HIV-1病毒样颗粒疫苗的构建及其免疫原性研究
发布时间：2018-08-10 16:44
【摘要】：一个成功的HIV-1 疫苗应该既能产生有效的中和抗体又能产生持久的细胞免疫,同时又要具有良好的安全性。传统的HIV-1 疫苗总是存在着这样或那样的欠缺,而VLP 疫苗以其安全性高、抗原性强等优点为HIV-1 疫苗的研究提供了一个新的思路。 本论文利用哺乳动物细胞系统构建HIV-1 病毒样颗粒(VLP)疫苗。该疫苗表达HIV-1 的主要结构蛋白gagpol 和env,并研究其免疫原性,为其成为新一代艾滋病的治疗性疫苗提供重要依据。 主要采用共转染及G418 抗性筛选的方法筛选单克隆细胞株,通过Western-blot 检测蛋白表达情况;经蔗糖垫密度梯度离心检测上清中的VLP 的分泌情况;SDS-PAGE 电泳及LOWRY 法初步确定分泌浓度;透射电镜观察VLP 的形态;免疫印迹及ELISA、CTL 及ELISPOT、细胞增殖等实验检测其动物体液及细胞免疫情况。 结果成功筛选出了高效、稳定表达HIV-1 结构蛋白gagpol 和env 的哺乳动物细胞株,该细胞株表达的结构蛋白可自我组装成病毒样颗粒(VLP)并分泌至培养上清中,透射电镜观察可见典型病毒样颗粒,该VLP(不加任何佐剂)在动物实验中表现了很好的免疫原性。在实验进程中分三级冻存了细胞种子库,初步确定了疫苗的质量控制标准,为今后的大规模疫苗生产打下了坚实的基础。 上述结果表明HIV-1 结构蛋白gagpol 和env 能够在哺乳动物细胞中高效、稳定表达,并能自我组装成VLP 分泌至上清中,动物实验证明了该HIV-1VLP 具有良好的免疫原性,可望成为新一代HIV-1 疫苗。
[Abstract]:A successful HIV-1 vaccine should produce both an effective neutralizing antibody and a sustained cellular immunity with good safety. The traditional HIV-1 vaccine is always lacking in one way or another, but the VLP vaccine provides a new idea for the research of HIV-1 vaccine because of its high safety and strong antigenicity. In this paper, HIV-1 virus-like granulocyte (VLP) vaccine was constructed by mammalian cell system. This vaccine expresses the main structural proteins of HIV-1, gagpol and env. and studies its immunogenicity, which provides an important basis for the new generation of AIDS therapeutic vaccine. The monoclonal cell lines were screened by co-transfection and G418 resistance screening, the protein expression was detected by Western-blot, the secretion of VLP in the supernatant was detected by density gradient centrifugation with sucrose pad, and the secretory concentration was determined by SDS-PAGE and LOWRY method. The morphology of VLP was observed by transmission electron microscope, and the humoral and cellular immunity was detected by immunoblotting, ELISAL, Elispot, cell proliferation and so on. Results mammalian cell lines with high efficiency and stable expression of HIV-1 structural proteins gagpol and env were successfully screened. The expressed structural proteins could self-assemble into virus-like particles (VLP) and secreted into the culture supernatant. The typical virus-like particles were observed by transmission electron microscope (TEM). The VLP (without any adjuvant) showed good immunogenicity in animal experiments. In the course of experiment, the cell seed bank was frozen in three stages, and the quality control standard of vaccine was preliminarily determined, which laid a solid foundation for the large-scale vaccine production in the future. These results suggest that HIV-1 structural proteins gagpol and env can be expressed efficiently and stably in mammalian cells and can self-assemble into VLP secreting supernatant. Animal experiments have proved that the HIV-1VLP has good immunogenicity and is expected to be a new HIV-1 vaccine.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392
本文编号：2175548
[Abstract]:A successful HIV-1 vaccine should produce both an effective neutralizing antibody and a sustained cellular immunity with good safety. The traditional HIV-1 vaccine is always lacking in one way or another, but the VLP vaccine provides a new idea for the research of HIV-1 vaccine because of its high safety and strong antigenicity. In this paper, HIV-1 virus-like granulocyte (VLP) vaccine was constructed by mammalian cell system. This vaccine expresses the main structural proteins of HIV-1, gagpol and env. and studies its immunogenicity, which provides an important basis for the new generation of AIDS therapeutic vaccine. The monoclonal cell lines were screened by co-transfection and G418 resistance screening, the protein expression was detected by Western-blot, the secretion of VLP in the supernatant was detected by density gradient centrifugation with sucrose pad, and the secretory concentration was determined by SDS-PAGE and LOWRY method. The morphology of VLP was observed by transmission electron microscope, and the humoral and cellular immunity was detected by immunoblotting, ELISAL, Elispot, cell proliferation and so on. Results mammalian cell lines with high efficiency and stable expression of HIV-1 structural proteins gagpol and env were successfully screened. The expressed structural proteins could self-assemble into virus-like particles (VLP) and secreted into the culture supernatant. The typical virus-like particles were observed by transmission electron microscope (TEM). The VLP (without any adjuvant) showed good immunogenicity in animal experiments. In the course of experiment, the cell seed bank was frozen in three stages, and the quality control standard of vaccine was preliminarily determined, which laid a solid foundation for the large-scale vaccine production in the future. These results suggest that HIV-1 structural proteins gagpol and env can be expressed efficiently and stably in mammalian cells and can self-assemble into VLP secreting supernatant. Animal experiments have proved that the HIV-1VLP has good immunogenicity and is expected to be a new HIV-1 vaccine.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392
【引证文献】
相关硕士学位论文 前1条
1 吴芬芳;新城疫病毒样颗粒免疫效力的初步研究[D];新疆农业大学;2007年
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