Study On The Immune Mechanism Mediated By Containing WAP Domain Protein And C-type Lectin Of The Weather Loach,misgurnus Anguillicaudatus

Misgurnus anguillicaudatus is a kind of freshwater fish distributed in Asia,known as"ginseng inwater"for its high nutritional and pharmacological value.The aquaculture of loach has attracted more and more attention and developed rapidly in recent years.However,with the expansion of loach breeding scale and the continuous deterioration of aquatic environment,loach aquaculture has been seriously threatened by the outbreak of various diseases.M.anguillicaudatus has both innate and acquired immune system,but its acquired immune system is not fully developed compared to that of higher vertebrate.It mainly relies on its own innate immune mechanism to resist the invasion of various pathogens.Therefore,the research on the innate immunity of M.anguillicaudatus has attracted more and more attention.In this study,we studied the immune function of a WAP domain containing protein and a C-type lectin in M.anguillicaudatus.1.Immune function of WAP domain containing protein（MaTWD）in M.anguillicaudatus.Whey acidic protein（WAP）exists in a variety of proteins in animals,and many whey acidic protein domain（WAPD）containing proteins participate in the immune response.The function of WAPD protein has been reported in mammals and invertebrates,especially extensively studied in crustaceans.However,there are few studies on the function of WAPD protein in fish.In this paper,we obtained a gene encoding three WAPDs in tandem,named MaTWD.Its cDNA sequence is composed of 760 bp in length,of which the opening read frame（ORF）is 507 bp.It encodes a peptide of 168 amino acids（aa）and the MaTWD protein predicted by SMART contains a signal peptide sequence（27 aa）and three WAPD arranged in tandem,namely TWD-1（40 aa）,TWD-2（44 aa）and TWD-3（43 aa）.The results of multiple sequence alignment and phylogenetic analysis demonstrate that MaTWD shares high similarity with TWD in teleost fish but low similarity with other animal phyla.The transcripts of MaTWD are mainly distributed in the gills and head kidney of the loach,and up-regulated in both tissues after bacterial immune stimulation.In vitro experiments show that MaTWD not only has antiprotease activity against bacterial proteases,but also has bacterial binding ability and antibacterial activity.More importantly,MaTWD can protect the loach from bacterial infection by reducing the mortality.In summary,we speculate that MaTWD participates in the antibacterial immunity of loach through its antiprotease and antibacterial activities.2.Functional analyses of a C-type lectin from M.anguillicaudatusC-type lectins（CTLs）have a significiant role in innate immunity as pattern recognition receptors.Transcriptome sequencing was performed on the liver tissue of the loach infected with the pathogen A.hydrophila,and a MaCTL gene was obtained.It contains a 478 bp ORF encoding 165 amino acids.The MaCTL protein contains a 27 amino acids signal peptide and a 120 amino acids C-type lectin domain（CTLD）.The CTLD contains a galactose-specific QPD motif and a conserved Ca²⁺binding site.After infection with A.hydrophila,the transcription level of MaCTL was significantly up-regulated.In vitro experiments with purified recombinant protein MaCTL（r MaCTL）showed that it has both hemorrhagic cell agglutination activity and bacterial agglutination activity in a Ca²⁺-dependent manner.Bacterial binding experiments and polysaccharide binding experiments show that MaCTL has the ability to bind to a variety of bacteria by binding to the bacterial polysaccharides including LPS and PGN.Moreover,MaCTL also shows antibacterial activity by inhibiting the growth of bacteria.The results of these studies together indicate that MaCTL is involved in the antibacterial defense of loach.