本文選題：丙型肝炎病毒　切入點：核心蛋白　出處：《上海交通大學(xué)》2009年碩士論文

【摘要】： 目的丙型肝炎病毒(hepatitis C virus,簡稱HCV)感染是當(dāng)前危害人類健康的重要傳染病之一,目前除干擾素聯(lián)合利巴韋林外尚無有效預(yù)防和治療手段。因此,尋找其他治療途徑,預(yù)防和清除HCV的持續(xù)感染很有必要。而將編碼HCV抗原的外源基因?qū)霗C體誘導(dǎo)產(chǎn)生特異性T細(xì)胞免疫反應(yīng)的HCV新型疫苗目前正在受到人們的重視。以重組復(fù)制缺陷型腺病毒(replication-deficient recombinant adenovirus,rAd)為載體的疫苗,能模擬天然病毒有效地將外源基因?qū)胨拗骷?xì)胞內(nèi)并使之高效表達(dá),誘導(dǎo)機體產(chǎn)生針對目的抗原的免疫應(yīng)答。為此,我們利用AdMaxTM腺病毒包裝系統(tǒng)構(gòu)建可表達(dá)HCV核心抗原Core的HCV重組腺病毒pAd-Core,并觀察其免疫小鼠后,誘導(dǎo)機體產(chǎn)生特異性免疫應(yīng)答的能力,為進一步研究防止HCV感染的基因疫苗和基因治療奠定基礎(chǔ)。 方法RT-PCR法從丙型肝炎患者血清中擴增出HCV Core基因,定向克隆到重組腺病毒AdMaxTM系統(tǒng)的穿梭質(zhì)粒pDC315的多克隆位點上,構(gòu)建重組穿梭質(zhì)粒pDC315-Core,在脂質(zhì)體介導(dǎo)下與腺病毒基因組骨架質(zhì)粒pBHGlox(delta)E1,3Cre共轉(zhuǎn)染至HEK293細(xì)胞,通過同源重組包裝產(chǎn)生復(fù)制缺陷型重組腺病毒pAd-Core;經(jīng)HEK293細(xì)胞擴增后,離子交換柱層析法純化病毒,測定病毒顆粒數(shù)和滴度;利用重組腺病毒體外感染人肝癌細(xì)胞株HepG2,經(jīng)免疫印跡(Western Blot)方法檢測目的蛋白的表達(dá),通過酶聯(lián)免疫吸附試驗(enzyme 1inked immunosorbent assay,ELISA)檢測免疫小鼠血清抗體水平,酶聯(lián)免疫斑點技術(shù)(enzyme-linked immunosorbent spot ,ELISPOT)檢測免疫小鼠特異性T淋巴細(xì)胞免疫反應(yīng)。 結(jié)果重組腺病毒pAd-Core經(jīng)PCR及序列測定證實Core基因插入正確,在HEK293細(xì)胞中具有良好的感染性;擴增純化后,重組腺病毒的比活性可達(dá)3.42IU/100VP,單細(xì)胞產(chǎn)量可達(dá)2.63×104VP/cell;Western Blot檢測結(jié)果表明pAd-Core在HepG2細(xì)胞可穩(wěn)定表達(dá)目的蛋白,免疫小鼠后可刺激機體產(chǎn)生特異性體液免疫應(yīng)答和細(xì)胞免疫應(yīng)答. 結(jié)論成功構(gòu)建了表達(dá)HCV核心抗原Core的HCV重組腺病毒pAd-Core,該重組腺病毒可在人肝癌細(xì)胞株HepG2細(xì)胞內(nèi)穩(wěn)定表達(dá)目的蛋白,并且能夠誘導(dǎo)機體產(chǎn)生有效的體液免疫應(yīng)答和細(xì)胞免疫應(yīng)答,具有成為丙肝疫苗的發(fā)展?jié)摿Α?br/>[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.
【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R512.63;R392

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