The Research Of Immune Evasion Protein Of R.anatipestifer Binding Host C4BP

Riemerella anatipestifer(RA)is one of the most important pathogenic bacteria that endangers the waterfowl breeding industry.It has many serotypes and lacks cross-immunity protection between each serotypes.RA is extremely prone to drug resistance,due to the limitations of existing vaccines and drugs in disease prevention and control,it has become one of the main pathogens that seriously e ndanger the duck industry.Studying the pathogenic mechanism of this bacterium is the basis for the development of new vaccines and drugs,and it is also a key scientific problem that must be solved to prevent and treat the disease.The complement system appears as an important component of the innate immune system,is composed of nearly 40 kinds of protein restriction proteolytic system,activated by classical pathway(CP),lectin pathway(LP)and alternate pathway(AP).After activation,the cleavage of the major complement molecules C3 and C5,as well as the assembly of the membrane attack complex(MAC),will proceed to the final cleavage of the target cell.As one of the liquid regulatory factors in the complement system,C4BP can competitively bind C4b with C2 molecules,inhibit the formation of C3 invertase.At the same time,it acts as a cofactor for factor I to promote the cleavage of C4b,shorten the half-life of C3 invertase in CP and LP,block the activation of the complement system.Previous studies have shown that some pathogenic bacteria could recruit C4BP to its surface by using their own outer membrane proteins to block the activation of complement system and mediate immune evasion.Pathogenic bacteria infecting the host need to escape the complement system,and the escape mechanisms of different bacteria are diverse.Therefore,escape from the complement system is an important part of the research on the virulence factors and pathogenic mechanisms of pathogenic bacteria.However,the research on RA-mediated complement immune evasion is still blank.This study based on the early stage of the laboratory,the prokaryotic expression of the complement regulatory domain SCRs of duck C4BP?was analyzed by biological information analysis.Using C4BP?as a bait protein,Combining His-pull down with LC-MS/MS Protein spectroscopy,we screened out the RA immune escape target protein that interacted with C4BP,identified its interaction region and preliminarily explored the biological function of the target protein.Through the above studies,we hope to provide a theoretical basis for further research on the pathogenic mechanism of RA and the exploration of new vaccine targets.The following research results have been obtained.1.Prokaryotic expression of duck C4BP gene and preparation of polyclonal antibodiesThe SCRs1-6 specific primers were designed based on the duck C4BP?sequence,and the recombinant cloning vector was obtained by T-A cloning.The target fragment size was1080bp.The plasmid p Cold-TF was used to construct a duck C4BP?prokaryotic expression vector,E.coli BL21(DE3)was used as the expression host strain,induced by IPTG,after sonication and SDS-PAGE detection,we obtained a soluble protein with a protein size of approximately 91.6 KDa.The disrupted supernatant was subjected to non-denaturing SDS-PAGE and Coomassie Brilliant Blue R-250 staining,and then the target protein gel band was cut out and treated as the immunoantigen.BALB/c mice were immunized three times,and blood was collected and serum separated 15 days after the third-immunizations.Western-bolt showed that the mouse anti-duck C4BP?could specifically react with the recombinant protein duck C4BP?.Indirect ELISA was used to determine the serum titer.When the mouse anti-duck C4BP?was diluted to 1:12800,the value of P/N was 5.239.2.Identification of immune evasion protein binding to duck C4BP?Dot immunoblot tests showed that RA strains with serotypes 1,2,4,and 14 were able to recruit C4BP in healthy duck serum.On this basis,the purified duck C4BP?recombinant protein was used as bait protein for His-pull down and LC-MS/MS protein spectroscopy identification.According to the comprehensive analysis of the protein peptide coverage,identification score and related functions in the identification results,three candidate outer membrane proteins that may interact with duck C4BP?were finally selected:ECE-1,SODs,and Omp62.Far-Western blot confirmed that only ECE-1 could interact with duck C4BP?.3.Identification of binding regions between immune evasion protein and duck C4BP?In order to explore the binding regions of ECE-1 and duck C4BP?,primers were designed for the ECE-1 functional domain:N-terminal,C-terminal and some truncated fragments to construct a prokaryotic expression vector.On the basis of N i²⁺affinity purification and his-tag removal by thrombin,Far-western blot was performed.The test results showed that only the full length of EC E-1 could interact with duck C4BP?.Similarly,in order to explore the binding regions of duck C4BP?and ECE-1,the prokaryotic expression vector was constructed for each SC R region o f duck C4BP?,and Far-western blot was performed using the recombinant protein of each SCR region as a probe.The results showed that the binding region of duck C4BP?and ECE-1 was located in SCRs 2 and SCRs 3.4.Determination of complement factor depositionTaking RA-AF strain as the research object,incubate ECE-1 antibody with RA in advance,and then incubate with healthy duck serum,proteins deposited on the surface of RA were eluted by eluent,and the contents of C3b,C4b and C4BP in eluent were detected by indirect ELISA.The results showed that when the serum concentration of healthy ducks was3.125%,EC E-1 antibody could significantly promote the deposition of complement factors C3b and C4b on the surface of RA(p<0.05).When the serum concentration o f healthy ducks was 6.25%,EC E-1 antibody could significantly inhibit the deposition of C4BP on the surface of RA(p<0.05).5.Determination of serum bactericidal activity and immune phagocytosisTaking the RA-AF strain as the research object,in antibody-mediated serum bactericidal test,the EC E-1 antibody was incubated with RA before co-incubation with healthy duck serum,and the relative survival rate was calculated using live bacteria counts.The results showed that EC E-1 antibodies could significantly promote the direct killing effect of complement(p<0.01).In addition,the promotion effect is dose-dependent with the antibody concentration.The highest effective dilution is 1:64(p<0.01).In the immunophagic opsonization test,ECE-1 antibody was incubated with RA before co-incubation with macrophage,and the relative survival rate was calculated using live bacteria counts.The results show that EC E-1 antibody could significantly promote macrophage opsonization,and the promotion effect is dose-dependent with the antibody concentration.The highest effective dilution is 1:16(p<0.05).In summary,this study successfully screened an RA immune evasion protein that binds to duck C4BP,ECE-1.the binding region of duck C4BP and EC E-1 was located in SCRs 2 and SCRs 3 of the C4BP?.After ECE-1 binds to C4BP,it could inhibit the deposition of C3b and C4b on the surface of RA.ECE-1 antibodies could mediate serum killing activity and immune phagocytosis.