Study On The Immune Function Of Novel Polyimmunoglobulin Receptor SIGR In Zebrafish

Polyimmunoglobulin receptor is a specific receptor of polyimmunoglobulin.Studies have reported that PIGR can exert antiviral and antibacterial functions.In this thesis,we discovered a novel immunoglobulin receptor containing the SRCR domain,named it SIGR,and explored its function in antibacterial immunity.SIGR contains a Scavenger receptor cysteine-rich（SRCR）domain and two IG domains.SIGR was expressed in normal tissues of zebrafish.After stimulation with Aeromonas hydrophila,SIGR expression was significantly up-regulated in all tested zebrafish tissues,it is speculated that it may participate in the antibacterial immune response of zebrafish.The SIGR recombinant protein was expressed and purified in vitro.It was found that SIGR could bind bacteria and cause bacteria to agglutinate by bacterial binding and agglutination experiments.By constructing truncated domain proteins SIGR-SRCR and SIGR-IG,bacterial binding and agglutination experiments on the two proteins were conducted,and it was found that the truncated SIGR-SRCR had weak agglutination and binding ability on Gram-negative bacteria.It is speculated that the SIGR-IG domain has a great influence on the binding and agglutination of Gram-negative bacteria,and the SRGR domain and IG domain have certain complementary functions in the ability to bind and agglutinate bacteria.In order to further study the antibacterial function sites of SIGR-SRCR and SIGR-IG,we constructed mutant SIGR^△51-55（missing SRCR conserved amino acid site"WGTVC"）,SIGR^△203-207（missing the first IG conserved amino acid site"GxYxC"）and SIGR^△310-314（missing the second IG conserved amino acid site"GxYxC"）.It was found that the agglutination ability of the mutants SIGR^△51-55and SIGR^△203-207against Gram-positive bacteria decreased.These results indicated that the conserved amino acid site"WGTVC"in the SRCR domain and the conserved amino acid site"GxYxC"in the first IG played an important role in SIGR agglutination of gram-positive bacteria.PIGR of human and flatfish was reported to be involved in NF-κB signaling pathway.SIGR was overexpressed in ZF4 cells,and the expression of NF-κB signaling pathway-related molecules MYD88,TRAF6,NF-κB（P65,P50）,TNF-αand its downstream antimicrobial peptide Defbl1 was up-regulated.Speculation SIGR may activate the NF-κB signaling pathway.In order to further determine the functional sites of SIGR activating NF-κB signaling pathway,the overexpression of mutants SIGR^△51-55,SIGR^△203-207and SIGR^△310-314were constructed,and SIGR^△51-55and SIGR^△203-207showed no significant changes in the upregulation of the above related factors.SIGR^△310-314significantly up-regulated the expression of these related factors.The results indicate that SRCR and IG1 are important functional sites of SIGR involved in NF-κB signaling pathway.Bacterial experiments with Defbl1 showed that Defbl1 could bind,agglutinate and inhibit bacteria.After incubation with Defbl1,the surface membrane structure of S.aureus and E.coli was severely damaged by scanning electron microscopy,indicating that Defbl1 could destroy the bacterial cell wall.In conclusion,SIGR can bind and agglutinate bacteria,up-regulate the NF-κB signaling pathway,and up-regulate the expression of the downstream antimicrobial peptide Defbl1,thus destroying the structure of the bacterial wall,clearing bacteria,and playing the antibacterial function.