欧洲类禽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 EUROPEAN AVIAN-LIKE H1N1 SUBTYPE SWINE INFLUENZA VIRUS

为了构建猪流感病毒A/swine/Tian Jin/6/2013(H1N1)的反向遗传操作技术平台,通过RT-PCR技术分别扩增了欧洲类禽H1N1亚型猪流感病毒的8个基因片段,并分别连接至双向转录表达载体p BD上。纯化8个重组质粒,共转染到293T细胞。54 h后收集上清,并接种到MDCK细胞。待细胞病变明显时,进行血凝试验检测收集到的上清,测得血凝效价为1∶32。测定拯救病毒的全基因组序列,结果显示,在核苷酸序列上,拯救病毒与野生病毒完全一致。细胞病变情况显示,拯救毒株与野生毒株无明显差别。本研究成功拯救了欧洲类禽H1 N1亚型猪流感病毒,为猪流感病毒的致病机理、基因功能研究以及H1N1亚型猪流感病毒新型疫苗的研发奠定了基础。

To rescue A/swine/TianJin/6/2013（H1N1） of Swine influenza virus, the eight gene segments of H1N1 were amplified by RT- PCR and cloned into the transcription/expression vector of pBD. The eight recombinant plasmids were purified and cotransfect into 293T cells. The supernatant of 293T cells was collected after 54 hours and inoculated into MDCK ceils. When cytopathie effects （CPE） appeared in MDCK cells, virus titer was determined at 1 ： 32 in hemagglutination test. The full genome of the rescued virus had no nucleic acid change, compared with the wild-type virus through sequence analysis. There were no obvious differences in CPE on MDCK cells between the rescued virus and the wild type virus. The establishment of reverse genetic system of European avian-like H1N1 subtype Swine influenza virus provided the foundation for further studies on gene function and new influenza vaccine.