Inhibition of hepatitis B virus replication in 2.2.15 cells by expressed shRNA.
Hepatitis B virus (HBV) infection is a worldwide health problem. To determine whether RNA interference (RNAi) could inhibit ongoing HBV replication in 2.2.15 cells, we constructed shRNA-producing vector pU6P based on the mouse U6 RNA promoter and cloned 12 targeted sequences against HBV into the vector, resulting in a series of pU6-siHBV vectors. The recombinant vectors were transfected into 2.2.15 cells, HBsAg and HBeAg in cultured media were assayed using enzyme-linked immunosorbent assay at various days after transfection. The amount of HBV DNA in the culture medium was quantitated by real-time polymerase chain reaction. HBsAg and HBeAg expression were inhibited by 72.8 +/- 5.4% (P = 0.00003) and 55.8 +/- 6.2% (P = 0.000026), respectively, 4 days after transfection with pU6-siHBV5. The greatest inhibition of HBV DNA was decreased by approximately 1.9-fold (P = 0.013) on day 6 post transfection with pU6-siHBV11 compared with that of empty vector. No change was found for HBV protein expression and DNA replication on pU6-siGFP (negative control) transfected cells. Our data demonstrate that the transfection of HBV-targeted shRNA-producing vector in 2.2.15 cells could inhibit the HBV protein expression and HBV DNA replication specifically. RNAi may be considered as a potential antiviral approach for human HBV infection.
Ren
X-R
XR
State Key Laboratory of Biocontrol, Department of Biochemistry, College of Life Sciences, Sun Yat-sen (Zhongshan) University, Guangzhou 510275, China.
Zhou
L-J
LJ
Luo
G-B
GB
Lin
B
B
Xu
A
A
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D004261:DNA Replication; D004279:DNA, Viral / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D015967:Gene Expression Regulation, Viral; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors / Q000494:pharmacology*; D006515:Hepatitis B virus / Q000235:genetics* / Q000502:physiology; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D014162:Transfection; D014764:Viral Proteins / Q000378:metabolism; D014779:Virus Replication
D004279:DNA, Viral; D014764:Viral Proteins