辐射敏感性新基因UHRF1真核表达载体的构建及鉴定  

Construction and Identification of Eukaryotic Expression Vector of pcDNA3-UHRF1

作　　者：李新莉[1] 孟庆慧[2] 朱然[1] 朱巍[1] 樊赛军[1] 

机构地区：[1]苏州大学医学部放射医学与公共卫生学院营养与食品卫生教研室江苏省放射医学与防护重点实验室,江苏苏州215123 [2]美国华盛顿特区乔治敦大学Lombardi肿瘤研究中心

出　　处：《苏州大学学报（医学版）》2011年第1期9-12,共4页Suzhou University Journal of Medical Science

基　　金：国家自然科学基金资助项目(81001185);苏州市社会发展基金资助项目(YJS0905);江苏省高校自然科学研究资助项目(10KJB310011);苏州市肿瘤放射生物学重点实验室基金资助项目(SZS0802)

摘　　要：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。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.

关 键 词：UHRF1 基因克隆 真核表达载体 脂质体 

分 类 号：R811.5[医药卫生—放射医学]