含内部核糖体进入位点的逆转录病毒载体介导多药耐药基因的高效表达

Efficient Expression of Human Multidrug Resistance 1 Gene Mediated by Retroviral Vector Containing Internal Ribosomal Entry Site

为研究内部核糖体进入位点(IRES)引导多药耐药基因(MDR1)的表达效率，利用pSXLC／pHa载体系统构建双顺反予载体pHa-IRES-MDR1(HaiMDR)。其中的MDR1基因翻译由脑心肌炎病毒IRES控制。通过脂质体法将载体HaiMDR转染入单嗜性包装细胞GP+E86而得到滴度2．0×10^5CFU／ml的病毒；用单嗜性病毒重复感染GP+env Am12细胞获得滴度约6．5×10^5CFU／ml的双嗜性病毒生产细胞，并命名为Am12／HaiMDR。两种病毒生产细胞均具经典MDR表型，与未转染的细胞相比，两者对长春新碱、柔红霉素和紫杉醇产生15—358倍耐药。采用聚合酶链反应，在两种生产细胞均证实有外源性MDR1基因的整合和转录。但同时也发现有异常剪接的转录本存在。利用P-糖蛋白(P-gp)特异性单克隆抗体UIC2结合流式细胞术分析。两种耐药细胞均表达高水平的P-gp。因而，MDR1基因可在IRES控制下以帽非依赖性方式进行有效表达，其在基因治疗研究中可有两种用途：在癌症患者化疗后用于保护干／祖细胞，或在双顺反子载体中作为体内显性选择性标志用于共表达治疗性基因。

To investigate the expression efficiency of human multidrug resistance 1 (MDR1) gene under control of an internal ribosomal entry site (IRES), the bicistronic vector HaiMDR was constructed based on the pSXLC/pHa system, in which the translation of MDR1 gene was controlled under an IRES from encephalomyocarditis virus. The vector HaiMDR was transfected into ecotropic GP + E86 cells by liposome method with titer of 2.0 × 105 CFU/ml. A high-titer (about 6.5 × 105 CFU/ml) amphotropic producer, designed as Aml2/HaiMDR, was generated by transducing GP + envAm 12 cells with ecotropic virus. These producers displayed a classical MDR phenotype with a 15-to 358-fold resistance to vincristine, daunorubicin, and Taxol in comparison with the parental cells. By means of polymerase chain reaction, the integration and overexpression of the exogenous MDR1 gene was confirmed in both producers, in which an aberrant splicing transcript of MDR1 gene was found simultanously. P-glycoprotein (P-gp) expression was verified using a monoclonal antibody UIC2 specific to human P-gp for flow cytometry analysis. In summary, the MDR1 gene can be efficiently expressed under control of IRES in a cap-independent manner, and it may be useful for gene therapy in two ways: to protect stem/progenitor cells from myelosuppression after chemotherapy in patients with cancer and to act as a dominant selectable marker in bicistronic vectors in vivo for the coexpression with therapeutic genes.