人死亡受体5全长基因的构建及转染胶质瘤细胞

Construction of full-length DR5 gene and its transfection into glioma cell line

目的构建人死亡受体5(death receptor5,DR5)全长基因真核细胞表达载体pcDNA3.1/DR5,pcDNA3.1/GFP/DR5,并观察其转染胶质瘤细胞U138的效果。方法通过重叠PCR获得DR5全长编码序列,构建pcDNA3.1/GFP/DR5,pcDNA3.1/DR5表达载体,利用脂质体转染试剂盒,分别将2种质粒pcDNA3.1/GFP/DR5、pcDNA3.1/GFP共转染胶质瘤细胞U138,转染后72h,半定量逆转录聚合酶链反应(RT-PCR)检测DR5mRNA的表达,流式细胞术检测DR5的表达强度、检测Anti-DR5对胶质瘤细胞U138生长的影响。结果获得了DR5全长编码序列,成功瞬时转染U138,RT-PCR、流式细胞术检测结果表明,转染后U138细胞DR5mRNA、蛋白水平的表达明显增加,Anti-DR5可以明显抑制U138细胞的生长。结论获得了DR5全长编码序列,探索到成功转染DR5的最佳方法,为稳定筛选高表达DR5的U138细胞提供依据。

Objective To construct two eukaryotic expression vectors of human DR5 full length gene which named pcDNA3.1/DR5 and pcDNA3.1/GFP/DR5,and observe the transfection efficiency after transfecting them into glioma cell line U138.Methods Full-length DR5 gene was constructed by using overlapping PCR techniques,for constructing recombinant plasmids of pcDNA3.1/DR5 and pcDNA3.1/GFP/DR5.Utilizing liposome transfection,pcDNA3.1/DR5 and pcDNA3.1/GFP/DR5 recombinant were transduced into U138,respectively.The levels of D...