目的：：构建Max作用蛋白1-0(Max interacting protein1-0,Mxi1-0)缺失突变体的真核表达载体并观察这些突变体在细胞内的定位情况,为研究Mxi1-0细胞内定位与其功能的关系奠定基础。方法：以野生型Mxi1-0真核表达质粒为模板,聚合酶链式反应扩增出5种类型Mxi1-0缺失突变体的基因片段,克隆至增强绿色荧光蛋白真核表达载体,经酶切和测序鉴定后,利用脂质体将重组质粒转染小鼠胚胎成纤维细胞株(NIH3T3)。 Western blot检测融合蛋白的表达,荧光显微镜下观察融合蛋白细胞内定位情况。结果：各种Mxi1-0缺失突变体经测序鉴定正确后,在NIH3T3中得到表达。免疫荧光结果表明,脯氨酸富集结构域( PRD)突变体在细胞质和细胞核中均有分布,与Sin3结合域高度同源结构域( tSID)突变体主要分布于细胞质,在细胞核中有少量分布；PRD-tSID突变体及△PRD突变体分布于细胞质,而△PRD-tSID突变体主要分布于细胞核。结论：成功构建了5种人Mxi1-0缺失突变体的真核表达载体,并初步观察这些突变体在细胞内的定位情况,为探寻Mxi1-0在细胞内定位机制及功能奠定了基础。%Objective:To construct the recombinant eukaryote expression vectors of Max interacting protein 1-0 ( Mxi1-0 ) truncation mutants and detect the intracellular localization of the mutants. Methods:Five kinds of truncation mutants were generated by polymerase chain reaction with a template of enhanced green fluorescent protein eukaryotic expression vector(pEGFP-N1) containing Mxi1-0 gene. Subsequently,the target sequences were subcloned into pEGFP-N1. The recombinant vectors were confirmed by restriction enzyme digestion and DNA sequencing. The mutants were transfected into mouse embryo fibroblast(NIH3T3). Fusion protein in NIH3T3 cells were detected by Western blot. The intracellular localization of mutants was investigated by immunofluorescence. Results:The recombinant eukaryote expression vectors of Mxi1-0 mutants were constructed successfully. The expression of these mutants in NIH3T3 cells could be detected by Western blot. In addition,the immunofluorescence results showed that in NIH3T3 cells,proline rich domain ( PRD) mutant was in cytosol and nuclei,putative Sin3-interacting domain( tSID) mutant was mainly in cytosol,little in nuclei. PRD-tSID mutant and △PRD mutant were mainly in cytosol,while △PRD-tSID mutant was mainly in nuclei. Conclusions:The truncation mutants of Mxi1-0 were constructed and expressed successfully in NIH3T3 cells,the localization in cells was also observed,which will be benefit for the future research on the mechanism of Mxi1-0 localization and function.
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