Prosaposin基因哺乳动物细胞表达质粒的构建及稳定转染细胞系的建立  

Construction of the Mammalian Expression Plasmid of Prosaposin and Establishment of its Stable Tranfected Cell Line

作　　者：郭芬[1] 李月琴[2] 李实骞[2] 罗志文[2] 张欣[2] 周天鸿[2] 

机构地区：[1]广东药学院生命科学与生物制药学院,510006 [2]暨南大学基因工程药物国家工程研究中心,510632

出　　处：《实用癌症杂志》2010年第1期13-16,共4页The Practical Journal of Cancer

基　　金：广东省自然科学基金项目(No.8151063201000013);"211"工程经费资助项目

摘　　要：目的构建前凋亡因子prosaposin的哺乳动物细胞表达质粒,转染NIH3T3细胞,建立稳定转染prosaposin的细胞系。方法采用PCR方法扩增prosaposin的开放阅读框,酶切后的PCR扩增产物和退火后的人工合成的c-Myc抗原表位标签,共同克隆至pcDNA3.1(+)哺乳动物细胞表达载体中,构建pcDNA-Psap-Myc融合表达质粒。用脂质体将经过验证、测序的pcDNA-Psap-Myc质粒转染NIH3T3细胞,通过G418筛选建立稳定过表达prosaposin的NIH3T3细胞系。采用RT-PCR和western blotting方法,检测prosaposin在稳定转染的NIH3T3细胞系中的表达。结果成功构建了pcDNA-Psap-Myc融合表达质粒,建立了稳定过表达prosaposin的NIH3T3细胞系。RT-PCR和Western blotting结果表明,构建的稳定细胞系中成功表达prosaposin-Myc融合蛋白。结论Prosaposin哺乳动物细胞表达质粒的成功构建和稳定转染NIH3T3细胞系的建立,为进一步体外研究prosaposin蛋白的功能及其与其他分子间的相互作用奠定了基础。Objective To construct the mammalian expression plasmid of the pro-survival molecule prosaposin and establish the stable transfected cell line. Methods The open reading frame of prosaposin was amplified by PCR. Annealed oligonucleotides containing a c-Myc antibody recognition sequence and an in-frame stop codon were cloned downstream of the prosaposin cDNA sequence and then subcloned into the pcDNA3. 1 （ ＋ ） expression vector to construct the mammalian expression plasmid pcDNA-Psap-Myc. The recomhiued plasmid was sequenced and then transfected into NIH3T3 cell by lipofectamineTM 2000. After screening by G418,stable transfected cell line was established, and the expression of prosaposin-Myc was identified by RT-PCR and western blotting assays. Results The mammalian expression plasmid pcDNA-Psap-Myc was constructed successfully and the stable transfected cell line Psap-Myc in NIH3T3 was established. RT-PCR and western blotting results indicated that prosaposin- Myc fusion protein was expressed successfully in NIH3T3 cell. Conclusion The construction of the recombinant eukaryotic expression pcDNA-Psap-Myc and the establishment of the stable transfected cell line Psap-Myc in NIH3T3 will facilitate further functional study on prosaposin and its interaction with other proteins.

关 键 词：prosaposin基因 哺乳动物细胞表达质粒 稳定转染细胞 

分 类 号：R73-351[医药卫生—肿瘤]