内在核糖体进入位点控制多药耐药基因mdr1表达的研究

Efficient expression of multidrug resistance gene mdr1 in retroviral vector under control of an internal ribosome entry site

目的：观察内在核糖体进入位点（ＩＲＥＳ）控制多药耐药基因ｍｄｒ１的表达能力。方法：以帽依赖性Ｈａｍｄｒ１载体为对照，利用ｐＳＸＬＣ／ｐＨａ系统构建依赖脑心肌炎病毒ＩＲＥＳ翻译的逆转录病毒载体ＨａＩＲＥＳｍｄｒ１（ＨａＩｍｄｒ１），脂质体转染法导入鼠源包装细胞ＧＰ＋Ｅ８６并获得长春新碱耐药细胞；用聚合酶链反应（ＰＣＲ）与流式细胞术（ＦＣＭ）检测ｍｄｒ１基因的转移与表达。结果：病毒生产细胞ＧＰ＋Ｅ８６／ＨａＩｍｄｒ１上清中逆转录病毒的滴度为２．０×１０５ｃｆｕ／ｍｌ，对长春新碱、柔红霉素与紫杉醇产生交叉耐药（２４～５２倍），而且具有多药耐药表型。ＰＣＲ证实ｍｄｒ１基因已稳定整合至ＧＰ＋Ｅ８６／ＨａＩｍｄｒ１细胞基因组；ＦＣＭ分析表明ＩＲＥＳ能引导ｍｄｒ１基因翻译成Ｐ糖蛋白而高效表达，程度略低于Ｈａｍｄｒ１对照。结论：ＩＲＥＳ可引导ｍｄｒ１基因进行有效的帽非依赖性翻译，ｍｄｒ１基因在双顺反子载体中可作为显性选择性基因用于基因治疗。

Objective: To test the efficiency of human mdr1 gene expression under the control of an internal ribosome entry site (IRES). Methods: The expression retroviral vector HaImdr1 was constructed from pSXLC/pHa system which contains an IRES from encephalomyocarditis virus. The vector was introduced into ecotropic packaging cells GP+E86 by liposome mediated transfection. The retrovirus producing cells were obtained by 50μg/L vincristine selection. The success of transfer and expression of mdr1 gene were determined by polymerase chain reaction (PCR) and flow cytometry (FCM). Results: Virus in the supernatant of the producer cells, in which the integration of mdr1 gene was confirmed by PCR, was titrated to 2.0×10 5 cfu/ml. The selected producer cells exhibited a 24～52 fold increase of resistance to vincristine, daunorubicin and taxol in comparison with control cells GP+E86. The expression of mdr1 gene was confirmed by both reverse transcription PCR at RNA level and FCM at protein level, although the HaImdr1 transfectants showed somewhat less expression of P gp when compared to that with cap dependent translation of Ha mdr1. Conclusion:mdr1 gene can be effectively translated and expressed under the control of IRES in cap independent manner , it may be used as a dominant selectable marker in bicistronic vectors for gene therapy.