在MDCK细胞上高产的乙型流行性感冒病毒株的筛选及其全基因组克隆  

Screening of the High-yield Influenza B Virus on MDCK Cell and Cloning of Its Whole Genome

作　　者：齐立[1] 吴昆昱[1] 李梓[1] 张烨[1] 董婕[1] 张立国[1] 张智清[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》2004年第3期195-199,共5页Chinese Journal of Virology

基　　金：科研院所技术开发研究专项基金(NCSTE-2000-JKZX-231);"973"计划传染病防治基础研究(973-G19999054108)。

摘　　要：流行性感冒(流感)病毒可引起世界范围的大流行,因此需要及时地生产流感疫苗来预防流感的流行。乙型流感病毒是疫苗的重要组成部分。传统制备疫苗的方法是使用鸡胚生产,存在很多缺陷,应用哺乳动物细胞生产流感疫苗是个更好的选择。但是,乙型流感病毒感染MDCK细胞后,很难得到持续的高产。此实验筛选到的乙型流感病毒在MDCK细胞连续传代15代后,PFU值从第1代的8×104到第15代2×109,TCID50从第1代6 5到第15代的8 0。将筛选到的毒株进行全基因组克隆,用两套引物获得乙型流感病毒的全部8个节段。此结果为利用反向遗传技术将高产毒株与流行毒株重配,进而在哺乳动物细胞上生产基因工程流感疫苗打下基础。Influenza virus may cause pandemic in the world.Thus, influenza vaccine should be produced timely and effectively.There are many advantages to produce influenza vaccine in mammalian cell culture than in chicken embryos.But it is difficult to obtain high titer of the virus on MDCK cell.We have passaged B/jingke/96/01 influenza virus strain on MDCK cell for 15 passages,the PFU reached from the first passage 8×10~4,to the fifteenth passage 2×10~9,the TCID_(50)/ml from 6.5 of the first passage to 8.0 of the fifteenth passage.Not only B/jingke/96/01 had high productive characteristics in MDCK cell,but also had a tendency of virulence increasing.We designed two sets of universal primers to amplify the whole genome of influenza virus B/jingke/96/01.In order to produce the recombinant influenza vaccine,this strain may be reassorted with the current influenza virus strain as a master strain by reverse genetic technique.

关 键 词：流行性感冒 病毒 乙型流感 毒株 通用引物 哺乳动物细胞 疫苗 全基因组克隆 

分 类 号：R373.13[医药卫生—病原生物学] Q78[医药卫生—基础医学]