Objective       To construct an eukaryotic expression vector of A20 mutant

which expresses A20 stably

and evaluate its effects on the expression of inflammatory factors and anti-apoptosis genes in prostate cancer PC-3M cells.       Methods       The domain of A20 in inhibiting NF-κB activity was designed based on the gene sequence of natural A20. Then the sequence of A20 mutant was subcloned into the eukaryotic expression vector pEGFP-C1

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

the constru

cted recombinant plasmid pEGFP-C1-hA20 mutant was transfected into COS 7 cells to replicate the plasmid

and then the expression of protein A20

IκB-α

pERK1/ERK2、pJNK and Bcl-2 were detected in PC-3M cells after transfection by Western blotting. The concentrations of NF-κB

TNF-α and IL-6 in the supernatant were determined by ELISA.       Results        Restriction enzyme digestion and DNA sequencing confirmed that the obtained sequence of mutant was correct. Western blotting indicated that the expression of protein A20 was enhanced after the pEGFP-C1 plasmid was transfected into the PC-3M cells. After stimulation with LPS

the degradation of IκB-α and phosphorylation of ERK1/ERK2 and JNK were inhibited after A20 mutant pretreatment. The expression of Bcl-2 was obviously increased

but decreased significantly after A20 mutant pretreatment. After stimulation with LPS for 24 h

the concentrations of NF-κB

TNF-α and IL-6 in the supernatants of PC-3M cells were obviously increased in the blank and control groups

but those levels were significantly reduced in mutant group (

P

0.05

P

0.01).       Conclusion       The eukaryotic expression vector of A20 mutant is successfully constructed

which expresses A20 stably and suppresses the expression of inflammatory factors and anti-apoptosis genes in PC-3M cells.