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目的构建肝型丙酮酸激酶启动子(LPKp)与人胰岛素基因(hINSg)逆转录病毒表达载体并鉴定。方法1)母本pMDN-SIN及父本p54质粒扩增、纯化、酶切鉴定;2)取相关片段构建pM54载体并扩增、纯化、鉴定;3)脂质体法转染pM54进入pT67等细胞系,p54等质粒做对照基因;4)用转染后细胞上清液感染3T3细胞;5)ELISA法检测转染、感染后上清液INS含量鉴定质粒。结果酶切鉴定质粒大小与预期相符;ELISA法检测转染、感染上清液中均含较高水平INS。对照结果均阴性。结论LPKp-hINSg逆转录病毒表达载体pM54构建成功。实验为基因治疗或基因结合干细胞治疗糖尿病的进一步相关研究奠定了基础。
Objective To construct and identify retroviral vector of hepatic pyruvate kinase promoter (LPKp) and human insulin gene (hINSg). Methods 1) The parental pMDN-SIN and paternal p54 plasmids were amplified, purified and identified by restriction enzyme digestion. 2) The pM54 vector was constructed and amplified, purified and identified. 3) Lipofectamine was transfected into pM67 Cell line, p54 and other plasmids as control gene; 4) transfected cells supernatant of 3T3 cells infected; 5) ELISA assay transfected, supernatant infection after identification of the plasmid. Results The plasmid size was confirmed by restriction enzyme digestion. The transfection was detected by ELISA, and the supernatant of infected cells contained higher INS. Control results were negative. Conclusion The LPKp-hINSg retrovirus expression vector pM54 was successfully constructed. Experiments lay the foundation for further relevant studies of gene therapy or stem cell-based therapy of diabetes.