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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 pEGFP-N1-ZIP2 eukaryotic expression vector and observe its expression in human T lymphocyte cell line Jurkat-E6-1 and human monocytic cell line THP-1 and to detect that ZIP2 is overexpressed in both cell lines And its impact on other zinc transporters. Methods Human ZIP2 cDNA sequence was amplified by RT-PCR from peripheral blood and ligated with pEGFP-N1. The expression of ZIP2 was detected by RT-PCR and the expression of ZIP1, ZIP6, ZIP8, ZIP10, ZnT1, ZnT5 and other zinc transporter expression changes. Results After double enzyme digestion and sequencing results showed that the size of the target gene, insertion direction are correct. In Jurkat-E6-1 cells, the overexpression of ZIP2 significantly decreased the expression of ZnT-1 (P <0.05), but over-expression of ZIP2 had no significant effect on the expression of other zinc transporters in THP-1 cells. Conclusions The eukaryotic expression vector pEGFP-N1-ZIP2 was successfully constructed and transiently transfected into Jurkat-E6-1 cells and THP-1 cells. The overexpression of ZIP2 in Jurkat-E6-1 cells down-regulated the expression of ZnT1, while the expression of ZIP2 Overexpression did not significantly affect selected other zinc transporters, and ZIP2 overexpression showed differences in the other members of the zinc transporter family in two different immune cell lines.