Primary Research On Thermostable Protective Agent Of Recombinant Adenovirus Vaccine Against BVDV

Bovine viral diarrhea（BVD）is one of the important diseases that seriously endanger the development of animal husbandry in China.It is highly contagious and has a high mortality rate.Currently,prevention is the main focus of the disease,and there is no specific drug treatment.The most effective measure to prevent and control the disease is mainly vaccination.Due to the limitations of traditional vaccines,their immune failure rate is also relatively high.At present,adenovirus vector vaccines have become the focus of scientists’attention.Adenovirus can serve as a vector to deliver exogenous genes,so it is widely used in gene therapy and vaccine development.Recombinant adenovirus vaccine vectors can stimulate the body to produce strong cellular and humoral immunity,with higher safety.The recombinant novel bovine viral diarrhea virus vaccine successfully constructed in this experiment has a good preventive effect on the disease.However,the poor thermal stability of adenovirus products poses significant difficulties in transportation and storage,greatly limiting their clinical application.To solve the problem of poor thermal stability of the recombinant adenovirus vector novel live vaccine for bovine viral diarrhea disease,the addition of heat-resistant protective agents during freeze-drying can effectively maintain the original physical and chemical properties and biological activity of the product,with minimal loss of active ingredients.In addition,due to the low water content of the vaccine,it is easy to preserve for a long time and has a loose porous structure,which can quickly and completely dissolve after adding water.At present,most live vaccines use 5%sucrose and skimmed milk as protective agents.This protective agent is simple to prepare,low in cost,poor in thermal stability,and requires high storage conditions.Generally,it needs to be stored and transported below-15℃.The development of new live vaccine protectors is of great significance in addressing the poor thermal stability and high transportation and storage conditions of traditional formulas,as well as prolonging the sustained efficacy of vaccines.1.Screening and optimization of heat-resistant freeze-drying agent formula for BVDV recombinant adenovirus vector live vaccine.Based on the protective mechanism of each component,a single factor experiment was conducted based on the physicochemical properties and biological activity of the adenovirus vector live vaccine.Trehalose,mannitol,sucrose,glycine,human serum albumin,dextran,sorbitol,gelatin,PEG,PVP,etc.were selected as small molecular components of the adenovirus heat-resistant freeze-drying protective agent,Add 0.1%to 5%of the added amount to 10%milk powder solution to prepare protective agent 1-10,and add protective agent 1:1 to the virus solution for freeze-drying.Determine which substances have good high-temperature resistance by conducting tests at a constant temperature of 37℃for 7 and 14 days.Three factors with significant freeze-drying heat resistance protection rates were selected using the Plackett Burman experimental design,and then analyzed using the Box Behnken experiment to determine the optimal level.The results showed that after the heat resistance test,trehalose,mannitol,sucrose,human serum albumin,dextran,and gelatin had a good protective effect on the virus;The Plackett Burman test identified three significant factors:trehalose was highly significant,mannitol and dextran were significant,with a maximum response value of 91.91%.The optimal formula was optimized by response surface methodology as follows:trehalose 3%,mannitol 6%,dextran 3%,sucrose 2%,human serum albumin 0.3%,gelatin 2%.The maximum response value of the virus after freeze-drying with the optimal formula is 92.6471%,and the virus titer can reach 6.31 log₁₀TCID50/m L.2.Stability（shelf life）test of BVDV recombinant adenovirus vector live vaccine protective agent.The optimized BVDV recombinant adenovirus vector live vaccine was stored in an environment of 2-8℃for 18 months.After physical properties,residual water content,vacuum degree,sterility test,mycoplasma test,virus titer measurement,and aging resistance test,all quality indicators met the requirements.Therefore,the shelf life of the vaccine was set at 18 months.In conclusion,this research successfully examined a freeze-dried thermostable protective agent suitable for the live adenovirus vector vaccine of BVDV,thus furnishing a benchmark for the production and conservation of such a vaccine in the future.