摘要
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.
出处
《生物技术通讯》
CAS
2007年第5期759-763,共5页
Letters in Biotechnology