不同片段的NPM1-EGFP融合表达载体的构建和表达

目的构建不同片段的NPM1与绿色荧光蛋白(EGFP)的真核融合表达质粒,检测其在人乳腺癌MDA-MB-231细胞中的表达和定位。方法 RT-PCR法从人乳腺癌MDA-MB-231细胞cDNA中扩增不同片段的NPM1,产物经电泳分离、切胶纯化后,分别采用XhoⅠ和EcoRⅠ进行酶切,然后与同样酶切后的pEGFP-C3载体进行连接反应,将连接产物转化大肠杆菌感受态细胞,挑取克隆,提取质粒并酶切鉴定。将酶切及测序鉴定后的pEGFP/NPM1质粒采用脂质体介导后将其转入MDA-MB-231细胞中,通过Western blot技术检测其蛋白的表达。倒置荧光显微镜观察其亚细胞定位。结果酶切鉴定和基因测序结果显示不同片段的pEGFP-NPM1融合表达质粒构建成功,并可在MDA-MB-231细胞内表达,倒置荧光显微镜下可以清晰显见融合蛋白的亚细胞定位。结论 NPM1及其不同结构域的真核重组质粒构建成功,在细胞中成功表达,并可以用于分析其亚细胞定位,从而为进一步研究NPM1在乳腺癌发生发展中的作用奠定基础。

Construction and expression of recombinant plasmid pEGFP-NPM1 and its different domain in human breast cancer cell line MDA-MB-231

Objective To construct recombinant plasmid pEGFP-NPM1 and detect its expression and location in human breast cancer cell line MDA-MB-231.Methods RT-PCR method was performed to amplify the entire fragment of NPM1 gene,the amplified NPM1 gene was inserted into eukaryotic expression vector pEGFP-C3 to construct pEGFP/NPM1 recombinant plasmid,and transfected into MDA-MB-231 cells by lipofectamine mediated gene transfection.The efficiency of transfection and distribution were determined by microscopy.Results The recombinant plasmid was identical as expected.The fusion protein was expressed in MDA-MB-231 cells.The green fluorescence of the protein could be observed by confocal laser microscope.Conclusions The encoding sequence of NPM1 gene and its different domain can be successfully cloned into the expressive vector and expressed in MDA-MB-231 cells,and analyze subcelluar location.The function of NPM1 protein can be used to further medical study.