【摘要】 目的:构建带有myc标签的核输入蛋白(importin)α/β的真核表达载体,转染人胚肾293T细胞并分析其表达。方法:以乳腺癌文库为模板PCR扩增核输入蛋白α/β基因,扩增产物插入真核表达载体p XJ-40-myc,经双酶切和测序鉴定;将空载体与重组质粒分别转染人胚肾293T细胞,通过Western印迹和免疫荧光技术检测核输入蛋白α/β的表达。结果:酶切鉴定与测序结果表明构建的myc-Importinα/β真核表达载体正确;Western印迹检测到重组质粒在293T细胞中的表达;免疫荧光检测到核输入蛋白α定位于细胞质和细胞核,而核输入蛋白β定位于细胞核。结论:构建了核输入蛋白α/β的真核表达载体,并确定了其在细胞中的表达定位。更多还原

【Abstract】 Objective:To construct the eukaryotic expression vectors of human importin α/β labeled with myc tag and detect intracellular localization of their expression product.Methods:Human importin α/β genes were obtained from breast cancer library by PCR and cloned into pXJ-40-myc vector.Human 293 T cells were transfected with the recombinant plasmids of myc-Importin α/β and the expression was detected by Western blotting and immunofluorescent techniques.Results:The constructed importin α/β eukaryotic expression vectors labeled with myc tag were confirmed by double enzyme digestion and sequencing.The expression of human importin α/β protein in human 293 T cells was identified by Western blotting.Immunofluorescence showed that importin α located in both cytoplasm and nucleus,while importin β located in nucleus.Conclusion:The eukaryotic expression vectors of myc-importin α/β have been constructed and their expression in human 293 T cells were determined.更多还原