重组pEGFP-C2-FOXC1-C真核表达质粒的构建与表达

Construction and expression of recombinant pEGFP-C2-FOXC1-C plasmid

目的:将人FOXC1-C基因定向连入pEGFP-C2质粒,使FOXC1-C蛋白可与绿色荧光蛋白在HeLa细胞内融合表达,为进一步研究FOXC1-C蛋白的功能及定位奠定实验基础。方法:采用EcoR I和NotI双酶切方法,从pcDNA3.1(+)-FOXC1-C质粒中获得FOXC1-C蛋白的cDNA羧基末端;连入pEGFP-C2质粒的C端。将构建成功的pEGFP-C2-FOXC1-C质粒转染入HeLa细胞,荧光显微镜下观察绿色荧光蛋白表达。结果:(1)将该质粒进行双酶切鉴定可见FOXC1-C片段;(2)转染重组质粒后可观察到绿色荧光蛋白的表达。结论:(1)FOXC1-C cDNA羧基末端成功连入pEGFP-C2质粒(;2)FOXC1-C蛋白可与绿色荧光蛋白在HeLa细胞中融合表达。

Objective： To construct eukaryotic green fluorescent protein （GFP） expressing recombinant plasmids, pEGFP-C2-FOXC 1- C ,which contain human FOXC1-C C-terminus cDNA. Methods： The FOXC1-C C-terminus cDNA was purified after pcDNA3.1 （＋）- FOXC1-C plasmid were digested by EcoR I and NotI.And then they were inserted into pEGFP-C2 fluorescent expressing vector. The FOXC1-C fragment were detected by double digestion.This pEGFP-C2-FOXC1-C recombinant plasmids was transfected into HeLa cell line and the expression of green fluorescent fusion protein was observed. Results： （ 1 ）The FOXC1 -C fragment was detected in the prod- ucts of the restriction enzyme digestion. （2）The green fluorescent protein could be observed by fluorescence microscope after the trans- fection. Conclusion： （1）The C-terminus of FOXC1-C cDNA fragment is inserted into the pEGFP-C2 vector. （2）The fluorescent ex- pressing recombinant plasmids pEGFP-C2-FOXC 1-C could express FOXC 1-C-GFP fusion proteins successfully.