Lentiviral vector-mediated RNA interference of HBs inhibits replication of HBV
10.3724/SP.J.1008.2009.00295
- Author:
Xiang-Ji LUO
1
Author Information
1. No. 1 Department of Billiary Surgery
- Publication Type:Journal Article
- Keywords:
Hepatitis B surface antigens;
Hepatitis B virus;
Lentivirus;
RNA interference
- From:
Academic Journal of Second Military Medical University
2010;30(3):295-299
- CountryChina
- Language:Chinese
-
Abstract:
Objective: To construct a lentiviral vector (LV) of RNA interference (RNAi) targeting HBs gene and to observe its effect on the replication of HBV and expression of antigens. Methods: The effective sequence of siRNA targeting HBs gene was confirmed in our previous study. The complementary DNA containing both sense and antisense Oligo DNA of the targeting sequence was designed, synthesized and cloned into the lentivirus vector (pGCLM-GFP). The resulting lentivirus vector containing HBs shRNA was named LVshHBs, and it was confirmed by PCR and sequencing. 293T cells were cotransfected with lentivirus vector pGCLM-GFP, pHelper 1.0 and pHelper 2.0. All virus stocks were produced by calcium phosphate-mediated transfection. The titer of virus was tested according to the expression level of GFP. HepG2. 2.15 cells were infected with LVshHBs and the supernatant of the cells was subjected to ELISA, Western blotting analysis and HBV DNA quantitative analysis. Results: PCR and DNA sequencing demonstrated that the LVshHBs-producing HBs shRNA was successfully constructed. The titer of concentrated virus was 5 × 108-2 × 109 TU/ml. The inhibitory effect was efficient and the corresponding viral transcript and expression of antigens were decreased after infection. The inhibitory effect was observed 4 days after infection and peaked 9 days after the initial treatment with RNAi. Secreted HBsAg was reduced by > 70% in cell culture compared with the negative control, which is also confirmed by Western blotting and real time PCR. After quantification of HBV DNA, the level of DNA relative to the controls was also significantly reduced after RNAi treatment(P<0.05). Conclusion: The lentivirus RNAi vector of HBs has been successfully constructed. The lentiviral microRNA-based RNAi targeting HBs can specifically mediate the down regulation of HBs expression, inhibiting HBV replication and antigen expression.
