RUNX3基因真核表达载体的构建及其在乳腺癌T47D细胞中的表达

Construction of eukaryotic expression vector pcDNA3.1(+)-RUNX3 and its expression in human breast cancer T47D cell line

目的:构建真核表达载体pcDNA3.1(+)-RUNX3,并在乳腺癌T47D细胞株中表达。方法:应用基因重组技术和限制性内切酶EcoRI和XhoI酶切,构建并鉴定pcDNA3.1(+)-RUNX3真核表达载体,经脂质体Lipofectamine2000介导质粒转染T47D细胞后应用逆转录-聚合酶链反应(RT-PCR)和Western blot实验,检测RUNX3在T47D细胞中的表达。结果:重组真核表达载体pcDNA3.1(+)-RUNX3经限制性内切酶EcoRI和XhoI酶切,电泳后显示1.3kb的RUNX3目的片段和5.4kb的pcDNA3.1(+)载体片段。测序证实酶切片段与gene bank中登记的RUNX3序列相同,证实pcDNA3.1(+)-RUNX3真核表达载体构建成功。经RT-PCR和Western blot检测,表明转染pcDNA3.1(+)-RUNX3的T47D细胞RUNX3阳性表达。结论:重组真核表达载体构建正确,并建立稳定表达RUNX3的T47D细胞系,从而为后续研究提供有用的细胞研究模型。

Objective： To construct the eukaryotic expression vector pcDNA3.1（＋）-RUNX3 and to express it in human breast cancer T47D cell line.Methods：Eukaryotic expression vector for pcDNA3.1（＋）-RUNX3 was constructed by use of recombinant DNA technique and was transfected into T47D cell by Lipofectamine 2000.The mRNA and protein expression of RUNX3 gene was detected by RT-PCR and Western blot methods.Results：The recombined eukaryotic expression vector for RUNX3 was digested with EcoRI and XhoI,and the electrophoresis of digested products showed two fragments： 1.3kb fragment for RUNX3 and 5.4kb fragment for pcDNA3.1（＋）.The sequence of DNA fragment was identified to that published in Genebank.The RUNX3 gene showed positive expression in transfected T47D cell line by RT-PCR and Western blot.Conclusion：Eukaryotic expression vector pcDNA3.1（＋）-RUNX3 was constructed and RUNX3 could be expressed in human breast cancer T47D cell line.This provides useful cell model for further research.