hAIF-1基因重组腺病毒的构建及其对A549细胞增殖和迁移的影响

Construction of Recombinant Adenovirus of h AIF-1 Gene and Its Effect on Proliferation and Migration of A549 Cells

h AIF-1是一种炎症因子,本研究通过构建h AIF-1基因的重组腺病毒,探讨h AIF-1对A549细胞增殖和迁移能力的影响.用PCR的方法克隆h AIF-1基因编码序列,并亚克隆到腺病毒穿梭质粒载体p DC315-EGFP上,构建穿梭质粒p DC315-EGFP-h AIF-1,将穿梭质粒与腺病毒骨架质粒p BHGloxdelta E13Cre共转染293细胞,利用Ad Max腺病毒载体包装系统包装腺病毒,TCID50法测定病毒滴度,利用RT-PCR和Western Blot分析检测目的基因表达,用MTT法检测A549细胞的增殖,细胞划痕和Transwell实验检测A549细胞的迁移.结果表明,成功构建了表达h AIF-1基因的重组腺病毒,所得病毒滴度为2.5×108pfu/m L;用h AIF-1重组腺病毒感染人A549细胞,RT-PCR和Western Blot鉴定显示细胞可过表达h AIF-1;MTT显示过表达h AIF-1可显著增强A549细胞增殖能力;细胞划痕和Tranwell实验证明h AIF-1能够促进A549细胞迁移.

hAIF-1 is an inflammatory factor. The study was supposed to explore the effect of h AIF-1 on the proliferation and migration of A549 cells by the preparation of the recombinant adenovirus with h AIF-1 gene. The h AIF-1 gene was cloned by PCR and subcloned into the shuttle vector p DC315-EGFP. The shuttle plasmid and adenovirus genomic plasmid p BHGloxdelta E13 Cre were co-transfected into 293 cells for packaging recombinant adenovirus with the help of the adenovirus packaging system Ad Max. The titer of the virus was measured by TCID50. The expression of the target gene was identified by RT-PCR and Western Blot,and the effect of the target gene on the proliferation and migration of A549 cells was examined by MTT,wound healing and Transwell assay. The results showed that the Ad DC315-EGFP-h AIF-1was successfully constructed and the titer of the virus was 2.5×108pfu/m L;Analysis of RT-PCR and Western Blot indicated that A549 cells could overexpress h AIF-1 after infected with the recombinant adenovirus;The MTT assay showed that h AIF-1 could increase the proliferative rate of A549 cells;The wound healing and Transwell assay proved that h AIF-1 could promote the migration of A549 cells.