AIF重组质粒的构建、表达和功能的研究

目的构建人凋亡诱导因子(AIF)基因功能片段的真核表达载体pcDNA3.1( )AIF,并初步研究AIF在细胞凋亡中的作用。方法从人细胞中提取总RNA,RTPCR扩增AIF基因片段;克隆到pMD18T载体上,通过酶切和测序进行鉴定;将AIF基因片段插入pcDNA3.1( ),构建真核表达载体;电转化淋巴母细胞Raji细胞,观察AIF的表达水平和功能。结果成熟AIF蛋白分子在真核细胞中表达,并转移到细胞核中,诱导Caspase非依赖性细胞凋亡。结论成功构建了pcDNA3.1( )AIF真核表达载体,AIF在Caspase非依赖性细胞凋亡中发挥重要作用。

Preliminary studies on construction, expression and function of recombinant AIF plasmid

Objective To construct eukaryotic expression vector carrying human Apoptosis-inducing factor(AIF) gene fragment,pcDNA3.1( )/AIF,and research the gene expression and function.Methods After total RNA was extracted from human cells,AIF gene fragment was amplified by RT-PCT,then cloned into pMD18-T vector,which was certified by restriction analysis and sequencing.The gene fragment was inserted into plasmid pcDNA3.1( ) and eukaryotic expression vector was constructed.The vector was transfected into Raji cells,and then the gene expression and function was studied.Results Eukaryotic expression vector,pcDNA3.1( )/AIF,was constructed and the gene was expressed in eukaryotic cells successfully.The expressed AIF protein was translocated into nucleus and induced caspase independent apoptosis.Conclusion AIF plays an important part in caspase-independent apoptosis.