两质粒系统重配甲型H1N1流感病毒株的研究  

Rescue of influenza N1N1 virus from two plasmid-system

作　　者：周珊珊[1,2] 杨晓岚 孙芳[2] 程晋霞[2] 谷宏婧 段跃强 杨鹏辉[2] 王希良[2] ZHOU Shanshan1,2, YANG Xiaolan2, SUN Fang2, CHENG Jinxia2, GU Hongjing2, DUAN Yueqiang2, YANG Penghui2, WANG Xiliang2(1. Graduate School, Anhui Medical University, HeFei 23060, China; 2. State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, Chin)

机构地区：[1]安徽医科大学研究生院,合肥230601 [2]军事医学科学院微生物流行病研究所免疫学研究室病原微生物生物安全国家重点实验室,北京100071

出　　处：《免疫学杂志》2018年第4期353-358,共6页Immunological Journal

基　　金：国家重点研发计划(2017YFC1200800)

摘　　要：目的构建两质粒冷适应流感病毒拯救系统,提高流感病毒的重配效率。方法本研究通过构建克隆载体pfastbac△plh HTB,将流感病毒A/Ann Arbor/6/60(H2N2)6个内部基因(PB2、PB1、PA、NP、M、NS)的双向转录表达元件定向克隆入此载体,获得pfastbac△plh HTBP6。同时将流感病毒A/California/7/2009(H1N1)表面基因(HA、NA)双向转录表达元件定向克隆入p ENTR-1A质粒,获得p ENTR-1AP2。将2个重组质粒共转染MDCK/COSI细胞,细胞上清接种10日龄SPF鸡胚,收取尿囊液,血凝实验筛选阳性株,并进行全面鉴定。结果本研究利用两质粒系统成功重配甲型H1N1流感病毒株,命名为TRG A/California/07/2009(H1N1)Vca,重配病毒株传代后HA效价为2~8,形态符合流感病毒典型特征,大小80~120 nm,具有冷适应、温度敏感表型。结论两质粒流感病毒拯救系统为高效重配流感疫苗株提供了新策略。This study was performed to develop a two-plasmid DNA transfection system for the rescue of infectious influenza A virus from cloned cDNA. The transcriptional expression elements of six internal genes were cloned into pfastbac △ plhHTB to obtain Pfastbac △ PlhHTBP6; the bi-directional transcriptional expression element of the surface gene derived from A/California/07/2009 were cloned into pENTR-1A plasmid to obtain pENTR-1AP2. Then the Pfastbac A PlhHTBP6 and pENTR-1AP2 plasmids were co-transfected into MDCK/COSI cells, and the supernatant was used to inoculate 10-day-old SPF chicken embryos. Allantoic fluid was collected, and the titer of hemagglutinin was measured. In this study, a influenza A （H1N1） termed as TRG A/California/07/ 2009 （H1N1） was successfully rescued with two-plasmid system. HA titer of reassortment virus is 2^8 and virus size is 80-120 nm. In addition, TRG A/California/07/2009 （H1N1） was cold-adaptive and temperature sensitive. In conclusion, the two-plasmid system provides a new strategy for reassorting the influenza vaccine virus.

关 键 词：反向遗传学 两质粒系统 流感病毒 

分 类 号：R392-33[医药卫生—免疫学]