張鴻民臺灣大學：食品科技研究所陳建男Chen, Jian-NanJian-NanChen2007-11-272018-06-292007-11-272018-06-292005http://ntur.lib.ntu.edu.tw//handle/246246/56295柳松菇（Agrocybe aegerita）是目前台灣常見的食用菇類，其低熱量、低脂肪且高蛋白質含量，長期以來被視為高級食材且不斷推廣。本研究以NCBI資料庫上已發表柳松菇凝集素之部分基因序列，設計引子進行北方雜合（Northern blot hybridization）反應，得知柳松菇凝集素之訊息RNA約為900 bp。此外，利用快速增殖cDNA 5’端鑑定柳松菇凝集素基因cDNA全長，得知轉錄子5’端轉錄起點位於ATG上游第39個核苷酸位置處，且柳松菇凝集素基因cDNA全長為672 bp，其中基因譯讀區序列共489 bp。再利用CLUSTAL_X程式將快速增殖cDNA 5’端鑑定所得到之柳松菇凝集素基因譯讀區序列各選殖片段DN序列，以及其預測性胺基酸組成序列進行分析比對，由於基因譯讀區序列及其預測性胺基酸序列之差異，推測柳松菇凝集素可能有兩種以上isotype形式存在。重組蛋白生產方面，藉由快速cDNA增殖5’/3’端結果，設計引子進行表現質體pET-28b構築並選殖入Escherichia coli表現系統進行表現。在1 mM IPTG誘導下，隨著培養時間（1-3小時）增加其表現量隨之增加，且大部分重組蛋白為可溶性蛋白。重組蛋白經純化後進行紅血球凝集反應測試，發現對蛋白酶處理過之2％兔子紅血球有凝集反應發生，但對未處理過之2％兔子紅血球並無凝集效果。Abstract Agrocybe aegerita (AA), one of popular duetary mushrooms in Taiwan, is well known for its good nutritional values and low calory content. Lectin of AA induced apoptosis, influenced DNase activity, and showed strong inhibition on growth of human tumour cell lines. In present study, primers were designed on the basis of the nucleotide sequence of AA lectin listed in NCBI to carry out Northern blot hybridization analysis and found that the mRNA of AA lectin was about 0.9 kb. In addition, the start site of cDNA of AA lectin was determined by 5’ RACE and found that retroposons of transcription start site was at 39 bp upstream to ATG and the full of cDNA was 672 bp, in which length of open reading frame was 489 bp. Then, nucleotide and deduced amino acid sequences of open reading frame of AA lectin were aligned by a program of CLUSTAL_X. Based on the differences of nucleotide and deduced amino acid sequences in the present sample from those listed in NCBI, we presumed that there were at least two isotypic forms of lectins in AA cultivation. For the production of recombinant protein of AA lectin, primers were designed to construct pET-28b expression vectored by nucleotide sequencing of result of 5’RACE, and then transformed into E. coli. The expression of recombinant protein by E. coli increased with the increasing incubation time for up to 3 hr, and the obtained recombinant protein was found mainly water-soluble. Hemagglutination assay revealed that recombinant protein expressed in E. coli induced agglutination with 2 % Pronase-treated rabbit RBC, while no effect was observed with normal rabbit RBC.目錄 頁碼 中文摘要……………………………………………………………… Ⅰ 英文摘要……………………………………………………………… Π 目錄…………………………………………………………………… Ⅲ 表次…………………………………………………………………… Ⅶ 圖次…………………………………………………………………… Ⅷ 第一章 文獻整理…………………………………………………….. 1 一、前言……………………………………………………….. 1 二、食藥用菇（Nutraceutical mushroom）……………………. 1 (一) 食藥用菇簡介…………………………………………….. 1 (二) 菇類之膳食功能……………………………………...….. 2 (1) 第一級功能營養成分…………………………………….. 3 (2) 第二級功能嗜好特性……………………………………. 3 (3) 第三級功能生理活性……………………………………. 4 (三) 菇類生理活性及相關研究……………………………… 4 (1) 抗腫瘤活性………………………………………………… 5 (2) 免疫調節…………………………………………………… 6 (3) 降血壓作用………………………………………………… 7 (4) 降血脂作用…………………............................................... 7 (5) 降血糖作用............................................................................ 8 (6) 抗氧化作用………………………………………………… 8 (7) 抗病毒物質………………………………………………… 9 (8) 抗生素……………………………………………………… 9 (9) 抗發炎作用………………………………………………… 9 (10) 抗菌作用………………………………………………….. 10 三、柳松菇介紹………………………………………………… 10 四、凝集素……………………………………………………… 11 (一) 凝集素之介紹…………………………………………….. 11 (二) 凝集素之分類…………………………………………….... 13 (1) 植物性來源………………………………………………… 14 (2) 動物性來源………………………………………………… 14 (3) 微生物來源………………………………………………… 14 第二章 材料與方法………………………………………………….. 16 一. 材料…………………………………………………………. 16 二. 實驗方法……………………………………………………. 16 1. 柳松菇RNA之萃取…………………………………………. 16 2. 北方雜合分析………………………………………………... 17 2-1柳松菇mRNA純化…………………………………………. 17 2-2 RNA甲醛洋菜膠體電泳……………………………………. 17 2-3柳松菇cDNA製備…………………………………………. 19 2-4松菇凝集素基因譯讀區專一性探針之製備……………….. 20 2-5北方雜合反應…………………………………….…………. 21 3.柳松菇凝集素基因譯讀區起始點鑑定………………..……... 22 4. 柳松菇凝集素基因譯讀區序列及其預測性胺基酸組成分 析………………………………………………..………..……… 23 4-1-1 柳松菇凝集素譯讀區選殖片段DNA序列及其預測性胺 基酸比對………………………………………………………… 23 4-1-2柳松菇與其他物種凝集素胺基酸序列比對………...…… 24 4-2柳松菇凝集素胺基酸組成分析…………………….………. 24 5 柳松菇凝集素重組蛋白質生產……………………………… 24 5-1 pET-28b表現質體的構築…………………………….…….. 24 5-2 以E. coli 表現柳松菇凝集素重組蛋白質………..……….. 25 6.柳松菇凝集素重組蛋白質分析…………………….…..…….. 27 6-1 西方轉漬分析……………………………….……………… 27 6-2柳松菇凝集重組蛋白活性分析-血液凝集反應……………. 28 第三章 結果與討論………………………………………………….. 31 1.柳松菇RNA之萃取……………………………………………. 31 2.北方雜合分析…......................................................................... 31 3.柳松菇凝集素基因譯讀區起始點鑑定 …………………….. 32 4. 柳松菇凝集素基因譯讀區序列及其預測性胺基酸組成析... 33 5. 柳松菇凝集素重組蛋白生產………………………………... 35 5-1. pET-28B表現質體之構築………………………………….. 35 5-2.重組蛋白之表現…………………………………………….. 36 6. 柳松菇重組蛋白活性分析-血球凝集反應測試…………….. 37 第四章 結論 ………………………………………………………… 38 第五章 參考文獻 …………………………………………………… 39 第六章 附錄……………………………………………………………64 附錄一. ………………………………………………………………....64 附錄二. 藥品與儀器………………………………………………….. 70 附錄三. 本論文所使用之引子序列表……………………………….. 74 附錄四. 質體pET-28b的限制圖譜………………………..………… 75 附錄五. 質體pGEM-T Easy的限制圖譜…………………………… 761127519 bytesapplication/pdfen-US凝集素柳松菇Agrocybe aegeritalectinthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/56295/1/ntu-94-R92641004-1.pdf