应用反向遗传学技术在哺乳动物细胞中产生甲型流感病毒  

Generation of Influenza A Virus from Separated Cloned cDNA Fragments

作　　者：陈稚峰[1] 张立国[1] 董婕[1] 吴昆昱[1] 陈爱珺[1] 孙梅生[1] 黄银霞[1] 齐立[1] 张智清[1] 郭元吉[1] 

机构地区：[1]中国疾病预防控制中心病毒病预防控制所,北京100052

出　　处：《病毒学报》2002年第3期193-197,共5页Chinese Journal of Virology

基　　金：国家科技部科研院所技术开发研究专项资金 (NC STE -2 0 0 0 -JKZX -2 3 1) ;"973"计划传染病防治基础研究课题( 973 -G199990 5 410 8)资助

摘　　要：在国内首次应用反向遗传学技术将多个质粒共转染哺乳动物细胞 ,得到了具有活性的甲型流感病毒。采用自行构建的含有 polⅠ和polⅡ启动子和终止子序列的pIVV2质粒 ,将A/PR/8/34(H1N1)流感病毒基因组全部 8个RNA节段的cDNA分别克隆到该质粒的两个启动子之间 ,得到了 8个可在真核细胞中复制和表达的质粒。将这 8个质粒共转染 2 93T细胞 ,培养 4 8h后吸取上清液 ,转接入MDCK细胞中 ,再经过 72h培养 ,发现在MDCK细胞中有明显的流感样细胞病变产生 ,测上清病毒血凝滴度为 1:10。将MDCK细胞冻融液继续接种鸡胚和MDCK细胞 ,培养后将鸡胚尿囊液和MDCK细胞冻融上清液进行血凝和血凝抑制实验 ,证明所得病毒为A/PR/8/34(H1N1)。In order to get whole active influenza A virus from separated cDNA fragments,we subcloned these cDNA fragments of influenza virus strain A/PR/8/34(H 1N 1) into PIVV2 plasmid which had promoter and terminator of RNA polymerase Ⅰ(polⅠ),promoter of RNA polymerase Ⅱ(polⅡ) and a polyadenylation site This entire polⅠ transcription unit was flanked by an RNA polymerase Ⅱ(polⅡ) promoter and a polyadenylation site The orientation of the two transcription units allowed the synthesis of negative RNA gene and positive mRNA from one viral cDNA template These eight plasmids were cotransfected into 293T cell simultaneously We transferred the supernatant into a cultured MDCK cell after incubation of 48 hours The apparent cell pathological effect(CPE) appeared after 72 hours,a titer of 1:10 was detected by HA experiment The supernatant was injected into chicken eggs and inoculated onto new MDCK cells HA and HI experiments were then performed The virus generated was confirmed to be A/PR/8/34 This is the first report in China that live virus is generated from eight separated cDNA fragments

关 键 词：反向遗传学技术 哺乳动物细胞 甲型流感病毒 质粒 共转染 

分 类 号：R373.13[医药卫生—病原生物学]