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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ＨａＩＲＥＳｍｄｒ１（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 observe the expression of multidrug resistance gene mdr1 regulated by internal ribosomal entry site (IRES). METHODS: The capsid-dependent Hamdr1 vector was used as a control. The retroviral vector HaIRIR-mdr1 (HaImdr1) was constructed by pSXLC / pHa system and transfected into murine packaging cells GP + E86 by lipofection The vincristine-resistant cells were obtained. The expression of mdr1 gene was detected by polymerase chain reaction (PCR) and flow cytometry (FCM). Results: The titer of retrovirus in GP + E86 / HaImdr1 supernatant of virus-producing cells was 2.0 × 105cfu / ml, which was cross-resistant to vincristine, daunorubicin and paclitaxel (24- to 52-fold) Multidrug resistance phenotype. PCR confirmed that the mdr1 gene has been stably integrated into the genome of GP + E86 / HaImdr1 cells; FCM analysis showed that IRES can transduce the mdr1 gene into P-glycoprotein for high expression, which is slightly lower than that of the Hamdr1 control. CONCLUSION: IRES can guide efficient cap-independent translation of mdr1 and mdr1 can be used as a dominant gene in gene therapy in dicistronic vectors.