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    Title: 人類顆粒細胞增生因子基因密碼在酵母菌中使用頻率的研究
    Other Titles: Codon optimization and expression of human granulocyte colony stimulating factor
    Authors: 汪恩慶;Wang, En-ching
    Contributors: 淡江大學化學學系碩士班
    簡素芳;Chien, Su-fang
    Keywords: 人類顆粒細胞增生因子;密碼偏好;Human Granulocyte Colony Stimulating Factor;hG-CSF;Codon usage bias
    Date: 2010
    Issue Date: 2010-09-23 16:10:47 (UTC+8)
    Abstract: 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)。
    酵母菌Pichia pastoris是常用於表現重組蛋白的宿主細胞之一,具有將蛋白質正確地折疊、轉譯後修飾以及將蛋白分泌至胞外等優點。在我們實驗室利用發酵槽誘導酵母菌可以獲得hG-CSF約71 mg/L。
    對宿主細胞而言的密碼使用頻率較高的基因可能會使蛋白產量有所提昇,因此本實驗依據酵母菌的密碼偏好,設計並合成出最佳化密碼的hG-CSF基因(syn hG-CSF),利用分析CAI值預測此段基因在大腸桿菌E. coli和酵母菌P. pastoris之中會有較高的蛋白表現,之後分別轉殖到大腸桿菌BL21(DE3)跟酵母菌SMD1168之中。
    目前已成功獲得大腸桿菌轉型株,在37℃下,使用搖瓶的方式加入IPTG誘導4個小時表現syn hG-CSF,以SDS-PAGE及西方點墨法(Western blotting)分析,發現所表現的蛋白分子量符合在22 kDa的位置,在500毫升的菌液當中大約有100毫克目標蛋白。
    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 and differentiation of neutrophils.
    Pichia pastoris has been extensively used as a cellular host for recombinant protein expression. It’s advantages are posttranslational modification, protein folding correction and protein secretion to the medium. In our laboratory, the expression of hG-CSF in P.pastoris,about 71 mg/L of recombinant protein in bioreactor.
    In theory, the higer the codon usage bias by th host cell, the more improved yield of protein.Therefore in this study, we designed and synthesized a new hG-CSF gene by using yeast preferred codon.The use of CAI values predicted this gene would yield higher expression level when transferred into E. coli BL21(DE3) and P.pastoris SMD1168.
    Up to the present, we have successfully cultured the colony of E. coli. after 4 hours of induction by IPTG at 37℃, the expression of the recombinant protein was confirmed by SDS-PAGE and Western blotting. The amount of recombinant protein was 100 mg in 500 ml LB medium approximately, and molecular weight was as predicted at 22 kDa corresponding to the commercial standard.
    Appears in Collections:[化學學系暨研究所] 學位論文

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