人同种异体移植炎症因子-1的基因克隆及其在小鼠成纤维细胞中的表达

Cloning of Human Allograft Inflammatory Factor-1 Gene and Its Expression in the Mouse NIH3T3 Cell Line

目的构建人同种异体移植炎症因子-1(allograft inflammatory factor-1,AIF-1)基因的真核表达载体并在小鼠成纤维细胞系(NIH3T3)中表达,为进一步研究AIF-1蛋白的功能奠定基础。方法利用基因重组技术,构建带有AIF-1基因的2种重组真核表达载体:pcDNA3.1(-)/AIF-1与pEGFP-C1/AIF-1。利用脂质体法将2种重组质粒分别转染NIH3T3细胞,用荧光倒置显微镜及Western blot方法观察及鉴定AIF-1在细胞中的稳定表达及瞬时表达。结果酶切分析及DNA序列测定证实,AIF-1基因片段被分别克隆入真核表达载体pcDNA3.1(-)与pEGFP-C1。荧光显微镜观察到NIH3T3细胞中有绿色荧光分布。Western blot结果表明,转染重组质粒的NIH3T3细胞中,AIF-1表达量明显增高。结论成功构建了2种重组真核表达载体:pcDNA3.1(-)/AIF-1与pEGFP-C1/AIF-1,经转染NIH3T3细胞株获得了AIF-1蛋白的瞬时表达及稳定表达。

Objective To construct two recombinant eukaryotic expression vectors of human allograft inflammatory factor-1（AIF-1）,and express two recombinant genes in the mouse NIH3T3 cell line respectively,laying a foundation for further study on biological functions of AIF-1.Methods By using gene modification techniques,two recombinant eukaryotic expression vectors of AIF-1 including pcDNA3.1（-）/AIF-1 and pEGFP-C1/AIF-1 were constructed.The recombinant vectors were transfected into NIH3T3 cells by lipofectamine.Stable and transient expression in cells was observed and identified by a fluorescent inverted microscope and Western blot method.Results Restrictive enzyme digestion analysis and DNA sequencing revealed that human AIF-1 gene was cloned into eukaryotic expression vectors pcDNA3.1（-）/AIF-1 and pEGFP-C1/AIF-1 respectively.The fluorescent inverted microscopy showed that green fluorescence was expressed in NIH3T3 cells.NIH3T3 cells transfected by recombinant vectors showed significantly increased expression of AIF-1 by Western blot.Conclusion The two recombinant eukaryotic expression vectors can be successfully constructed.The AIF-1 protein can be stably and transiently expressed in NIH3T3 cells.