Construction Of Eucaryotic Expression Vector Of The Antisense Gene To Core Promotor Of HBV

BackgroundHepatitis B virus(HBV) infection remains a worldwide public healthy problem.HBV is the important etiological factor for acute and chronic hepatitis,liver cirrhosis and hepatocellular carcinoma. antiviral therapy is an important method for hepatitis B.Currently Antiviral drugs for HBV include interferon-alpha and nucleotide analogs and their therapeutic effects are limitid.Therefore,more and more researches focus on the development of gene therapy.The keys of gene therapy are selecting transgenic vector,proper gene segment and the self-modulating expression of objective gene.Eucaryotic expression vector are used widely in genetic engineering, such as virus vectors.These vectors can carry gene segment into cells and make them express stably.We select pEGFP-C1 as the tools for transforming gene segment.This vector is hepfull for observing the effect of supressing HBV replication in cells and in vivo.Targeting gene is also important for gene therapy.We select core promotor of HBV as the targeting gene segment,because this promotor direct to transcribe for pregenome RNA and pc mRNA.We construct the antisense gene of core promotor of HBV genome and this vector may transcribe the antisense RNA of core promotor. This antisense RNA binding to core promtor segment in pregenome RNA may prevent reverse transcription and decrease the level of pregenome RNA translation. These may affect replication and package of HBV and may supress HBV replication long term.ObjectiveCloning core promotor of HBV genome and construction of eucaryotic expression vector of the antisense gene of core promotor to probe new target of gene therapy for chronic hepatitis B.Methods1. Extraction HBV genome:select chronic hepatitis B patients whose virus load in blood are more than 106IU/ml and extract HBV genome in serum with virus extraction kit.2. Amplification core promotor of HBV genome by PCR:Select nucleotides from 1636 to 1860 in HBV genome as targeting gene (core promotor is included in this selected region).Core promotor of HBV genome amplified by polymerase chain reaction (PCR) with Taq DNA polymerase and the amplification production identified by agarose gel electrophoresis to assure primers and templates available.Then amplify targeting gene with Pfu DNA polymerase to increase fidelity of targeting gene.and to construct eucaryotic expression vector.3. Construction and identification of the pEGFP-C1-cp recombinant vector:Both core promotor and pEGFP-C1 eucaryotic expression vector is digested by restriction endonuclease Sacâ…¡and Hindâ…¢.Ligate them with T4DNA ligase to get recombinant vector pEGFP-Cl-cp and transform competent E.coli DH5a.Afer that,we screen positive clones to identify them by PCR,restriction endonuclease Sacâ…¡and Hindâ…¢digestion and sequencing.Results1. Amplifying core promotor of HBV genome by PCR:Amplification production with Taq DNA polymerase and Pfu DNA polymerase are identified by agarose gel electropherisis.The results show a specific limpid strap at 225bp respectively.2. Identification of the pEGFP-C1-cp recombinant plasmid:The pEGFP-C1-cp recombinant plasmid is identifid by colony PCR and amplification production is identifid by agarose gel electropherisis.The results show a specific limpid strap at 225 bp.The pEGFP-C1-cp recombinant plasmid is digested by restriction endonuclease Sacâ…¡and Hindâ…¢.The products are identified by agarose gel electropherisis and the result show that there is a specific limpid strap at 225bp and 4700bp respectively.The positive recombinant plasmid is chosen for sequencing.The result is consistent with the published sequence in GeneBank and the homology is up to 97%.Conclusion:The core promotor of HBV genome is cloned successfully.The eucaryotic expression vector of the antisense gene of core promotor of HBV is constructed successfully.