六株不同地区分离的H7N9流感病毒假病毒制备与生物学特性研究  

Preparation and characteristic analysis of six influenza A （H7N9） pseudovirus derived from different districts of China

作　　者：黄保英[1] 李善琴 齐香荣[1] 任皎[1] 宋敬东[1] 谭文杰[1] 田厚文[1] 阮力[1] Huang Baoying Li Shanqin Qi Xiangrong Ren Jiao Song Jingdong Tan Wenfie Tian Houwen Ruan Li(Key Laboratory for Medical Virology, Ministry of Health. National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China)

机构地区：[1]中国疾病预防控制中心病毒病预防控制所卫生部医学病毒和病毒病重点实验室,北京102206

出　　处：《中华实验和临床病毒学杂志》2017年第4期281-286,共6页Chinese Journal of Experimental and Clinical Virology

基　　金：国家传染病科技重大专项（2014ZX10004002）;国家863计划（2006AA02A203）;国家自然科学基金（31200127）;国家重点研发计划（2016YFD0500300、2016YFC1200200）

摘　　要：目的 选择不同地区分离的H7N9亚型流感病毒代表株,制备各代表株的假病毒,为H7N9疫苗对不同地区毒株的免疫保护效果评价奠定基础.方法 通过对29株H7N9流感病毒血凝素（Hemagglutinin,HA）的氨基酸序列分析,获得6株不同地区H7N9亚型流感病毒代表株病毒;采用基于慢病毒载体的假病毒包装系统,以HA与神经氨酸酶（Neuraminidase,NA）作为包膜质粒,包装获得6株带有荧光素酶报告基因的假病毒;通过电镜负染、Western blot检测、感染MDCK后的荧光素酶读值和血凝试验等了解6株假病毒的生物学特性,通过血清交叉中和试验了解其在中和抗体检测中的应用情况.结果 筛选获得6株不同地区分离的H7N9流感病毒代表株并制备了相应的假病毒颗粒,电镜下可观察到典型的流感病毒形态,Western blot可检测到HA、NA和P24的表达;滴度测定结果显示6株假病毒对MDCK细胞的感染力为104- 105TCID50/50μl,血凝活性可达64- 512;SH1上海株H7N9 HA疫苗免疫血清针对6株流感假病毒100TCID50剂量的中和效力不同,提示6株假病毒可用于疫苗的交叉免疫效果评价.结论 成功获得6株H7N9不同地区代表株流感假病毒,为疫苗的免疫效果评价奠定了基础.Objective To prepare strains of influenza A （H7N9） pseudovirus derived from different districts of China for vaccine efficacy evaluation.Methods Phylogenetic tree was built based on hemagglutinin （HA） amino acid sequence analyses from 29 influenza A （H7N9） virus strains and 6 influenza A （H7N9） virus strains with HA determinants variation were selected.293FT cells were cotransfected with plasmid pNL4-3-Luc.R-E-,pVRC-HA and pVRC-NA with codon-optimized hemagglutinin （HA） and neuraminidase （NA） derived from the six influenza A （H7N9） virus strains,respectively.Transmission electron microscopy assay and Western blot analysis were performed to demonstrate morphology and specificity of these particles,luciferase activity assay and hemagglutinin titers detection were used to determine their infectivity and hemagglutinin activity.And finally,pseudovirus-based neutralization assays were evaluated with HA immunized mice serum.Results Six influenza A （H7N9） peseudovirus particles derived from different districts of China were selected and prepared.All of the particles bearing HA and NA were characterized with classic influenza virus morphology,with TCID50 titer ranged from 104TCID50/50 μl to 105TCID50/50 μl and with hemagglutinin activity ranged from 64 to 512.Neutralization efficacies on influenza A/Shanghai/1/2013 （H7N9） HA vaccine serum against 100TCID50 dose of these pseudovirus particles indicated their potential application in the vaccine cross-protective evaluation in future.Conclusions Six influenza A （H7N9） pseudovirus derived from different districts of China with potential antigenic variation on HA were constructed successfully,established foundation for their further application in vaccine cross-reactive efficacy evaluation.

关 键 词：H7N9流感病毒 假病毒 血凝素 抗原特性 中和试验 

分 类 号：R392[医药卫生—免疫学]