Expression And Immune Function Of ScFREP-1 And ScFREP-2 In Sinonovacula Constricta

Sinonovacula constricta is an important cultured shellfish in tidal flats of China.Like other invertebrates,shellfish lack immunoglobulin and only rely on innate immune mechanisms to recognize microbial pathogens.Under the current high-density culture mode,shellfish are less likely to suffer large-scale disease in the process of culture than shrimp,crab and fish.How shellfish can effectively defend off pathogen invasion in the environment where microbes are widely distributed has aroused researchers’ interest in the study of innate immunity of shellfish.Fibrinogen-related proteins(FREPs)are a class of proteins that contain fibrinogen-related domains(FRED)in their C-terminal.In recent years,many studies have found that fibrinogen-related proteins of invertebrate act as pattern recognition receptors(PRRs)to participate in innate immunity.In this study,two fibrinogen-related protein genes from S.constricta were involved in the recognition of polysaccharides and the recognition and agglutination of microorganisms,which may play an important role in shellfish immunity.1.Full length c DNA cloning and sequence analysis of two fibrinogen-associated protein genesTwo incomplete fragments of fibrinogen-related proteins were obtained from the S.constricta transcriptome data previously established in our laboratory.After verifying their accuracy,the full-length c DNA sequences of these two genes were obtained by rapid amplification of c DNA ends(RACE).The total length of ScFREP-1gene was 2065 bp,containing an 825 bp open reading frame(ORF),encoding 274 amino acids,and the predicted molecular weight is 30.28 k Da.The full length of ScFREP-2gene was 1675 bp,with an open reading frame length of 882 bp,encoding 293 amino acids and a predicted molecular weight of 33.08 k Da.The C terminal of both genes contained a fibrinogen-related domain(FRe D),so they were named ScFREP-1 and ScFREP-2.The signal peptide prediction analysis showed that ScFREP-1 had no signal peptide,and ScFREP-2 had classical secreted signal peptide.2.Expression pattern analysis in early development stages and healthy tissues,together with immune response patternThe expression level of ScFREPs m RNA in early development stages together with adult healthy tissues of S.constricta were detected using qRT-PCR method.The expression of ScFREP-1 and ScFREP-2 in hemolymph and liver at different time points after infection by different bacteria were also detected.The highest expression of ScFREP-1 was detected in gills.The highest expression of ScFREP-2 was detected in mantle,followed by hemolymph.After infection with Staphylococcus aureus and Vibrio angularis,the expression levels of ScFREP-1 and ScFREP-2 m RNA showed different degrees of change,indicating that both genes were immune-related genes of S.constricta,and the two genes may have different reaction mechanisms.3.Recombinant protein expression and functional verification of the ScFREPsThe recombinant proteins ScFREP-1 and ScFREP-2 were expressed.The ability of the two recombinant proteins to bind polysaccharides and bacteria and to agglutinate bacteria was studied.rScFREP-1 binds and agglutinates to both Grampositive and Gram-negative bacteria.Studies on the binding ability of different PAMPs indicate that rScFREP-1 has a strong binding ability to N-acetylglucosamine(NAG).rScFREP-2 binds and agglutinates to both Gram-positive and Gram-negative bacteria,and also shows significant affinity for NAG.These results indicate that ScFREPs are important immune proteins,which function through its unique molecular structure recognized by its FReD.