臺灣博碩士論文加值系統

中文摘要

在亞洲地區米在主食能量提供上佔非常重要角色。米中14-16 kDa過敏蛋白屬於鹽溶性蛋白部分，彼此蛋白質屬於同源性基因產物具有相似性構造
，對人類唾液中α-澱粉酶有抑制作用。其胺基酸序列與小麥α-澱粉酶抑制劑、大麥胰蛋白酶抑制劑及篦麻子之貯藏蛋白具相似性。目前已證實這些蛋白質是造成穀類過敏病人之主要過敏蛋白。研究顯示:米中14-16、26、33及56 kDa蛋白質會與米過敏病人體內IgE產生過敏反應，其中14-16 kDa蛋白質會與90-95% 米過敏病人的IgE產生反應。因此，14-16 kDa米過敏蛋白被認為是米中的主要過敏蛋白質。目前因環境污染及飲食問題造成更多過敏體質病人，顯示探討過敏蛋白對人類生活之影響是一重要研究課題。

關於食物過敏原之檢測工具，所需要的是針對過敏原所發展出的專一性抗體，鑑於米14-16 kDa過敏蛋白之專一性抗體取得不易，故本論文目的在以分子轉殖技術將米之14-16 kDa過敏蛋白基因擴增轉殖於表現載體，利用大腸桿菌表現宿主表現目標蛋白質，進而純化將之製備成專一性抗體。利用此抗體做為檢測米中14-16 kDa過敏蛋白之工具。

本研究第一部分是利用逆轉錄聚合酶連鎖反應由米中mRNA擴增出米過敏蛋白基因- RA17之cDNA，並構築成pET-29a(+)-RA17表現載體，利用Escherichia coli BL21表現宿主得到RA17重組過敏蛋白，以此蛋白質製備多株抗體做為檢測植酸酶轉基因米與母本米(台農67號)中14-16 kDa主要過敏蛋白含量之工具。由西方轉漬法測得的結果顯示，植酸酶轉基因米所含的主要過敏蛋白之含量為母本米之96.3%；酵素免疫分析法分析結果，14-16 kDa主要過敏蛋白含量在植酸酶轉基因米為1.66 mg/g ± 0.08 mg/g、母本米為1.73 ± 0.09 mg/g。顯示植酸酶轉基因米中主要過敏蛋白，並不會因外來植酸酶基因而造成其過敏蛋白含量的增加。

本研究第二部分目的在分離純化稻米中14-16 kDa 過敏蛋白，利用純化之過敏蛋白做進一步特性分析。首先，利用1M NaCl溶液萃取鹽溶性蛋白後，利用硫酸銨沉澱法、DEAE-Sepharose 離子交換層析管柱得到四個主要波峰(Ⅰ、Ⅱ、Ⅲ、Ⅳ)。經由SDS-PAGE電泳及免疫轉漬法分析將得到14-16 kDa明顯條帶之波峰部分(Ⅳ)，再以分子篩Sephadex G-50凝膠過濾層析管柱做進一步分離純化，得到四個主要波峰部分，經電泳及免疫轉漬確認得到分子量在14-16 kDa目標蛋白；經LC-MS-MS質譜鑑定證實：分子篩層析得到之主要目標蛋白分別為米過敏蛋白RA14/RA14B (FDEAE-Peak I)、RA14C/RAG2 (FDEAE-Peak II)、 similar to RA17 precursor (FDEAE-Peak III ) 、RA14/RA14B (FDEAE-Peak IV)。將此四個主要波峰蛋白做模擬胃液反應，發現RA14/RA14B、RA14C/RAG2在5分鐘後即消化完全；但similar to RA17 precursor經反應60分鐘後仍穩定存在。在模擬腸液反應中RA14/RA14B、RA14C/RAG2與 similar to RA17 precursor經反應60分鐘後仍穩定存在，不受腸液酵素所分解。在100 °C耐熱試驗，發現RA14/ RA14B、RA14C/RAG2與similar to RA17 precursor 加熱60分鐘後經免疫轉漬分析均可測得目標蛋白存在，顯示米過敏蛋白RA14/RA14B、RA14C/RAG2與 similar to RA17 precursor對熱安定。

本研究第三部分是利用本實驗製備的米過敏原抗體及純化得到的過敏蛋白做標準品，用於檢測市售米及糯米中14-16 kDa米過敏蛋白之含量。發現九種市售米中其含量分別為2.36 ± 0.12 mg/g (台稉2號)、2.16 ± 0.13 mg/g (台稉4號)、1.91 ± 0.11 mg/g (台稉9號)、2.26 ± 0.10 mg/g (桃園3號)、2.61 ± 0.07 mg/g (台南11號)、1.81 ± 0.11 mg/g (高雄139號)、2.26 ± 0.09 mg/g (高雄145號)、2.12 ± 0.10 mg/g (台秈22號)、1.95 ± 0.09 mg/g (進口泰國秈米) ，1.69 ± 0.08 mg/g (溪湖長糯米)、1.67 ± 0.12 mg/g (新化長糯米)、1.73 ± 0.11 mg/g (溪湖圓糯米)與1.80 ± 0.10 mg/g (西螺圓糯米)。在檢測的稉米及秈米中14-16 kDa過敏蛋白含量以台南11 號米最高、高雄139 號米含量最低，泰國秈米之含量與台稉9 號米相近。而在4種糯米中其含量彼此間無顯著差異。結果顯示：分析的國產米中14-16 kDa過敏蛋白含量的不同與產地無相關性，且稉米及秈米中14-16 kDa過敏蛋白含量在有些品種中會有差異。經模擬胃液試驗發現：檢測的米樣品中14-16 kDa過敏蛋白在2分鐘後即可被胃蛋白酶完全消化分解，且分解後之小分子蛋白即不再具有抗原特性。

Rice is the staple food in Asia, it plays an important role in providing energy to general population within the region. Rice 14-16 kDa allergenic proteins, a salt-soluble protein fraction, were the products of homologous genes with similar structures. The 14-16 kDa allergenic proteins have been reported as human salivary α-amylase inhibitor, barley trypsin inhibitor and storage protein of grate asako. It was demonstrated that these proteins were the major allergens which caused allergic reactions with consumption of cereals. It was reported that the 14-16, 26, 33 and 56 kDa proteins in rice could react with IgE of rice allergic patients in which the 14-16 kDa proteins, especially, were found to be recognized by IgE antibodies from 90-95% of rice allergic patients. Therefore, 14-16 kDa allergens are thought to be the main allergy proteins in rice. Environmental pollution and dietary factors on human allergenicity has drawn much attention lately. More information in this regard is needed.

To develop an allergen-specific antibody is the first step for food allergen detection. It is difficult to obtain the specific antibodies against rice 14-16 kDa allergens. The objectives of this study were to utilize the molecular transgenic technology to amplify rice 14-16 kDa allergen genes and transfer the genes into the expression vector followed by expressing the target recombinant protein in Escherichia coli expression host, to purify target recombinant protein, and to study the use of purified protein on detection of rice 14-16 kDa allergens.
pET-29a(+)-RA17 Expression vector of rice was constructed by using the RT-PCR method to amplify the cDNA of RA17 (one of rice 14-16 kDa allergens) from rice mRNA. Recombinant RA17 (rRA17) was expressed in Escherichia coli expression host as a histidine-tag fusion protein and was purified to its homogeneity. The polyclonal antibody was then prepared from the rRA17 protein to be further used as a tool to analyze the content of 14-16 kDa allergens in phytase-transgenic rice (GR) and non-transgenic rice (NR) (wild-type, Oryza sative L. cv. Tainung 67). Result of western blotting showed that the content of major allergenic proteins in GR was 96.3% of that in NR, which were 1.66 ± 0.08 mg/g and 1.73 ± 0.09 mg/g for GR and NR, respectively. It appeared that transferring phytase gene into rice plant did not affect the content of rice major allergenic proteins significantly.

The 14-16 kDa allergens of rice was purified and was further analyzed and characterized. Salt-souble proteins of rice were first extracted with 1M NaCl solution followed by precipitation with 90% saturated ammonium sulfate solution. The precipitate was dialyzed and then subjected to DEAE-Sephadex ion-exchange chromatography. Four fractions (I-IV) were obtained. Each fraction was further purified by Sephadex G-50 gel-filtration. The purified fractions were identified as RA14/RA14B (FDEAE-Peak I), RA14C/RAG2 (FDEAE-Peak II), similar to RA17 precursor (FDEAE-Peak III ) and RA14/RA14B (FDEAE-Peak IV) by LC-MS-MS analysis. In a simulated gastric fluid (SGF) test, both RA14/RA14B and RA14C/RAG2 were fully digested in 5 min whereas similar to RA17 precursor was stable up to 60 min. In contrast, both RA14/RA14B and RA14C/RAG2 were relatively stable when subjected to a simulated intestinal fluid (SIF) test. Besides, RA14/RA14B, RA14C/RAG2 and similar to RA17 precursor remained to be stable when heated at 100°C for 60 min.

Contents of 14-16 kDa allergens in 9 studied rice samples were as follows: 2.36 ± 0.12 mg/g (Oryza sative L. cv. Taikeng 2), 2.16 ± 0.13 mg/g (Oryza sative L. cv. Taikeng 4), 1.91 ± 0.11 mg/g (Oryza sative L. cv. Taikeng 9), 2.26 ± 0.10 mg/g (Oryza sative L. cv. Taoyuan 3), 2.61 ± 0.07 mg/g (Oryza sative L. cv. Tainan 11), 1.81 ± 0.11 mg/g (Oryza sative L. cv. Kaohsiung 139), 2.26 ± 0.09 mg/g (Oryza sative L. cv. Kaohsiung 145), 2.12 ± 0.10 mg/g (Oryza sative L. cv. Taisen 22), 1.95 ± 0.09 mg/g (Imported Thailand Indica rice), 1.69 ± 0.08 mg/g (Sihu long waxy rice), 1.67 ± 0.12 mg/g (Xinhua long waxy rice), 1.73 ± 0.11 mg/g (Sihu round waxy rice) and 1.80 ± 0.10 mg/g (Siluo round waxy rice). Data indicated that there was no correlation between contents of 14-16 kDa allergens and growing areas of rices. Furthermore, the 14-16 kDa allergen contents were different in some japonica and indica rice strains. In SGF test, the 14-16 kDa allergens in tested rices could be completely digested in 2 min, and all fragments after SGF digesting showed no allergic activity when subjected to Western blotting.

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頁次 i
圖次 v
表次 ix
中文摘要 x
英文摘要 xii
第一章、文獻整理 1
一、食物過敏 1
二、食物過敏盛行率 1
三、食物過敏症的機轉 2
四、食物過敏之臨床表現 2
(一)、腸胃道食物過敏反應 2
(二)、皮膚食物過敏反應 3
(三)、呼吸道過敏反應 3
五、食物過敏原的特性及種類 4
六、稻米種類、特性及組成 5
(一)、稻米種類 5
(二)、稻米之特性 5
(三)、稻米之組成 6
七、米過敏原 6
八、轉基因食物之過敏性 7
九、轉基因食物之過敏性評估方法 7
(一)、序列同源性比對 8
(二)、消化穩定性試驗 8
十、偵測食品過敏原之方法 9
十一、加工處理對穀類過敏原之影響 10
(一)、加工處理對麥類過敏原之影響 10
(二)、加工處理對米類過敏原之影響 11
十二、消化對穀類過敏原之影響 11
(一)、消化對麥類過敏原之影響 11
(二)、消化對米類過敏原之影響 12
十三、重組過敏蛋白 12
(一)、重組過敏蛋白製備方法 12
1.原核生物表現系統 12
2.真核生物表現系統 13
(二)、重組過敏蛋白之研究與應用 14
十四、質譜分析與蛋白質鑑定 15
論文章節 30
實驗動機與目的 31
第二章、稻米14-16 kDa 過敏蛋白之基因選殖與重組蛋白之表現 33
壹、實驗設計 34
貳、材料與方法 35
一、實驗材料 35
(一)、儀器設備 35
(二)、軟體 35
(三)、藥品 36
(四)、菌株 37
(五)、質體 37
(六)、稻米材料 37
二、米14-16 kDa 過敏蛋白cDNA的製備 37
(一)、稻米Total RNA 之製備 37
(二)、米14-16 kDa 過敏蛋白第一股cDNA之合成 38
(三)、RT-PCR擴增RA17 DNA片段 38
三、純化PCR 產物 RA17 DNA 39
四、pGEM-T Easy Vector之接合反應 39
五、載體DNA之轉形 40
(一)、勝任細胞之製備 40
(二)、大腸桿菌之電轉形作用 40
(三)、轉形基因庫之篩選(藍白篩選) 40
六、大腸桿菌微量質體DNA之抽取 41
七、DNA之剪切、回收及黏合 41
(一)、DNA之剪切 41
(二)、DNA之剪切後之回收 41
(三)、DNA經限制酶剪切後之黏合 42
八、目標基因在大腸桿菌中之表現 42
(一)、表現載體pET29a-RA17之構築 42
(二)、大腸桿菌來源重組蛋白之誘生 42
(三)、大腸桿菌來源重組蛋白表現方式之偵測 42
九、Ni2+-NTA金屬螯合樹脂管柱純化重組蛋白 43
十、重組蛋白抗體之製備 44
十一、蛋白質濃度測定 44
十二、SDS-PAGE 蛋白質電泳分析 44
十三、西方轉漬分析 44
十四、酵素聯結免疫分析 45
十五、米鹽溶性蛋白之萃取 45
參、結果與討論 46
一、非轉基因與植酸酶轉基因米Total RNA 抽取方法之建立 46
二、米Total RNA 經RT-PCR反應逆轉錄成 cDNA 46
三、篩選轉基因米及非轉基因米中過敏蛋白cDNA之TA cloning 轉
形菌株 47
四、構築過敏蛋白RA17表現載體 47
五、RA17重組蛋白在表現宿主E. coli BL21之表現 47
六、大腸桿菌中R A17重組蛋白之分離與純化 48
七、重組過敏蛋白RA17抗體之製備 48
八、非轉基因米與轉植酸酶基因米之14-16 kDa 過敏蛋白之檢測 48
肆、結論 50
第三章、稻米14-16 kDa 過敏蛋白之純化與特性分析 67
壹、實驗設計 68
貳、材料與方法 69
一、實驗材料 69
(一)、儀器設備 69
(二)、藥品 69
(三)、稻米材料 70
二、米鹽溶性蛋白質萃取 70
三、硫酸銨沉澱 70
四、DEAE離子交換樹脂管柱層析 71
五、分子篩膠體過濾層析 71
六、蛋白質質譜鑑定 71
七、蛋白質濃度定量 72
八、Tricine SDS-PAGE蛋白質電泳 72
九、西方轉漬分析 72
十 、模擬胃液消化試驗 72
十一、模擬腸液消化試驗 72
十二、100oC熱穩定性試驗 73
參、結果與討論 74
一、米14-16 kDa過敏蛋白之純化 74
二、14-16 kDa過敏蛋白純化後之鑑定 75
三、過敏蛋白之模擬胃液消化試驗 77
四、過敏蛋白之模擬腸液消化試驗 77
五、過敏蛋白在100 oC下熱穩定性試驗 78
肆、結 論 79
第四章、不同品種米中14-16 kDa過敏蛋白之檢測 99
壹、實驗設計 100
貳、材料與方法 101
一、實驗米樣品 101
二、米鹽溶性蛋白質之萃取 101
三、蛋白質濃度定量 101
四、Tricine SDS-PAGE蛋白質電泳 101
五、西方轉漬分析 101
六、模擬胃液消化試驗 101
參、結果與討論 102
一、市售米之鹽溶性蛋白含量 102
二、市售米中14-16 kDa 過敏蛋白之偵測 102
三、市售米中14-16 kDa 過敏蛋白含量之檢測 102
四、市售米中14-16 kDa 過敏蛋白之模擬胃液消化試驗 103
肆、結論 105
第五章、參考文獻 118

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