Abstract:

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 48h, 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.

Key words: ZIP2 eukaryotic expression vector; Zinc transporter; Over expression; Polymerase chain reaction