| 摘要: |
| 为了建立用于流感病毒拯救的反向遗传操作系统,用RT-PCR方法扩增得到A型流感病毒流行株A/shanghai/7/99的HA和NA基因,将其克隆到pGEM-T载体上并测序,挑选阳性克隆分别用BsmBⅠ、BsaⅠ酶切,连接到以BsmBⅠ酶切的双向转录/表达载体pAD3000上,获得重组质粒pAD-HA和pAD-NA,分别与含A/PR/8/34流感病毒7个基因的阳性质粒共转染COS-1细胞,拯救了重组病毒R-HA-PR8和R-NA-PR8,说明构建的重组质粒pAD-HA和pAD-NA是正确的,将pAD-HA和pAD-NA与含A/PR/8/34流感病毒6个基因的阳性质粒共转染COS-1细胞,培养48 h后吸取上清液及COS-1细胞,接种10日龄SPF鸡胚,孵育96 h后,收获鸡胚尿囊液进行血凝和血凝抑制试验。结果显示,鸡胚尿囊液中重组病毒H1N1的血凝效价和血凝抑制效价均为29,鸡胚半数感染剂量为10-8.5～10-9。病毒的成功拯救为进一步深入研究流感病毒的基因功能以及流感新型疫苗的研发奠定了基础。 |
| 关键词: 流感病毒 反向遗传学 8质粒系统 重组病毒 |
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| Construction of reassortant H1N1 influenza A virus by eight-plasmid system |
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| Abstract: |
| To establish the reverse genetic system for influenza virus,Hemagglutinin(HA) and Neuraminidase (NA) gene of A/shanghai/7/99 subtype H1N1 were amplified by RT-PCR,then were cloned into the vector pGEM-T and sequenced. The positive clone was digested by BsmBⅠand BsaⅠ,seperately,then inserted into the bi-directional expression vector pAD3000 to construct recombinant plasmids:pAD-HA and pAD-NA.They were transfected into COS-1 cells in combination with plasmids incorporating the other 7 genes of A/PR/8/34 respectively to generate reassortant virus R-HA-PR8 and R-NA-PR8,which showed that recombinant plasmids:pAD-HA and pAD-NA had been correctly constructed.pAD-HA and pAD -NA were transfected into COS-1 cells in combination with plasmids incorporating the inner genes of A/PR/8/34 for 48 hours after transfection,then the supernatant and COS-1 cells transfected were inoculated into the allantoic cavity of 10-day-old specific-pathgen-free(SPF) chicken eggs.The HA and HI titer were determined，The HA titer is 29 and EID50 is between 10-8.5－10-9.The result provided the basis for further research on gene function and novel vaccine candidate of human influenza virus. |
| Key words: influenza virus reverse genetics eight-plasmid system reassorted virus |
