Construction And Immunogenicity Of Hepatitis B Core Antigenvirus-like Particles Carrying PEDV S1 Epitope

The pathogen of porcine epidemic diarrhea(PED)is porcine epidemic diarrhea virus(PEDV),which can cause intestinal infectious diseases in piglets.The main cause is that the mortality rate of newborn piglets is extremely high,even reaching 100%,which can cause symptoms such as watery diarrhea,dehydration and vomiting of newborn piglets.However,it generally does not cause death in adult pigs.Since PED has occurred in my country in 1973,it broke out in the United States in 2013,and has a large-scale outbreak in my country in 2010,which has caused limited and regional impacts on China’s pig industry,and has caused a serious blow to my country’s pig industry.In order to prevent and control the occurrence of PEDV,the main method is to vaccinate pigs.However,the commercially available inactivated vaccines and attenuated live vaccines that have been widely used on the market cannot provide complete protection for newborn piglets.Traditional inactivated vaccines are mostly used.Classic strains,which cause the vaccine’s immune protection capacity to vary in different regions;Although the immune effect of the attenuated live vaccine is far better than that of the traditional inactivated vaccine,its research and development cost and time are greatly increased,and there is also a certain risk of dispersing the virus.Based on current research and clinical observations,it has been found that there is a difference between the attenuated live vaccine and the traditional inactivated vaccine.The phenomenon of genetic recombination of epidemic strains occurs.Therefore,it is particularly important to develop a safe and effective new PEDV vaccine to prevent and control the outbreak ofPEDV.In this study,hepatitis B core antigen(HBcAg)was used as a carrier to present PEDV S1 epitope,and virus-like particles(VLPs)of PEDV S1 epitope were constructed.Among them,the spike protein S is one of the main structural proteins of PEDV?which can efficiently induce the body to produce specific neutralizing antibodies.Accordingly,in this experiment,the 1-74aa of the hepatitis B core antigen and the 270bp containing the B cell epitope of PEDV S1 were connected by design fusion PCR.After double enzyme digestion and sequencing verification,the PET-32a(+)-HBcAg(1-74aa)-PEDV S1 recombinant plasmid.Then it was connected with HBcAg(83-144aa),that is,the PEDV S1 B cell epitope was inserted into the immunodominant region(MIR)of HBcAg,and the PET-32a(+)-HBcAg-PEDV S1 recombinant plasmid was successfully constructed after double enzyme digestion verification.The recombinant plasmid was transformed into BL21,the protein expression was induced by IPTG,and the refolded protein was purified after expression,and the successful formation of virus-like particles was detected by transmission electron microscopy,indicating that PEDV S1 virus-like particles were successfully constructed using the E.coli expression system.It provides further reference for the subsequent development of PEDV vaccine.Construct an immunogenic HBcAg-PEDV S1 candidate vaccine,and conduct a preliminary evaluation of the vaccine’s immunogenicity through a mouse immunization test.The mouse epidemic diarrhea virus antibody(PEDV Ab)ELISA test kit was used to detect the level of specific antibodies in the serum of mice.The results showed that HBcAg-PEDV S1 VLPs can stimulate the body to produce PEDV-specific antibodies in the second week after the first immunization.After immunity,the antibody level increased in the first week,and then gradually increased in the second week.After the third immunization,the PEDV antibody level rose to the highest value,but the antibody titer was lower than that of the vaccine group.In the second week of the third immunization There is not much difference between the two;Mouse lymphocyte proliferation test and cytokine IL-4,IFN-γ detection results According to data analysis,immunization with HBcAg-PEDV S1 VLPs can increase the expression level of IL-4 in the body;at the same time,IFN-γ levels continue to rise after immunization.In summary,HBcAg-PEDV S1 VLPs have good immunogenicity,which lays the foundation for the further development of PEDV candidate vaccines in the future,and also provides a data reference for the PEDV ontology animal experiment.