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    Please use this identifier to cite or link to this item: http://ir.cnu.edu.tw/handle/310902800/9073


    標題: 於大腸桿菌中表現與純化蘭花之疑似甘露糖結合凝集素
    Expression and purification of a putative mannose-binding lectin from orchid in E. coli
    作者: 陳信丞
    Shin-Cheng Chen
    貢獻者: 張竣凱
    黃怡仁
    嘉南藥理科技大學:生物科技研究所
    關鍵字: 抗菌試驗
    抗蟲試驗
    重組蛋白表現
    蝴蝶蘭
    cytotoxity
    gene expression
    mannose-binding lectin
    Orchidaceae
    日期: 2005
    上傳時間: 2008-11-24 17:00:50 (UTC+8)
    摘要: 甘露糖結合凝集素(mannose-binding lectin, MBL),也稱為甘露糖結合
    蛋白(mannose-binding protein, MBP) 可以在鈣離子的作用下結合一些糖分
    子,如N-乙醯葡萄糖胺(GlcNAc)或甘露糖…等。由蝴蝶蘭基因庫選殖的一
    段cDNA,包含522 個胺基酸,經由基因庫比對後,發現與已知的MBL 有
    73%的相似度。但是這段基因的產物是否具有MBL 的生物活性,目前還無
    法得知。對於這個未知基因的產物暫時命名為甘露糖結合凝集素蛋白
    (mannose-binding lectin protein, MBLP)。本研究的目的是異源表現這段基
    因,以便探討這段基因產物的弁遄C以GST 及IMPACT 兩個蛋白質表現系
    統進行MBLP 基因表現時,提升誘導溫度到42℃之後,這個基因才可以成
    左熙Q表現出來,不過效果不佳。由於表現效率不佳,所以我們改用Wheat
    Germ CECF Kit 真核蛋白質表現系統,此系統以in vitro 方式合成出MBLP
    這個蛋白質,並以此蛋白進行抗昆蟲細胞及微生物的活性試驗。實驗結果
    顯示,MBLP 具有有抑制昆蟲細胞(SF9)和疑似軟腐病微生物的弁遄C另外,
    以reverse transcription PCR(RT-PCR)得知,MBLP 這個基因在蝴蝶蘭葉和花
    均有表現。
    Mannose-binding lectin (MBL), also called mannose-binding protein
    (MBP), binds to mannose or N-acetylglucosamine (GlcNAc) in a
    calcium-dependent manner. A cDNA obtained from Orchidaceae species,
    encoding a polypeptide with 140 amino acid has a 73% homology to known
    MBL. Whether the gene product is a ture MBL is not clear. In this study, we
    intented to overexpress the gene product and to investigate its function(s). Base
    on the results from SDS-PAGE and Western blotting analyses the putative MBL
    gene can be expressed in E. coli. However, the yield of the protein synthesis was
    not satisfied. To investigate whether the low efficience of protein expression in
    E. coli is due to different codon usage between E. coli and plant, we switched
    over to Wheat Germ CECF Kit (Roche), witch is an eukaryotic protein
    expression system. The putative MBL was successfully translated in virto.
    Biological analysis showed that the putative MBL had cytotoxic effect to insect
    cell (Spodoptera frugiperda, SF9) and bacteria. Reverse transcriptase-PCR were
    performed to investigate the putative gene expression in diffent part of tissue of
    Orchidaceae species
    Appears in Collections:[生物科技系(所)] 博碩士論文

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