| tPA突变体在哺乳动物细胞中的表达 |
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| 引用本文： | 于芳,李朝,周晓巍,黄培堂.tPA突变体在哺乳动物细胞中的表达[J].生物技术通讯,2005,16(3):278-279,286. |
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| 作者姓名： | 于芳 李朝 周晓巍 黄培堂 |
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| 作者单位： | 军事医学科学院,生物工程研究所,北京,100071 |
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| 摘 要： | 利用携带有二氢叶酸还原酶(dhfr)基因的pCI载体，实现tPA突变体(FrGGI)在CHO-dhfr^-细胞中的高效表达，获得高表达细胞株。采用分子克隆常规技术，将去除3’端非蛋白编码区的tPA突变体cDNA与pCI载体连接，构建真核表达载体pCI—tPA；采用阳离子脂质体转染法转染CHO-dhfr^-胞。经酶切及测序鉴定，证明所构建的质粒正确，转染CHO—dhfr细胞后，经过MTX加压筛选，得到了10株表达水平较高的细胞株，其活性可达每106细胞4000U／24h。以上结果为进行tPA突变体工程细胞株的筛选奠定了基础。
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| 关 键 词： | tPA 突变体 哺乳动物细胞 二氢叶酸还原酶 真核表达载体 脂质体转染法 4000U 工程细胞株 pCI 高效表达 常规技术 分子克隆 cDNA 高表达 编码区 阳离子 CHO 构建 筛选 基因 蛋白 测序 质粒 |
| 文章编号： | 1009-0002(2005)03-0278-02 |
The Expression of tPA Mutant in Mammalian Cell |
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| YU Fang,LI Chao,ZHOU Xiao-wei,HUANG Pei-tang.The Expression of tPA Mutant in Mammalian Cell[J].Letters in Biotechnology,2005,16(3):278-279,286. |
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| Authors: | YU Fang LI Chao ZHOU Xiao-wei HUANG Pei-tang |
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| Institution: | YU Fang,LI Chao,ZHOU Xiao-wei,HUANG Pei-tang Institute of Biotechnology,Academy of Military Medical Sciences,Beijing 100071,China |
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| Abstract: | One expressing plasmid pCI-dhfr , containing the tissue-type plasminogen activator (tPA) mutant cDNA gene (removed of 3' end none coding region protein ), to realize a expression of high level in the mammalian cell. The resultive recombination vector was verified by restriction analysis and gene sequencing method. After tranfection with CHO-dhfr- cell line, fibrinolysis activity was detected in the cultivate supernatant. The high-lever expression cell line was obtained with expression lever 4 000 U/24 h per 106 cell. This work have set base to high level expression study of tPA mutant in mammalian cell lines. |
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| Keywords: | t-PA mutant CHO-dhfr- cell expression |
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