真核表达载体pEGFP-N1-ZIP10的构建及其对人乳腺癌细胞中其他锌转运体基因表达的影响  

Construction of the recombinant plasmid pEGFP-N1-ZIP10 and its effect on expressions of other zinc transporters in breast cancer cells

作　　者：郭鲁[1] 胡晓燕[1] 蒋雅丽[1] 徐同福[1] 王品[2] 李铭[2] 张莲英[1] 

机构地区：[1]山东大学医学院生物化学与分子生物学研究所,济南250012 [2]山东大学医学院临床医学七年制学生,济南250012

出　　处：《山东大学学报（医学版）》2010年第11期29-32,共4页Journal of Shandong University:Health Sciences

摘　　要：目的构建pEGFP-N1-ZIP10表达载体,观察其在乳腺癌MCF-7和MDA-MB-231细胞中的表达,并检测ZIP10过表达对其他锌转运体的影响。方法从外周血经RT-PCR扩增ZIP10cDNA序列,并将其定向克隆至真核表达质粒pEGFP-N1中,pEGFP-N1-ZIP10经酶切及测序鉴定后,转染乳腺癌MCF-7和MDA-MB-231细胞,RT-PCR检测ZIP10的表达及其他锌转运体ZnT1、ZIP1、ZIP6等表达的变化。结果酶切和测序证实目的基因片段大小、方向均正确,转染MCF-7和MDA-MB-231细胞,RT-PCR检测到ZIP10在mRNA水平过表达,并发现使ZIP1的表达显著降低。结论成功构建真核表达载体pEGFP-N1-ZIP10,并在乳腺癌MCF-7和MDA-MB-231细胞瞬时表达成功,转染后使细胞中ZIP1的表达明显降低,为进一步研究ZIP10在乳腺癌发生发展中的作用奠定基础。Objective To construct the pEGFP-N1-ZIP10 expression vector and observe its expression in human breast cancer M CF-7 and M DA-M B-231 cell lines,and also to detect expressions of other zinc transporters when ZIP10 is over-expressed. Methods The target sequence of ZIP10 was obtained and amplified from human blood by RT-PCR. Then,the cDNA segment was cloned into eukaryote plasmid pEGFP-N1. The pEGFP-N1-ZIP10 was identified by restriction enzyme digestion and checked by DNA sequence analysis. M CF-7 and M DA-M B-231 cells were transiently transfected,and expressions of ZIP10 and other zinc transporters were detected by RT-PCR. Results Identification of pEGFP-N1-ZIP10 by enzyme digestion and PCR showed that the length,location of insertion and direction of the target gene inserted into the recombinant were correct. After the transfection,over-expression of ZIP10 was found in human breast cancer M CF-7 and M DA-M B-231 cells,while expression of ZIP1 was significantly reduced in the transfected cells. Conclusion The eukaryotic expression plasmid pEGFP-N1-ZIP10 has been successfully constructed and it can be expressed transiently in M CF-7 and M DA-M B-231 cells. The decreased expression of ZIP1 in the transfected cells may indicate the role of ZIP10 in breast carcinogenesis and development.

关 键 词：乳腺癌细胞 ZIP10 ZIP1 表达载体 

分 类 号：R737.9[医药卫生—肿瘤]