[Inhibition of HBsAg and HBeAg secretion by RNA interference of the polymerase gene sequence of hepatitis B virus: an experimental study].
OBJECTIVE
To investigate the effects of small interfering RNA (siRNA) targeting the polymerase (P) gene sequence of hepatitis B virus (HBV) on the replication and antigen secretion of HBV.


METHODS
From the 29 base sequences of the HBV in the HepG2.2.15 cells that accord with the demands of siRNA designing five sequences targeting the P gene of HBV were selected and cloned into the siRNA expressing vector pGE-1. Then the plasmid pGE-HBVP was transfected into the cultured HepG2.2.15 cells. Chemiluminescent immunoassay was used to determine the levels of HBsAg and HBeAg in the supernatant of culture medium 24, 48, 72, and 96 hours after the transfection and the expression of HBsAg in the 2.2.15 cells 24 hours after the transfection so as to observe the inhibitory effects. Untransfected cells and cells transfected with blank pGE-1 vector were used as controls.


RESULTS
Five vectors expressing the siRNAs targeting the HBV P region, pGE-HBVP1-pGE-HBV5 were successfully constructed. The efficiency of transfection of the vectors into the 2.2.15 cells were 30% to 40%. 24, 48, 72, and 96 hours after the transfection of pGE-HBVP2, the strongest inhibitor among the five, the inhibitory rates of HBsAg secretion in the supernatant were 28.88%, 32.28%, 29.10%, and 18.42% respectively; and the inhibitory rates of HBeAg secretion were 38.33%, 27.50%, 33.41%, and 12.60% respectively. In view of the transfection efficiency of 30%-40%, the actual inhibitory rate of HBV antigen secretion might reach 80% and over. 24 hours after the transfection the expression rate of HBsAg in the 2.2.15 cells transfected with pGE-HBVP2 was 50%, significantly lower than that in the cells transfected with the blank vector pGE-1 (82%).


CONCLUSIONS
siRNAs targeting the HBV P gene effectively prevent the HBV gene expression and replication and may play an important role in the clinical anti-viral treatment.
Liu
Shun-ai
SA
Beijing Ditan Hospital, Beijing 100011, China.
Wei
Hong-shan
HS
Dong
Qing-ming
QM
Guo
Jing-jing
JJ
Qin
Jing
J
Zhang
Qian-ying
QY
Liu
Zhi-ying
ZY
Yan
Jie
J
Cheng
Jun
J
D003086:Bacteriocin Plasmids / Q000235:genetics; D045744:Cell Line, Tumor; D015684:Gene Products, pol / Q000235:genetics / Q000378:metabolism*; D006514:Hepatitis B Surface Antigens / Q000378:metabolism*; D006513:Hepatitis B e Antigens / Q000378:metabolism*; D006515:Hepatitis B virus / Q000201:enzymology / Q000235:genetics / Q000276:immunology; D006801:Humans; D007118:Immunoassay; D034622:RNA Interference; D014162:Transfection
D015684:Gene Products, pol; D006514:Hepatitis B Surface Antigens; D006513:Hepatitis B e Antigens; C066027:P protein, Hepatitis B virus