Objective      To construct an eukaryotic expression vector of A20 artificial zinc finger-artificial transcription factor (ZF-ATF)

which expresses A20 stably

and evaluate its anti-inflammation effects on  human umbi

lical vein endothelial cells(HUVEC).         Methods      A20 artificial ZF-ATF was designed based on the gene sequence of A20. Then the sequence of ZF-ATF was subcloned into the eukaryotic expression vector PCDNA-NLS-P65-Flag

which was identified and confirmed by restriction enzyme digestion and DNA sequencing. Subsequently

the PCDNA-NLS-ZF-ATF-P65-Flag plasmid was transfected into HUVEC cells

and then the expression of protein A20 was detected by Western blotting. The expression of NF-κB p65 was detected in HUVEC cells after LPS treatment by Western blotting. The concentrations of TNF-α

IL-6 and IL-8 in the supernatants were determined by cytokine ELISA kits.        Results      Restriction enzyme digestion and DNA sequencing confirmed that the obtained sequence of ZF-ATF was correct. Western blotting indicated that the expression of protein A20 was upregulated in HUVEC cells in 24 h after recombinant vector transfection. After stimulation with LPS for 24 h

the concentrations of TNF-α

IL-6 and IL-8 in the supernatants of HUVEC cells were obviously increased in the blank and control groups

but those had no change in ZF-ATF group

which were significantly lower in the blank and control groups(

P

<

0.05

P

<

0.01). Western blotting indicated that LPS stimulation resulted in an significant decrease of NF-κB p65 in ZF-ATF group (19.8±4.5) than in blank (90.2±5.9) and control groups (96.3±3.3

both

P

<

0.01).       Conclusion      The eukaryotic expression vector of ZF-ATF is successfully constructed

which expresses A20 stably and A20 could play crucial protective roles in anti-inflammation of HUVEC cells against LPS stimulation. This research could be a foundation for our study the molecular function of A20.