| Egg yolk antibody(IgY)has been widely used in livestock production in China in recent years.Compared with antibiotics,IgY had the advantages of safety,non-residue,and non-drug resistance in the prevention and treatment of animal bacterial diseases,and could significantly improve the production performance of animals.According to the foreign studies,IgY could be digested into Fab' and pFc' fragments by pepsin under appropriate conditions.The Fab' fragment after enzymolysis still had antigen-binding activity,while the pFc' fragment could continue to be digested into pFc' small molecule fragments without any biological activity.Domestic researchers have further explored the optimal IgY digestion conditions by changing the pH of IgY digestion,the mass ratio of pepsin and IgY,and the reaction time.Compared with IgY,Fab' had more application value in animal disease control.The research content of this experiment was divided into the following two parts:Experiment I Optimized the digestion conditions of Fab' fragment from infectious bursal disease IgY by response surfaceIn this experiment,pepsin was used to digest infectious bursal disease IgY(IBD IgY).The effects of three factors on the amount of Fab' fragments were investigated by examining the pH value,the mass ratio of pepsin and IgY,and the time of enzymolysis.SDS-PAGE and Western blotting were used to determine the appropriate conditions for preparing Fab' fragments.The results showed that IgY could be completely digested by 8 hours of reaction when the mass of pepsin:IgY mass? 0.2.In order to optimize the preparation process of Fab' fragments,two factors(the mass ratio of pepsin to IgY,and the time of enzymolysis)were selected to perform a response surface center combination design test.The results showed that the optimized preparation process of Fab' fragment was pH 4.0,temperature 37?,mass ratio of pepsin and egg yolk antibody 0.54:1(reaction ratio 432:1 U/mg),and enzymolysis time 8.5 h,respectively.Under these conditions,the maximum amount of Fab' fragments could be obtained.1.36 g of IgY lyophilized powder was obtained under optimized conditions.SDS-PAGE analysis of the enzymatic hydrolysis products showed that IgY was completely decomposed into Fab' and inactive pFc' small molecule fragments.The BCA protein detection kit was used to detect the protein concentration of the enzymolysis solution.The results showed that the protein concentration of the enzymolysis solution was 2.40 mg/mL,and the protein content in the Fab' solution was 1.308 g,which accounted for 96.18%of the original lyophilized powder.It was used for the preparation of Fab' injections with different concentrations.Experiment II Clinical trial of Fab' fragments against IBDV in chickensThe lyophilized powder was dissolved in physiological saline to make Fab' injections with different concentrations(20 mg/mL,10 mg/mL and 5 mg/mL)for IBDV clinical trials.The results showed that 10 mg/mL Fab' injection could treat IBDV infection in chickens by intramuscular injection.Compared with the virus group,it could significantly increase the bursal index and reduce the spleen index(P<0.01).20 mg/mL high-dose Fab' injection could be used to prevent cyst enlargement caused by early IBDV infection in chickens.Compared with the normal group,the bursal index and spleen index were not significantly different(P>0.05),respectively.In summary,the maximize the yield of Fab' fragments were obtained under the influence of various factors.This method reduced resource waste,improved the process efficiency of preparing Fab' fragments,and laid a foundation for large-scale production of Fab' fragments.At the same time,the Fab' lyophilized powder was used in this study to prove that a certain amount of Fab' fragments could prevent and treat chicken infectious bursal virus infection.This study provided a clinical basis for the clinical application of Fab'. |
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