甲型H1N1流感重组病毒模型的建立及应用

Establishment and application of a pseudotyped virus assay for anti-H1N1 influenza virus

目的:针对流感病毒感染宿主机体的作用机制,建立以甲型H1N1流感A/California/04/2009(H1N1)为靶点的重组病毒模型,并对其应用进行探索。方法:通过将表达流感病毒血凝素蛋白HA、神经氨酸酶NA及敲除外壳基因、复制基因并带有荧光素酶报告基因的HIV^(-1)质粒共转染至293T包装细胞,产生以HA[A/California/04/2009(H1N1)]和NA[A/California/04/2009(H1N1)]为膜蛋白,HIV^(-1)(p NL4-3.Luc.R-E-)为核心的重组病毒。通过该重组流感病毒模型评价阳性对照化合物(奥斯他韦、奥斯他韦酸)、中和血清以及板蓝根药材提取液对重组病毒活性的抑制作用。结果:奥司他韦、奥司他韦酸抑制重组病毒p NL+HA04+NA04的IC50为1.07×10-8 mol·L^(-1)和1.82×10-9 mol·L^(-1);中和血清的IC50为1∶320~1∶640;中药板蓝根提取液的IC50为1.91×10-5 g·m L^(-1)。结论:奥斯他韦、奥斯他韦酸、中和血清及板蓝根提取液对于重组病毒的活性有抑制作用,提示所构建的重组病毒模型可用于上述作用机制药物的筛选和评价。

Objective：To establish a method based on a pseudotyped virus assay for A/California/04/2009（H1N1） targeting and drug screening of anti-H1N1 influenza virus,and evaluate its application. Methods： A model of HIV（-1）（p NL4-3.Luc.R-E-） based pseudovirions packed with HA [hemagglutinin,A/California/04/2009（H1N1）] and NA [A/California/04/2009（H1N1）] was established,then the compounds activities（oseltamivir and oseltamivir acid）,serum neutralization and Radix Isatidis extract were evaluated. Results： IC50 of oseltamivir and oseltamivir acid was 1.07×10-8 mol·L（-1） and 1.82×10-9 mol·L（-1）;IC50 of neutralization serum was 1∶320（-1）∶640;IC50 of Radix Isatidis extract of traditional Chinese medicine was 1.91×10-5 g·m L（-1）. Conclusion： Due to the similar mechanism of action of oseltamivir,oseltamivir acid,neutralization serum and Radix Isatidis extract,the constructed pseudotyped virus model can be used for target screening and evaluation of above mentioned drugs.