Study On Thermostable Stabilizer In Canine Distemper Of Live Attenuated Vaccine

Biological products are susceptible to temperature and need to be kept at low temperatures during storage and transportation.However,relying on high-cost cold chain transportation has always been a huge problem faced by vaccine distribution.If the vaccine cannot be effectively preserved,it will bring huge economic losses such as immunization failure.According to the2001 edition of Quality Standards for Veterinary Biological Products in China,the effective shelf life of lyophilized live virus vaccine at 2-8?is 3-9 months.Limited shelf life limits the sustained efficacy of vaccines,but it also brings many inconveniences to remote areas where good preservation conditions cannot be provided in China.The stable preservation of biological products mainly depends on the addition of protective agents.At present,the traditional formula of freeze-drying protectant in our country include gelatin,sucrose,lactose,skim milk and other basic ingredients-which has many problems,such as simple formula,poor protection function and so on.Therefore,the development of a new type of live virus protectant is of great significance to solve the poor stability of traditional formulations and prolong the sustained efficacy of vaccines.Canine distemper?CD?is a highly contagious animal disease caused by Canine distemper virus?CDV?.The case fatality rate of CD is over 80%.CD is one of the most important diseases threatening pet health,fur economic animal breeding and wildlife conservation.Vaccination is the most effective to prevent CD.Most of the Ondersteport strains used in CD vaccine are Vero cell-adapted strains,which are relatively mature standard attenuated vaccine strains.They are commonly used in commercial vaccines at present.Lower titer and no visual cytopathy are the major barrier for culturing CDV in vitro.Meanwhile,CDV is a single negative-stranded RNA virus,whose characterization is very sensitive to temperature and external environment,and is not easy to be preserved effectively for a long time.Based on the above problems,it is necessary to develop a heat-resistant freeze-drying protectant for the stable preservation of CDV vaccine.Part ?:Optimization of Culture Conditions of Canine Distemper Virus?Ondersteport Strain?.Firstly,the effects of different doses on the growth kinetics curves of CDV influenced by different MOI were tested.The viral titer was detected after infecting Vero cells with multiple infection?MOI?of 0.5,0.1,0.01 and 1%,and 2%inoculation of Vero cells.Vero cells were infected with 1%and 2%of the inoculated dose,and the growth kinetics curves were plotted for1 to 5 days.Freeze-thaw and ultrasonic fragmentation were used to compare the results of effects of toxicity methods on CDV titer were studied.The best conditions of each group were used to establish the basic seed batch toxicity.The results showed that the highest titer of virus liquid harvested by 2%inoculation dose was 10^6.38 TCID₅₀/mL.The growth curve showed that the highest CDV titer was 10^6.681 TCID₅₀/mL on the 4th day after exposure.The traditional freeze-thaw method is superior to the ultrasonic method,and its titer is 10^7.38 TCID₅₀/mL.Three batches of basic seed virus were constructed by large-scale cultivation institute.Their aseptic test and mycoplasma test were qualified,which provided material basis for subsequent screening and optimization of protective agents.Part ?:Screening and deeply optimization of heat-resistant freeze-drying stabilizers for attenuated canine distemper vaccine.Based on the protective mechanism of various components,four formulations of A1,B2,C3 and D4 were designed according to the basic principles of the protective agents;The optimum basic formula was screened by aging test at 37?for 7 d;the significance of each component content in the optimal basic formula affecting the survival rate of virus was determined by Plackett-Burman test?PB test?;Regression analysis was established.Minitab 18software was used to determine the optimal combination of components under the maximum response value and to verify the freeze-drying.The results showed that the highest survival rate of C3 formula virus was 72.82%.The model of PB test?N=12?was not significant,and the maximum response value was 97.12%.The titer of the virus reached 10^6.335 TCID₅₀/mL after freeze-drying.In summary,a heat-resistant freeze-drying protectant for CDV?Ondersteport strain?has been successfully developed in this study,which provides a reference for the development and preservation of CDV vaccine.