HCV重组腺病毒的构建、鉴定及初步免疫学效果观察

发布时间：2018-04-05 02:28

本文选题：丙型肝炎病毒　切入点：核心蛋白　出处：《上海交通大学》2009年硕士论文

【摘要】： 目的丙型肝炎病毒(hepatitis C virus,简称HCV)感染是当前危害人类健康的重要传染病之一,目前除干扰素联合利巴韦林外尚无有效预防和治疗手段。因此,寻找其他治疗途径,预防和清除HCV的持续感染很有必要。而将编码HCV抗原的外源基因导入机体诱导产生特异性T细胞免疫反应的HCV新型疫苗目前正在受到人们的重视。以重组复制缺陷型腺病毒(replication-deficient recombinant adenovirus,rAd)为载体的疫苗,能模拟天然病毒有效地将外源基因导入宿主细胞内并使之高效表达,诱导机体产生针对目的抗原的免疫应答。为此,我们利用AdMaxTM腺病毒包装系统构建可表达HCV核心抗原Core的HCV重组腺病毒pAd-Core,并观察其免疫小鼠后,诱导机体产生特异性免疫应答的能力,为进一步研究防止HCV感染的基因疫苗和基因治疗奠定基础。方法RT-PCR法从丙型肝炎患者血清中扩增出HCV Core基因,定向克隆到重组腺病毒AdMaxTM系统的穿梭质粒pDC315的多克隆位点上,构建重组穿梭质粒pDC315-Core,在脂质体介导下与腺病毒基因组骨架质粒pBHGlox(delta)E1,3Cre共转染至HEK293细胞,通过同源重组包装产生复制缺陷型重组腺病毒pAd-Core;经HEK293细胞扩增后,离子交换柱层析法纯化病毒,测定病毒颗粒数和滴度;利用重组腺病毒体外感染人肝癌细胞株HepG2,经免疫印迹(Western Blot)方法检测目的蛋白的表达,通过酶联免疫吸附试验(enzyme 1inked immunosorbent assay,ELISA)检测免疫小鼠血清抗体水平,酶联免疫斑点技术(enzyme-linked immunosorbent spot ,ELISPOT)检测免疫小鼠特异性T淋巴细胞免疫反应。结果重组腺病毒pAd-Core经PCR及序列测定证实Core基因插入正确,在HEK293细胞中具有良好的感染性;扩增纯化后,重组腺病毒的比活性可达3.42IU/100VP,单细胞产量可达2.63×104VP/cell;Western Blot检测结果表明pAd-Core在HepG2细胞可稳定表达目的蛋白,免疫小鼠后可刺激机体产生特异性体液免疫应答和细胞免疫应答. 结论成功构建了表达HCV核心抗原Core的HCV重组腺病毒pAd-Core,该重组腺病毒可在人肝癌细胞株HepG2细胞内稳定表达目的蛋白,并且能够诱导机体产生有效的体液免疫应答和细胞免疫应答,具有成为丙肝疫苗的发展潜力。
[Abstract]:Objective Hepatitis C virus (HCV) infection is one of the most important infectious diseases that endanger human health. There is no effective prevention and treatment except interferon and ribavirin.Therefore, it is necessary to find other therapeutic ways to prevent and eliminate persistent infection of HCV.However, the new HCV vaccine which induces specific T cell immune response by introducing foreign gene encoding HCV antigen into the body is being paid more and more attention.The recombinant replication-deficient recombinant adenovirusrAd-based vaccine can effectively transfer foreign genes into host cells and express them efficiently, and induce immune response to the target antigen.Therefore, we used AdMaxTM adenovirus packaging system to construct the recombinant adenovirus pAd-Core, which can express HCV core antigen Core, and to observe the ability of inducing specific immune response after immunizing mice with the recombinant adenovirus pAd-Core.It lays a foundation for further research on gene vaccine and gene therapy to prevent HCV infection.Methods the HCV Core gene was amplified by RT-PCR from the sera of patients with hepatitis C and cloned into the shuttle plasmid pDC315 of the recombinant adenovirus AdMaxTM system.The recombinant shuttle plasmid pDC315-Corewas constructed and co-transfected into HEK293 cells with adenovirus genomic skeleton plasmid pBHGloxx delta-E1O3Cre mediated by liposome. The replication-deficient recombinant adenovirus pAd-Corewas produced by homologous recombination packaging, and the recombinant adenovirus pAd-Corewas amplified by HEK293 cells.The virus was purified by ion exchange column chromatography, the number and titer of virus particles were determined, the recombinant adenovirus was used to infect human hepatoma cell line HepG2 in vitro, and the expression of the target protein was detected by Western blot.The serum antibody level of immunized mice was detected by enzyme 1inked immunosorbent assaysa, and the specific T lymphocyte immunoreactivity of immunized mice was detected by enzyme-linked immunosorbent spot (Elisa) with enzyme linked immunosorbent assay (Elisa).Results PCR and sequencing of the recombinant adenovirus pAd-Core confirmed that the Core gene was inserted correctly and had good infectivity in HEK293 cells.The specific activity of recombinant adenovirus was 3.42 IUR / 100VPand the single cell yield was 2.63 脳 104VP / cell Blot. The results showed that pAd-Core could stably express the target protein in HepG2 cells. After immunizing mice, specific humoral and cellular immune responses were produced.Conclusion the recombinant adenovirus pAd-Core of HCV expressing HCV core antigen Core was successfully constructed. The recombinant adenovirus could stably express the target protein in HepG2 cells and induce effective humoral and cellular immune responses.It has the potential to become hepatitis C vaccine.
【学位授予单位】：上海交通大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R512.63;R392

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