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    題名: 人類顆粒細胞增生因子在酵母菌的表現與純化
    其他題名: Expression and purification of recombinant human granulocyte colony stimulating factor (hG-CSF).
    作者: 簡名揚;Chien, Ming-yang
    貢獻者: 淡江大學化學學系碩士班
    簡素芳;Chien, Su-fang
    關鍵詞: 人類顆粒細胞增生因子;嗜甲基酵母菌;Human Granulocyte Colony Stimulating Factor;hG-CSF;Pichia pastoris
    日期: 2008
    上傳時間: 2010-01-11 02:35:44 (UTC+8)
    摘要: 人類顆粒細胞增生因子(Human Granulocyte Colony Stimulating Factor;hG-CSF)能刺激嗜中性白血球前驅細胞生長,並促進其增殖、分化,同時具有促進嗜中性白血球由骨髓釋出及增強成熟嗜中性白血球的機能,它被廣泛的應用在癌症化療和骨髓移植後,所引起的嗜中性白血球缺乏症(neutropenia)。本實驗將hG-CSF基因整合到pPIC9K質體,轉殖到酵母菌SMD1168,以4 mg/ml G-418抗生素篩選出multicopy inserted clones,並誘導使其表現hG-CSF。
    使用搖瓶及發酵槽方式大量誘導表現hG-CSF,並以SDS-PAGE及西方點墨法(Western blottnig)分析,發現表現之hG-CSF分子量確實在20 kDa。比較以搖瓶和發酵槽方式表現,以發酵槽方式誘導之hG-CSF表現量為搖瓶方式的7倍。
    將誘導後之濃縮培養基,以FPLC-DEAE Sepharose Fast Flow與Sephadex G-50管柱純化,回收率分別為76.5 %及65.7 %,純化倍率分別為3.6及10.4。之後以此hG-CSF樣品對HL-60細胞增生的測試,採用1.計算細胞總數及 2.定量細胞內酵素活性(succinate dehydrogenase),結果是1.hG-CSF確實有助於HL-60細胞增生,添加100 ng/ml hG-CSF於培養7天後,所增生之細胞數目為未添加hG-CSF的2倍;2.細胞內之succinate dehydrogenase酵素活性也增加,在培養7天後,加100 ng/ml hG-CSF約為加2 ng/ml濃度時的9~10倍;細胞所增生的數目,亦隨著hG-CSF添加量的增加而增加。
    The recombinant human granulocyte colony stimulating factor (hG-CSF) is a hematopoietic cytokine that has been widely used for the treatment of neutropenia after chemotherapy and bone marrow transplantation. It can stimulate both proliferation, differentiation of neutrophils. In this study, we cloned the G-CSF encoding gene into pPIC9K vector and transformed it into SMD1168 yeast cells. The transformed cells were selected on the plate containing 4 mg/ml of G-418.
    The recombinant hG-CSF was found to be secreted in the medium, and molecular weight was as predicted at 20 kDa on SDS-PAGE, corresponding to the commercial standard. Comparing with bioreactor and shake flask, the specific hG-CSF protein production increased 7 times.
    After medium was concentrated, both DEAE Sepharose Fast Flow and Sephadex G-50 column chromatography were used to purify the hG-CSF protein. It resulted in the recovery of 76.5 % and 65.7 % respectively, and the purification of 3.6 and 10.4 fold respectively.
    By using HL-60 cell (Human promyelocytic cell), the bioactivity of hG-CSF can be observed. For one: The effect of the purified hG-CSF to promote the total cell numbers to 2 fold when 100 ng of the purified hG-CSF was added to the cells. The proliferation rate was hG-CSF-dependent. The most efficient was at the inoculation size of 1 × 105 cells/ml for 100 ng of purified hG-CSF. The other was the MTT assay to estimate the stimulation of the cellular succinate dehydrogenase activity. When the addition of 2~100 ng purified hG-CSF to the cells, the enzyme activity increased 9~10 times.
    顯示於類別:[化學學系暨研究所] 學位論文

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