Liu Li-Qi, Dong Jie, Bo Hong, Wen Le-Ying, Xin Li, Zhang Ye, Li Zi, Dong Li-Bo, Wang Min, Guo Yuan-Ji, Shu Yue-Long
Chinese Influenza Center, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100052, China.
Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi. 2009 Feb;23(1):41-3.
To provide a technology platform for vaccine development as well as the research on transmission and pathogenesis, the reverse genetic system for H9N2 avian influenza virus was established.
Eight full-length cDNAs of avian influenza virus A/Guangzhou/333/99 (H9N2) were amplified by RT-PCR and separately cloned into the transcription/expression vector, pCI-polI. The 8 plasmids DNA was cotransfected into 293T cell, the cell supernatant was collected and inoculated into embryonated eggs, the rescued virus from the allantoic fluid was identified by hemagglutinination assay.
The avian influenza H9N2 virus was successfully rescued by 8 plasmids co-transfection in 293T cells. The hemagglutinination titer of the rescued virus is up to 2(9)/50 microl and its growth curve remained relatively as to the wild-type virus.
The reverse genetic for avian influenza H9N2 subtype virus has been established successfully.
为疫苗研发以及病毒传播与致病机制研究提供技术平台，构建H9N2禽流感病毒反向遗传系统。
通过RT-PCR扩增禽流感病毒A/广州/333/99（H9N2）的8个全长cDNA，并分别克隆至转录/表达载体pCI-polI。将8种质粒DNA共转染至293T细胞，收集细胞上清液并接种至鸡胚，通过血凝试验鉴定尿囊液中拯救出的病毒。
通过8种质粒在293T细胞中共转染成功拯救出禽流感H9N2病毒。拯救出的病毒血凝效价高达2(9)/50微升，其生长曲线与野生型病毒相对一致。
已成功建立禽流感H9N2亚型病毒的反向遗传系统。
