P100蛋白及其片段重组质粒构建与表达

Construction and Expression of Recombinant Plasmids Containing p100 and its Fragments

目的分别将人类p100基因，p100的SN基因片段和TD片段定向连入pERFP-CI质粒，使它们可与红色荧光蛋白在HeLa细胞内融合表达，从而为进一步研究P100蛋白及其片段的定位、功能及与其它蛋白的相互关系奠定实验基础。方法PCR分别扩增出P100蛋白全长，SN片段和TD片段基因的序列，定向克隆至真核表达载体pERFP-CI，构建相应的3种重组质粒。将构建成功的质粒转染入HeLa细胞，荧光显微镜下可观察红色荧光融合蛋白表达。结果①PCR法获得P100基因序列，长度为2659bp，SN基因片段1918bp，TD基因片段741bp；②将重组质粒直接进行双酶切鉴定可见P100片段，将经过蓝白斑筛选后的重组子经双酶切再与pERFP-CI载体连接并酶切得到SN片段和TD片段；③转染重组质粒后可观察到红色荧光蛋白的表达。结论3种外源片段成功载人pERFP-CI质粒；P100全长、SN片段、TD片段均可与红色荧光蛋白在HeLa细胞中融合表达。

Objective To construct eukaryotic red fluorescence protein expressing recombinant plasmids pERFP-CI-p100, pERFP-CI-SN and pERFP-CI-TD, which contain human p100 full length eDNA, SN fragment eDNA, and TD fragment cDNA, respectively. Methods The p100 full length eDNA, SN fragment cDNA, and TD fragment eDNA were amplified by PCR and cloned into eukaryotic expression vector pERFP-CI, and then correspondingly inserted into pEGFP-CI fluorescent ex- pressing vector. The resultant pERFP-CI-p100 recombinant plasmids were transfected into HeLa cell line, and the expression of fluorescent recombinant proteins was observed under fluorescence microscopy. Results①The full-length of p100 and the fragment of SN and TD were confirmed in the products of restriction enzyme double digestion.②The red fluorescent proteins was observable under fluorescence microscope after the transfection. Conclusion The fluorescent expressing recombinant plasmids, which encode p100-RFP, SN-RFP and TD-RFP fusion proteins respectively, were successfully constructed.