猪流行性腹泻病毒变异株CH/GX/750A/2015在Vero细胞上转瓶培养条件的优化试验  

Optimization of Large-scale Rotary Bottle Culture Conditions forPorcine Epidemic Diarrhea Virus Mutant CH/GX/750A/2015

作　　者：卢冰霞[1] 陶明华 赵硕 秦毅斌[1] 刘磊[1,4] 陈忠伟 何颖[1] 段群棚[1] 梁家幸[1] 周英宁[1] 李斌[1] 蒋冬福[1] 杨思仪[1] 赵武[1] LU Bingxia;TAO Minghua;ZHAO Shuo;QIN Yibin;LIU Lei;CHEN Zhongwei;HE Ying;DUAN Qunpeng;LIANG Jiaxing;ZHOU Yingning;LI Bin;JIANG Dongfu;YANG Siyi;ZHAO Wu(Guangxi Key Laboratory of Veterinary Biotechnology,Guangxi Veterinary Research Institute,Nanning 530001,China;Jiangxi Shangrao Animal Husbandry and Veterinary Science Research Institute,Shangrao 334000,China;Guangxi University,Nanning 530005,China;Guangxi Agricultural Vocational and Technical College,Nanning 530007,China)

机构地区：[1]广西兽医研究所,广西兽医生物技术重点实验室,南宁530001 [2]江西省上饶市畜牧兽医科学研究所,上饶334000 [3]广西大学,南宁530005 [4]广西农业职业技术学院,南宁530007

出　　处：《中国畜牧兽医》2020年第5期1506-1513,共8页China Animal Husbandry & Veterinary Medicine

基　　金：广西科技重大专项(桂科AA17204057、桂科AA18118051);广西自然科学基金项目(2017GXNSFBA198092);广西兽医生物技术重点实验室开发基金课题(17-259-36-B-2、16-380-45-B-3);广西基本科研业务费专项(桂科专项17-2)。

摘　　要：本研究旨在确定猪流行性腹泻病毒(PEDV)变异株CH/GX/750A/2015株在Vero细胞上转瓶培养的最佳条件,为大规模生产高效PEDV变异株疫苗提供技术支撑。试验以10 L转瓶培养病毒,对接毒时细胞维持液中胰酶浓度(0、2、4、6、8和10μg/mL)、细胞密度(40%、60%、80%、100%、200%)、接毒剂量(MOI分别为1、0.1、0.01和0.001)、吸附时间(0、30、60、90、120、240 min)、收毒时间(接毒后12、24、36、48、60、72 h)、维持培养温度(35、36、37和38℃)和转瓶转速(6、8、10、12、14、16 r/h)7个条件进行优化。结果显示,PEDV CH/GX/750A/2015株在10 L转瓶中的最佳培养条件为:细胞刚铺满单层时接毒,接毒量为MOI=0.01,维持液(无血清DMEM培养液)中胰酶质量浓度为6μg/mL,不需吸附,维持培养温度为37℃,12 r/h旋转培养48 h后收获病毒液可获得较高效价的病毒液,效价可稳定达到106.50 TCID50/0.1 mL。本试验为PEDV变异株大量培养提供了参考,为变异株疫苗研发奠定了基础。The purpose of this study was to determine the optimal conditions for the rotation culture of CH/GX/750A/2015 strain of porcine epidemic diarrhea virus(PEDV)on Vero cells,and provide technical support for the mass production of high-efficiency vaccine.The virus was cultured on Vero cells in a 10 L spinner flask.The conditions of trypsin concentrations(0,2,4,6,8,10μg/mL),cell density(40%,60%,80%,100%and 200%),virus inoculation dose(MOI=1,0.1,0.01 and 0.001),virus adsorption time(0,30,60,90,120 and 240 min),virus harvest time(12,24,36,48,60,72 h after virus inoculation),culture temperature after virus inoculation(35,36,37 and 38℃)and spinner speed(6,8,10,12,14 and 16 r/h)were optimized.The results showed that the best culture condition of PEDV CH/GX/750A/2015 strain in 10 L transfer flask was to inoculate the virus when the cell was just covered with a single layer,the dose of virus was MOI=0.01,the concentration of trypsin in the culture medium after virus inoculation was 6μg/mL,without adsorption,the culture temperature after virus inoculation was 37℃,the best spinner speed 12 r/h,and the best virus harvest time was 48 h after virus inoculation.After optimization,the virus titer can reach 106.50 TCID50/0.1 mL.This study provided a reference for the large-scale culture of PEDV mutant strain,and laid a foundation for the development of high-efficiency vaccine of PEDV mutant strain.

关 键 词：猪流行性腹泻病毒 变异株 转瓶 培养条件 

分 类 号：S852.65[农业科学—基础兽医学]