【摘要】 目的建立H IV-1 Tat蛋白结合Tar的抗H IV-1药物筛选模型用于抗H IV药物筛选。方法构建表达H IV-1Tat蛋白的真核表达质粒(pCDNA3.1(+)-Tat)和H IV-1 LTR-luc荧光素酶报告基因,采用脂质体转染法共转染HeLa细胞,荧光仪检测Tat蛋白促进荧光素酶在HeLa细胞内的表达情况,建立H IV-1Tat蛋白结合Tar的细胞模型,用于抗H IV-1的药物筛选。以DRB(5,6-二氯-1-β-呋核亚硝脲-苯并咪唑)为阳性对照,进行模型的建立及条件的优化。结果通过反复试验表明,目的基因和报告基因的配比、转染时培养液中小牛血清的含量、细胞浓度、转染前细胞状态以及药物作用时间的长短等对模型的稳定性和敏感性有影响。结论应用优化的细胞模型对文冠果、紫苏等浸提物进行筛选及结果的分析。更多还原

【Abstract】 Objective To establish HIV-1 Tal binding to Tar model.Methods HIV-1 Tat protein eukaryotic expression plasmid(pCDNA3.1(+)-Tat)and luciferase report gene HIV-1 LTR-luc(LTR-LUC) were constructed and transfected into HeLa cells with lipofectamine 2000.The expression of HIV-1 Tat protein in HeLa cells was detected with Iuminometer.HIV-1 Tat protein binding tar model for screening HIV-1 inhibitors was thereby constructed.Results The abstractions of Xanthoceras sorbifolia and Perilla frutescens and so on were detected and DRB(5,6-dichloro-1-β-D-robofuranosylbenzimidazole) was positive control.The stability of the model was tested and regulated.Conclusion The repeated experiments indicate that the matching of purpose gene and report gene,the rate of new calf serum in medium during transfection,density of the transfected cells,condition of the untransfected cells and the action time of drugs affect the stability and sensitivity of the model.更多还原