H1N1亚型猪流感病毒的拯救  

The Rescue of H1N1 Subtype Swine Influenza Virus

作　　者：彭亚平[1] 周红波[1] 李春[1] 金梅林[1] 

机构地区：[1]华中农业大学动物医学院农业微生物学国家重点实验室,武汉430070

出　　处：《生物工程学报》2008年第5期857-861,共5页Chinese Journal of Biotechnology

基　　金：国家"973"计划项目(No.2005CB523003);国家"十五"科技攻关项目(No.2004BA519A65)资助~~

摘　　要：利用8质粒拯救系统成功拯救出了猪流感病毒毒株A/Swine/TianJin/01/2004(H1N1)(A/S/TJ/04)。将猪流感病毒8个基因节段经RT-PCR合成cDNA后,分别克隆到RNA聚合酶I/II双向表达载体PHW2000中,构建成8个重组质粒。用8个重组质粒共转染COS-1细胞,30h后加入TPCK-胰酶至终浓度0.5μg/mL。共转染48小时后收获COS-1细胞及其上清,经尿囊腔接种9日龄SPF鸡胚。收获死亡鸡胚尿囊液并继续用SPF鸡胚传3代,得到有感染性的病毒。经血凝、血凝抑制验、测序分析、电镜观察等均证实了A/S/TJ/04猪流感病毒的成功拯救。这是目前国内首次报道拯救出H1N1亚型猪流感病毒,为进一步研究猪流感病毒基因组结构与功能的关系、流感跨种传播的机制以及构建新型猪流感疫苗株奠定了基础。The swine influenza virus （SIV） strain A/Swine/TianJin/01/2004（H1N1） （A/S/TJ/04） was rescued successfully by an eight-plasmid rescue system. The cDNAs of SIV 8 gene segments were synthesized by RT-PCR and cloned into the RNA polymerase I/II bidirection expression vector PHW2000 independently, resulting in 8 recombinant plasmids. The 8 recombinant plasrnids were cotransfected, xi into COS- 1 cell, 30 h later TPCK-trypsin was added to 0.5 μg/mL. The COS-1 cell and supernatant were harvested 48 h after cotransfection and were inoculated into the allantoic cavity of 9-day-old specific-pathgen free （SPF） chicken eggs. The allantoic fluid of dead eggs was harvested and passaged 3 generations in SPF chicken eggs to get infective virus. The successful rescue of A/S/TJ/04 SIV was identified by hemagglutination assay, hemagglutination inhibition assay, sequence analysis and electron microscope observation. The successful rescue of SIV built a platform for the research of the relationship between genome structure and function of SIV, the mechanisms of trans-species transmission of influenza virus and for the generation of new SIV as vaccine.

关 键 词：猪流感病毒 双向表达载体 拯救 共转染 

分 类 号：S852.65[农业科学—基础兽医学]