【作者】 李华；

【导师】 余祖江；

【作者基本信息】 郑州大学 ， 内科学， 2010， 硕士

【摘要】 背景HBV感染是一个严重威胁到人类健康的全球性问题,HBV是引起急慢性肝炎、肝硬化和原发性肝细胞肝癌的重要致病因素。抗病毒治疗是目前慢性乙型肝炎的关键性治疗措施,现有的抗病毒药物干扰素-α和核苷类似物的疗效有限,基因治疗日益成为目前的研究热点之一。基因治疗的关键在于选择良好的转基因载体、合适的目的基因和实现目的基因的可控性表达。基因工程中应用最多的载体是真核细胞表达载体,如病毒类载体,它们能将目的基因导入宿主细胞并稳定表达。本实验选择真核表达载体pEGFP-C1,为在细胞水平和体内观察其抑制HBV复制效果打下基础,同时选择HBV core启动子(core promoter, cp)作为目的基因(因为该启动子指导HBV pgRNA和pc mRNA转录的正确启动),构建HBV cp的反义真核表达载体,该载体可以转录出cp的反义RNA,反义RNA与pgRNA中的cp部分结合有可能使pg RNA逆转录为负链DNA受阻,同时可能导致pg RNA翻译蛋白的水平下降,从而使HBV合成和包装下降,最终有可能使HBV的复制长期受到抑制。目的HBV核心区启动子的克隆及其反义真核表达载体的构建,为乙型肝炎的基因治疗探索新的治疗靶点。方法1.提取HBV DNA:选取外周血HBV DNA载量大于106IU/ml的慢性乙型肝炎患者,用体液病毒提取试剂盒提取HBV DNA。2.PCR扩增HBV核心区启动子序列：选取HBV全长序列中nt1636-nt1860作为扩增片段(该片段包含有HBV核心区启动子),用Taq DNA聚合酶进行PCR反应扩增HBV核心区启动子,琼脂糖凝胶电泳鉴定PCR扩增产物,初步确定引物及提取的HBV DNA模板的可实用性；用Pfu DNA聚合酶PCR扩增HBV核心区启动子序列,以增加扩增基因的保真性,扩增产物进行载体构建。3.重组质粒pEGFP-C1-cp的构建及鉴定：将扩增的HBV核心区启动子和真核表达载体pEGFP-C1经限制性内切酶SacⅡ和HindⅢ分别酶切,用T4DNA Ligase连接后得到重组质粒pEGFP-C1-cp。将重组质粒pEGFP-C1-cp转化感受态大肠杆菌(escherichia coli, E.coli)DH5α,选取阳性克隆进行PCR、酶切鉴定,鉴定后测序。结果1.HBV核心区启动子的PCR扩增：用Taq DNA聚合酶和Pfu DNA聚合酶扩增后的PCR产物经琼脂糖凝胶电泳,在225bp处有特异性条带出现。2.重组质粒pEGFP-C1-cp的鉴定：重组质粒pEGFP-C1-cp PCR反应后电泳,结果显示在225bp处有特异性条带出现；重组质粒pEGFP-C1-cp经SacⅡ和HindⅢ酶切后电泳,结果显示在约225bp和4700bp处均有特异性条带出现；重组质粒pEGFP-C1-cp中HBV核心区启动子测序结果与GeneBank公布的序列相一致,同源性为97%。结论本实验成功克隆了HBV核心区启动子序列,并构建了HBV核心区启动子的反义真核表达载体pEGFP-C1-cp。更多还原

【Abstract】 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 SacⅡand HindⅢ.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 SacⅡand HindⅢdigestion 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 SacⅡand HindⅢ.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.更多还原