以新霉素抗性基因突变体为筛选标志的真核表达载体的构建  

Construction of Eukaryotic Expression Vector Using Neomycin-resistanceGene Mutant as Selectable Marker

作　　者：高川[1] 朱旭东[2] 周晓巍[2] 于芳[2] 卢柏松[2] 黄培堂[2] 

机构地区：[1]北京药物化学研究所,北京102205 [2]北京生物工程研究所,北京100071

出　　处：《生物工程学报》2002年第3期308-312,共5页Chinese Journal of Biotechnology

摘　　要：新霉素抗性基因 (neo)是真核表达载体的常用筛选标志 ,neo基因编码新霉素磷酸转移酶Ⅱ(NPTⅡ) ,能催化G418、卡那霉素等多种氨基糖苷抗生素分子磷酸化而使之失去抗菌活性。通过对真核表达载体的筛选标志基因neo进行定点突变 ,以降低NPTⅡ的活性 ,然后用含neo突变体的真核表达载体pmDNA构建荧光素酶表达质粒 ,稳定转染CHO K1细胞 ,发现表达荧光素酶的阳性细胞比例达到 95 % 。Neomycin-resistance gene is widely used as a selectable marker in eukaryotic expression vector. It codes neomycin phosphotransferaseⅡ(NPTⅡ) which confers resistance to various aminoglycoside antibiotic such as G418 and kanamycine. In this work ,by site-directed mutagenesis the neo gene mutant was obtained. The expression vector pmDNA using the neo gene mutant as selectable marker has been constructed. After inserting interest luciferase gene, the expression plasmid pmDNAluc+ was stably transfected CHO-K1 cells. As a result, the expression positive ratio reaches to approximate 95% and the ratio of high expression colonies is apparently higher than the controls.

关 键 词：新霉素 抗性基因突变体 筛选标志 真核表达载体 

分 类 号：Q782[生物学—分子生物学]