Preliminary Exploration On The Technology Parameters Of Suspension Culture Of Mink Canine Distemper Virus Vaccine

Canine Distemper(CD)is a highly contagious infectious disease,which has become one of the diseases that seriously affect the development of mink breeding industries in China.The disease occurs in the United States,Britain,France,Italy,Spain and other developed countries.The disease has been effectively controlled in some developed countries,and the disease causes serious economic losses to the mink breeding industries in China every year.The main measure to prevent the disease is to vaccinate in minks.The common vaccines common in the domestic market are attenuated Canine Distemper virus of chick embryo and Vero call.Both of these attenuated virus vaccines are all produced by the rotation bottle culture process,and the suspension cultivation techniques of biological products are still not applied in the development of canine distemper.Based on findings,the parameters are still not determined of the suspension cultivation techniques in laboratory and the canine distemper virus vaccines are not commercial.In this study we carried out an optimized analysis on this issue and cultured Vero cells with microcarrier suspension cultivation techniques in bioreactors to screen their parameters,including microcarrier concentration,culture medium,serum concentration,cell seeding density,and culture method,furtherly to optimize different parameters for culturing canine distemper-toxic strain(CDV3-CL)with microcarrier suspension cultivation technology,including culture temperature,virus multiplicity,and virus-collection time.The control variate method was used to screen the relevant parameters for Vero cell growth and the results showed that the carrier utilization rate can reach a higher level with the concentration of 5g/L in 1L bioreactor culture system;DMEM medium was more suitable for Vero Large-scale culture in cell microcarrier bioreactors;Vero cells could adhere to microcarriers well and began to proliferate in medium with concentration of 10% for fetal bovine serum;with inoculum concentrations,30 to 40 cells for per microcarrier,the cells could grow over the surface of the entire microcarrier after the logarithmic growth phase.Perfusion culture was more suitable for Vero cells.On the basis of the optimization of attenuated mink distemper virus vaccine training process,the results indicated that temperature 33℃,was more suitable for viral replication.Vaccines could be produced massively at the virus infection complex of 0.03.The viral titers could reach a high level at 108 hours post inoculation,which was the best time to harvest the virus.In the present study,we conducted safety trails in minks with vaccines produced as optimized parameters and processes and the results indicated that the vaccine was safe,which provided basic data for the large-scale production of CDV3-CL vaccines in bioreactor.