三源重组H1N1亚型猪流感病毒反向遗传操作平台的建立

ESTABLISHMENT OF REVERSE GENETICS SYSTEM OF TRIPLE-REASSORTANT H1N1 SUBTYPE SWINE INFLUENZA VIRUS

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。

The eight gene segments of triple-reassortant H 1N 1 subtype Swine influenza virus A/swine/Tianjin/10/2013（H 1N 1） were amplified in RT-PCR and individually cloned into the transcription/expression vector pBD that was used to transfect 293T cells. The supernatant samples of transfected 293T cells were collected after 48 h and inoculated into MDCK cells. The rescued viruses was determined to Swine influenza virus in hemagglutination test. The full genome of the rescued virus was confirmed no nucleic acid change as compared with the wild-type virus through sequence analysis. The measured results of growth curves showed no significant differences in virus titer between two strains. Therefore, it concluded that the virus was rescued successfully. The establishment of reverse genetic system of H1N1 subtype Swine influenza virus settles the foundation for future research on biological characteristics and production of recombinant influenza vaccine of H1N1 swine influenza.