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利用RT-PCR技术扩增三源重组H1N1亚型猪流感病毒A/Swine/Tianjin/10/2013(H1N1)的8个基因片段,分别克隆至双向转录/表达载体p BD上。将8个重组质粒纯化后共转染293T细胞,收取转染48 h后的细胞上清并接种MDCK细胞,成功拯救出有血凝活性的病毒。全基因序列测定结果表明,拯救病毒与野生病毒的核苷酸序列完全一致。生长曲线的测定结果表明,不同时间点野生毒株与拯救毒株在MDCK细胞上的病毒滴度没有明显差异。三源重组H1N1亚型猪流感病毒反向遗传操作平台的成功建立为进一步开展猪流感病毒的生物学特性研究奠定了基础,同时也为H1N1亚型猪流感疫苗的研制开辟了新的途径。
Eight gene fragments of the three-source recombinant swine influenza A / Swine / Tianjin / 10/2013 (H1N1) of H1N1 were amplified by RT-PCR and cloned into p BD. Eight recombinant plasmids were co-transfected into 293T cells, and the supernatants were harvested 48 h after transfection and inoculated with MDCK cells to successfully rescue the hemagglutinating activity of the virus. The results of whole genome sequencing showed that the nucleotide sequences of the rescue virus and the wild virus are exactly the same. The results of the growth curve showed that there was no significant difference in virus titer between wild-type strain and rescue strain in MDCK cells at different time points. The successful establishment of the three-source recombinant H1N1 subtype swine influenza virus reverse genetic platform has laid the foundation for the further study on the biological characteristics of swine influenza virus and also opened a new way for the development of H1N1 subtype swine influenza vaccine.