LPKp-人胰岛素基因逆转录病毒载体的构建与鉴定

Construction and identification of retroviral vector liver-type pyruvate kinase promoter with human insulin gene

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 the retroviral vector liver-type pyruvate kinase （LPK） promoter with human insulin gene （hINSg） in gene or stem cell therapy for diabetes mellitus. Methods Parent （pMDN-SIN, p54） and pM54 （LPKp-hINSg） plasmids were amplified, purified and identified with restriction enzyme. The pM54, p54 and pCMVI3Gal plasmids were transfected into pT67 and Phoenix E package cell lines with the FuGENE method. 3T3 cells served as controls. Supernatants were collected 48 or 72 hours after transfection with DNA. Seventy hours after 3T3 cells were infected, supernatants were collected. Transfection ratio was calculated with trypan blue staining, and INS expression was detected by ELISA. Results The retroviral vector pM54 （LPKp-hINSg） was successfully constructed and transfected into pT67 and infected 3T3 cells. The human insulin gene expression was upregulated. Conclusion The retroviral vector pM54 we constructed lays a vital foundation for further study of gene therapy or gene therapy in combination with stem cells for diabetes mellitus.