The Role Of SVWC In Antibacterial Immunity Of Musca Domestica

Innate immunity is an important immune route for invertebrates.Invertebrates rely on Pattern Recognition Receptors(PRRs)to accurately realize non-self-recognition.This is the basic step of insect defense against pathogens and the key to triggering subsequent immune responses.Single von Willebrand factor C-domain proteins(SVWC)is a family of proteins mainly found in arthropods.It is characterized by a single von Willebrand factor C-domain protein with 8 of the 10 conserved cysteine residues in the canonical VWC domain.VWC family proteins will produce corresponding stress effects when animals are exposed to bacterial stimulation and nutritional status changes.This study took Musca domestica as the main research object and identified a new SVWC gene,named MdSVWCIA-1.MdSVWCIA-1 was significantly increased in the transcriptome of Musca domestica larvae after bacterial stimulation,suggesting that MdSVWCIA-1 may participate in the antibacterial immunity of Musca domestica,but the specific mechanism of action is not clear.We conducted a systematic bioinformatics analysis of the Musca domestica SVWC family,and explored the function of MdSVWCIA-1 in innate immunity from both gene and protein aspects.The research results are as follows:1.Bioinformatics analysis revealed that MdSVWCIA-1 is a small secreted protein with SVWC domain and 8 conserved cysteine residues.2.The spatiotemporal expression profile analysis based on q PCR found that MdSVWCIA-1 is mainly expressed in the fat body and intestine of larvae;after being stimulated by Escherichia coli or Staphylococcus aureus,the transcription level of MdSVWCIA-1 in larvae increased significantly.3.After knocking down the expression of larval MdSVWCIA-1 using RNAi technology,inject bacteria for immune stimulation.Compared with the control group(ds GFP),the larvae of the interference group had lower survival rate and phenoloxidase activity.At the same time,the expression levels of antimicrobial peptides(muscin,diptericin,attacin,cecropin)and NF-?B-like transcription factor(dorsal)genes were also significantly reduced,indicating that MdSVWCIA-1 has a regulatory effect on humoral immunity.4.Obtain the recombinant protein rMdSVWCIA-1 using E.coli expression system.Coating E.coli with rMdSVWCIA-1 will significantly enhance the Musca domestica's ability to eliminate bacteria injected into the body.In vitro experiments have found that rMdSVWCIA-1 has binding activity to a variety of bacteria and yeast,Ca²⁺-dependent agglutination activity,and phagocytosis of blood cells.Further ELISA experiments confirmed that rMdSVWCIA-1has a broad-spectrum binding ability to LPS,Lys-PGN,DAP-PGN,galactose,mannose,glucan,etc.(PAMPs).Immunofluorescence experiments have found that rMdSVWCIA-1 can bind to the surface of blood cells.It is speculated that there is a specific receptor forMdSVWCIA-1 on the blood cell membrane,and it regulates phagocytosis together with MdSVWCIA-1.In conclusion,this study found that MdSVWCIA-1 may be a new type of pattern recognition receptor,which mediates pathogen recognition through PAMP binding ability,and then participates in cellular and humoral immune regulation through functions such as agglutination,opsonizing phagocytosis,and promoting antimicrobial peptide synthesis,plays a role in the process of host antibacterial immunity.