【作者】 谢桂煌；

【导师】 陈朝银；

【作者基本信息】 昆明理工大学 ， 生物化工， 2011， 硕士

【摘要】 随着人类对艾滋病致病机制的深入研究,越来越多新的抗HIV药物进入临床试验阶段,但大量抗HIV药物的使用也导致了耐药性问题的出现。因此,建立高通量快速的抗HIV药物初步筛选平台对于研究新的抗HIV药物非常重要。针对传统抗HIV药物细胞水平筛选模型对实验室条件要求高,并且具有较高危险性等缺点,本论文通过构建HIV假病毒和应用体外药物靶向筛选方法来研究抗HIV药物的初步筛选,以建立快速方便且危险性较低抗HIV药物筛选和评价系统,用于新的抗HIV药物的开发。主要的研究内容如下：(1)构建HIV假病毒,为进一步建立基于HIV假病毒的抗HIV药物筛选细胞模型提供基础。实验过程中成功构建出了具有感染活性的HIV假病毒。但由于其感染活性较低,尚不能应用于抗HIV药物的筛选。需要进一步对假病毒构建的实验过程进行优化,以提高假病毒的感染活性,(2)利用SPR技术,建立抗HIV药物的筛选和分析方法,获得不同样品对HIV包膜蛋白rgp120 MN结合能力和平衡解离常数等数据,得出三个多糖样品中Za24-70-5-1002#与rgp120 MN的结合作用非常明显。动力学／亲和分析结果显示,Za24-70-5-1002#与rgp120 MN之间平衡解离常数KD(M)为1.172E-13M。两者之间具有很高的亲和力,因此Za24-70-5-10023可能具有较高的抗HIV活性,有必要对其药理作用进行进一步的研究。Za48-70-×100 J与rgp120 MN之间也具有比较好的亲和性,它们之间的平衡解离常数KD(M)为5.752E-6M。(3)应用ELISA法建立抗HIV药物初步筛选方法,以细胞表面受体CD4作为研究靶位,研究多糖样品对gp120与CD4结合的抑制作用。通过实验测得13个不同样品对gp120与CD4结合的抑制效果,计算出不同样品对gp120与CD4结合抑制的IC5o值,得到不同样品的IC5o值在1.150μg/mL之间。为进一步的药物筛选和分析提供依据。更多还原

【Abstract】 With increasingly understanding of mechanism of HIV infection, more and more new anti-HIV agents were developed and used in clinical experiments. However, the abuse of anti-HIV drugs was causing serious drug-resistance problem, so it is very important to develop new high-throughput screening systems for drug discovery. For the traditional anti-HIV drugs screening system based on cell model has high requirements on laboratory conditions and high risk of exposure to HIV, this dissertation study on anti-HIV drugs screening by constructing pseudovirus and using drug targeting screening in vitro, which are used for establishing high-thoughput and less dangerous anti-HIV drug evaluation systems. The main results can be listed as follows:(1) The pseudotyped Human Immunodefieiency Virus-1(HIV-1) was constructed successfully and may lay foundations for establishing a pseudovirous-based system for anti-HIV drugs screening. Pseudovirous with infection activity have been successfully constructed. But the infection activities are low, so the pseudovirous can not be used in anti-HIV drug screening yet. Further research is needed to optimize the experimental conditions for improving the infection activity of pseudovirous.(2) Establishing screening and evaluation system by surface plasmon resonance analysis is an effective way to develop new anti-HIV drugs. Three polysaccharide samples were been analyzed by this system. The equilibrium dissociation constant of interaction between rgp120 MN and Za24-70-5-1002# is 1.172E-13M by kinetics/affinity analysis through Biacore X100 evaluation software. The result shows that a strong interaction is exist between the two molecules. Therefore, this sample may have high anti-HIV activity. Otherwise, the equilibrium dissociation constant of interaction between rgp120 MN and Za48-70-×100 J is 5.752E-6M.(3) Establishing screening system by ELISA can be also a good method to discover new anti-HIV drugs. Using the cell surface receptor CD4 as a research target, the inhibitory effects of the binding between gp120 and CD4 by polysaccharide were analyzed. The inhibitory effects of 13 different samples were detected by ELISA. The IC50 values of all samples were calculated out and they are 1-150μg/mL. Some samples may have anti-HIV activity. And it can be proved and analyzed in further experiments.更多还原