tPA突变体在哺乳动物细胞中的表达  

The Expression of tPA Mutant in Mammalian Cell

作　　者：于芳[1] 李朝[1] 周晓巍[1] 黄培堂[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100071

出　　处：《生物技术通讯》2005年第3期278-279,286,共3页Letters in Biotechnology

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-细胞。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。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.

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 

分 类 号：Q78[生物学—分子生物学] TQ323.4[化学工程—合成树脂塑料工业]