Inhibition of hepatitis B virus surface antigen expression by small hairpin RNA in vitro.
OBJECTIVE
To explore the anti-hepatitis B virus effect of RNA interference (RNAi) using small hairpin RNA (shRNA) expression vector.


METHODS
Hepatitis B virus surface antigen green fluorescent protein (HBs-GFP) fusion vector and shRNA expression vectors were constructed and cotransfected transiently into HepG2 cells. mRNAs extracted from HepG2 cells were detected by real-time PCR. Fluorescence of HBs-GFP protein was detected by fluorescence-activated cell sorting (FACS). The effective shRNA expression vector was transfected into HepG2.2.15 cells. HBsAg and HBeAg in HepG2.2.15 cells were analyzed by radioimmunoassay (RIA) method.


RESULTS
FACS revealed that shRNA targeting at HBsAg reduced the GFP signal by 56% compared to the control. Real-time PCR showed that HBs-GFP mRNA extracted from HepG2 cells cotransfected with pAVU6+27 and HBs-GFP expression plasmids decreased by 90% compared to the empty vector control. The expressions of HBsAg and HBeAg were also inhibited by 43% and 64%, respectively.


CONCLUSIONS
RNAi using shRNA expression vector can inhibit the expression of HBsAg, providing a fresh approach to screening the efficient small interfering RNAs (siRNAs).
Yang
Zheng-Gang
ZG
Institute of Infectious Diseases, First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, Zhejiang Province, China.
Chen
Zhi
Z
Ni
Qin
Q
Xu
Ning
N
Shao
Jun-Bin
JB
Yao
Hang-Ping
HP
D006528:Carcinoma, Hepatocellular; D045744:Cell Line, Tumor; D015967:Gene Expression Regulation, Viral; D049452:Green Fluorescent Proteins / Q000235:genetics; D006514:Hepatitis B Surface Antigens / Q000235:genetics*; D006513:Hepatitis B e Antigens / Q000235:genetics; D006515:Hepatitis B virus / Q000235:genetics*; D006801:Humans; D066298:In Vitro Techniques; D008113:Liver Neoplasms; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D014162:Transfection
D006514:Hepatitis B Surface Antigens; D006513:Hepatitis B e Antigens; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D049452:Green Fluorescent Proteins