| 重组人KGF制备工艺和活性检测方法的建立 |
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| 引用本文： | 王希菊,李剑凤,孙丽霞,艾丽静,刘克玲,王克波,王红洁,薛彤彤,王晶翼.重组人KGF制备工艺和活性检测方法的建立[J].生物技术,2011,21(3). |
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| 作者姓名： | 王希菊 李剑凤 孙丽霞 艾丽静 刘克玲 王克波 王红洁 薛彤彤 王晶翼 |
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| 作者单位： | 齐鲁制药有限公司药物研究院,山东济南,250100 |
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| 摘 要： | 目的:在毕赤酵母中实现了KGF的高效表达,并初步建立了生物学活性检测方法.方法:合成5'端缺失69个核苷酸的KGF基因序列,克隆入pPIC9并转化毕赤酵母菌株GS115中,经诱导表达.发酵液上清采用脱盐层析和阳离子交换层析进行分离纯化.利用貂肺上皮细胞(Mv-1-Lu)检测其生物学活性.结果:表达水平达到了110mg/L发酵液;表达产物经一步离子交换层析就能得到有效分离,总收率在50mg/L发酵液以上;纯化的rhKGF生物学活性与KepivanceTM相当.结论:rhKGF制备工艺和检测方法的建立将为该因子的规模化生产和进一步的临床应用提供良好基础.
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| 关 键 词： | 重组人角质细胞生长因子 毕赤酵母 高效表达 纯化 生物学活性 |
Preparation of Recombinant Human Keratinocyte Growth Factor and Its Biological Activity Study |
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| WANG Xi-ju,LI Jian-feng,SUN Li-xia,AI Li-jing,LIU Ke-ling,WANG Ke-bo,WANG Hong-jie,XUE Tong-tong,WANG Jing-yi.Preparation of Recombinant Human Keratinocyte Growth Factor and Its Biological Activity Study[J].Biotechnology,2011,21(3). |
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| Authors: | WANG Xi-ju LI Jian-feng SUN Li-xia AI Li-jing LIU Ke-ling WANG Ke-bo WANG Hong-jie XUE Tong-tong WANG Jing-yi |
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| Institution: | WANG Xi-ju,LI Jian-feng,SUN Li-xia,AI Li-jing,LIU Ke-ling,WANG Ke-bo,WANG Hong-jie,XUE Tong-tong,WANG Jing-yi*(Research and Development Institute of Qilu Pharmaceutical Limited Company,Jinan 250100,China) |
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| Abstract: | Objective:High level and industrial preparation of recombinant rhKGF was achieved in Pichia pastoris.Method:The KGF coding sequence lacking the 23 N-terminal amino acid residues from the origin sequence of KGF(FGF7) in the Genebank was synthesized chemically,cloned into the pPIC9K vector and integrated into Pichia pastoris strain GS115.The rhKGF expression was induced by using methanol.The rhKGF was purified from the supernatant of culture by desalination and cation exchange column chromatography.MTT method... |
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| Keywords: | recombinant human keratinocyte growth factor Pichia pastoris efficient expression purification biological activities |
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