论文部分内容阅读
本文旨在建立以2009新型甲型H1N1流感病毒神经氨酸酶(neuraminidase,NA)为靶点的神经氨酸酶抑制剂(neuraminidase inhibitors,NAIs)细胞水平评价体系。NA有促进流感病毒释放的作用,本研究应用重组病毒技术,通过将表达NA[A/California/04/2009(H1N1)]的质粒、表达流感病毒血凝素蛋白HA(hemagglutinin)的质粒以及表达敲除外壳基因并带有荧光素酶报告基因的HIV-1基因组共转染至病毒生成细胞,产生以HA、NA为外壳蛋白包裹HIV-1核心的重组病毒。在病毒释放前加入不同浓度的化合物,收集病毒上清液感染细胞,通过测定感染率来反映化合物对重组病毒NA的抑制作用。经用阳性对照药物奥司他韦及其羧酸盐证实本模型能够反映化合物对病毒NA的抑制作用;在此基础上,本研究还建立了奥司他韦耐药株评价模型。本研究所建立的体系可用于针对新型甲型H1N1流感病毒及其临床耐药株神经氨酸酶抑制剂的寻找和评价。
This article aims to establish a neuraminidase inhibitor (NAIs) cell level evaluation system targeting the 2009 new type A H1N1 influenza virus neuraminidase (NA). NA has the effect of promoting the release of influenza virus. In this study, recombinant virus technology was used to express the influenza virus hemagglutinin by expressing a plasmid expressing NA [A / California / 04/2009 (H1N1)], The HIV-1 genome knocked out of the coat gene and carrying the luciferase reporter gene was co-transfected into virus-producing cells to produce a recombinant virus that enveloped HIV-1 core with coat protein of HA and NA. Different concentrations of compounds were added before the virus was released, and the virus supernatant was collected to infect the cells. The infection rate was measured to reflect the inhibitory effect of the compound on the recombinant virus NA. The positive control drug oseltamivir and its carboxylate confirmed that the model can reflect the inhibitory effect of the compound on virus NA. On this basis, the evaluation model of oseltamivir resistant strain was also established in this study. The system established in this study can be used to find and evaluate neuraminidase inhibitors against the new type A H1N1 influenza virus and its clinical drug-resistant strains.