柳松菇凝集素之基因選殖及其重組蛋白活性評估

Gene Cloning and Evaluation Activity of Recombinant Lectin Isolated from Agrocybe aegerita

陳建男(Jian-Nan Chen)

指導教授 ： 張鴻民

國立臺灣大學/生物資源暨農學院/食品科技研究所/碩士(2005年)

https://doi.org/10.6342/NTU.2005.01857

摘要

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。

關鍵字

凝集素 ； 柳松菇

並列摘要

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.

並列關鍵字

Agrocybe aegerita ； lectin

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