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

doi:10.16431/j.cnki.1671-7236.2020.05.025

摘要/Abstract

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10 μg/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6 μg/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10^6.50 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。

关键词: 猪流行性腹泻病毒; 变异株; 转瓶; 培养条件

Abstract: 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(0,2,4,6,8,10 μg/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 6 μg/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 10^6.50 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.

Key words: porcine epidemic diarrhea virus; variant strain; rotary bottle; culture condition

中图分类号:

S852.65

卢冰霞, 陶明华, 赵硕, 秦毅斌, 刘磊, 陈忠伟, 何颖, 段群棚, 梁家幸, 周英宁, 李斌, 蒋冬福, 杨思仪, 赵武. 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[J]. 中国畜牧兽医, 2020, 47(5): 1506-1513.

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. Optimization of Large-scale Rotary Bottle Culture Conditions for Porcine Epidemic Diarrhea Virus Mutant CH/GX/750A/2015[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(5): 1506-1513.

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