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.
出处
《现代生物医学进展》
CAS
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)
关键词
Rhox5
真核表达质粒
稳定转染细胞
Rhox5
Eukaryotic expression plasmid
Stable transfected cell line
