摘要禽流感病毒是养禽业危害最严重的病原微生物之一。为探讨小干涉RNA(siRNA)对A型禽流感病毒复制的干扰作用,以H5亚型AIV PB2基因为靶序列,设计合成了4对编码siRNAs的DNA序列,将其克隆到psiRNA-hH1neo载体中,构建siRNAs表达载体,鉴定正确后将重组质粒转染MDCK细胞,采用G418筛选建立抗性细胞系,用血凝(HA)试验和real time RT-PCR试验检测抑制效果, 在细胞水平筛选出具有高效抑制AIV复制的2个靶位点PB2-1154、PB2-342、为AIV的基因功能研究,抗病毒药物的开发和转基因动物的研究奠定了基础。
Abstract：Avian Influenza virus (AIV) is one of the most harmful pathogens in poultry industry. Because of its antigenic shift and drift, the efficacy of current vaccines and drugs therapy is limited. Here we show that four recombinant plasmids which can express four different kinds of short hairpin RNAs (shRNA) targeting at the polymerase II gene of the avian influenza virus in cell were successfully constructed. The recombinant plasmids and vacancy plasmid were transfected into MDCK cells respectively and five cell lines were established. The hemagglutinantion assay and real time RT-PCR were used to detect the inhibition efficacy. PB2-1154 , PB2-342 were proved that they can specifically inhibit the virus production in MDCK cells.