Rhox5基因真核表达质粒的构建及其在NIH3T3细胞中的稳定表达  

Construction of the eukaryotic expression plasmid of Rhox5 gene and its stable expression in NIH3T3 cell

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,广东广州510006 [2]暨南大学生命科学技术学院,广东广州510632

出　　处：《现代生物医学进展》2010年第1期66-69,共4页Progress in Modern Biomedicine

基　　金：教育部高等学校博士学科点专项科研基金(No20060559006);广东省自然科学基金(No8151063201000013)

摘　　要：目的:构建同源异性框基因Rhox5的真核表达质粒,转染NIH3T3细胞,建立稳定过表达Rhox5的细胞系。方法:PCR方法扩增Rhox5的全长cDNA序列,PCR产物双酶切后和人工合成的HA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-Rhox5-HA融合表达质粒。脂质体法将经过测序成功的pcDNA-Rhox5-HA融合质粒和pcDNA3.1空载体分别转染NIH3T3细胞,潮霉素B筛选后建立阴性对照pcDNA3.1 in NIH3T3和稳定过表达Rhox5的Rhox5-HA in NIH3T3细胞系。RT-PCR和western blotting方法检测Rhox5-HA在稳定转染细胞系中的表达情况。结果:成功构建了pcDNA-Rhox5-Myc重组质粒,获得稳定过表达Rhox5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Rhox5-HA融合蛋白。结论:Rhox5基因真核表达质粒的构建及其在NIH3T3细胞中的稳定表达为进一步体外研究Rhox5蛋白单独的功能及其与其他分子间功能性相互作用奠定了实验基础。Objective：To construct the recombinant eukaryotic expression plasmid of the homeobox gene Rhox5 and establish the stable transfected cell line.Methods：The full length cDNA sequence of Rhox5 was amplified by PCR.Annealed oligonucleotides containing a HA antibody recognition sequence and an in-frame stop codon were cloned downstream of the Rhox5 cDNA sequence and then subcloned into the pcDNA3.1（-） expression vector to construct the recombinant eukaryotic expression plasmid pcDNA-Rhox5-HA.The recombinant plasmid and the pcDNA3.1 vector were transfected into NIH3T3 cell by lipofectamineTM 2000 respectively.After screening by hygromycin B,stable transfected cell line was established,and the expression of Rhox5-HA was identified by RT-PCR and western blotting assays.Results：The eukaryotic expression plasmid pcDNA-Rhox5-HA was constructed successfully and the stable transfected cell line Rhox5-HA in NIH3T3 was established.RT-PCR and western blotting results indicated that Rhox5-HA fusion protein was expressed successfully in NIH3T3 cell.Conclusion：The construction of the recombinant eukaryotic expression pcDNA-Rhox5-HA and its stable expression in NIH3T3 cell will facilitate further functional study on Rhox5 and its interaction with other proteins.

关 键 词：Rhox5 真核表达质粒 稳定转染细胞 

分 类 号：Q291[生物学—细胞生物学] Q75