低氧诱导因子-1的病毒载体制备及感染效果

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1.2×106个HEK293细胞,2 d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300 ml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1.8×1011PFu/ml.病毒以MOI=50感染表皮细胞,2 d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.

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

Objective To prepare the recombinant adenovirus vector containing human HIF-I ( 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 × 106 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-I. Cells and supernants (300 m]) were collected for purification and titer detection of the adenovirus. Final]y, epidermal keratinocytes were infected with Ad/IIIF-1 prepared above to evaluate the infection efficiency. Results The recombinant p]asmids 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 × 1011 PFU/ml. To evaluate the infection rate of epidermal keratinocytes,thc adenovirus was added to the culture medium. The virus applied at a titer of SO multiplicity of infection (MOI) resulted in almost 97.46% infection rate. Conclusion Ad/HIF-1 was successfully generated,and could be expressed in epidermal keratinocytcs with high infection rate.