低氧诱导因子-1的病毒载体制备及感染效果

Preparation of recombinant adenovirus vector containing human HIF-1 and infective evaluation of the adenovirus on epidermal keratinocytes

目的制备低氧诱导因子（HIF）-1腺病毒载体并评价其对表皮角质细胞的感染效果。方法Ad／HIF-1质粒经酶切鉴定后，以4μg质粒转染1．2×10^6个HEK293细胞，2d后荧光显微镜下观察转染效率。1周后收集细胞及上清并感染HEK293细胞进行病毒扩增，待扩增的细胞及上清量达300ml时进行病毒纯化及滴度测定。最后用制备的病毒载体感染表皮角质细胞，流式测定其感染效果。结果Ad／HIF-1质粒酶切鉴定正确；转染或感染HEK293细胞后有绿色荧光蛋白（GFP）表达；获得的腺病毒滴度为1．8×10^11PFU／ml。病毒以MOI=50感染表皮细胞，2d后GFP阳性细胞的比例为97．46％。结论成功制备了HIF-1腺病毒载体，且感染表皮角质细胞后能得到较高的感染效率，从而为腺病毒介导的基因功能研究提供理论基础。

Objective To prepare the recombinant adenovirus vector containing human HIF-1 （ Ad/HIF-1 ） and evaluate the infective effects of the adenovirus on epidermal keratinocytes. Methods Ad/HIF-1 plasmids were identified by restriction endonuclease digestion with Pac I , and then transfected into 1.2 × 10^6 HEK293 cells with 4 p.g plasmids. After 2 days, transfection efficiency was monitored by GFP green fluorescence microscopy. After one week, transfectants were collected and HEK293 cells were infected to amplify the Ad/HIF-1. Cells and supernants （300 ml） were collected for purification and titer detection of the adenovirus. Finally, epidermal keratinocytes were infected with Ad/HIF-1 prepared above to evaluate the infection efficiency. Results The recombinant plasmids pAd/HIF-1 were designed. Two days after HEK293 cells were transfected with above plasmids, cells began to express GFP. After detection, the titer of the generated adenovirus was about 1.8 × 10^11 PFU/ml. To evaluate the infection rate of epidermal keratinocytes, the adenovirus was added to the culture medium. The virus applied at a titer of 50 multiplicity of infection （MOI） resulted in almost 97.46% infection rate. Conclusion Ad/HIF-1 was successfully generated, and could be expressed in epidermal keratinocytes with high infection rate.