禽源H9N2亚型猪流感病毒反向遗传操作技术平台的建立

摘要

Eight gene fragments of avian-like H9N2 subtype Swine influenza virus A/Swine/Guangxi/7/2007(H9N2) were amplified by RT-PCR and separately cloned into the vector of pBD. Then recombinant plasmids were purified and co-transfected into 293T cells. The supernatant of 293T cells was collected after 54 h and inoculated into MDCK cells. Virus titer was determined in hemagglutination test after the rescued virus was proliferated into MDCK cells for three times, which was comparable to the wild type strain. Sequence analysis demonstrated that eight genes of the rescued virus were identical to the original virus, indicating that the virus was rescued successfully. The establishment of reverse genetics system of avian-like H9N2 subtype Swine influenza virus settles the foundation for the research on pathogenesis, transmission mechanism and gene function in the future.%利用RT-PCR技术扩增了禽源H9N2亚型猪流感病毒A/Swine/Guangxi/7/2007(H9N2)的8个目的基因片段,分别克隆至pBD双向转录表达载体.将8个重组质粒纯化后共转染293T细胞,54 h后收集上清,接种MDCK细胞.当拯救的病毒在MDCK细胞上增殖至第3代时,收集的细胞上清经检测具有血凝效价.经过测序分析,确定获救病毒的8个基因片段序列与野生株完全一致,表明病毒拯救成功.禽源H9N2亚型猪流感病毒反向遗传操作技术平台的成功建立为探索猪流感病毒致病机理、传播机制与基因功能研究奠定了基础.