可表达新型甲型H1N1流感病毒血凝素蛋白辅助病毒依赖型腺病毒载体的构建及初步鉴定

发布时间：2017-12-31 02:23

本文关键词：可表达新型甲型H1N1流感病毒血凝素蛋白辅助病毒依赖型腺病毒载体的构建及初步鉴定　出处：《北京交通大学》2012年硕士论文　论文类型：学位论文

【摘要】：目的：新型甲型H1N1流感病毒是威胁人类健康的重要新发传染病,虽然已有可用于该病毒预防和治疗的疫苗及药物,但现有的神经氨酸酶抑制剂类抗病毒药物和以传统方法制备的流感病毒裂解疫苗均存在一定的局限性。近期研究表明,以重组腺病毒制备流感疫苗具有明显的优越性。本研究以全基因合成的方法获得甲型H1N1流感病毒血凝素(Hemagglutinin, HA)基因,并以辅助病毒依赖型腺病毒(Helper-dependent adenoviral vector, HDAd)载体系统为基础,构建可表达HA的重组HDAd,为进一步的体内免疫效果和免疫保护作用研究奠定基础。方法：根据GenBank上登录的甲流H1N1流感病毒美国加利福尼亚毒株(A/California/07/2009(H1N1))基因序列,采用全基因合成的方法合成由1701个碱基组成的HA编码基因。经核酸序列分析证实后,将HA基因克隆至HDAd穿梭载体pSC11-CMV,经限制性内切酶分析后,进一步将含有HA基因的表达盒克隆至HDAd骨架质粒pSC15B,构建pSC15B/HA重组质粒,并以多组限制性内切酶进行分析鉴定。Pme I消化pSC15B/HA去除原核复制元件及抗性基因,获得HDAd/HA DNA分子,经磷酸钙共沉淀法转染293Cre4细胞,16h后感染辅助病毒,收获HDAd/HA载体粗提液,随后以HDAd/HA粗提液及辅助病毒连续共感染293Cre4细胞直至HDAd/HA达到复制极限。随后,以HDAd/HA和辅助病毒感染293Cre4细胞大量制备HDAd/HA, CsCl密度梯度及等密度梯度两次超速离心纯化,利用透射电子显微镜观察病毒形态,分子生物学技术体外鉴定HDAd/HA表达情况。结果：成功构建了可表达甲流H1N1流感病毒HA蛋白的HDAd/HA载体,通过与辅助病毒共感染293Cre4细胞及CsCl梯度法超速离心制备了大量纯化的HDAd/HA载体,透射电子显微镜观察到腺病毒,体外感染293细胞,RT-PCR检测到HA基因有转录结论：应用HDAd系统,成功构建了HDAd/HA重组腺病毒,并完成了该病毒的大量制备和纯化,为体内免疫学效力试验奠定了初步基础。
[Abstract]:Objective: the new influenza A (H1N1) virus is an important emerging infectious disease threatening human health, although there are vaccines and drugs available for the prevention and treatment of the virus. However the existing neuraminidase inhibitors antiviral drugs and traditional methods of influenza virus lytic vaccine have some limitations. The preparation of influenza vaccine by recombinant adenovirus has obvious superiority. In this study, the hemagglutinin (HAN) gene of influenza A (H1N1) virus was obtained by the method of full gene synthesis. It is based on Helper-dependent adenoviral vector system of adjuvant virus-dependent adenovirus. The construction of recombinant HDAds expressing HA could lay a foundation for further study on immunological effects and immunoprotective effects in vivo. Methods: according to the gene sequence of A / A / 7 / 07 / 2009 H1 / N _ (1) of the H1N1 influenza virus A / A / 7 / 2009 which was registered on GenBank. The HA encoding gene was synthesized by whole gene synthesis. The HA gene was confirmed by nucleic acid sequence analysis and cloned into HDAd shuttle vector pSC11-CMV. After restriction endonuclease analysis, the expression cassette containing HA gene was further cloned into HDAd skeleton plasmid pSC15B to construct pSC15B/HA recombinant plasmid. The pSC15B/HA was digested by multiple restriction endonuclease to remove the prokaryotic replication elements and resistant genes, and the HDAd/HA DNA molecule was obtained. The 293Cre4 cells were transfected with calcium phosphate coprecipitation for 16 hours and then infected with covirus. The crude extract of HDAd/HA vector was harvested. Subsequently, 293Cre4 cells were co-infected with HDAd/HA extract and auxiliary virus until HDAd/HA reached the replication limit. HDAd- / HA, CsCl density gradient and isodensity gradient were purified from 293Cre4 cells infected with HDAd/HA and covirus respectively. The morphology of the virus was observed by transmission electron microscope (TEM) and the expression of HDAd/HA was identified by molecular biology in vitro. Results: the HDAd/HA vector expressing HA protein of H1N1 influenza virus was successfully constructed. A large number of purified HDAd/HA vectors were prepared by co-infection with auxiliary virus 293Cre4 and CsCl gradient centrifugation. The adenovirus was observed by transmission electron microscope. Transcription of HA gene detected by RT-PCR in 293 cells infected in vitro Conclusion: the recombinant adenovirus of HDAd/HA was successfully constructed by using HDAd system, and the preparation and purification of the recombinant adenovirus were completed, which laid a preliminary foundation for the in vivo immunological potency test.
【学位授予单位】：北京交通大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R373

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