| Chronic hepatitis B virus (HBV) infection is recognized worldwidely as a leading cause of cirrhosis and hepatocellular carcinoma. Its prevalence approaches 10% in such hyperendemic areas as China. Current therapies, including immune modulators such as interferon Alfa, or nucleoside analogs such as lamivudine or adefovir have provided some cures but could not clear HBV entirely.RNA interference is a recently discovered antiviral mechanism present in plants. C. elegans. Drosophila and fungi that is induced by double-stranded RNA (dsRNA) and leads to sequence-specific gene silencing at the post-transcriptional level. Unlike invertebrate cells, most mammalian cells respond to dsRNAs longer than 30 bp by inducing imerferon a and B expression and activating dsRNA-dependent protein kinase (PKR and 2. 5' oligoadenylate synthetase and non-specific degradation of mRNA by RNAs L. Hamessing RNA to study gene function in mammals seemed potentially problematic. Recently, luschl and colleagues have demonstrated that RNAi can be provoked in numerous mammalian cell lines. This process is mediated by 21-23 nucleotides. called by short interfering RNAs (siRNAs), cleaved dsRNA. RNAi exhibitssome advantages such as effectiveness, exquisite sequence specificity, quickness and non-side effects. The use of RNAi in antivirus and gene function is well established in mammals. To date, in vitro studies suggest that in human cells, this progress can more effectively inhibit replications of HIV-1, poliomyelitis virus and influenza virus than antisense and ribozyme technologies.The life cycle of HBV is characterized by the synthesis of a -3 kb partially double-stranded, relaxed-circular DNA (rcDNA) genome by reverse transcription of an RNA intermediate, the pregenome. Since the process of pregenomic RNA and other RN A,s take piace in cellular plasma. RNAs are attacked by siRNA easily.Aim to explore the anti-hepatitis B virus effect of RNA interference (RNAi) using small hairpin RNA (shRNA) expression vector. First as an assay for the siRNAs. we fused HBV S gene (HBs) to enhanced green fluorescent protein (EGFP) to provide a reporter system for monitoring siRNA function. Genes encoding a siRNA targeted to HBs gene were inserted behind the Pol III U6+27 snRNA promoter of pAVU6+27 vectors, To choose target sequences for the siRNAs, we subjected the cells to fluorescence microscopic analyses and fluorescence-activated cell sorting (FACS). Second we study the effect of RNAi in FlepG2.2.15 cells which are derived from HepG2 cells and produce all HBV proteins.Part One: Selection and validation of RNAi target sites!. Construction and identification of HBs-EGFP fusion gene plasmid Full length cDNA fragment of MBsAg was obtained from pcDNA3.1+S. and amplified by Pyrobest DNA Polymerase. The fragment was inserted into a eukaryotic report vector pEGFPN-1. The fusion gene vector, named as. was transformed into H.coli strain DH5a and identified by restricted endonuclease digestion. PCR and DNA sequences anatysis. The results were shown as expected. The fluorescence of GFP in HepG2 ceils which was transfected with pEGFP-S plasmid DNA was detected successfully.2. Construction and identification of different shRNA expression vectorsThe small hairpin DN A (shDNA) sequences targeted to HBV S gene for the siRNAs were determined by using the Ambion web-based criteria and were synthesized. Then the annealed shDNA was inserted into the pAVU6+27 vector. The positive clones were identified by PCR and DNA sequences analysis.3. The changes of HBs-HGFP images detected by FACS and microscopyDifferent shRNA expression vectors were respectively transfected into AD293 cells with pEGFP-HBs. The fluorescence intensity of HBs-EGFP in cells treated shRNA expression vectors trended to decrease and reached the lowest degree at 72 h after transaction. The relative fluorescence intensity (RF1) of HBs-EGFP in cells transfected with pAVU6+4sh239. pAVU6-4sh359. pAVU6+4sh579 and pAVU6+4sh691 DNA reduced by 56.2%. 38.7%. 69.8% and 63.1% compared... |
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