Screening and Identification of Outer Membrane Proteins of Riemerella anatipestifer Recruited Duck C4b-binding Protein

The purpose of this experiment was to screen and identify the outer membrane proteins of Riemerella anatipestifer (R. anatipestifer) interacting with duck C4b-binding protein (C4BP). The preserved R. anatipestifer was resuscitated, then the outer membrane proteins of R. anatipestifer were extracted and His pull-down and LC-MS/MS were conducted by using duck C4BPα as the bait protein, and the candidate outer membrane proteins that might interact with duck C4BP were screened out. The candidate proteins were cloned, prokaryotic expression was conducted and the polyclonal antibodies were prepared by immunizing the mice. Far-western blot was conducted to verify the outer membrane proteins of R. anatipestifer interacting with duck C4BP. The clone and prokaryotic expression of functional domains of candidate proteins and C4BPα were conducted, and Far-western blot was used to identify the interaction sites between candidate proteins and C4BP. The deposition of complement C3b, C4b and C4BP on the surface of R. anatipestifer were detected by ELISA to verify the function of the candidate proteins. The results showed that a total of 3 outer membrane proteins of R.anatipestifer interacting with duck C4BP were screened out by His pull-down and LC-MS/MS, namely ECE-1, SODs and Omp62. The polyclonal antibodies of 3 outer membrane proteins were successfully prepared, the titers of 3 polyclonal antibodies were more than 1∶6 400 by ELISA, and the result of Western blot showed that 3 polyclonal antibodies could specifically react with corresponding recombinant proteins. The results of Far-western blot showed that only ECE-1 could interact with C4BP, and only the full length of ECE-1 could interact with duck C4BP, and the interaction region between duck C4BP and ECE-1 was located in SCR 2 and SCR 3 of C4BPα. Anti-ECE-1 antibody could significantly increase the C3b and C4b deposition on the surface of R. anatipestifer using 3.125% normal duck serum (NDS, P<0.05), while anti-ECE-1 antibody could significantly decrease the deposition of C4BP on the surface of R. anatipestifer using 6.25% NDS (P<0.05). The study successfully screened out and identified one outer membrane protein (ECE-1) of R. anatipestifer interacting with duck C4BP, which provide a basis to further study the mechanism of R. anatipestifer immune escape.