Expression And Immunogenicity Analysis Of Receptor Binding Domain Protein Of Porcine Delta Coronavirus

Porcine delta coronavirus(PDCoV)is a newly discovered porcine intestinal pathogenic coronavirus in 2012,which can infect pigs of all ages,especially in lactating piglets.The clinical symptoms are acute watery diarrhea,vomiting and dehydration,which have strong pathogenicity and can cause the death of suckling piglets.Diarrhea caused by PDCoV broke out in many places in the United States in 2014,which spread and spread widely around the world in the following years,causing serious economic losses to the global pig industry.The positive rate of PDCoV in a pig farm in Jiangxi Province in China in 2016 was as high as 33.1%.More importantly,PDCoV is often co-infected with porcine epidemic diarrhea virus(PEDV),which not only aggravates the harm to pigs,but also brings difficulties to the diagnosis and prevention of the two viruses.At the same time,PDCoV has the ability of cross-species transmission and a wide range of host tropism,can infect a variety of mammals and birds,including human beings,and has potential harm to food safety and public health.However,there is no commercial vaccine and effective treatment to prevent and control PDCoV.Inspired by the same type of vaccine related to the receptor binding domain(RBD)of Severe acute respiratory syndrome coronavirus(SARS-Co V-2),this study took the PDCoV receptor binding domain protein as the research object,expressed PDCoV receptor binding domain protein in mammalian cells and insect animal cells,and prepared polyclonal antibodies against PDCoV receptor binding domain protein in mice.An indirect ELISA method for mouse PDCoV receptor binding region was established based on polyclonal antibody serum.At the same time,based on the PDCoV receptor binding domain proteins expressed in two systems,the PDCoV receptor binding domain subunit vaccine was prepared.After immunizing mice and piglets,the related indexes were detected and its immunogenicity was evaluated.The soluble PDCoV receptor binding domain protein(MPDR)was successfully expressed in mammalian cell system.The purity of the protein purified by Strep-Tactin XT affinity chromatography was more than 98%.The polyclonal antibody serum with good reactivity was successfully prepared after immunizing mice with this antigen,and the titer was more than 102400.A mouse indirect ELISA method was established by using mouse polyclonal serum as positive samples.The conditions such as protein coating concentration,serum dilution multiple and action time,enzyme labeled second antibody dilution concentration and action time,color time were optimized,and the specificity,sensitivity and repeatability of the established method were evaluated.Finally,the optimal reaction condition of ELISA detection was determined:the optimal antigen coating concentration was 10μg/m L.The best condition of serum was incubated with 1:400 for 30min,the best condition of enzyme labelled second antibody was incubated with 13000 HV for 30min,and the best color developing time of substrate was 15min.The critical value for the evaluation of specific antibody level was0.2169.There was no cross reaction with SARS-Co V-2,TGEV,PEDV and other positive sera.The sensitivity,intra-assay coefficient of variation and inter-assay coefficient of variation were 2.93%,3.84%and 2.41%,respectively.The indirect ELISA method of mouse PDCoV receptor binding domain protein antibody was successfully established.It can be used to evaluate and analyze the antibody level of PDCoV receptor binding domain protein in mice.The PDCoV receptor binding domain protein(BPDR)with reactivity was successfully expressed in insect cell expression system,and BPDR protein was concentrated by ultracentrifugation.Purified MPDR protein and concentrated BPDR protein were mixed with adjuvant(aluminum salt+Cp G)to prepare MPDR-AC and BPDR-AC subunit vaccines to immunize mice.The changes of body weight,specific antibody level and titer,neutralizing antibody level,cell subsets and cytokines were measured,and the immunogenicity of the two subunit vaccines were evaluated comprehensively.The results showed that after immunization,the body weight of mice in all groups except SF9-AC control group showed an upward trend within 31 days,and the specific antibodies of MPDR-AC and BPDR-AC immunized groups reached a higher level on the 31st day,and the titers of specific antibodies in MPDR-AC and BPDR-AC immunized groups were both 51200 and BPDR-AC,and the average neutralizing antibody levels on the 31st day of immunization group were 1:107 and 1:56,respectively.In T lymphocyte proliferation test,the SI of MPDR-AC immunized group was 3.84 times higher than that of PBS control group,the percentage of CD3~+CD4~+lymphocyte subsets increased significantly in MPDR-AC group,and IL-4 cytokines increased to some extent in MPDR-AC group and BPDR-AC group after PDCoV stimulation.Compared with PBS,AC and BSF9-AC control groups,MPDR-AC and BPDR-AC immunization groups could induce a certain level of immune response,and MPDR-AC group showed better immune effect than BPDR-AC group,so MPDR protein with better immune effect was selected for piglet immunity and challenge test.The subunit vaccine was prepared by mixing MPDR protein with adjuvant(aluminum salt+molecular adjuvant+Cp G).The body weight and body temperature of immunized piglets were monitored.At the same time,the weekly changes of specific antibody level of piglets were analyzed,and the serum with the highest specific antibody level was detected.The results showed that the body weight of piglets increased continuously and the body temperature did not fluctuate significantly after immunization,and the level of specific antibody increased as a whole and reached the peak on the 31st day,and the specific antibody titer of 4 piglets in the MPDR-ACF group was between 12800 and 102400 on the 31st day,and the neutralizing antibody in the serum MPDR-ACF immunized group was 3 times higher than that in the ACF control group on the 31st day.The body weight,body temperature and fecal virus load of the piglets after second immunization were detected.The results showed that the body temperature of the two groups was stable in the normal range,the body weight of the MPDR-ACF immunized group increased as a whole,one piglet in the ACF control group showed symptoms of weight loss and diarrhea,the fecal viral load of the MPDR-ACF immunized group showed a low level,and the overall viral load of the ACF control group was higher than that of the MPDR-ACF immunized group.The results showed that MPDR subunit vaccine had a certain protective effect on piglets infected with PDCoV.The MPDR subunit vaccine prepared in this study can induce a high level of immune response in mice and piglets,and can provide some protection to piglets,which lays a foundation for the study of PDCoV receptor binding domain proteins and the development of new PDCoV vaccines.