慢性粒细胞白血病MGMT基因的克隆及逆转录病毒表达载体的构建

Cloning of a Human MGMT Gene from Patient with Chronic Myeocytic Leukemia and the Construction of A Retrovirus Vector with It

目的 :从慢性粒细胞白血病病人外周血中克隆人MGMT基因 ,并进行逆转录病毒表达载体的构建。方法 :用RT PCR方法从慢性粒细胞白血病病人外周血白细胞中克隆得到长为 6 84bpMGMTcDNA。将该片段与 pGEM T载体连接 ,转化大肠杆菌DH5后得到重组质粒 pGEM T MGMT ,进行限制性内切酶酶切分析、PCR及测序鉴定。再将MGMTcDNA亚克隆到G1Na逆转录病毒载体EcoRⅠ和xhoⅠ位点之间 ,并进行酶切、PCR鉴定。结果 :成功克隆了人MGMT基因 ,经酶切分析、PCR初步筛选及测序鉴定证实序列正确 ,并成功构建到逆转录病毒载体上。结论 :通过克隆人MGMT基因 ,并构建逆转录病毒载体G1Na MGMT ,为探讨将耐药基因导入造血干细胞对烷化剂类化疗药物抗性的研究奠定了基础。

Objective: To isolate human O6 methylguanine DNA methyltransferase (MGMT) from peripheral blood leukocyte of patient with chronic myeocytic leukemia and construct a retrovirus vector with the gene. Methods: The cDNA encoding the Human MGMT was isolated from peripheral blood leukocyte of patient with chronic myeocytic leukemia using RT PCR method. The fragment was cloned into pGEM T vector and further subcloned into the G1Na retrovirus vector. Result: The human MGMT was successfully cloned and constructed into retrovirus vector G1Na. They were verified by restriction endonuclease analysis?PCR amplify and DNA sequencing. Conclusion: This study provides a foundation for transferring a drug resistance gene MGMT into hematopoietic progenitor cells to improve tolerance to alkylating agents.