携带反义多药耐药相关蛋白的重组腺病毒载体的构建

目的　构建携带反义多药耐药相关蛋白 (MRP)的重组腺病毒载体以用于肝癌耐药的基因治疗研究。方法　将 MRP基因片段反向克隆到腺病毒载体质粒 p Ad Track- CMV上 ,与骨架质粒在大肠杆菌 BJ5 183胞内进行同源重组 ,经 2 93细胞包装、扩增后得到携带反义 MRP的重组腺病毒 Ad Easy- GFP- ASmrp。结果　成功地构建了携带反义 MRP的重组腺病毒载体系统 ,经测定病毒滴度可达 2 .5× 10 9。结论　构建的重组腺病毒 Ad Easy- GFP-ASm rp可望有效地将反义 MRP导入人肝癌耐药细胞株 ,为进一步研究肝癌耐药机制及其逆转方式提供实验基础

Construction of the Recombinant Adenovirus Vector Carrying Antisense Multidrug Resistance-associated Protein

OBJECTIVE: The recombinant adenovirus vector carrying antisense multidrug resistance-associated protein (MRP) was constructed for use in gene therapy to overcome drug resistance in hepatocellular carcinoma. METHODS: The fragment of MRP gene encoding 5' region was cloned reversely into the shuttle plasmid pAdTrack-CMV, with the resultant plasmid and the backbone plasmid pAdEasy-1, the homologous recombination took place in the E. coli BJ5183 and the recombinant adenoviral plasmid was generated. The adenoviruses were packaged and amplified in the 293 cells. Then the viral titer was checked by GFP. RESULTS: The recombinant adenovirus vector carrying antisense MRP was constructed successfully. The viral titer was 2.5 x 10(9). CONCLUSION: The recombinant adenovirus vector constructed by us could introduce the antisense MRP into the human drug-resistant hepatocellular cell line effectively, which would provide experimental basis for the mechanisms and reversal methods of the multidrug resistance in human hepatocellular carcinoma.