Study On The Immune Efficacy Of Chicken Infectious Anemia Virus VP1/VP2 Protein Combined With Antiviral Protein RSAD2

Chicken infectious anemia(CIA)is a type of immunosuppressive disease in poultry caused by chicken infectious anemia virus(CIAV)infection,which can cause anemia and death in chicks.Immune vaccines are an important means of prevention and control for CIA.However,foreign CIA vaccines are developed by continuously passing the virus through cells or chicken embryos to weaken its virulence.These vaccine viruses have not completely lost their pathogenicity,and there is a risk of virulence regression in vaccinated chickens.There is no commercially available CIA vaccine in China yet.VP1 protein is an immunogenic protein that plays a major role in CIAV genome.The presence of VP2 protein is a necessary condition for VP1 protein to form the correct conformation.Numerous studies have shown that the simultaneous presence of VP1 and VP2 proteins can induce the body to produce antibodies against CIAV.RSAD2(radio S-adenosylmethionine domain containing2),as an interferon stimulating gene,can recognize viral nucleic acids and promote the expression of type I IFN by regulating the Toll like receptor 7(TLR7)and TLR9 in cells,making it an important innate antiviral immune molecule in the body.This study mainly utilized the expression system of baculovirus to express the VP1,VP2 proteins,and RSAD2 proteins of CIAV in eukaryotic cells,and further evaluated the immune efficacy of combined immunization on SPF chickens infected with CIAV virulent strains.In order to construct polyclonal antibodies that can specifically recognize CIAV VP1,VP2 proteins and RSAD2 proteins,amplify CIAV VP1,VP2 genes and chicken RSAD2 genes,clone them into prokaryotic expression vectors,and obtain VP1,VP2,RSAD2 recombinant proteins after IPTG induction,the protein was immunized into mice to prepare polyclonal antibodies and identified by indirect immunofluorescence(IFA).The results showed that the VP1 and VP2 of CIAV and the RSAD2 gene of chicken were successfully cloned,and the prokaryotic expression plasmids of p ET-28a-VP1,p ET-28a-VP2 and p GEX-6p-RSAD2 were constructed,and the induced expression sizes were 33KD,32KD and 43KD respectively.The polyclonal antibody prepared after immunizing mice can specifically recognize VP1,VP2 and RSAD2 proteins.In order to prepare recombinant baculoviruses expressing CIAV VP1,VP2 and RSAD2proteins and to study the effect of RSAD2 proteins on the replication of CIAV at the cellular level,the CIAVVP1 and VP2 genes were cloned into the p Fast Bac Dual eukaryotic expression vector,which was obtained after transposition and blue-white screening Recombinant baculovirus plasmid Bacimd-VP1/VP2,the supernatant after the recombinant bacmid transfected sf9 cells is the recombinant baculovirus,named r Bac-VP1/VP2,IFA and Western blotting to identify the protein expression of VP1 and VP2.The recombinant baculovirus expressing chicken RSAD2 protein was constructed in the same way,and the expression of RSAD2 protein was identified with the prepared polyclonal antibody.After purification of the recombinant RSAD2 protein produced by insect cells,MDCC-MSB1 cells were incubated and infected with CIAV.The cells were harvested 12,24,and 36 hours after infection to extract DNA,and the CIAV viral load was determined by fluorescent quantitative PCR.The results showed that recombinant baculoviruses expressing CIAV VP1,VP2 and chicken RSAD2 proteins were constructed,and the prepared polyclonal antibodies could react with sf9 cells infected with r Bac-VP1/VP2 and r Bac-RSAD2,and emit specific bright green fluorescence.Further Western blotting results showed that VP1,VP2 proteins and RSAD2proteins were successfully expressed in sf9 cells infected with the recombinant virus.Compared with the control group,RSAD2 protein could significantly inhibit the proliferation of CIAV on MSB1 cells(P<0.05).In order to evaluate the immune effect of CIAV VP1 and VP2 eukaryotic expression proteins on SPF chickens infected with SD1520,80 1-day-old SPF chickens were randomly divided into 5 groups,and the prepared RSAD2 protein,VP1/VP2 protein and Freund’s adjuvant were used to emulsify separately or mix immune SPF chicken.Group 1 was RSAD2protein and VP1/VP2 protein mixed immunization group;Group 2 was VP1/VP2 protein immunization group;Group 3 was RSAD2 protein immunization group;Group 4 was CIAV challenge control group and Group 5 was blank control group.After immunizing the protein,the CIAV SD1520 strain was injected intramuscularly at the age of 21 days,and the immune organ index,Hct level,antibody level,changes in cytokine expression,and viral load in various tissues of SPF chickens were detected at different ages after challenge to evaluate the immunity.Protective effects of different proteins on SPF chickens.At the same time,in order to evaluate the change of antibody level,an ELISA detection method of VP2 protein was established.The results showed that the atrophy of the bursa and thymus appeared in all groups after the challenge,and the atrophy was the most serious in the challenge group,and the levels of immune proteins in each group were significantly lower than those in the challenge group(P<0.05).The blood of the VP1/VP2 protein immunization group and the mixed immunization group was collected at different periods after immunization to measure the VP2 antibody titer.The results showed that the antibody titer of the mixed immunization group was higher than that of the VP1/VP2 protein immunization group in the early stage of immunization,indicating that RSAD2 protein had an early effect on VP2 antibody.The production of VP1/VP2 protein may have a promoting effect,and the antibody titer of the VP1/VP2 protein immunization group began to increase in the later stage.The results of viral load in each tissue showed that compared with the challenge group,the viral load of each group decreased after immunization with protein,among which the immunized VP1/VP2protein group and the mixed immunization group could significantly inhibit virus proliferation(P<0.05),and the mixed immunization group most notably.Detecting the expression of cytokines in the spleens of each group found that the expression of IL-1β,IL-6,and IL-2 showed a trend of decreasing first and then increasing after infection with the virus,and the expression of IFN-γcontinued to increase.The expression of IFN-γin the mixed immunization group was significantly higher than that in other groups(P<0.05),indicating that RSAD2 protein can promote the expression of IFN-γand improve the immune function of chickens.The results of flow cytometry showed that 5 days after the challenge,there was no significant difference between the CD4~+T cells in the mixed immunization group and the blank group(P>0.05),and the CD4~+T cells in the other groups were significantly down-regulated(P<0.05),and the CD8~+T cells Significantly up-regulated(P<0.05),indicating that combined immunization of VP1/VP2 and RSAD2 protein significantly reduced the immunosuppression caused by CIAV.In summary,this study successfully constructed recombinant baculoviruses capable of expressing CIAV VP1,VP2 proteins,and chicken RSAD2 proteins and prepared specific polyclonal antibodies.It is confirmed that RSAD2 protein can inhibit the replication of CIAV.Further animal experiments show that CIAV VP1 and VP2 proteins have good immune effects on SPF chickens infected with CIAV,and RSAD2 protein can significantly improve their immune efficacy.This study provides a solid material basis for the research on the immune prevention and control of CIAV,and has important scientific significance for the research on the antiviral function of RSAD2.