可得不含紅色化合物之IFAF。此純化出之蛋白質樣品同樣包含66 kDa及14 kDa兩種蛋白,但它們可以藉由等電聚焦法進一步分離。測定IFAF加熱後與酵素水解後的活性,以及抗體中和試驗,結果顯示IFAF中的活性成分為14 kDa之蛋白質,因此將此蛋白命名為IPAF(immunomodulatory protein from A. formosanus)。IPAF是金線連中重要之生物活性物質,可提升宿主之免疫反應,且具醫藥以及臨床之發展潛力。
IFAF(immunomodulatory fraction from Anoectochilus formosanus Hayata) was purified by ammonium sulfate precipitation and DE-52 / HiTrap Q ion-exchange chromatography from A. formosanus. IFAF includes two proteins with molecular weight of 66 kDa and 14 kDa and an unclear red compound. Neither is IFAF a glycoprotein nor does it agglutinate mouse red blood cells. IFAF activates RAW 264.7 macrophages by enhancing the production of nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha), and also increases the expression of mRNAs of iNOS, TNF-alpha, IL-1 beta, and IL-18 by the cells. Additionally, IFAF also activates murine splenocytes. It stimulates the proliferation of murine splenocytes, increases the gamma-interferon (IFN-gamma) secretion, and promotes the mRNA expression of IL-2, IL-5, and IFN-gamma. Moreover three hybridoma clones that secrete monoclonal antibodies recognizing different protein components of IFAF are obtained. In order to understand the component of IFAF, the proteins are precipitated by acetone to exclude the red compounds, and the proteins with molecular weight of 66 kDa and 14 kDa are found in the acetone-precipitant. These proteins are further separated using isoelectric focusing techniques. The active component of IFAF is determinated by autoclave, trypsin hydrolysis and antibody neutralization. The results support that the 14 kDa protein has immunomodulatory ability. Therefore, the 14 kDa protein is further named as IPAF (immunomodulatory protein from A. formosanus Hayata). IPAF is an important biofunctional component of A. fomosanus, with medicinal capability and could strongthen the immunity of the host.
中文摘要..................................................1
英文摘要..................................................2

第一章 研究背景
第一節 序言..............................................3

第二節 植物中具生理活性的成分
一、植物生理活性成分的分類................................4

第三節 金線連的生理活性
一、金線連之分類與種原....................................9
二、台灣金線連之型態.....................................10
三、台灣金線連之營養成分.................................11
四、台灣金線連的已知生理活性.............................11
五、毒性測試.............................................15

第四節 免疫調節與健康
一、免疫功能之防護作用...................................15
二、發炎反應.............................................18
三、癌細胞之增生、分化與凋亡.............................19

第五節 研究動機與目的...................................24

第二章 材料與方法
一、金線連蛋白之萃取.....................................26
二、金線連蛋白之生化特性分析.............................31
三、免疫調節活性試驗.....................................39
四、利用單株抗體評估金線連免疫調節蛋白...................52
五、IFAF之化性與活性之探討...............................60

第三章 結果
第一節 金線連蛋白IFAF的純化與生化特性...................63
一、金線連蛋白IFAF之純化(硫酸銨沈澱法).................63
二、金線連蛋白IFAF之純化(丙酮沈澱法)...................64
三、等電點分析...........................................65
四、醣蛋白分析與血球凝集活性試驗.........................65

第二節 IFAF之免疫調節活性...............................67
一、對巨噬細胞之影響.....................................67
A、IFAF可活化RAW 264.7巨噬細胞產生一氧化氮...............67
B、IFAF可活化RAW 264.7巨噬細胞產生細胞激素TNF-alpha......68
C、IFAF可活化RAW 264.7巨噬細胞產生細胞激素IL-1 beta、IL-18.......................................................69

二、對小鼠脾臟細胞之影響.................................71
A、IFAF可促進小鼠脾臟細胞的增生效應(lymphocyte proliferation)..........................................71
1、細胞代謝活性分析(MTT / XTT assay)...................71
2、BrdU攝取分析..........................................72
B 、IFAF可活化小鼠脾臟細胞產生細胞激素IFN-alpha、
IL-2、IL-5...............................................73

第三節 金線連免疫調節蛋白單株抗體之製作.................75
一、單株抗體細胞株之製作.................................76
二、陽性融合細胞株之篩選.................................77
三、單株抗體之生產.......................................77
四、西方轉漬分析.........................................78

第四節 IFAF之化性與活性之探討...........................79
一、排除LPS污染IFAF之可能性..............................79
A、熱處理試驗............................................79
B、LPS抑制物polymyxin B(PMB)試驗.......................80

二、排除IFAF之活性成分為醣類之可能性.....................81
A、蛋白質水解試驗........................................81
B、分析IFAF蛋白質 / 醣類含量與活性之間的關連性...........82

三、排除IFAF之活性成分為紅色化合物之可能性...............83

四、確認具有免疫活性之蛋白分子量.........................83
A、等電分離試驗..........................................83
B、蛋白質中和試驗........................................84

第四章 討論.............................................86

第五章 參考文獻.........................................91


圖表目錄
Fig. 1 Preparation of IFAF from Anoectochilus formosanus. ............................................103
Fig. 2 UV / VIS absorbsion spectrum of IFAF. ..........104
Fig. 3 Elution profile of IFAF from a DEAE 52 cellulose column..................................................105
Fig. 4 SDS-PAGE analysis of IFAF. .....................106
Fig. 5 FPLC purification of IFAF. .....................107
Fig. 6 Tricine SDS-PAGE analysis of IFAF. .............108
Fig. 7 SDS-PAGE analysis of IFAF. .....................109
Fig. 8 IEF analysis of IFAF. ..........................110
Fig. 9 Isoelectric purified IFAF was purified using Bio-Rad Rotofor system with pH 4 - 6 ampholyte. ............111
Fig. 10 SDS-PAGE analysis isoelectric focused fraction.112
Fig. 11 Schiff’s staining of IFAF. ...................113
Fig. 12 Hemagglutination assay of IFAF toward mouse red blood cells. ...........................................114
Fig. 13 IFAF promotes nitrite accumulation in the culture supernatants of RAW 264.7 macrophages. .................115
Fig. 14 Immunomodulatory protein IFAF, isolated from A. formosanus, enhance iNOS mRNA expression in macrophage cell-line RAW 264.7. ........................................116
Fig. 15 Induction of TNF-alpha release from RAW 264.7 murine macrophages by IFAF. ...................................117
Fig. 16 Immunomodulatory protein IFAF, isolated from A. formosanus, enhance TNF-alpha mRNA expression in macrophage cell-line RAW 264.7. ...................................118
Fig. 17 Immunomodulatory protein IFAF, isolated from A. formosanus, enhance IL-1 beta mRNA expression in macrophage cell-line RAW 264.7. ...................................119
Fig. 18 Immunomodulatory protein IFAF, isolated from A. formosanus, enhance IL-18 mRNA expression in macrophage cell-line RAW 264.7. ...................................120
Fig. 19 Immunomodulatory protein IFAF, isolated from A. formosanus, enhance IL-6 mRNA expression in macrophage cell-line RAW 264.7. ........................................121
Fig. 20 IFAF induces cell proliferation of mouse splenocytes. ...........................................122
Fig. 21 IFAF induces cell proliferation of mouse splenocytes. ...........................................123
Fig. 22 IFAF induces cell proliferation of mouse splenocytes. ...........................................124
Fig. 23 IFAF enhances gamma-interferon (IFN-gamma) production by mouse splenocytes. ..................................125
Fig. 24 Immunomodulatory protein IFAF, isolated from A. formosanus, enhance IL-2, IL-5, and IFN-gamma mRNA expression in mouse splenocytes. .......................126
Fig. 25 Immunomodulatory protein IFAF, isolated from A. formosanus, enhance IL-4 mRNA expression in mouse splenocytes. ...........................................127
Fig. 26 Culture of myeloma and hybridoma production. ..128
Fig. 27 Specificity of mAb I1, J1, and H1 toward IFAF by western blotting analysis. .............................129
Fig. 28 Heat treatment decrease the activity of IFAF in RAW 264.7 macrophages. .................................130
Fig. 29 Macrophages activation of IFAF in the presence of LPS-inhibitor PMB. .....................................131
Fig. 30 Macrophage activity of IFAF in the presence of trypsin.................................................132
Fig. 31 Correlation between protein / carbohydrate content and their activity of isoelectric focused IFAF fractions...............................................133
Fig. 32 Induction of NO production by ammonium sulfate precipited and acetone precipited in RAW 264.7 macrophages. ...........................................134
Fig. 33 SDS-PAGE analysis isoelectric focused fraction of IFAF. ...............................................135
Fig. 34 The activity of isoelectric focused IFAF fractions. .............................................136
Fig. 35 Macrophages activation of IFAF was not decreased under the treatment with the mAb H1. ...................137
Table 1 Proximate chemical composition of Anoectochilus flrmosanus polysaccharide fractions. ...................138
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