Technological Study On Domestication Of PEDV By Animal Cell Suspension Culture

Objective:The XJ-DB2 strain of porcine epidemic diarrhea virus was acclimated and subcultured on ST suspension cells,and the strain was obtained to adapt to the whole suspension culture technology,providing reference data for virus production process amplification.Methods:Firstly,the XJ-DB2 strain of porcine epidemicdiarrheaviruswas cultured and subcultured to improve the virus content.Then,the optimized adherent culture seed virus was inoculated on ST suspension cells to study the growth characteristics through blind a cclimation.The main experimental contents include:(1)The strain was continuously acclimated and subcul tured in three different sources of adherent Vero cells.The most sensitive and highly expressed adherent cell strain was obtained by comparing the pathological changes,virus content and harvest time after inoculation.(2)The optimized adherent cytotoxic suspension cells were cultured in shake flasks for preliminary sensitiv ity acclimation and passage.The technical parameters of shake flasks suspension culture were obtained by s creening suspension sensitive cells,virus maintenance solution,virus exposure process,infection dose and cell culture time.(3)According to the technical parameters obtained in the shake flask culture mode,the pr eliminary scale- up culture was carried out in the bioreactor culture mode,and the difference between the rapid flow reactor and the tank reactor was tested to verify the stability of the acclimated suspension virus.Results:(1)After adaptive passage,XJ-DB2 strain was subcultured in ZJ Vero cells,and the virus content reached 10^7.5TCID50/ml in p21 generation.The results showed that the sensitivity and adaptability of the isolated strainweredifferentin Vero cell lines from different sources,and the pathological changes and expression levels in the passage pro cess were quite different.(2)After 3 days of culture,the cells were inoculated with the virus.Centrifugation wasusedtochangetheculturemedium,and Celkey^?CD ST 258 growth liquid was used to inoculate the virus,and the infection dose was 0.002 MOI.A fter 48 hours of culture,the virus expression reached 10^7.5-8.0 TCID₅₀/ml.(3)The culture parameters of ST suspension cells infected with PEDV virus were obtained,that is,the cells were inoculated on the second day,and the cells were diluted to 3.0×10⁶cells/m L,the titer of suspension seed was 10^7.0 TCID₅₀/ml(2-5 generations).The amount of exposure was in accordance with the standard 0.04 MOI.The concentration of trypsin was 5 mg/L.serum-free medium Celkey^?CD ST 258 was used in both growth and production stages,adjust the temperature to 35?after inoculation.According to the above process parameters,the titer of TCID₅₀ of ST suspension cells infected by PEDV could be maintained above 10^7.5 TCID₅₀/ml in shake flask and 50 L torrent bioreactor.