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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 construct the eukaryotic expression vector of UHRF1 and verify its expression in breast cancer cell line MDA-MB-231. Methods The cDNA fragment of 2.3 kb UHRF1 gene was amplified by RT-PCR from the total cDNA of breast cancer cell line MCF-7. The recombinant plasmid was digested with KpnⅠand XhoⅠand cloned into the eukaryotic expression vector pcDNA3, the recombinant plasmid pcDNA3 (+) - UHRF1 was constructed and the recombinant plasmids were identified by restriction endonuclease digestion analysis and DNA sequence analysis. The recombinant plasmids were successfully constructed and transfected into MDA-MB- 231 cells. The positive clones were screened by G418. The expression of UHRF1 mRNA and protein was detected by RT-PCR and Western blot. Results The full length of UHRF1 gene fragment of about 2.3 kb was obtained. The recombinant plasmid was digested with restriction endonucleases Xho I and Kpn I. After electrophoresis, the 2.3 kb UHRF1 gene fragment and the 5.4 kb pcDNA3 vector fragment, ie, the cDNA of the UHRF1 gene The results of RT-PCR and Western blot showed that the mRNA and protein levels of UHRF1 were highly expressed in breast cancer cell line MDA-MB-231. Conclusion The eukaryotic expression vector of UHRF1 gene was successfully constructed, which laid the foundation for further study on the function of this gene.