【摘要】 目的构建新型转录因子Mdfic和c-Myc标签融合表达的真核表达载体pcDNA3.1-Mdfic-Myc,实现其在成肌细胞C2C12中的表达。方法 PCR方法扩增Mdfic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标签共同克隆至pcDNA3.1(+)真核表达载体中,构建pcDNA3.1-Mdfic-Myc融合表达质粒。重组质粒经过PCR、双酶切和测序鉴定后,脂质体法转染C2C12细胞。RT-PCR和Western blotting方法检测转染后细胞中Mdfic-Myc的表达。结果成功构建了pcDNA-Mdfic-Myc真核表达质粒;RT-PCR和Western blotting结果表明,Mdfic-Myc融合蛋白在成肌细胞C2C12中获得高效表达。结论 pcDNA3.1-Mdfic-Myc融合表达质粒的成功构建及其在成肌细胞中的表达为进一步体外研究Mdfic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基础。更多还原

【Abstract】 Objective To construct an eukaryotic expression plasmid carrying the novel transcriptional factor Mdfic with c-Myc tag. Methods The full length cDNA sequence of Mdfic was amplified by PCR. Annealed oligonucleotides containing a c-Myc antibody recognition sequence and an in-frame stop codon were cloned downstream of the Mdfic cDNA sequence and then subcloned into the pcDNA3.1 (+) expression vector to construct the eukaryotic expression plasmid pcDNA-Mdfic-Myc. The recombined plasmid was sequenced and then transfected into C2C12 cell by lipofectamineTM 2000. The expression of Mdfic-Myc was confirmed by RT-PCR and Western blotting assays. Results The eukaryotic expression plasmid pcDNA-Mdfic-Myc was constructed successfully. Results from RT-PCR and Western blot indicated that the Mdfic-Myc fusion protein was expressed successfully in C2C12 cell. Conclusion The recombinant eukaryotic expression pcDNA-Mdfic-Myc plasmid can be used in the functional study of Mdfic and its interacting proteins.更多还原