【摘要】 为了构建重组禽流感病毒,试验选取具有代表性的禽流感毒株A/chicken/Shanghai/10/01(H9N2),通过RNA提取和RT-PCR获取HA、NA完整基因,并将其克隆至经SapⅠ酶切、Klenow(大片段)处理、小牛碱性磷酸酶(CIAP)处理后的载体质粒pBD中,构建pBD-HA和pBD-NA质粒;将克隆产物分别进行EcoRⅠ+BamHⅠ(pBD-HA)和EcoRⅠ+NotⅠ(pBD-NA)双酶切鉴定及序列测定。结果表明:所克隆的基因大小和序列均与理论值相符,说明感染性克隆构建成功。更多还原

【Abstract】 To construct a recombinant avian influenza virus,the test selected a typical strain A/chicken/Shanghai/10/01(H9N2)to obtain the full-length HA and NA genes by using RNA extraction and RT-PCR.After digested by SapⅠ and treated with Klenow fragment and CIAP,the two genes were cloned into vector plasmid named pBD to obtain the products named pBD-HA and pBD-NA,and then the cloned product was respectively digested by EcoRⅠ+BamHⅠ(pBD-HA)and EcoRⅠ+NotⅠ(pBD-NA),at last,the sequences of cloned HA and NA genes were determined.The results showed that the experimental values were conformed with the theoretical values for the size and sequence analysis of the cloned genes,it was proved that the full-length cDNA infectious clones had been successfully constructed.It concludes that the infectious cDNA clones may serve as basis for further research to prepare for constructing a recombinant virus with reverse genetics technique.更多还原