流感病毒在MDCK细胞上培养条件的优化分析  

Optimization and analysis of culture condition for cultivation of influenza virus on MDCK cell

作　　者：周大宇[1] 李春[2] 赵晓辉[3] 孙佩龙[1] 张旭[1] 毛玲玲[4] 

机构地区：[1]锦州市疾病预防控制中心,辽宁锦州121000 [2]渤海大学,辽宁锦州121000 [3]辽宁医学院附属第一医院,辽宁锦州121001 [4]辽宁省疾病预防控制中心,辽宁沈阳110000

出　　处：《现代预防医学》2015年第18期3368-3370,3383,共4页Modern Preventive Medicine

基　　金：国家重大传染病防治科技重大专项项目(2012ZX10004209-001);食源性致病微生物的生物信息学研究(LT2014024)

摘　　要：目的 优化甲、乙型流感病毒在MDCK细胞上的培养条件,提高流感病毒的分离效果。方法 将5种流感病毒液（甲型流感病毒：甲型H1N1、季节性H1N1、季节性H3N2和乙型流感病毒：Victoria、Yamagata）分别接种于MDCK细胞,通过比较不同的病毒接种量（30μl/cm2、60μl/cm2、90μl/cm2、120μl/cm2、150μl/cm2）、不同的病毒孵育时间（0.5 h、1.0 h、1.5 h、2.0 h、2.5 h、3.0 h）、不同的TPCK-胰酶添加浓度（1.0μg/ml、2.0μg/ml、3.0μg/ml、4.0μg/ml、5/0μg/ml）以及不同的MDCK细胞增毒代次（连续盲传5代）对5种流感病毒易感性的影响,确定其最佳分离效果;对分离后的病毒以红细胞凝集（HA）方法检测病毒滴度,结果 通过对比病毒HA滴度结果,5种流感病毒（甲型H1N1、季节性H1N1、季节性H3N2、Victoria和Yamagata）的最佳病毒接种量分别为：90μl/cm2、90μl/cm2、60μl/cm2、120μl/cm2、120μl/cm2;最佳病毒孵育时间：甲型流感病毒为1.0~1.5 h,乙型流感病毒为2.0~2.5 h;胰酶最佳使用浓度分别是：2.0μg/ml、2.0μg/ml、2.0μg/ml、3.0μg/ml、3.0μg/ml;最适宜的细胞增毒传代次数为：1代、3代、2代、2代、2代;甲型流感病毒经培养后病毒的HA滴度低于乙型流感,甲型H1N1流感病毒经培养后HA滴度较低。结论 甲型流感病毒对MDCK细胞的易感性低于乙型流感病毒;甲型H1N1流感病毒的分离效果低于季节性H1N1和季节性H3N2;Victoria与Yamagata的分离条件基本相同。Objective The aim of this study was to optimize the cultivation condition of influenza virus A and B on MDCK cells, in order to improve the separation rate of the influenza virus. Methods The virus specimens from five kinds of the influenza virus were cultivated on MDCK cells. The viruses included swine-origin H1N1, seasonal H1N1, seasonal H3N2, Victoria and Yamagata. The most suitable condition was made by comparing the different inoculum size （30 μl/cm2, 60 μl/cm2, 90 μl/cm2, 120 μl/cm2, and 150 μl/cm2）, the different virus incubate time （0.Sh,1.0h, 1.Sh, 2.0h, 2.5h, and 3.0h）, and the different concentration of TPCK-Trypsin （1.0μg/ml, 2.0 μg/ml, 3.0 μg/ml, 4.0 μg/ml, and 5.0 μg/ml）. In addition, the different susceptibility of cell generations for serial passage culture was analyzed for five generations of these five influenza viruses, The efficiency of hemadsorption for isolated influenza virus was then tested. Results Throughout the comparison the five kinds of the influenza virus, swine-origin H1N1, seasonal H1N1, seasonal H3N2, Victoria and Yamagata had the most suitable inoculum size of 90 μl/cm2, 90 μl/cm2, 60 μl/cm2, 120 μl/cm2, 120 μl/cm2, the most suitable concentration of TPCK-Trypsin of 2.0 μg/ml, 2.0 μg/ml, 2.0 μg/ml, 3.0 μg/ml, 3.0 μg/ml, and the most incubate time 1.0h - 1.Sh （influenza virus A） and 2.0h - 2.5h （influenza virus B） and the most suitable generations of cell 1, 3, 2, 2, 2 respectively. The hemagglutination inhibition （HI） of influenza virus A was lower than the influenza virus B. And swine-origin H 1N 1 had the weak HI on MDCK cells after cultivated. Conclusion The separating effect of the influenza virus B on the MDCK cell was better than influenza virus A. The effect of the swine-origin H1N1 virus on the MDCK cell was inferior to the seasonal H 1N 1 and seasonal H3N2. And the culture condition of Victoria and Yamagata were roughly similar.

关 键 词：流感病毒 MDCK细胞 分离培养 

分 类 号：R183.3[医药卫生—流行病学]