HIV-1病毒样颗粒疫苗临床前期的实验研究

发布时间：2018-02-03 19:44

本文关键词： HIV-1疫苗 DNA疫苗病毒样颗粒疫苗 CTL:细胞毒性T细胞 ELISPOT:酶联免疫斑点法中和抗体　出处：《吉林大学》2007年博士论文　论文类型：学位论文

【摘要】： 自1981年发现第一例病例以来,艾滋病(AIDS)一直以惊人的速度在全球蔓延,成为世界上危害人类健康最严重的病毒性疾病,迫切需要研制安全、有效、价廉的艾滋病疫苗预防艾滋病病毒(HIV)的传播,或降低HIV病毒载量,延缓HIV感染者发病。艾滋病疫苗必须通过科学设计的临床试验,考察其安全性、免疫原性及保护性。随着人类对艾滋病认识的不断提高,越来越多的证据显示阳性CD8介导的CTL在控制艾滋病毒感染中起举足轻重的作用,有效地诱导针对HIV-Gag等较保守区域的CTL是设计有效的艾滋病疫苗重要手段。另一方面,诱导产生有效的中和抗体是一个疫苗成功与否的重要指标,也是十几年来艾滋病疫苗研究的主攻方向之一,这是因为中和抗体的产生在阻止病毒感染过程中起着关键的作用。本研究首次利用两种质粒共转染的方法,成功构建并筛选出了高效、持续表达HIV-1结构蛋白Gagpol和Env的哺乳动物细胞株,监测了细胞培养基中病毒样颗粒(virus like particles,VLPs)的分泌情况,分离纯化后确立了成品的质量控制标准,检测了VLPs诱导小鼠机体产生细胞免疫的能力和中和抗体的水平,同时完成了HIV-1病毒样颗粒疫苗初步的安全性评价。结果表明:①获得的高效表达HIV-1结构蛋白Gagpol和Env的重组293细胞系能够持续分泌两种目的蛋白;②分泌在培养上清中的目的产物经分离纯化后,透射电镜观察其能够组装成病毒样颗粒(VLP);③该病毒样颗粒能够诱导机体产生良好的体液免疫和细胞免疫水平;④安全性评价表明,HIV-1病毒样颗粒疫苗无明显的毒副作用。
[Abstract]:Since the first case was discovered in 1981, HIV / AIDS AIDShas been spreading at an alarming rate all over the world, and has become the most serious viral disease in the world, which urgently needs to be developed safely. Effective and inexpensive AIDS vaccine to prevent the spread of HIV / AIDS, or reduce the load of HIV virus, to delay the onset of HIV infection. AIDS vaccine must be scientifically designed clinical trials. Its safety, immunogenicity and protection were investigated. With the increasing awareness of AIDS, more and more evidence shows that CTL mediated by positive CD8 plays an important role in the control of HIV infection. Effective induction of CTL targeting more conservative regions such as HIV-Gag is an important means of designing an effective AIDS vaccine. Induction and production of effective neutralizing antibodies is an important indicator of the success of a vaccine, but also one of the main directions of AIDS vaccine research in the past ten years. This is because the production of neutralizing antibodies plays a key role in preventing virus infection. In this study, we successfully constructed and screened mammalian cell lines with high efficiency and persistent expression of HIV-1 structural proteins Gagpol and Env by two plasmid cotransfection methods. The secretion of virus-like granulovirus like particlesus VLPsin cell culture medium was monitored and the quality control standard of the finished product was established after isolation and purification. The ability of VLPs to induce cellular immunity and the level of neutralizing antibody in mice were measured. At the same time, the preliminary safety evaluation of HIV-1 virus-like granular vaccine was completed. The results showed that:. 1Recombinant 293 cell line which highly expressed HIV-1 structural protein Gagpol and Env could continuously secrete two kinds of target proteins; (2) the target product secreted in the culture supernatant was isolated and purified, and it was observed by transmission electron microscope that the product could be assembled into virus-like particles. (3) the virus-like particles can induce good humoral and cellular immunity; 4 the safety evaluation showed that the HIV-1 vaccine had no obvious side effects.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R392

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