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  • 學位論文

以米麴菌表達由小孢子靈芝選殖之免疫調節蛋白質GMI

Expression of the immunomodulatoryprotein, GMI, from Ganoderma microsporum in Aspergillus oryzae

指導教授 : 黃慶璨
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摘要


Aspergillus屬表達系統目前已廣泛地應用於工業蛋白質生產,特別是A. oryzae等高產量菌株。然而,此表達系統面臨最大的瓶頸是異源蛋白質產量過低的問題。近年來,已發展出一些可以提升異源蛋白質產量的策略,包括建立蛋白質水解酶缺陷突變株、使用強力啟動子、基因融合策略、利用5’端非轉譯區(5’UTR)調控轉譯效率,以及調控蛋白陪伴分子(chaperone)表現量以提升異源蛋白質之分泌等。 本研究利用上述策略中的強力啟動子與基因融合策略,搭配電穿孔轉形系統以米麴菌異源表達由小孢子靈芝(Ganoderma microsporum)選殖之免疫調節蛋白質GMI,並利用缺乏C端澱粉結合區的葡萄澱粉酶(GLaA1-511)以基因融合方式將目標蛋白質分泌至胞外。結果共篩選到2株GLA::GFP與6株GLA::GMI轉形株,經南方氏雜合分析結果顯示質體插入染色體的套數為1至多個不等。由西方雜合法得知異源蛋白質確實可以藉由基因融合策略分泌至胞外。此外,融合蛋白質斷裂的現象可能發生在胞內或培養基中,而且以固態基質培養的分離效果較佳。另外,附著於GFP轉形株菌絲邊緣水珠發出綠色螢光的現象,可能是胞內GFP蛋白質透過非古典分泌途徑分泌到胞外所造成。此外,純化後得到的重組蛋白質GMI可以活化人類T細胞,促使其分泌介白素2(IL-2),顯示米麴菌表達系統生產的GMI具有免疫調節活性。 關鍵字:米麴菌、表達系統、基因融合、GMI、免疫調節蛋白質、電穿孔轉形法。

並列摘要


Aspergillus expression system is generally used for the production of industrial proteins, especially those high yield species, such as A. oryzae. However, the main bottleneck of this system is the low yield for heterologous protein expression. In recent years, several strategies have been developed for improving the heterologous protein production, including the use of protease-deficient strains, strong promoters, gene fusions and 5’UTR for improving the translation efficiency as well as the modulation of chaperone gene expression for improving the secretion. In this study, we used the strong promoter and gene fusion strategies in conjunction with electroporation to express GMI, an immunomodulatory protein cloned from Ganoderma microsporum in A. oryzae. We also investigated the feasibility of secreting the target proteins using a truncated glucoamylase gene(glaA1-511)fusion. After electroporation, two GLA::GFP and six GLA::GMI transformants were selected. Southern blot analysis of transformants showed that the target genes were integrated into chromosome by single or multiple copies. In addition, the heterologous proteins were secreted into medium by gene fusion strategy, and fusion proteins might be cleaved in cytoplasm or medium. Drops with green fluorescence on the filaments of GFP transformant might result from GFP secretion via non-classical secretion pathway. The immunoactivity analysis of recombinant GMI indicated that human Jurkat T cell could be stimulated to secrete IL-2 by recombinant GMI. Keywords:Aspergillus oryzae, expression system, gene fusion, GMI, immunomodulatory protein, electroporation

參考文獻


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被引用紀錄


謝宛伶(2014)。小孢子靈芝免疫調節蛋白質 GMI 部分片段之生理活性分析〔碩士論文,國立臺灣大學〕。華藝線上圖書館。https://doi.org/10.6342%2fNTU.2014.01271
蔣友邦(2012)。小孢子靈芝免疫調節蛋白質GMI雙體化對其免疫調節功能之影響〔碩士論文,國立臺灣大學〕。華藝線上圖書館。https://doi.org/10.6342%2fNTU.2012.02604
翁瑞芸(2011)。小孢子靈芝免疫調節蛋白質GMI之功能與結構分析〔碩士論文,國立臺灣大學〕。華藝線上圖書館。https://doi.org/10.6342%2fNTU.2011.02191
吳明玥(2008)。利用熱休克蛋白質5'端非轉譯區片段調控免疫調節蛋白質GMI於米麴菌表達系統之產量〔碩士論文,國立臺灣大學〕。華藝線上圖書館。https://doi.org/10.6342%2fNTU.2008.01984

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