H3N2亚型犬流感病毒反向遗传操作系统的建立

发布时间：2018-11-09 13:56

【摘要】：H3N2亚型犬流感病毒(Canine influenza virus,CIV)可以在犬中迅速的传播,甚至引起犬的死亡,也可感染其他哺乳动物。目前该病毒致病的分子机制尚不清楚。为此,本研究采用RT-PCR技术扩增犬流感病毒A/Canine/Zhejiang/01/2010(H3N2)(简称ZJ2010)的PB2、PB1、PA、HA、NP、NA、M和NS 8个基因片段,并分别克隆至双向表达载体p BD上。采用8质粒系统共转染293T细胞,转染48 h后加入TPCK胰酶作用2 h,将上清和细胞一同接种9~11日龄SPF鸡胚,并检测血凝效价。结果显示,本研究成功拯救获得了病毒株r ZJ2010。r ZJ2010株的8个基因片段的序列均与亲本ZJ2010株的序列相同。r ZJ2010株与ZJ2010株在鼠的肺和鼻中复制水平接近。这些结果证实,本研究已成功建立H3N2亚型犬流感病毒反向株遗传操作系统,为该病毒的致病机理、传播机制以及跨宿主传播机制研究等奠定了技术平台。
[Abstract]:H3N2 subtype (Canine influenza virus,CIV can spread rapidly in dogs, even cause death in dogs and infect other mammals. The molecular mechanism of the disease is unclear. In this study, the PB2,PB1,PA,HA,NP,NA,M and NS fragments of canine influenza virus A/Canine/Zhejiang/01/2010 (H3N2) (ZJ2010) were amplified by RT-PCR and cloned into bidirectional expression vector p BD. 293T cells were co-transfected with 8 plasmids. After 48 h of transfection, the supernatants and cells were incubated with TPCK trypsin for 2 h. The supernatants and cells were inoculated with SPF chicken embryos at the age of 9 and 11 days, and the hemagglutination titers were detected. The results showed that the sequence of 8 gene fragments of r ZJ2010.r ZJ2010 strain was the same as that of parent ZJ2010 strain, and the replication level of r ZJ2010 strain and ZJ2010 strain was similar in lung and nose of mice. These results confirm that the genetic operating system of H3N2 subtype canine influenza virus reverse strain has been successfully established in this study, which provides a technical platform for the study of the pathogenic mechanism, transmission mechanism and cross-host transmission mechanism of the virus.
【作者单位】：黑龙江大学;中国农业科学院上海兽医研究所;
【基金】：国家自然科学基金项目(31302115,31472206) 国家重点研发计划(2016YFD0500204) 上海市科技兴农重点攻关项目(沪农科攻字(2015字)第1-11字号)
【分类号】：S852.655

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