【摘要】 目的 构建葡萄糖激酶(Glucokinase，GK)基因重组逆转录病毒，为将GK基因应用于糖尿病的基因治疗打下基础。方法 质粒pCMV4-GKZ1经EcoR1/BamH1双酶切后亚克隆至逆转录病毒载体PLXSN，构建成重组逆转录病毒表达载体PLX-GK，采用酶切及测序对重组体进行鉴定。而后脂质体转染PLX-GK至包装细胞PA317，检测培养上清病毒滴度，挑选滴度较高的稳定产毒细胞系并对其行PCR及免疫组化鉴定。结果 构建了GK基因逆转录病毒表达载体PLX-GK，经酶切及序列分析证实目的基因插入位点和读码框架正确、无突变;建立了稳定产毒细胞系PA317/GK，其培养上清平均病毒滴度为6.8×10 5 cfu/ml，最高为1.8×10 6 cfu/ml，PCR及免疫组化证实GK基因整合入PA317/GK细胞基因组并有GK的表达。结论 成功构建了GK基因重组逆转录病毒。

    关键词 葡萄糖激酶基因 逆转录病毒

    Construction of a recombinant retroviral containing GK gene

    Zheng Hongting，Deng Huacong，Jian Rui，et al.

    Department of Endocrinology，Affiliated Xinqiao Hospital of Third Millitary University of Medical Science， Chongqing400037.

    【Abstract】 Objective To establish a recombinant retroviral containing GK gene，which provides the basis of application of GK gene in treating dia betes.Methods The plasmid pCMV4-GKZ1was cut by EcoR1/BamH1reˉstriction，and subcloned to retroviral vector PLXSN，construct recombinate plasmid PLX-GK.The recombinant GK exˉpression vector was evaluated by using enzyme cutting and sequencing.Applying the Lipofectamine 2000 ......