The Optimiztion Of Production Process Of Chicken Marek's Disease Bivalent Vaccine (CVI988/Rispens+HVT)

The CEF cells were prepared with the same preparation method and culture condition, comparing with the cell quantity and growth complexion,there are no significant difference between SPF and normal chicken embryos.200-240 million CEFs(vivid cells)per embryo were prepared by magnetic mixing method,3-4 times of mixing by pipette or glass pearls,and the quantity of per embryo CEF were remarkably increased.4 ml 0.25%trypsin per embryo,six times of 400rpm centrifugation,1% FBS-Hanks digestion terminating media,5%FBS H10-m199 growth media are the best method of CEFs preparation. After being thawed,the CVI988 virus seeds were inoculated to revived first generation cells(F1),F1 inoculated to second generation cells(F2),F2 inoculated to vaccine generation,then harvested after 72 hrs culture and frozen to working virus seeds. Lyophilized HVT virus seeds were inoculated to F1 cells layer,F1 to F2,F2 to vaccine, then harvested as working virus seeds,which being inoculated into roller bottles directly to produce HVT vaccine.Comparative experiments on different thawing temperature were tested,F1 inoculating dosage,each roller bottle inoculating dosage, inoculation methods.Results show,when CVI988 virus seeds were thawed rapidly in 60s between 27 to 37℃,the loss of virus is less than 6%;inoculation dosages of CV1988 and HVT are optimum in 200,000 and 300,000 PFU each flask respectively; inoculation dosages of CVI988 and HVT are optimum in 12,000,000 and 8,000,000 PFU each roller bottle respectively;in the batch production,viruses are inoculated into roller bottler asynchronously,and the infected cells were cultured and harvested, the HVT and CVI988 are mingled together in the ratio 1:2.The yields of CVI988 and HVT virus were 50,000 and 100,000 PFU per roller bottle,and virus titer is up to the standards of procedure.F10-M199 with 1%fetal bovine serum(FBS)is the optimum medium for virus preservation,which could promote the growth of infected cells and reduce production cost highly. During the procedure of harvest and freeze of infected cells,the ration of trypsin and EDTA,concentration of DMSO of the freezing preserver,and freezing method are some principal factors to affect the virus titer.We did three comparative experiments and the results are as follows.Trypsin-EDTA in the ratio of 1:4 is good for the dispersion of cells,10%DMSO in the freezing preserver can hold back the harm of freeze to the cell membrane,and the freezing program of 4℃30min→1℃/min(-4℃)→25℃/min(-40℃)→15℃/min(-12℃)→1℃/min(-40℃)→10℃/min(-90℃)can hardly affect the virus titer.