ABSTRACT
Objective To investigate the regulating effect of hepatitis B virus(HBV)SPⅠbinding protein 1(SBP1)on inducible nitric-oxide synthase(iNOS)gene promoter activity.Methods Polymerase chain reaction(PCR)technique was employed to amplify the coding sequence of iNOS promoter DNA by using HepG2 genomic DNA as template,and 3 deletion mutants were amplified. The products were cloned into pGEM-T vector,respectively.The iNOS gene and 3 deletion mutants were cut from T-iNOS by KpnⅠand XhoⅠ,and then were cloned into pCAT3-Basic.The construc- ted vectors were named as p1-iNOSp,p2-iNOSp,p3-iNOSp and p4-iNOSp,respectively.Each of the vectors containing different iNOSp DNA fragments was transfected into the HepG2 cell line and cotransfected into HepG2 cells with pcDNA3.1(-)-SBP1 by FuGENE 6 transfection reagents.The HepG2 cells transfected with pCAT3-Basic were used as negative control.The activity of chloram- phenicol acetyltransferase(CAT)in transfected HepG2 cells was detected by an enzyme-linked immu- nosorbent assay(ELISA)kit after 48 hours,which would reflect the regulating effect of SBP1 on iNOS gene promoter activity.Results The expression vector pcDNA3.1(-)-SBP1 and report vector pCAT3-iNOS were constructed and confirmed by restriction enzyme digestion and sequencing.The expression of pcDNA3.1(-)-SBP1 in HepG2 cells up-regulated the activity of p1-iNOSp and down- regulated the activity of p3-iNOSp.The inhibiting rate was 31.3%.Conclusions It is suggested that SBP1 can regulate iNOS gene promoter bidirectionally by influencing the binding sites of nuclear factor (NF)-IL6,A activator domain binding site and NF-?B in iNOS gene promoter.