【作者】 冯婷； 涂文伟； 黄娟； 李晋蓉； 殷仲伟； 范营营； 李虹；

【Author】 Feng Ting,Tu Wenwei,Huang Juan,Li Jingrong,Yin Zhongwei,Fan Yingying,Li Hong.The Joint Research Center of West China Second University Hospital of Sichuan University and Faculty of Medicine,the University of Hong Kong, Chengdu 610041,China

【机构】 四川大学华西第二医院港大联合研究中心； 香港大学李嘉诚医学院儿科及青少年医学系；

【摘要】 目的优化流感病毒HlNl在MDCK细胞上的培养条件,高效扩增流感病毒。方法对MDCK细胞培养流感病毒时添加的TPCK-Trypsin的浓度和培养基的种类进行了筛选;比较了不同细胞代次的MDCK对流感病毒的易感性;按MOI值0.001、0.01、0.1于MDCK细胞上接种H1N1,CPE>75%收获病毒上清,并按MOI值0.001接种H1N1后连续培养5天,分别于1、2、3、4、5天收获病毒上清,HA试验检测上清滴度,上清浓缩纯化后行TCID50检测,分别选取最佳的接种MOI值和收毒时间;最后对浓缩纯化病毒的红细胞吸附方法进行检验。结果通过对比,TPCK-Trypsin的最佳作用浓度是0.25ug/ml,MEM为培养病毒的最佳培养介质;代次低的MDCK对流感病毒的易感性明显强于代次高的MDCK细胞;于MDCK细胞按MOI值0.001接种H1N1,且第3天收获的上清病毒滴度最高;红细胞吸附法能高效浓缩纯化流感病毒。结论优化了在MDCK细胞上培养及纯化流感病毒的方法 。更多还原

【Abstract】 Objective To optimize the cultivation condition of influenza virus(H1N1) on MDCK cells and expand H1N1 efficiently.Methods Compare the HA titer of H1N1 cultivation supernatants with different concentration of TPCK-Trypsin as an addition,different culture medium and also the effect of cell generation on sensitivity of influenza virus;Seed H1N1 as an inoculum on MDCK cells at MOI 0.001,0.01,0.1 and harvest the supernatants when CPE is over 75%;Seed H1N1 as an inoculum on MDCK cells at MOI 0.001 and harvest the supernatants at day 1,2,3,4,5;Compare the titer of hemagglutination of the supernatants and the TCID50 of the purified supernatants;Then,test the efficiency of hemadsorption method for purification of H1N1.Results the optimized TPCK-Trypsin concentration is 0.25ug/ml, and MEM supports the most efficient expansion of H1N1;MDCK of low generation has much more higher sensitivity of influenza virus than that of MDCK of high generation.0.001 is the favorable MOI for H1N1 as an inoculum on MDCK cells, and the titer both of hemagglutination and TCID50 of the supernatants harvested on day 3 are the highest.Hemadsorption method for purification of H1N1 is efficient.Contusion We have achieved the optimized culture condition of H1N1 on MDCK cells and the purification method.更多还原