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
Keywords: hAIF-1, 腺病毒, 增殖, 迁移hAIF-1, adenovirus, proliferation, migration
Abstract:
hAIF-1是一种炎症因子，本研究通过构建hAIF-1基因的重组腺病毒，探讨hAIF-1对A549细胞增殖和迁移能力的影响.用PCR的方法克隆hAIF-1基因编码序列，并亚克隆到腺病毒穿梭质粒载体pDC315-EGFP上，构建穿梭质粒pDC315-EGFP-hAIF-1，将穿梭质粒与腺病毒骨架质粒pBHGloxdeltaE13Cre共转染293细胞，利用AdMax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WesternBlot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hAIF-1基因的重组腺病毒，所得病毒滴度为2.5×108pfu/mL；用hAIF-1重组腺病毒感染人A549细胞，RT-PCR和WesternBlot鉴定显示细胞可过表达hAIF-1；MTT显示过表达hAIF-1可显著增强A549细胞增殖能力；细胞划痕和Tranwell实验证明hAIF-1能够促进A549细胞迁移.hAIF-1isaninflammatoryfactor.ThestudywassupposedtoexploretheeffectofhAIF-1ontheproliferationandmigrationofA549cellsbythepreparationoftherecombinantadenoviruswithhAIF-1gene.ThehAIF-1genewasclonedbyPCRandsubclonedintotheshuttlevectorpDC315-EGFP.TheshuttleplasmidandadenovirusgenomicplasmidpBHGloxdeltaE13Crewereco-transfectedinto293cellsforpackagingrecombinantadenoviruswiththehelpoftheadenoviruspackagingsystemAdMax.ThetiteroftheviruswasmeasuredbyTCID50.TheexpressionofthetargetgenewasidentifiedbyRT-PCRandWesternBlot，andtheeffectofthetargetgeneontheproliferationandmigrationofA549cellswasexaminedbyMTT，woundhealingandTranswellassay.TheresultsshowedthattheAdDC315-EGFP-hAIF-1wassuccessfullyconstructedandthetiteroftheviruswas2.5×108pfu/mL；AnalysisofRT-PCRandWesternBlotindicatedthatA549cellscouldoverexpresshAIF-1afterinfectedwiththerecombinantadenovirus；TheMTTassayshowedthathAIF-1couldincreasetheproliferativerateofA549cells；ThewoundhealingandTranswellassayprovedthathAIF-1couldpromotethemigrationofA549cells
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