人肌纤生成调节因子1真核表达载体的构建及其在乳鼠心肌细胞中的表达

Construction of Human Myofibrillogenesis Regulator-1 Gene Eukaryotic Vectors and Its Expression in Neonatal Rat Cardiomyocytes

目的构建人肌纤生成调节因子1全长的真核表达质粒,并观察其在HEK293T细胞系及Sprague-Daw-ley乳鼠心肌细胞中的表达。方法从NCBI GenBank数据库中克隆得到人肌纤生成调节因子1基因(AF417001)全长序列,与真核表达载体质粒pcDNA3.1/Myc-His(-)B连接并转化大肠杆菌XL1-Blue,筛选阳性克隆,T7引物测序,转染细胞后以逆转录聚合酶链反应、Western Blotting方法检测人肌纤生成调节因子1的表达。结果pcDNA3.1/Myc-His(-)B-hMR-1质粒经测序证实目的基因序列正确,无碱基突变。该质粒转染到HEK293T细胞系和乳鼠心肌细胞后人肌纤生成调节因子1的转录水平及表达水平明显增高。结论成功构建了人肌纤生成调节因子1全长的真核表达载体并确定了简便有效的乳鼠心肌细胞瞬时转染方法。

Aim To construct the human myofibrillogenesis regulator-1（hMR-1） full length eukaryotic expression plasmid and measure its expression in HEK293T cell lines and Sprague-Dawley neonatal rat cardiomyocytes.Methods The whole hMR-1 coding gene was cloned from NCBI GenBank database（AF417001）,recombined with plasmid pcDNA3.1/Myc-His（-）B and transformed into Escherichia coli XL1-Blue.Positive clone was selected and sequenced by T7 primers.The recombined plasmid pcDNA3.1/Myc-His（-）B-hMR-1 was transfected by Lipofectamin2000 to cells,reverse transcription polymerase chain reaction（RT-PCR） and Western Blotting was employed for measuring the expression of hMR-1.Results Plasmid pcDNA3.1/Myc-His（-）B-hMR-1 was verified by sequencing that the target gene was correct,without any mutation;the expression level at mRNA and protein were both increased significantly after hMR-1 transfection.Conclusion A full-length hMR-1 coding gene eukaryotic vector was successfully constructed,based on which,a convenient and effective transient transfection method in neonatal rat cardiomyocytes was established as well.