Study On Micro-carrier Culture Of MDV And Protective Efficacy Of Vaccine

Marek's disease(MD)is an immunosuppressive disease caused by Marek's disease virus(MDV)serotype 1,which is characterized by lymphoid tissue hyperplasia,causing serious economic losses to the poultry industry.At present,vaccination is the primary means of preventing the occurrence of Marek's disease.In china,the production of the Marek's virus vaccine is usually carried out using chicken embryo fibroblasts as a material in glass rotary bottle-based culture system.Due to the high cost of using SPF chicken embryo,the time required for primary cell preparation and the high risk of contamination in cell preparation process,traditional vaccine preparation methods have been incompatible with the current large-scale suspension of animal culture.In order to study the more efficient culture of Marek's virus vaccine,in this study,DF-1 cells were used instead of traditional chicken embryo fibroblasts,and the Marek's virus vaccine was cultured by microcarrier suspension culture instead of the traditional glass rotary bottle-based to provide a reference for the mass production of Marek's virus vaccine by bioreactor.In traditional bottle-based culture,DF-1 cell culture effect were best grown at 37°C and p H7.2 with DMEM/F12 medium containing 10%neonatal bovine serum but no HEPES buffer in traditional bottle based culture.In cell passage,the cells were inoculated at an area of adherence per square centimeter 8×10~4-12×10~4 DF-1 cells are more conducive to cell culture.In microcarrier culture,using 20r/min continuous stirring of the initial cell culture,microcarrier concentration was 3-7g/L,Fifty DF-1 cells were inoculated with each microcarrier to obtain the best suspension culture effect.The results showed that MDV(CVI988/Rispens strain)can proliferate well on DF-1 cells by optimizing the proliferation conditions.in traditional bottle based culture,Using simultaneous poisoning,MOI0.02,24h replacement of maintenance solution,72h after poisoning in the DF-1 cells can harvest the most virus.The microcarrier culture of MDV(CVI988/Rispens strain)test showed that the best virus culture effect was achieved by using the method of simultaneous poisoning,150-200 cells on the surface of each microcarrier,MOI 0.02.Immune protection test shows that the SPF chickens were immunized with the microcarrier suspension culture of the MDV(CVI988/Rispens strain)vaccine,Five days after immunization,chicks were challenged with Md5 strain.The results showed that MDV(CVI988/Rispens strain)vaccine which culturedby microcarrier culturesystem could achieve the same protectio n efficiency as commercial vaccine CVI988/Rispensstrain,reaching more than 90%.Vaccine immunization had no significant effect on chicken growth status,body weight and immune organs.In a word,this study in this paper provided a new way for the large-scale production of MDV vaccines and other biological products,which has great theoretical and practical value.