Construction Of Pseudotyped HIV And Study On Anti-HIV Drugs Screening By SPR And ELISA

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.