非受体型酪氨酸激酶JAK1基因真核表达质粒的构建与真核表达

Construction of eukaryotic expression vector for nonreceptor tyrosine kinase-JAK1 gene and its expression

[目的]构建非受体型酪氨酸激酶JAK1(nonreceptor tyrosine kinase-JAK1)基因真核表达质粒pcDNA4.0-HisJAK1,并转染293T细胞进行真核表达。[方法]从Hela细胞中提取总的RNA,通过RT-PCR获得JAK1基因全长c DNA序列,并将其克隆至真核表达载体pcDNA4.0-His中,构建重组质粒pcDNA4.0-His-JAK1。将真核重组表达质粒转染293T细胞,转染48 h后,在荧光显微镜下观察转染情况。免疫印迹法检测JAK1蛋白转染24 h、36 h与48 h在293T细胞中的表达。[结果]经双酶切与质粒PCR鉴定质粒克隆正确,测序鉴定序列中第2199位碱基A突变为G,为同义突变,不影响氨基酸序列。转染293T细胞48 h后,间接免疫荧光实验显示在293T细胞中检测到绿色荧光。免疫印迹检测可见大小约为133 kDa的目的蛋白。[结论]成功获得JAK1基因全长序列,并成功构建pcDNA4.0-HisJAK1真核重组表达质粒,并在真核细胞293T中获得有效表达,为进一步研究JAK1蛋白质相互作用奠定了基础。

[ Objective] To construct a eukaryotic expression vector for nonreceptor tyrosine kinase -JAK1 gene and express in 293T cells. [ Methods] Total RNA was extracted from Hela cells ,with which JAK1 gene was amplified by RT - PCR and inserted into eukaryotic expression vector pcDNA4.0 - His. 293T cells were transfected with the constructed recombinant plasmid pcDNA4.0 -His -JAK1, observed by fluorescent microscopy 48 hours later, and determined for expression level of JAK1 by Western Blot 24 hours,36 hours and 48 hours later. [ Results] Restriction analysis and sequencing proved that pcDNA4.0 - His - JAK1 was a correct recombinant plasmid. Green fluorescence was observed in 293T cells 48 h after transfection with the plas- mid. The target protein band by Western Blot. [ Conclusion ] Eukaryotic expression vector pcDNA4.0 - His - JAK1 was successfully constructed and expressed effectively in 293T cells, which laid a foundation of further research on protein -protein interaction of JAK1.