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ISSN
1672-9145
Acta Biochim Biophys Sin
2005, 37(5): 310–316
CN 31-1940/Q
Establishment of a Screening System for Selection of siRNA Target
Sites Directed against Hepatitis B Virus Surface Gene
Xiu-Min ZHOU1,3, Ju-Sheng LIN1, Yi SHI3,
De-An TIAN2, Huan-Jun HUANG2, He-Jun ZHOU1,
and You-Xin JIN3*
1Institute of Liver Diseases, Tongji Hospital, Tongji Medical
College, Huazhong University of Science and Technology, Wuhan 430030, China;
2Department of Gastroenterology, Tongji Hospital, Tongji Medical
College, Huazhong University of Science and Technology, Wuhan 430030, China;
3State Key Laboratory of Molecular Biology, Institute of Biochemistry
and Cell Biology, Shanghai Institutes for Biological Sciences,
Chinese Academy of Sciences, Shanghai 200031, China
Abstract To study the inhibitory effects of plasmid-derived small interfering
RNA (siRNA) and synthetic siRNA on the expression of the hepatitis B virus
surface (HBs) gene, three plasmid-derived siRNAs and one synthetic siRNA
that complement the coding region of the HBs gene were prepared. The HBs
expression plasmid pHBs-EGFP was also constructed. HeLa cells were
co-transfected with pHBs-EGFP and the above siRNAs. The HBs mRNA quantities
were measured by reverse-transcription PCR, and the level of HBs-EGFP fusion
protein was quantified by fluorescent microscope. The concentrations of the
hepatitis B virus surface antigen (HBsAg) derived from the culture supernatant
of transfected HepG2.2.15 cells were measured by an enzyme-linked immunosorbent
assay (ELISA) kit. The results showed that the three plasmid-derived siRNAs and
the synthetic siRNA can effectively reduce the quantities of HBs mRNA and
protein. The plasmid-derived siRNA psiRNA1 was found to be the most effective
inhibitor of HBs expression. It can inhibit HBs-EGFP expression by 63.3% and
suppress HBs mRNA by 75.6%. To further substantiate the above observations,
psiRNA1 was transfected into HepG2.2.15 cells (an HBV secreting cell line). The
transfections resulted in almost complete blockage of HBsAg production, whereas
control vector-transfected cells secreted high levels of HBsAg 7 days
post-transfection. In conclusion, our data suggests that RNA interference
(RNAi) is an efficient approach for reducing the level of HBs transcripts and
proteins and for suppressing HBsAg production.
Key words RNA interference (RNAi); small interference RNA (siRNA); hepatitis B
virus surface gene (HBs gene)
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Received: October 27, 2004 Accepted: March 3, 2005
This work was supported by the grants from the National Natural
Science Foundation of China (No. 30270311, No. 30330680)
*Corresponding author: Tel, 86-21-54921222; Fax, 86-21-54921011;
E-mail, [email protected]