腺病毒-甲病毒杂合载体的构建

A novel adenovirus-alphavirus hybrid vector

目的将腺病毒Ad5/F11p造血细胞靶向和甲病毒复制子载体目的基因高效转录的特点相结合,构建腺病毒-甲病毒杂合载体,以提高目的基因在造血细胞的表达水平。方法基本实验过程包括骨架质粒构建,穿梭质粒构建,同源重组产生杂合病毒基因组,杂合病毒在包装细胞的拯救和扩增,以及杂合病毒对造血细胞基因转移效率的初步评价。具体步骤如下:利用重叠延伸PCR技术将pFiber5/11p质粒上Ad5的E4区部分删除产生骨架质粒pAdEasy/F11pDE4orf3。在pShuttle质粒的多克隆位点处分别插入造血细胞特异性启动子(CD11a promoter,CD11Ap)、甲病毒载体元件(nsP)、目的基因(GFP)、PolyA加尾信号构建杂合穿梭质粒pSh-SFV-GFP。骨架质粒与穿梭质粒在大肠杆菌BJ5183菌株中同源重组产生杂合病毒质粒pAd5/F11p-SFV-GFP,经与辅助病毒质粒Ad5/f11p-HV共转染293细胞后拯救出杂合病毒Ad5/F11p-SFV-GFP,经CsCl密度梯度离心法纯化,得到的病毒滴度为3×1010pfu/ml。与对照病毒Ad5-GFP相同感染复数(100MOI)分别感染U937细胞,利用流式细胞术检测GFP+细胞比例。结果pAdEasy-1/F11p-E4orf3和pSh-SFV-GFP经相应酶切和测序鉴定,与预期设计一致;CsCl密度梯度离心纯化得到杂合病毒;100MOI Ad5/F11p-SFV-GFP感染U937细胞2 d后,GFP+细胞比例为55.79%,而对照病毒Ad5-GFP的感染效率为2.42%。结论成功构建了腺病毒-甲病毒杂合载体,为进一步评价其对造血细胞的基因转移效率奠定了基础。

Objective To construct an adenovirus-alphavirus hybrid vector to enhance transgene expression in hematopoietic cells.Methods The main experimental procedure involved construction of the backbone and hybrid shuttle plasmids,rescue and propagation of the hybrid virus in packaging cells,and evaluation of gene transfer efficiency of the established vector.With the method of overlap extension PCR,E4 region of pAdEasy/F11p was partially deleted to generate the new backbone plasmid pAdEasy/F11pDE4orf3.Hematopoietic cell-specific promoter（CD11a promoter,CD11Ap）,alphaviral vector elements（nsP）,the target gene（GFP） and PolyA signal sequence（pA） were inserted in the multi-cloning site of pShuttle to construct the hybrid shuttle plasmid pSh-SFV-GFP.E.coli BJ5183 competent cells were co-transformed with pAdEasy/F11pDE4orf3 and linearized pSh-SFV-GFP to produce the hybrid virus plasmid pAd5/F11p-SFV-GFP by homologous recombination.Linearized pAd5/F11p-SFV-GFP was co-transfected to 293 cells with the helper virus plasmid pAd5/f11p-HV to rescue the hybrid virus Ad5/F11p-SFV-GFP.After being proliferated in 293 cells,Ad5/F11p-SFV-GFP was purified by CsCl density gradient centrifugation and then titrated.U937 cells were infected with Ad5/F11p-SFV-GFP or control Ad5-GFP viruses at a multiplicity of infection（MOI） of 100,and GFP expression was determined by flow cytometry assay 2 days post infection.Results pAdEasy-1/F11p-E4orf3 and pSh-SFV-GFP plasmids were confirmed by restriction enzyme digestion and sequencing.The hybrid virus was successfully rescued and purified,which reached a titer of 3×1010 IU/ml.GFP-positive cells were 55.79% or 2.42% for U937 cells infected by Ad5-GFP or Ad5/F11p-SFV-GFP,respectively.Conclusion Adenovirus-alphavirus hybrid vector is successfully constructed,which can facilitate further evaluation of gene transfer efficiency of the novel vector in hematopoietic cells.