基因重組 : 人類白血球顆粒細胞增生因子

淡江大學機構典藏 > 理學院 > 生命科學研究所 > 學位論文 > Item 987654321/32618

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题名:	基因重組 : 人類白血球顆粒細胞增生因子
其它题名:	Molecular cloning for human granulocyte colony stimulating factor (hG-CSF) in Pichia pastoris
作者:	邱俊瑋;Chou, Chun-wei
贡献者:	淡江大學生命科學研究所碩士班簡素芳;Chien, Su-fang
关键词:	嗜甲基酵母菌;人類白血球顆粒細胞增生因子;人類白血球顆粒細胞;Pichia pastoris;hG-CSF;Human granulocyte
日期:	2006
上传时间:	2010-01-11 02:27:43 (UTC+8)
摘要:	人類白血球顆粒細胞增生因子（Human granulocyte colony stimulating factor；hG-CSF）是人體嗜中性白血球（neutrophilic granulocyte）生長、分化所必須的細胞激素，是由174個氨基酸所構成。主要是應用在化療所引起之嗜中性白血球缺乏症（neutropenia）。本實驗以基因工程的方法，將hG-CSF基因轉殖到酵母菌，如酵母菌可使hG-CSF分泌到細胞外，即可簡化之後之純化程序。實驗中，我們以聚合酶連鎖反應(PCR) 從pET25b-hG-CSF中擴增hG-CSF，並轉殖此因子之基因片段。此DNA片段有522 bp，並且在5’端設計有6個組織氨酸（histidine tag）和可被腸激酶所辨識（enterokinase cleavage site）的序列，需要時可將此酵素切割成hG-CSF蛋白質。PCR之後挑選clone帶有hG-CSF基因，並且將此基因再接到表現質體（pPIC9K）上，完成質體pPIC9K-hG-CSF的建構。最後將質體轉殖到酵母菌SMD1168內，挑選出生長於含有4 mg/ml G-418的YPDS固態培養基上的菌落，這可能是含有high copy number的轉型菌。將誘導後的細胞及培養基，分別以SDS-PAGE及西方點墨法（Western blotting）來鑑定結果。可發現不管是在細胞內或培養基都有hG-CSF，而分泌到細胞外的hG-CSF約為每公升10毫克，表現的hG-CSF確實其分子量在20 kDa左右。將經由FPLC分子篩液相層析管柱（HiPerp Sephacryl S-200）初步純化後的Fraction I（12~19）和Fraction II（21~34），各分別添加到人類白血病細胞株（HL-60）中，發現Fraction II有助於HL-60細胞增生，而Fraction I卻不能使細胞增生，因此可判斷Fraction II具有生物活性。添加40 ng/ml的Fraction II，在第７天時，可以觀察到細胞數目比對照組增加了２倍；添加Fraction II由1 ng ~ 40 ng/ml有顯著增生，而在40 ng ~ 150 ng/ml達到最高的細胞數。 Human granulocyte colony stimulating factor (hG-CSF) is a 18.7 kDa glycoprotein , consisting 174 amino acids. It can stimulate granulocyte colony formation and affects proliferation, differentiation and activation of mature neutrophilic granulocytes. It was widely for treatment of neutropenia in cancer therapy. We cloned the hG-CSF gene into P. pastoris and hope to see the recombinant hG-CSF can secrete into medium. It will make the purification procedure much easiler. We amplified the hG-CSF gene from pET25b-hG-CSF by polymerase chain reaction (PCR). The size of the hG-CSF DNA contains 522 bp, and histidine tag (His6) and enterokinase cleavage site (Asp4Lys) were added at the N-terminus of the protein. The PCR product was first cloned to pOPtima™ cloning vector (TA- Cloning). The “insert” was further cloned into pPIC9K vector. pPIC9K-hG-CSF plasmid was integrated into the alcohol oxidase region of the SMD1168 genome. We selected the transformants that were selected from medium contains 4 mg/ml of G-418. Based on it can be selected from the plate contains 0.2 mg/ml of G-418 having 1 copy of insertion. We can predict this is a multicopy clone. Under on culturing condition, the recombinant hG-CSF was able to be secreted into the medium, 10 mg/L of culture medium. After the medium was concentrated, we obtained the protein from FPLC-gel filtration column (Hiperp Sephacryl S-200). There were two fractions . 「Fraction I and Fraction II」 From SDS-PAGE, both were located at 20 kDa. But from the biological activity study for stimulation of granulocyte. We only found Fraction II has the activity. We concluded that Fraction I was obtain in the early portion of the gel filtration column. So it may exist as the aggregated form.
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