Rhox5基因真核表达质粒的构建及其在NIH3T3细胞中的稳定表达

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.