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选择冷适应、温度敏感、减毒的A/Ann Arbor/6/60(H2N2)流感病毒株作为骨架病毒,对其6个内部基因片段进行了全基因合成,同时人工引入5个氨基酸突变(PB1-391E,581G,661T,PB2-265S,NP-34G).HA和NA来源于2006～2007当年流行株A/New Caledonia/20/99(H1N1).8个基因片段通过与改造后的转录载体pAD3000连接,构建8个基因的拯救载体,经测序获得序列准确的拯救质粒:pMDV-A-PB2、pMDV-A-PB1、pMDV-A-PA、pMDV-A-NP、pMDV-A-M、pMDV-A-NS、pMDV-A-HA、pMDV-A-NA.6质粒与当年流行株的表面基因HA和NA进行“6+2”组合的病毒拯救,8个重组质粒共转染COS-1细胞,成功拯救出了具有血凝性的冷适应减毒的重组A型人流感病毒.鸡胚尿囊液中重组病毒的血凝效价为1∶29～1∶210.构建的A/AA/6/606个内部基因的病毒骨架拯救系统,为深入研究冷适应减毒人流感病毒的基因功能和新型疫苗研发奠定了基础.
A cold-adapted, temperature-sensitive, and attenuated A / Ann Arbor / 6/60 (H2N2) influenza virus strain was used as a skeleton virus to perform genome-wide DNA synthesis of its six internal gene fragments. At the same time, five amino acid mutations -391E, 581G, 661T, PB2-265S, NP-34G) .HA and NA originated from the epidemic strain A / New Caledonia / 20/99 (H1N1) pMDV-A-PA, pMDV-A-NP, pMDV-AM, pMDV-AM and pMDV-A-NP were successfully constructed. A-NS, pMDV-A-HA and pMDV-A-NA.6 plasmids were rescued with the “6 + 2” combination of the surface genes HA and NA of the current year strains. 1 cells successfully rescued the clotting-tolerant cold-adapted attenuated recombinant human influenza A virus.The hemagglutination titer of recombinant virus in chick embryo allantoic fluid was 1:29 ~ 1: 210. The constructed A / The AA / 6/606 virus skeleton rescue system of internal genes laid the foundation for further study on the gene function of cold-adapted attenuated human influenza virus and the development of new vaccines.