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目的 研究含内在核糖体进入位点 (IRES)的逆转录病毒双顺反子载体介导的醛脱氢酶基因 (ALDH1 )与多药耐药基因 (mdr1 )转导和共表达。方法 以携带ALDH1与mdr1基因的重组逆转录病毒双顺反子G1Na ALDH1 IRES mdr1 (G1Na AIM)为载体 ,电穿孔法转导双嗜型包装细胞PA3 1 7,长春新碱筛选 ;所得病毒生产细胞PA3 1 7 AIM与单嗜型包装细胞GP +E86乒乓感染提高病毒滴度 ;以重组病毒上清转染K562细胞 ,应用聚合酶链反应 (PCR)和Southernblot检测转移基因的整合 ,流式细胞术(FCM)及MTT分析基因表达 ,集落培养法测定转导效率。结果 双嗜型病毒上清滴度达 1 .0× 1 0 5cfu ml。基因修饰细胞K562 AIM经PCR和Southernblot证实双基因稳定整合至基因组 ,对 4 氢过氧化环磷酰胺 ( 4 HC)及长春新碱 (VCR)耐药 (提高 3～ 1 0倍 ) ,FCM及集落法检测基因转导效率为 62 %～70 %。结论 逆转录病毒IRES双顺反子载体能引导不同类型的耐药基因有效共表达 ,可用于扩大耐药范围 ,进行体内显性选择。
Objective To study the transduction and coexpression of ALDH1 and MDR1 gene by retroviral bicistronic vector with internal ribosome entry site (IRES). Methods The recombinant retroviral bicistronic G1Na ALDH1 IRES mdr1 (G1Na AIM) carrying ALDH1 and mdr1 genes was used as a vector to transduce the double-type packaging cells PA3 1 7 by electroporation. Vincristine was screened. The resulting virus-producing cells PA3 1 7 AIM and monophilic packaging cells GP + E86 pingpong infection to increase the virus titer; K562 cells transfected with recombinant virus supernatants, polymerase chain reaction (PCR) and Southern blot detection of transfer gene integration, flow cytometry (FCM) and MTT analysis of gene expression, colony culture method for the determination of transduction efficiency. Results Double-addicted virus supernatant titer 1.0 × 105cfu ml. The genetically modified cells K562 AIM confirmed by PCR and Southern blot that the double gene was stably integrated into the genome and resistant to 4-hydroperoxide cyclophosphamide (4 HC) and vincristine (VCR) (3 to 10 times higher), FCM and colonies Law detection of gene transduction efficiency of 62% to 70%. CONCLUSION: The retroviral IRES bicistronic vector can guide efficient co-expression of different types of drug resistance genes and can be used to expand the range of drug resistance and make dominant selection in vivo.