辐射敏感性新基因UHRF1真核表达载体的构建及鉴定

目的构建UHRF1的真核表达载体,并验证其在乳腺癌细胞MDA-MB-231中的表达。方法采用RT-PCR方法,从乳腺癌细胞MCF-7的总cDNA中扩增出2.3kb的UHRF1基因的cDNA片段,经限制性内切酶KpnⅠ与XhoⅠ双酶切,定向克隆到真核表达载体pcDNA3中,构建重组质粒pcDNA3（＋）-UHRF1,利用限制性内切酶双酶切分析和DNA序列分析鉴定重组质粒;构建成功的重组质粒,经脂质体Lipofactamin2000介导转染MDA-MB-231细胞,G418筛选阳性克隆,以RT-PCR和Western blot检测UHRF1的mRNA和蛋白的表达。结果获得全长约为2.3kb的UHRF1基因片段;重组质粒经限制性内切酶XhoⅠ和KpnⅠ酶切、电泳后显示2.3kb的UHRF1目的片段和5.4kb的pcDNA3载体片段,即UHRF1基因的cDNA已正确克隆到真核细胞表达载体pcDNA3中;UHRF1转染乳腺癌MDA-MB-231细胞后的RT-PCR和Western blot的结果显示：UHRF1的mRNA和蛋白水平均呈现高表达。结论成功构建UHRF1基因的真核表达载体,为进一步研究该基因的功能奠定基础。

Construction and Identification of Eukaryotic Expression Vector of pcDNA3-UHRF1

Objective To generate eukaryotic expression vector of pcDNA3-UHRF1（ubiquitin-like, containing PHD and RING finger domains 1,UHRF1 ） and testify its expression in breast cancer cells MDA-MB-231.Methods A 2.3 kb cDNA fragment was amplified from the total RNA of the human breast cancer cells MCF-7 by the RT-PCR method and was cloned into the plasmid pcDNA3.The vector was identified by the double digestion with restriction enzymes Kpn I and Xho I and was sequenced.The cDNA of UHRF1 was transfected into human breast cancer cells MDA-MB-231 by Lipofactamin2000.The positive clones were selected by G418.The expression of the UHRF1 was detected by RT-PCR and Western blot analysis.Results The recombinant eukaryotic expression vector pcDNA3-UHRF1 was digested with Kpn I and BamH I,and the electrophoresis of the digested products showed two fragments;2.3kb fragment of UHRF1 and 5.4 kb fragment of pcDNA3,and the sequence inserted was identical to the published sequence.The MDA-MB-231 cells transfected with the pcDNA3-UHRF1 plasmid expressed a high level of the UHRF1 mRNA and protein.Conclusion The recombinant eukaryotic cell expression vector of pcDNA3-UHRF1 is constructed successfully.The recombinant plasmid pcDNA3-UHRF1 can provide a very useful tool and lay an important foundation for the research on the function of UHRF1.