用于筛选抑制丙型肝炎病毒内部核糖体进入位点活性药物的整合型细胞药物筛选模型的构建和评价

Establishment and Evaluation of a Cell-Based Drug Screening Model Used for Screening HCV-IRES Inhibitor

目的:构建一个能用于筛选抑制丙型肝炎病毒内部核糖体进入位点(HCV-IRES)活性药物的整合型细胞药筛模型。方法:构建携带HCV5'-UTR调控报告基因分泌型碱性磷酸酶(SEAP)基因的质粒pHCV5'-SEAP和用作筛选标记的pNeoR,线性化后共转染于Huh-7细胞中,经G418筛选后得到抗性细胞克隆,进行SEAP定量检测筛选后,得到能用于筛选抑制HCV-IRES活性药物的整合型细胞药筛模型;连续培养24代,用MTT法测细胞相对活力评价该药筛模型的生长稳定性,用SEAP定量检测法评价该药筛模型的SEAP表达稳定性,用反义寡聚核苷酸抑制试验评价模型的药筛灵敏度。结果:G418筛选后得到26个抗性细胞克隆;对其中5个抗性细胞克隆进行SEAP定量检测筛选后,得到3个能表达SEAP的阳性细胞克隆;连续培养24代过程中,该药筛模型的生长稳定性大于80%,SEAP表达稳定性达95%。结论:构建的细胞模型具有良好的稳定性和药筛灵敏度,符合作为药物筛选模型的要求。

Objective： To establish a drug screening model which be used for screening HCV-IRES inhibitor, and to evaluate its stability and sensitivity. Methods： The plasmid pHCV5＇-SEAP carrying the HCV 5＇-UTR adjusting and controlling reporter gene SEAP （secreted form of alkaline phosphatase） and the plasmid pNeoR with NeoR gene were constructed; co-transfected linearied pHCV5＇-SEAP and pNeoR into Huh-7 cell and screened positive clone with 500 μg/mL G418, 26 positive clones were acquired. Verified 5 positive clones at random with SEAP checkout-testing, 3 clones were integrating with pHCVS＇-SEAP and pNeoR and had ability on expressing SEAP steadily; relative vigor stability of the cell models was test by using NTT; drug screening sensitivity was evaluated by using ASODN test. Results： The relative vigor stability of the cell models reached 80%; the expressing SEAP steadily reached 95%. Conclusion： The cell model was acceptable for drug screening was draw.