知识与财富的链接
目的将人类PSF基因的不同功能片段定向连入pEGFP—C2质粒，使PSF蛋白的各功能片段与绿色荧光蛋白在HeLa细胞内融合表达，观察其在HeLa细胞中的表达及定位。方法以重组质粒pEGFP—C2-PSF为模板，PCR法扩增出目的基因，将扩增片段双酶切后连接到质粒pEGFP—C2上，构建重组质粒pEGFP—C2-PSF（I—V）。将构建成功的pEGFP—C2-PSF（I—V）质粒脂质体法转染HeLa细胞，Western印迹检测融合蛋白的表达，并在荧光显微镜下观察融合蛋白的定位与分布。结果成功构建质粒pEGFP—C2-PSF（I～V），并在HeLa细胞中实现表达；Western印迹检测到融合蛋白GFP—PSF（I～V）；在激光共聚焦显微镜下观察到绿色的融合蛋白表达和定位。结论人类PSF基因的不同功能片段的重组质粒pEG—FP—C2-PSF（I～V）构建成功，可用于标记PSF蛋白的不同功能片段，为进一步研究PSF在信号转导中的作用机制以及其生物学功能奠定基础。
Objective To construct eukaryotic green fluorescent protein （GFP） expressing recombinant plasmids, pEGFP-C2-PSF （ I - V ）, which contain differential human PSF fragments. Methods The fragments of PSF gene were amplified by PCR from the recombinant pEGFP- C2-PSF plasmid and the amplified PSF fragments were then subcloned into pEGFP-C2. The recombinant expression vector pEGFP-C2-PSF （ I - V ） was transfected into HeLa cells. The expression of the fusion proteins was examined by fluorescent microscopy and Western blot. Results The expression vector pEGFP- C2-PSF （ I - V ） was successfully constructed. The fusion proteins of GFP-C2-PSF （ I - V ） were detected in HeLa cells by Western blot. In addition, the localization of these fusion proteins was detected by laser scanning confocal microscope. Conclusion These re combinant eukaryotic plasmids of pEGFP-C2-PSF （ I - V ） may provide tools for further study on molecular mechanism, signaling transduction and biologic function of PSF.