中文摘要：

目的利用慢病毒载体系统构建稳定表达HIF1α的A549细胞系。方法以NCBI人HIF1αa基因编码序列为模板，设计并合成引物，PCR法扩增HIF1α。酶切回收的HIF1α片段与制备的慢病毒载体HBLV—RFP—Puro重组反应。PCR和基因测序鉴定重组质粒。重组质粒和辅助质粒共转染293T细胞。包装好的病毒经过滤、浓缩后，利用稀释计数法测定病毒滴度。制备好的慢病毒感染A549细胞，采用药物筛选稳定转染细胞系。荧光显微镜及Westernblot检测观察转染及筛选效果。结果PCR扩增HIF1α片段经鉴定成功，重组质粒经PCR、基因测序鉴定构建成功。成功包装获得高滴度LV—HIF1α。LV-HIF1α感染A549细胞后经药物筛选，荧光显微镜下细胞带有红色荧光，Westernblot结果显示LV-HIF1α组HIF1α的表达水平明显高于对照组。结论慢病毒载体介导HIF1α稳定表达的A549细胞系构建成功。

英文摘要：

Objective To establish the A549 cell line with stable expression of HIF1α by using lentiviral vector system. Methods Primers were designed and synthesized with human HIF1α gene coding sequence by the National Center of Biotechnogical Information（NCBI） as the template. HIF1α was amplified by PCR. The HIF1α fragment recycled by enzyme digestion was recom- bined with prepared lentiviral vector HBLV-RFP-Puro. The recombinant plasmid was identified by PCR and gene sequencing. The recombinant plasmid and the auxiliary plasmid were co-transfect into 293T cell. After filtration and concentration of packaged virus, the viral titer was detected by using the dilution counting method. The prepared lentivirus was infected A549 cells. The drug screen- ing was adopted to stabilize the transfected cell line. The transfection effect was detected and observed by fluorescence microscope and Western blotting. Results The HIF1α fragment amplified by PCR was successfully verified and the recombinant plasmid was successfully constructed by PCR and gene sequencing identification. High-titer LV-HIF1α was obtained by successful package. Af- ter LV-HIF1α infecting A549 cells, the cells showed the red fluorescence by fluorescence microscope. The expression level of HIF1α in the LV-HIF1α group was significant higher than that in the control group by Western blot. Conclusion The 549 cell line with HIF1α stable expression mediated by lentivirus is constructed successfully.