设计带有BsmBI、BsaI或AarI酶切位点的引物,用RT-PCR扩增H9N2亚型禽流感病毒(AIV)的8个基因全长片段,克隆入双向转录/表达载体pHW2000,并在PB2、PB1和NA基因中共引入了3个沉默突变标签。将其2个表面基因(HA和NA基因)加上任意1个内部基因,而其它5个内部基因来自A/WSN/33,进行了6种3+5组合形式的基因重排,把相应组合的转录/表达质粒共转染COS-1细胞,均产生了预期组合、有感染性的H9N2亚型流感病毒,表明亲缘关系遥远的流感病毒可以互相获取基因片段产生重组病毒,提示表面结构基因和单个内部基因不足以限制H9N2AIV在哺乳动物细胞上的宿主范围,同时也验证了构建的8个转录/表达载体均能有效工作,为进一步研究H9N2亚型AIV基因结构与功能、AIV与宿主之间的关系打下了基础。

Eight full-length cDNAs of H9N2 Avian influenza virus (AIV) genes were amplified and separately cloned into the transcription/expression vector, pHW2000.A total of three genetic tags of silent mutations were introduced into PB2, PB1 and NA genes,respectively. Six 3+5 reassortants were generated by reverse genetics, each containing three genes, HA, NA and any one of the internal genes, from the H9N2 virus, and the remaining five internal genes from A/WSN/33. Thereby, the six transfectants were all designed as H9N2 subtypes. The results showed that influenza A virus could obtain one or more gene segments from another virus which was remotely related, and suggested that the surface genes and a single internal gene were not enough to exhibit host range restriction for the H9N2 virus on COS-1 mammalian cells. The results also showed that each of the eight constructs worked efficiently. This reverse genetics system would be a useful tool for further studies on the structure and function of H9N2 influenza virus gene