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研究生:吳佳恬
論文名稱:分離並鑑定台梗九號稻米蛋白質和其抗白血病的免疫能力
論文名稱(外文):Protein isolation and identification from a Japonica rice (Oryza sative) MT9 with promoting activity of anti-leukemia immunity
指導教授:廖慧芬廖慧芬引用關係
學位類別:碩士
校院名稱:國立嘉義大學
系所名稱:生化科技研究所
學門:生命科學學門
學類:生物科技學類
論文出版年:2009
畢業學年度:97
語文別:中文
中文關鍵詞:稻米
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稻米是一種有益健康、不易引起過敏現象且容易消化的穀類,根據研究顯示稻米的成分及其衍生物,具有各種藥理及生物活性。先前研究亦指出,米中蛋白質或醣蛋白為重要的活化抗腫瘤免疫能力的成分。因此本研究目的是分離台粳九號蛋白質並且使用蛋白質體技術進行分析。藉由二維電泳與質譜分析白米(胚乳)和米糠中的蛋白質,我們鑑定出幾個有差異的蛋白質點,包含代謝、運輸、儲存、抗氧化、抵抗疾病和發育相關蛋白。其中,儲存蛋白可能是重要的調控免疫反應物質。我們進一步分別使用二次水、鹽溶液,鹼溶液和酒精分離出四種儲存蛋白,包含:白蛋白(albumin)、球蛋白(globulin)、穀蛋白(glutelin)和醇溶蛋白(prolamin)。經由抗白血病的免疫能力分析後,顯示prolamin對於刺激人類周邊血液單核球細胞(human mononuclear cells, MNC)並抑制人類白血病U937細胞生長,具有高度活性。且抗體的中和反應也證明稻米的prolamin是一個主要的活性成分。此外,發現在小麥的gluten和gliadin萃取成分中有表現會引起腹腔過敏疾病(celiac disease)的gliadin蛋白,但在稻米蛋白中並未被測得。因此我們認為稻米是個理想的不含gluten的食物,它不會引起乳糜瀉等相關疾病。總結,本研究證明稻米的prolamin在抗白血病免疫功能方面可能扮演重要的角色。
中文摘要................................................................................................... 1
英文摘要................................................................................................... 2
前言........................................................................................................... 4
台粳九號米............................................................................................4
植物種子蛋白質....................................................................................5
稻米儲存性蛋白質................................................................................6
Prolamin的消化特性.............................................................................8
小麥儲存性蛋白質................................................................................8
Gluten引起的麩質敏感症或乳糜瀉.....................................................9
利用蛋白質體學的方法研究稻米蛋白質..........................................10
研究目的..................................................................................................15
實驗材料..................................................................................................16
實驗方法..................................................................................................25
稻米之萃取與製備..............................................................................25
胚乳的粗萃取..................................................................................25
蛋白質定量......................................................................................25
總糖含量測定..................................................................................26
二維電泳及SDS-PAGE...........................................................................26
等電點電泳分析..............................................................................26
Strip平衡..........................................................................................27
SDS-膠體電泳分析.........................................................................28
蛋白質膠體染色..............................................................................28
二維電泳膠體影像分析..................................................................29
膠內蛋白質水解............................................................................. 29
稻米蛋白質之萃取與製備..................................................................30
稻米的四種儲藏性蛋白質和總蛋白質之萃取..............................30
抗體之製備......................................................................................31
膠體電泳分析和西方墨點法..........................................................32
Mini SDS-PAGE電泳分析..............................................................32
Coomassie blue 染色.......................................................................33
醣蛋白電泳分析..............................................................................33
Mini SDS-PAGE蛋白質轉漬分析...................................................34
抗體分析..........................................................................................35
統計分析............................................................................................36
結果..........................................................................................................37
糙米、白米和米糠萃取物對白血病U937細胞的生長抑制率..........37
稻米胚乳和米糠的蛋白質表現..........................................................37
Spot 1和GluA1、GluA2、GluA3、GluB1、GluB2和GluB4的胺基酸的親源關係..................................................................................38
分析稻米四種儲存性蛋白對於U937細胞之生長抑制情形.............39
分析四種儲存性蛋白質和經由粗萃取出來物中的醣類和蛋白質含量......................................................................................................39
電泳分析四種儲存蛋白質..................................................................40
Anti-prolamin的抗體中和反應...........................................................41
Western blot分析prolamin..................................................................41
Gliadin在稻米與小麥蛋白質表現......................................................41
討論..........................................................................................................43
未來展望..................................................................................................55
參考文獻..................................................................................................56
圖表..........................................................................................................64
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