MDR1启动子调控的双自杀基因真核表达载体的构建及其在K562/A02细胞中的表达

目的：构建多药耐药基因（MDR1）启动子调控的CD-TK双自杀基因真核表达载体用于耐药白血病的靶向性基因治疗。方法：采用PCR方法从K562/A02细胞基因组DNA中扩增MDR1启动子，将其连接到双自杀基因CD TK的上游，构建pcDNA3 MDR1 Promoter CD-TK真核表达载体，用脂质体介导法将构建好的载体转染入K562、K562/A02细胞，PCR鉴定CD、TK基因的整合，RT-PCR鉴定CD、TK基因的表达。结果：PCR克隆出MDR1启动子经DNA测序证实序列正确，通过连接成功构建含正确目的基因的表达载体pcDNA3-MDR1-Promoter-CD-TK。转染入细胞后PCR结果显示，CD、TK基因均整合入K562、K562/A02细胞中，RT-PCR结果显示,562/A02/CD TK细胞CD、TK基因有特异性条带表达，而K562/CD TK细胞无特异性条带。结论： MDR1启动子调控的CD TK双自杀基因真核表达载体的成功构建及其在K562/A02细胞中的特异性表达为耐药白血病的靶向性基因治疗提供了实验基础。

Construction of expressive vector containing double suicide genes controlled by MDR1 promoter and its expression in K562/A02 cell

Objective: To construct expressive vector containing double suicide genes targeted by MDR1 promoter for the purpose of targeted gene therapy for MDR leukemia.Methods: The DNA fragment of MDR1 promoter was amplified from genome DNA of K562/A02 cells with PCR and was inserted into the upstream of CDTK to construct pcDNA3-MDR1-Promoter-CD-TK.This recombinant vector was transfected into K562,K562/A02 cells by means of liposome.PCR and RT-PCR were resorted to identify the integration and expression of CD and TK genes.Results: The length and sequence of MDR1 promoter amplified by PCR were confirmed by DNA sequencing.The pcDNA3-MDR1-Promoter-CD-TK expression vectors were constructed successfully.PCR proved double suicide genes were integrated into K562/A02 and K562 cells.RT-PCR revealed that CD and TK genes expressed in K562/A02/CD-TK cells, whereas not in K562/CD-TK cells.Conclusion: Construction of expressive vector containing double suicide genes targeted by MDR1 promoter and its specific expression in K562/A02 cell provide a sound basis for targeted gene therapy for MDR leukemia.