pEGFP-N1-ZIP2真核表达载体的构建及对人T淋巴和单核细胞系锌转运体基因表达的影响

Construction of the recombinant plasmid pEGFP-N1-ZIP2 and its effect on expression of other zinc transporters in human T lymphocyte and monocyte cell lines

目的构建pEGFP-N1-ZIP2真核表达载体,观察其在人T淋巴细胞系Jurkat-E6-1和人单核细胞细胞系THP-1中的表达,并检测ZIP2在两种细胞系中过表达及其对其他锌转运体的影响。方法利用外周血经RT-PCR扩增获得人ZIP2 cDNA序列,将其与pEGFP-N1定向连接,构建完成转染细胞48 h后,通过RT-PCR检测ZIP2过表达情况,并检测ZIP1、ZIP6、ZIP8、ZIP10、ZnT1、ZnT5等锌转运体表达变化。结果经过双酶切鉴定以及测序结果显示目的基因大小、插入方向均正确。在Jurkat-E6-1细胞中,ZIP2过表达使ZnT-1表达显著降低(P<0.05),但在THP-1细胞中,ZIP2过表达对其他锌转运体表达没有显著影响。结论成功构建pEGFP-N1-ZIP2真核表达载体,瞬时转染Jurkat-E6-1细胞和THP-1细胞,Jurkat-E6-1细胞中ZIP2过表达使ZnT1表达下调,而THP-1细胞中ZIP2过表达并未对选定的其他锌转运体产生显著影响,两种不同的免疫细胞系中ZIP2过表达对锌转运体家族其他成员影响表现出差异。

Objective To construct the pEGFP-N1-ZIP2 expression vector,observe its expression in the human T lymphocyte cell line Jurkat-E6-1 and human mononuclear cell line THP-1,and detect whether expression of other zinc transporters changed when ZIP2 is over-expressed.Methods The target cDNA sequence of ZIP2 was obtained from human blood by RT-PCR.The target cDNA segment was ligated into eukaryote plasmid pEGFP-N1.The plasmids were checked by double restriction enzyme digestion and DNA sequence analysis.Jurkat-E6-1 and THP-1 cell lines were transiently transfected for 48 h,and expressions of ZIP2,ZIP1,ZIP6,ZIP8,ZIP10,ZnT1 and ZnT5 were detected by RT-PCR.Results Identification of pEGFP-N1-ZIP2 by double enzyme digestion and DNA sequence analysis showed that the length and direction of the target gene inserted into the recombinant plasmid were correct.After transfection of pEGFP-N1-ZIP2,ZIP2 over-expression was found in Jurkat-E6-1 and THP-1 cell lines.Expression of ZnT1 was significantly reduced in the transfected Jurkat-E6-1.However,on change was found in other zinc transporters in the transfected THP-1.Conclusion The eukaryotic expression plasmid pEGFP-N1-ZIP2 was successfully constructed.Jurkat and THP-1 cell lines were transfected with the plasmids.ZIP2 was up-regulated and ZnT1 was down-regulated in transfected Jurkat-E6-1,while no changes of other zinc transporter genes were found in transfected THP-1.Over-expression of ZIP2 has different effects on other zinc transporter genes in Jurkat-E6-1 and THP-1 cell lines.