CCR5,CXCR4双靶区反义RNA重组腺病毒载体的构建

Construction of recombinant adenoviral vectors carrying antisense RNA to dual-target chemokine receptors CCR5 and CXCR4

目的 :构建趋化因子受体CCR5 ,CXCR4双靶区反义RNA重组载体并获取重组腺病毒以用于抗HIV 1基因治疗的研究 .方法 :用RT PCR法从健康人外周血单个核细胞中分别扩增出趋化因子受体CCR5 ,CXCR4 5′端翻译起始区 6 5 3bp和6 36bp的cDNA片段 ,将其反向插入腺病毒穿梭载体质粒pAd Track CMV中 ,再与包装质粒pAdEasy 1共转染BJ5 1 83细菌同源重组 ,卡那抗性培养基筛选阳性克隆 ;同源重组的载体用脂质体转染剂转染 2 93细胞包装、扩增 ,荧光显微镜下观察细胞中绿色荧光蛋白 (GFP)以及PCR法鉴定、氯化铯密度梯度离心法纯化重组腺病毒 .结果 :成功构建了CCR5 ,CXCR4双靶区反义RNA重组腺病毒载体 ,并于 2 93细胞中包装、扩增得到高滴度的重组腺病毒 ,其滴度为 :7.2× 1 0 1 2 PFU/mL .结论 :CCR5 ,CXCR4双靶区反义RNA重组腺病毒的构建为研究双靶位辅助受体反义RNA抗HIV

AIM: To construct the recombinant adenoviral vector carrying antisense RNA to chemokine receptors CCR5 and CXCR4 and to obtain recombinant adenovirus, which will be used to resist HIV 1 infection. METHODS: The 653 bp and 636 bp DNA fragments targeted to the initial part of CCR5 and CXCR4 mRNA 5′ sides translation were obtained by RT PCR from peripheral blood mononuclear cells (PBMCs) and were conversely inserted into adenoviral vector pAdTrack CMV. The homologous recombination with adenovirus backbone plasmid pAdEasy 1 was performed in BJ5183 bacteria and the recombinant vector was selected by anti kanamycin plate. The recombinant vector was packaged and amplified in 293 cells and the expression of GFP was observed by fluorescence microscope. The recombinant adenovirus was detected by PCR and purified by CsCl density gradient centrifugation. RESULTS: The recombinant adenoviral vector carrying antisense RNA to CCR5 and CXCR4 had been constructed and recombinant adenovirus had been obtained and accurately detected. Its tier was 7.2×10 12 PFU/mL. CONCLUSION: The construction of the recombinant adenovirus carrying antisense RNA to CCR5 and CXCR4 lays the basis for the study of its inhibitive effect on HIV 1 infection.