犬黑皮质素受体4真核表达载体的构建及在COS-7细胞中的表达

目的]构建犬黑皮质素受体4真核表达载体并在COS-7细胞中进行瞬间表达。方法]以犬MC4R线性DNA为模板,进行引物设计,PCR特异性扩增,扩增产物连接到pMD18-T载体上,酶切鉴定后测序。将该片段亚克隆到真核表达载体pcDNA3.1(+)。重组体pcD-NA3.1(+)-MC4R经过酶切和测序鉴定。采用FuGENE HD介导转染技术将pcDNA3.1(+)-MC4R导入COS-7细胞,提取细胞内总RNA,RT-PCR扩增犬MC4R基因的全长cDNA编码序列。结果]克隆犬MC4R基因的全长cDNA序列,并以pcDNA3.1(+)表达载体为骨架,构建真核表达载体,测序的扩增片段与GenBank公布的模板序列经Blast比对相似性为99%。采用G418筛选,获得带有pcDNA3.1(+)-MC4R的COS-7细胞。结论]成功构建犬真核表达载体pcDNA3.1(+)-MC4R,重组体能在真核细胞中表达。

Construction of Canis Melanocortin Receptor-4 Eukaryotic Expression Vector and its Expression in COS-7 Cells

Objective]To Construct the pcDNA3.1( )-Canis MC4R expression plasmid and investigate its transient expression in COS-7 cells.Methods]Using the canis MC4R DNA as template,designed the specific primers.The fragment of MC4R gene was amplified and then cloned into pMD18-T vector.The eukaryotic expression vector pcDNA3.1( )-MC4R was constructed by ligating canis MC4R gene with pcDNA3.1( ) vector.The recombinant pcDNA3.1( )-MC4R was digested by double restriction enzymes and sequenced.Then it was transfected into the COS-7 cells by FuGENE HD transfection reagent,from which total RNA was extracted.CDS of the canis MC4R was amplified by RT-PCR.Results]The CDS of canis MC4R gene was cloned and it has a similarity of 99% with the reported sequence in GenBank.Through G418 filtration,We obtained the positive COS-7 cells.Conclusion] The construction and the expression of eukaryotic plasmid pcDNA3.1( )/MC4R have been achieved successfully.