China Animal Husbandry and Veterinary Medicine ›› 2020, Vol. 47 ›› Issue (5): 1506-1513.doi: 10.16431/j.cnki.1671-7236.2020.05.025

• Preventive Veterinary Medicine • Previous Articles     Next Articles

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

LU Bingxia1, TAO Minghua2, ZHAO Shuo3, QIN Yibin1, LIU Lei1,4, CHEN Zhongwei1, HE Ying1, DUAN Qunpeng1, LIANG Jiaxing1, ZHOU Yingning1, LI Bin1, JIANG Dongfu1, YANG Siyi1, ZHAO Wu1   

  1. 1. Guangxi Key Laboratory of Veterinary Biotechnology, Guangxi Veterinary Research Institute, Nanning 530001, China;
    2. Jiangxi Shangrao Animal Husbandry and Veterinary Science Research Institute, Shangrao 334000, China;
    3. Guangxi University, Nanning 530005, China;
    4. Guangxi Agricultural Vocational and Technical College, Nanning 530007, China
  • Online:2020-05-20 Published:2020-05-18

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 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.

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

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