流感病毒在狗肾上皮细胞上的最优培养条件  

Optimal culture conditions of influenza virus in renal epithelial cells of dog

作　　者：马萍[1] 李欣玥 

机构地区：[1]陕西省疾病预防控制中心病毒室,陕西西安710054 [2]渭南市疾病预防控制中心,陕西渭南714000

出　　处：《现代预防医学》2017年第23期4326-4331,共6页Modern Preventive Medicine

基　　金："十二五"国家科技重大专项(2013ZX10004202-001-002)

摘　　要：目的优化不同亚型流感病毒在狗肾上皮细胞(MDCK)上最优培养条件,提高不同亚型流感病毒在MDCK细胞上的分离效果。方法将甲型H1N1、H3N2、Victoria、Yamagata四种亚型的流感病毒以不同的培养条件:不同培养温度、不同培养基、不同的牛血清浓度、不同L-1(对-甲苯磺酰)苯丙胺酰氯甲酮(TPCK)胰酶浓度、不同的接种量、不同的接种时间接种到MDCK细胞上,在24h、48h、72h、96h取培养物上清测血凝滴度,比较不同亚型流感病毒在MDCK细胞病变速度和病毒含量的差异。结果甲型H1N1流感病毒的最优培养温度为35℃,DMEM培养基,无牛血清,TPCK胰酶浓度为1-3μg/ml,接种300ml,不吸附或吸附1.5-3.0小时;H3N2病毒的最优培养温度为34℃,DMEM培养基,无牛血清,TPCK胰酶浓度为1μg/ml,吸附1.5小时,接种300ml;Victoria流感病毒的最优培养温度为35℃,DMEM培养基,无牛血清,TPCK胰酶浓度为0.5-2.0μg/ml,接种200ml,吸附1.5小时;Yamagata流感病毒的最优培养温度为35℃,DMEM培养基,无牛血清,TPCK胰酶浓度为1μg/ml,接种200ml,吸附1.5小时。结论根据流感病毒不同亚型来选择流感病毒的培养条件可获得更好的实验结果。Objective To optimize the culture conditions of different subtypes of influenza virus on MDCK cells and to improve the isolation of different subtypes of influenza virus on MDCK cells. Methods The virus specimen which came from Influenza A （ H1 N1 ） ,H3N2 ,Victoria and Yamagata were cultivated by MDCK cells in different culture conditions ： under different culture temperature, different culture medium, different concentrations of bovine serum, different concentrations of TPCK trypsin, different inoculum size, different inoculation time. We measured Hernagglutination titer at 24h,48h ,72h and 96h and compared the time of CPE and viral content of different subtypes of influenza virus. Results The optimal culture condition of influenza A （H1 N1 ） virus was the temperature of 35℃, DMEM medium, no bovine serum, concentration of TPCK trypsin of 1 -3 ~g/ml, inoculated with 300ml, no adsorption or adsorption for 1.5 -3hours. H3N2 virus optimal culture was the temperature of 34℃, DMEM medium, no bovine serum, concentration of TPCK trypsin of 1μg/ml, adsorption 1.5 hours, inoculation 300ml. Victoria influenza virus optimal culture was the temperature of 35℃, DMEM medium, no bovine serum, TPCK trypsin concentration of 0.5 -21xg/ml,inoculated with 200ml, adsorption for 1.5 hours. Yamagata influenza virus optimal culture was the temperature of 35℃,DMEM medium,no bovine serum, TPCK trypsin concentration of 1μg/ml, inoculation of 200ml, adsorption 1.5 hours. Conclusion We should select the culture conditions according to the different subtypes of influenza virus.

关 键 词：流感病毒 MDCK细胞 分离培养 

分 类 号：R113[医药卫生—公共卫生与预防医学]