Basic Research On Preparation And Immune Effect Of Infectious Bursal Of Chicken Subunit Vaccine

Infectious bursal disease(IBD)is an acute,highly contagious infectious disease caused by Infectious Bursal Disease Virus(IBDV).It can lead to a certain degree of immunosuppression in infected chickens,which further increases the economic loss.So far,with the emergence of mutant strains and super-virulent strains,the role of traditional vaccines has gradually decreased,so a A new type of vaccine has important practical significance.Among them,VP2 protein is a protective antigen of infectious bursal disease virus and has become the preferred target protein for the development of new vaccines such as genetic engineering subunit vaccines.So far,subunit vaccines based on VP2 protein have not been widely used in production practice.The main reasons are whether soluble expression proteins can be formed,the cost of processing technology and the protection rate of vaccines formulated.Therefore,this paper develops a related experimental study on a genetic engineering subunit vaccine with low cost and high protection rate.In this experiment,the yeast-derived SUMO gene and VP2 gene were ligated into the prokaryotic expression vector pETDuet-1 to construct a recombinant plasmid,which was transformed into different competent cells,and the correct expression was carried out,and the competent cells were selected.Optimal culture temperature,induction concentration and time to select the optimal expression strain for preparation for subsequent experiments;then use the selected optimal strains for large-scale culture to obtain sufficient bacterial colonies,resuspended in PBS The high-pressure homogenizer was used to crush the bacterial liquid,and the obtained crushed bacterial liquid was purified by two different methods of affinity chromatography nickel column and ammonium sulfate precipitation.The protein solution obtained by purification of the affinity chromatography nickel column is subjected to ultrafiltration treatment,and the protein solution obtained by precipitation and purification of ammonium sulfate is subjected to dialysis treatment,and the two processed protein solutions are further subjected to endotoxin by using Triton X-114.Remove the test and find out the suitable conditions for removing endotoxin.At the same time,the inactivation process was explored,and the traditional method of formaldehyde and pyrrole(BEI)was used to inactivate the protein solution.Finally,the best method was used to inactivate the protein solution and the corresponding oil adjuvant according to a certain ratio.The vaccine is prepared,and the prepared vaccine is subjected to stability test and sterility test.Finally,the prepared vaccine is immunized with SPF chickens of 21 days old for animal test,and the best titer protein solution is selected for subsequent test by safety test.The titer of the protein solution for the potency and the protective test for the challenge were used to fully verify the immune effect of the self-made vaccine,and to test whether the chicken body has side effects and weight gain.The study found that the protein expressed by the best strain Duet-2 was a soluble protein,which was confirmed by Western-Blot.Through the exploration of the purification process,the affinity protein used in the test and ammonium sulfate precipitation can be used to obtain the target protein.After repeated treatment of the sample with Triton X-114,the endotoxin content is less than 1000 EU/mL,which meets the relevant quality standards and proves to be utilized.The method can effectively remove endotoxin and has no significant effect on protein titer.After the comparison of the two inactivation tests,formaldehyde was finally used for the inactivation test,and the inactivated protein solution and the oil adjuvant were mixed in a certain ratio to prepare a vaccine to verify the stability.The sterility test was established and can be used for subsequent experiments.Finally,using the vaccine prepared in this paper for animal experiments,it was found that the protection ratio of 1:16(crude pure)vaccine group and 1:16 vaccine group can reach 80%to have a good protective effect.In summary,this strain obtained the soluble expression of the strain Duet-2,and the SPF chicken can be protected by the preparation of the vaccine with the target protein expressed by the strain,of which 1:16(coarse)The protection rate of the vaccine group and the 1:16 vaccine group were both 80%,and the 1:16(coarse)can greatly reduce the production cost.At the same time,the purification operation by ammonium sulfate precipitation is simple and the requirements for the required equipment are low.Subsequent testing of the 1:16(coarse)vaccine group can be carried out in a deep,multifaceted,and reproducible test.Provide theoretical support for the widespread application of genetic engineering subunit vaccines.