重组真核质粒pERFP-C1-hTudor-SN-SN(1～4)的构建和表达

Construction and Expression of pERFP-C1-hTudor-SN-SN (1-4) Recombinant Eukaryotic Plasmids

目的:将人类Tudor-SN蛋白SN(1～4)基因片段分别定向连入pERFP-C1质粒,使Tudor-SN蛋白SN各功能片段与红色荧光蛋白在HeLa细胞内融合表达。方法:以重组质粒pSG5-Tudor-SN-flag为模板,PCR法扩增出目的基因,利用EcoRⅠ和BamHⅠ双酶切法将目的片段连接到pERFP-C1载体上,再将构建成功的pERFP-C1-Tudor-SN-SN(1～4)重组质粒转染入HeLa细胞内,在荧光显微镜下观察红色荧光蛋白的表达。结果:以单/双酶切及基因测序法鉴定构建的重组质粒均无误,荧光显微镜下观察到红色融合蛋白的表达。结论:重组pERFP-C1-h Tudor-SN-SN(1～4)质粒构建及表达成功。

Objective： To construct eukaryotic red fluorescent protein （RFP） expressing recombinant plasmids,pERFP-C1-hTudor-SN-SN（1-4）,which contain SN（（1-4） fragments of human Tudor-SN.Methods： The genes of Tudor-SN fragments were amplified by PCR from the recombinant pSG5-Tudor-SN-flag plasmid and inserted into pERFP-C1 fluorescent expressing vector with EcoRⅠ and BamHⅠ site.These recombinant pERFP-C1-hTudor-SN-SN （1-4） plasmids were transfected into HeLa cells and the expression of red fluorescent fusion proteins was examined by fluorescence microscope.Results： The SN（1-4） fragments of Tudor-SN were sequenced correctly and detected in the products of the restriction single/double enzyme digestion.The red fluorescent fusion proteins were observed in HeLa cell after the transfection.Conclusion： These recombinant eukaryotic plasmids of pERFP-C1-Tudor-SN（1-4） were constructed successfully and expressed effectively.