H7N9流感假病毒制备条件的优化及其在中和抗体检测中的初步应用

Optimization of the Package Condition for H7N9 Influenza Pseudovirus and its Utilization in Neutralization Assay

目的:优化A/Anhui/1/2013(H7N9)流感假病毒的制备条件。方法:采用基于慢病毒载体的假病毒包装系统,通过对质粒组合方式、骨架质粒与包膜质粒配比、质粒复合物与转染试剂配比、换液时间点、维持液血清浓度、假病毒收获时间点等条件的探索,优化H7N9假病毒的包装条件;通过对4种假病毒纯化方案的比较,确定纯化方法;对纯化的假病毒进行Western印迹鉴定和电镜形态观察,了解其生物学特征;通过中和试验了解其在中和抗体检测中的应用情况。结果:假病毒包装条件优化结果表明,以血凝素(HA)与神经氨酸酶(NA)作为膜质粒,采用骨架质粒与膜质粒质量比3:1、质粒复合物与转染试剂质量体积比1:2混合,转染293FT细胞后10 h换含2%胎牛血清的维持液,培养48 h后收获上清,可高效获得流感假病毒AHop-H7N9pp;不同纯化方式获得的假病毒对感染H7N9的雪貂血清的中和抗体检测结果表明,假病毒纯化方法对中和抗体检测灵敏度的影响没有显著性差异;生物学特征分析显示,纯化的假病毒在电镜下具有典型的流感病毒形态,Western印迹能检测到HA和P24蛋白;多种流感疫苗小鼠血清中和试验结果表明该假病毒具有高度特异性,可用于H7N9中和抗体检测。结论:获得了包装有A/Anhui/1/2013(H7N9)HA与NA的假病毒,并确定了高效制备H7N9假病毒的各条件参数,为基于H7N9流感假病毒的中和抗体检测平台的建立奠定了基础。

Objective：To optimize the package condition of H7N9 influenza pseudovirus.Methods：Use the lentiviral-based system for pseudovirus produce,we investigated different envelope plasmid combination,different ratio between backbone and envelope plasmid,different ratio between plasmid compound and transfection reagent,different FBS serum concentration,different time point for medium change and different time point for harvest to optimize the package conditions of the H7N9 influenza pseudovirus.Based on the optimized package conditions,we used four methods to purify the pseudvirus.Luciferase activity assay,Western blot assay and transmission electron microscopy assay were used to evaluate infectivity of pseudovirus in supernatant and the morphology of purified pseudovirus,and finally pseudovirus-based neutralization assays were evaluated.Results：The pseudovirus AHopH7N9pp was packaged at high efficience with optimized conditions,the ratio of transfection backbone plasmid with envelope plasmid was at 3：1,plasmid mixture and transfection reagent was at 1：2,the medium was replaced with DMEM contain 2％ FBS at 10 h after transfection,the time of harvesting AHop-H7N9pp was at 48 h after transfection,and purification of pseudovirus was by centrifuge at 2500 r/min for 5 min,then 24 000 r/min for 2 h with 20％ sucrose cushion.AHop-H7N9pp bearing HA and P24 protein showed classic influenza virus morphology.Based on the data of neutralizing antibody assay with AHop-H7N9pp,we found that AHop-H7N9pp was high ly train specificity and could be applied to neutralization assay.Conclusion：Pseudovirus bearing HA and NA from A/Anhui/1/20l3（H7N9） with high infectivity was constructed with optimized conditions.It established foundation for H7N9 pseudovirus-based neutralization assay platform.