慢病毒载体介导HIF1α稳定表达A549细胞系构建.docx

慢病毒载体介导HIF1α稳定表达A549细胞系构建

邹斌;周学亮;詹宇亮;陈紫晴;赖松青;吴霞;刘季春

【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HIF1α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.%ObjectiveToestablishtheA549celllinewithstableexpressionofHIF1αbyusinglentiviralvectorsystem.MethodsPrimersweredesignedandsynthesizedwithhumanHIF1αgenecodingsequencebytheNationalCenterofBiotechnogicalInformation(NCBI)asthetemplate.HIF1αwasamplifiedbyPCR.TheHIF1αfragmentrecycledbyenzymedigestionwasrecombinedwithpreparedlentiviralvectorHBLV-RFP-Puro.TherecombinantplasmidwasidentifiedbyPCRandgenesequencing.Therecombinantplasmidandtheauxiliaryplasmidwereco-transfectinto293Tcell.Afterfiltrationandconcentrationofpackagedvirus,theviraltiterwasdetectedbyusingthedilutioncountingmethod.ThepreparedlentiviruswasinfectedA549cells.Thedrugscreeningwasadoptedtostabilizethetransfectedcellline.ThetransfectioneffectwasdetectedandobservedbyfluorescencemicroscopeandWesternblotting.ResultsTheHIF1αfragmentamplifiedbyPCRwassuccessfullyverifiedandtherecombinantplasmidwassuccessfullyconstructedbyPCRandgenesequencingidentification.High-titerLV-HIF1αwasobtainedbysuccessfulpackage.AfterLV-HIF1αinfectingA549cells,thecellsshowedtheredfluorescencebyfluorescencemicroscope.TheexpressionlevelofHIF1αintheLV-HIF1αgroupwassignificanthigherthanthatinthecontrolgroupbyWesternblot.ConclusionThe549celllinewithHIF1αstableexpressionmediatedbylentivirusisconstructedsuccessfully.

【期刊名称】《重庆医学》

【年(卷),期】2017(046)020

【总页数】4页(P2744-2746,2750)

【关键词】HIF1α;慢病毒载体;稳定表达;A549

【作