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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。
Objective To construct the eukaryotic expression vector pcDNA3.1 (+) AIF of human apoptosis-inducing factor (AIF) gene and to study the role of AIF in apoptosis. Methods The total RNA was extracted from human cells. The AIF gene fragment was amplified by RTPCR, cloned into pMD18T vector and identified by restriction enzyme digestion and sequencing. The AIF gene fragment was inserted into pcDNA3.1 (+) to construct the eukaryotic expression vector. Raji cells of lymphoblastoid cells, observe the expression level and function of AIF. Results The mature AIF protein was expressed in eukaryotic cells and transferred to the nucleus to induce caspase-independent apoptosis. Conclusion The eukaryotic expression vector pcDNA3.1 (+) AIF was constructed successfully. AIF plays an important role in the apoptosis of Caspase-independent cells.