Construction Of Recombinant Adenovirus Expressing PEDV S Protein Antigen Region And IFN-γ Recombinant Adenovirus And Preparation And Evaluation Of Yolk Antibody Effect

Porcine epidemic diarrhea(PED)is one of the most harmful diseases in the pig industry.The main symptoms are diarrhea,vomiting,dehydration and death.Porcine epidemic diarrhea virus(PEDV)can infect pigs at all stages,among which large pigs are usually recessively infected or transiently infected,while newborn piglets often die or form stiff pigs due to dehydration due to incomplete development of the immune system and poor ability to correct water and electrolyte disorders.The morbidity and mortality are about 90 %-100 %.In production practice,the prevention method of the disease is mainly to use inactivated vaccine and attenuated vaccine to carry out artificial active immunization of sows.However,traditional vaccines inoculated by injection are often difficult to stimulate sows to produce reliable mucosal immunity and sufficient s Ig A.Once piglets cannot obtain sufficient s Ig A from breast milk to neutralize the virus in the intestine(intestine-mammary gland-s Ig A axis),a series of serious symptoms such as diarrhea and dehydration will occur.In addition,PEDV S gene is prone to insertion,deletion and mutation,resulting in a decrease in the cross-immune protection of the vaccine,which is also one of the difficulties in the prevention and control of PED.PEDV S protein is a key target for the study of subunit vaccines and vector vaccines.The replication-defective adenovirus type 5 vector has the advantages of high transduction efficiency and good immune effect,and is one of the most promising platforms for the development of new potent vaccines.Yolk antibodies have been shown to have significant effects on a variety of infectious intestinal diseases in humans and animals.Therefore,in order to solve the current difficulties in the prevention and control of PED,this study intends to use PEDV S protein antigen region and IFN-γ fusion expression recombinant adenovirus vaccine to immunize laying hens,harvest purified egg yolk antibody(Immunoglobulin of yolk,Ig Y),and use passive immunization program to prevent PEDV infection,in order to significantly reduce the mortality of piglets infected with PEDV and the economic losses of pig farms.The specific contents and results of this study are as follows :1.Isolation and identification of PEDV-NX-2022 strain and sequence analysis of S geneA PEDV strain was successfully isolated and identified from the intestinal samples of PEDV-positive sick piglets detected by RT-PCR,which was named PEDV-NX-2022.Phylogenetic analysis based on the full-length S gene showed that the isolate had the closest genetic distance with the GIIb representative strains CH / HNXX / 2016(MT338517.1)and CH / HNZZ47 / 2016(KX981440.1),and belonged to the GIIb epidemic strain.2.Construction and characterization of rADM-IFN-G-ped recombinant adenovirusBased on the Ad Max packaging system,the PEDV S protein fusion epitope region and IFN-γ gene were cloned on the adenovirus vector to construct a recombinant adenovirus.The recombinant adenovirus rADM-IFN-G-ped was successfully constructed by indirect immunofluorescence and Western blotting.3.Preparation and immune effect evaluation of anti-PEDV yolk antibodyThe rADM-IFN-G-ped recombinant adenovirus was used as immunogen to immunize laying hens.The egg yolk antibody with anti-PEDV neutralization activity was successfully extracted from eggs by PEDG-6000 precipitation method.The neutralization titer of antiPEDV yolk antibody was 1:96 by using PEDV Ningxia isolate.The artificial infection treatment test of piglets showed that the mortality rate of piglets in the control group was100 %,and the mortality rate of Ig Y treatment group was 50 %,which proved that the yolk antibody had a certain protective effect on the infection of PEDV epidemic strain.In summary,this study successfully isolated and identified a PEDV GIIb epidemic strain from a large-scale pig farm in Zhongwei,Ningxia,and named it PEDV-XN-2022.The rADMIFN-G-ped recombinant adenovirus was successfully constructed and used to immunize HyLine Brown laying hens.The high purity anti-PEDV yolk antibody was extracted from eggs by PEG-6000 precipitation method.The cure rate was 50 % in the artificial infection treatment test,which proved that the prepared yolk antibody had certain protective effect on the infection of PEDV epidemic strain.This study provides a basis for exploring the prevention and control of PED by passive immunization,and provides technical support for the comprehensive prevention and control of PED.