摘要
为了构建H9N2亚型禽流感病毒A/Chicken/Shandong/LY1/2017的反向遗传操作平台,通过RT-PCR技术分别扩增该毒株的8个基因片段,并分别克隆至双向转录表达载体PHW2000中.8个质粒共转染293T/MDCK混合细胞,96 h后将细胞板反复冻融3次收获细胞悬液,将其接种10日龄SPF鸡胚,96 h后收集尿囊液并将拯救毒株命名为rH9N2,测定其HA效价为1:256.rH9N2在HA、MDT、EID50、TCID50以及病毒生长曲线、空斑等方面保持了与亲本毒株一致的生物学特性.本研究成功构建了A/Chicken/Shandong/LY1/2017的感染性克隆,为今后通过基因替换、定点突变技术研究H9N2亚型禽流感病毒致病性的分子机制奠定了基础.
To rescue A/Chicken/Shandong/LY1/2017(H9N2)of Avian influenza virus,eight gene segments of the subtype H9N2 were amplified by RT-PCR and cloned into the transcription vector of PHW2000.Then,these eight recombinant plasmids were purified and transfected into 293T/MDCK cells.The resulting virus fluid was inoculated into 10-day-old SPF chicken embryos.The allantoic fluid was harvested at 96 hours post inoculation and tested for HA activity.The rescued virus was determined to have HA titer at 1:256 and designated as the strain rH9N2.The strain rH9N2 maintained the same biological characteristics as its parent strain,such as MDT,EID50,TCID50,virus growth curve and plaque size and so on.The successful establishment of the reverse genetics system of A/Chicken/Shandong/LY1/2017 would enable us to further study the molecular basis of pathogenesis of Avian influenza virus.
作者
李梦娇
徐凯
杨彬
刘伟
仇旭升
孙英杰
谭磊
廖瑛
宋翠萍
刘炜玮
张小荣
丁铲
孟春春
吴艳涛
LI Meng-jiao;XU Kai;YANG Bin;LIU Wei;QIU Xu-sheng;SUN Ying-jie;TAN Lei;LIAO Ying;SONG Cui-ping;LIU Wei-wei;ZHANG Xiao-rong;DING Chan;MENG Chun-chun;WUYan-tao(College of Veterinary Medicine,Hangzhou University,Yangzhou 225009,China;Shanghai Veterinary Research Institute,CAAS,Shanghai 200241,China)
出处
《中国动物传染病学报》
CAS
北大核心
2020年第3期49-54,共6页
Chinese Journal of Animal Infectious Diseases
基金
上海市兽医生物技术重点实验室开放基金(Klab201702)。
