人源H3N2亚型猪流感病毒反向遗传操作技术平台的建立  

Development of Reverse Genetics System of H_(3)N_(2) Subtype Swine Influenza Virus

作　　者：姚云 张毅峰 王帅勇 虞凌雪[1] 朱世强 王娟 单同领[1] 周艳君[1] 童武[1] 郑浩[1] 李国新[1] 童光志[1] 于海[1,2] YAO Yun;ZHANG Yifeng;WANG Shuaiyong;YU Lingxue;ZHU Shiqiang;WANG Juan;SHAN Tongling;ZHOU Yanjun;TONG Wu;ZHENG Hao;LI Guoxin;TONG Guangzhi;YU Hai(Shanghai Veterinary Research Institute,CAAS,Shanghai 200241,China;Jiangsu Coinnovation Center for the Prevention and Control of Important Animal Infectious Disease and Zoonoses,Yangzhou 225009,China)

机构地区：[1]中国农业科学院上海兽医研究所,上海200241 [2]江苏省动物重要疫病与人兽共患病防控协同创新中心,扬州225009

出　　处：《中国动物传染病学报》2022年第6期13-18,共6页Chinese Journal of Animal Infectious Diseases

基　　金：国家自然科学基金面上项目(31970175);中央级公益性科研院所基本科研业务费项目(2019JB05);中国农业科学院创新工程“猪病毒性繁殖障碍综合症团队”项目。

摘　　要：为了构建人源H3N2亚型猪流感病毒的反向遗传操作技术平台,利用RT-PCR扩增出流感病毒的8个基因片段,酶切后连接到双向转录表达载体p BD上,将8个重组质粒纯化后转染至293T细胞于54 h后收取上清液并接种MDCK细胞直至出现细胞病变,取上清液检测出有血凝活性。全基因序列测定结果表明,拯救毒株与野生毒株核苷酸序列比对结果一致,病毒拯救成功。人源H3N2亚型猪流感病毒的成功拯救为猪流感病毒的生物学特性、分子致病机制及疫苗研发奠定了基础。In order to construct a reverse genetic operation technology platform for H3N2 subtype swine influenza virus, 8 genes of Infl uenza virus were amplifi ed by RT-PCR, and then separately cloned into the transcription expression vector PBD, and the resulting 8recombinant plasmids were purifi ed and co-transfected into 293T cells. The supernatant samples were collected after 54 h for inoculation into MDCK cells and performance of hemagglutination. The rescued viruses were amplified into 8 fragments for identification and sequencing, which were consistent with sequences of the wild strain, indicating the success of virus rescue. The availability of the rescued H3N2 subtype swine infl uenza viruses laid the foundation for biological characteristics, molecular mechanism and vaccine development.

关 键 词：RT-PCR 猪流感 H3N2亚型 反向遗传操作技术 

分 类 号：S852.65[农业科学—基础兽医学]