[A study of the down-regulation effect of hepatitis B virus pre-protein S2 on inducible nitric oxide synthase gene promoter].
OBJECTIVE
To investigate the regulating effect of HBV pre-S2 protein on iNOS gene promoter and the molecular biological mechanisms of pre-S2 protein in HBV pathogenicity.


METHODS
Polymerase chain reaction (PCR) technique was employed to amplify the sequence of iNOS promoter and 3 deletion mutants using HepG2 genomic DNA as the template, and the products were cloned into the pGEM-T vector. The iNOS gene and 3 deletion mutants were cut from T- iNOS by Kpn I and Xho I, and then cloned into pCAT3-Basic. The resulting vectors were named p1-iNOSp, p2-iNOSp, p3-iNOSp, and p4-iNOSp. Each of the reporter vectors was transfected into the HepG2 cell line and cotransfected into HepG2 cells with pcDNA3.1(-)-pre-S2 by FuGENE 6 transfection reagents. The HepG2 cells transfected with pCAT3-Basic were used as a negative control. The activity of CAT in HepG2 cells transfected was detected by an ELISA kit 48 hours after the transfection, which reflected the regulating effect of HBV pre-S2 protein on iNOS gene promoter activity.


RESULTS
The expressive vector pcDNA3.1(-)-pre-S2 and report vector pCAT3-iNOSp were constructed and confirmed by restriction enzyme digestion and sequencing. The expression of pcDNA3.1(-)-pre-S2 in HepG2 cells could down-regulate the activity of p1-iNOSp, p3-iNOSp, and the inhibition rate was 54.7% and 79.5%, respectively. The expression of pcDNA3.1(-)-pre-S2 in HepG2 cells had no regulatory effects on p2-iNOSp and p4-iNOSp.


CONCLUSIONS
It is suggested that HBV pre-S2 protein can down-regulate iNOS gene promoter.
Guo
Feng-jin
FJ
Institute of Infectious Diseases, Ditan Hospital, Beijing 100011, China.
Cheng
Jun
J
Ji
Dong
D
Liu
Yan
Y
Zhang
Li-ying
LY
Song
Fang-zhou
FZ
D015536:Down-Regulation; D006514:Hepatitis B Surface Antigens / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D052247:Nitric Oxide Synthase Type II / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011498:Protein Precursors / Q000096:biosynthesis* / Q000235:genetics; D015533:Transcriptional Activation; D014162:Transfection
D006514:Hepatitis B Surface Antigens; D011498:Protein Precursors; C050807:presurface protein 2, hepatitis B surface antigen; D052247:Nitric Oxide Synthase Type II