| Canine distemper virus(CDV)infection can cause mass mortality in a variety of carnivores and is currently one of the major viruses afflicting the pet industry.In order to carry out effective immunization prevention and control,the epidemic of the disease is mainly prevented at home and abroad by vaccination with attenuated vaccines.Although the prevalence of canine distemper has been greatly reduced by vaccination,immunocompromised animals still cannot resist virulent attacks and thus infect the disease.Patients can only be treated with specific hyperimmune serum,monoclonal antibodies,immunoglobulins combined with antiviral drugs.The cure rate of sick dogs treated in the early stage of infection can reach 70%,while the highest cure rate in the middle and late stages of infection is only 50%,and the above treatment methods are expensive to treat and have a low prevalence in small and medium-sized towns,so they cannot be widely used.As another type of immunoglobulin,yolk antibody has the advantages of low production cost,high output efficiency,high stability and less damage to animal welfare,which provides a new idea for the clinical development of affordable biological agents for the treatment of canine distemper.In this study,we combined the safe and long-acting advantages of recombinant adeno-associated virus(r AAV)with yolk antibodies,established a new method for the preparation of yolk antibodies,and prepared yolk antibodies against canine distemper,followed by the determination of neutralizing antibody titers and the study of factors affecting the activity of yolk antibodies,and mainly obtained the following results:1.Establish a new method for the preparation of yolk antibody against canine distemper using recombinant adeno-associated virusThis study is based on the previously constructed plasmids of adeno-associated viruses carrying the H gene and F gene of canine distemper virus in the laboratory,recombinant adeno-associated viruses(r AAV-CDV-H-HA,r AAV-CDV-F-HA)with target antigens were obtained by virus packaging.After the antigen gene carried by r AAV can be stably expressed at the cellular level,the virus is packaged in large batches and white feather laying hens that are in the peak laying period are immunized.2.Preparation and examination of anti-canine distemper yolk antibodyAfter immunization of laying hens,the yolk antibody concentrations of r AAV-CDV-H and r AAV-CDV-F extracted by polyethylene glycol precipitation were high,and the yolk antibody concentrations measured ranged from 13 to 26 mg/m L,with the yolk antibody concentration of r AAV-CDV-H generally higher than that of r AAV-CDV-F yolk antibody.SDS-PAGE electrophoresis showed that the relative quality of yolk antibody was consistent with the theory and the purity was high.Indirect immunofluorescence showed that the specificity of yolk antibody was good.3.Neutralization titer determination of anti-canine distemper yolk antibody and its influencing factorsThe results of neutralization test showed that the r AAV-CDV-H experimental group was able to produce higher levels of neutralizing antibodies against canine distemper virus.With the gradual booster of immunization,the neutralizing antibody level gradually increased and reached 1:300 at the highest;however,the neutralizing antibody level was lower in the r AAV-CDV-F experimental group,all below 1:50.The results of the study on the influencing factors of yolk antibody activity showed that the sensitivity of yolk antibody to temperature was low;while the sensitivity to p H was high.Yolk antibody can not resist the digestion of pepsin in the simulated artificial gastric juice environment,but has a strong resistance to the digestion of trypsin in the simulated artificial intestinal juice environment.In summary,immunization of white-feathered laying hens with recombinant adeno-associated virus as a vector can effectively activate the immune system of white-feathered laying hens and produce high levels of neutralizing antibodies,indicating that the method of preparing yolk antibodies using recombinant adeno-associated virus was successfully established.Through the detection and evaluation of anti-canine distemper yolk antibody,it is further indicated that yolk antibody with good specificity,immune effect and stability can be obtained by this method.This study provides a new method for the preparation of efficient,low-cost and highly stable yolk antibodies,and explores the method of making antibodies for further study of yolk antibodies that can be used for the prevention and treatment of canine distemper. |
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