HIV-1中和抗体快速安全的评价体系的研究

发布时间：2018-04-01 00:02

本文选题：HIV-1　切入点：共转染　出处：《吉林大学》2006年硕士论文

【摘要】：艾滋病的流行已成为人类健康和社会稳定的大敌,我国已进入了艾滋病快速增长期,因此迫切需要一种有效的疫苗来阻止艾滋病的流行。现在,已经设计出很多能诱导广泛有效中和抗体的候选疫苗。疫苗研究的进展要求在体外准确计量中和抗体的中和水平,以评价抗体和疫苗在实验动物及人体内的保护效果,所以,传统的中和实验技术必须进行改进,以满足理论研究进展的需要。一个准确、可重复性的体外中和实验对于研究病毒中和的机制,评价候选疫苗诱导中和反应能力都是至关重要的。本论文研究是利用荧光素酶作为报告基因,表达假病毒进行中和抗体评价,即构建中国流行株各亚型HIV-1 envelope基因的真核表达质粒和带有荧光素酶报告基因的pNL-Luc-E—R—质粒共转染293T细胞上,培养收获含有假病毒。假病毒和抗体或血清共孵育后感染表达CD4受体和一个辅助受体(CCR5 /CXCR4)的HOS细胞,培养3天后,用PBS洗涤细胞,将细胞裂解,检测荧光素酶含量。并进一步建立稳定表达HIV-1假病毒的细胞系,批次大量获取假病毒,以适用于大批量的疫苗临床受检样品。这种方法大大降低了传统方法中宿主细胞( PBMC)的差异而引起的结果变异,是一种具有可重复性的极有发展前景的体外中和实验方法。
[Abstract]:The AIDS epidemic has become a major enemy of human health and social stability. Our country has entered a period of rapid growth of AIDS. Therefore, an effective vaccine is urgently needed to stop the spread of AIDS. Many candidate vaccines have been designed to induce a wide range of effective neutralizing antibodies. Progress in vaccine research requires that neutralization levels of neutralizing antibodies be accurately measured in vitro to evaluate the protective effects of antibodies and vaccines in laboratory animals and humans, so, The traditional neutralization experiment must be improved to meet the needs of theoretical research. An accurate and repeatable in vitro neutralization experiment is needed to study the mechanism of virus neutralization. Evaluating the ability of candidate vaccines to induce neutralization is essential. In this study, luciferase was used as a reporter gene to evaluate the neutralization antibody of pseudovirus. The eukaryotic expression plasmid of HIV-1 envelope gene of Chinese epidemic strain and pNL-Luc-E-R- plasmid with luciferase reporter gene were cotransfected into 293T cells. Cultured and harvested HOS cells containing pseudovirus. co-incubated with antibodies or serums, infected with HOS cells expressing CD4 receptor and a coreceptor CCR5 / CXCR4. After 3 days of culture, the cells were washed with PBS, and the cells were lysed. The luciferase content was detected, and the cell lines stably expressing HIV-1 pseudovirus were further established, and lots of pseudoviruses were obtained. This method can greatly reduce the variation caused by the difference of host cells (PBMCs) in traditional methods and is a reproducible and promising in vitro neutralization method.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392

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