烷化剂耐药基因真核表达质粒的构建及在K562细胞中的表达

目的克隆烷化剂耐药基因MGMT，构建真核表达质粒并导入真核细胞进行表达，筛选稳定表达的细胞系。方法从人肝组织中克隆MGMT基因后重组构建真核表达粒质pIRES2-MGMT—EGFP并鉴定。通过脂质体法将目的基因导入K562细胞，应用RT—PCR及Western blot检测目的基因的表达并筛选稳定转染的细胞克隆。结果成功克隆MGMT并构建了真核表达质粒pIRES2-MGMT—EGFP。转染细胞后MGMT在mRNA及蛋白水平均有表达，而对照组则为阴性。结论含烷化剂耐药基因MGMT真核表达质粒的构建及转染后表达的成功为烷化剂耐药基因的相关研究奠定了良好的基础。

Construction of mammalian expression plasmid containing alkylating-resistant gene and its expression in K562 cell line

Objective To clone the alkylating agent-resistant gene MGMT and to construct mammalian expression plasmid to be transferred into eukaryotic cells for expression. Methods MGMT gene was cloned from human hepatocytes and inserted into pIRES2-EGFP to construct mammalian expression plasmid. Reconstructed.plasmid and empty plasmid were transferred into K562 cells via liposome, and RT-PCR plus Western blot were used to evaluate the expression of MGMT gene. Results Mammalian expression plasmid containing MGMT was constructed and the transferred gene expressed at mRNA and protein levels while the control groups showed negative results. Conclusion The successful construction and expression of plasmid containing alkylating-resistant gene makes a good foundation for further studies related to alkylatingresistant gene.