Identification And Antimicrobial Functions Analysis Of Peptidoglycan Recognition Protein(PGRP)gene Family In Sinonovacula Constricta

Peptidoglycan recognition protein（PGRP）plays a vital role in invertebrate innate immunity system as a specific pattern recognition receptor for peptidoglycan.Bivalves possess various PGRP systems for self-defense,however,it has not been characterized in Sinonovacula constricta.S.constricta is an economically important mollusk.However,it is often attacked by bacterial diseases in recent years.Therefore,it is of great significance to study the ScPGRP genes for the understanding of its innate immune system mechanism.The main contents of this study were as follows:the PGRP genes were identified from the genome of S.constricta,and their sequence analysis,multiple sequence alignment,phylogenetic tree analysis and chromosome location were carried out,and its protein tertiary structure model was also predicted.The tissue distribution of ScPGRP genes and the temporal expression after bacterial stress and PAMPs stimulation were analyzed by q RT-PCR.Moreover,the recombinant protein was purified and the antibody was synthesized.The expression changes of the peptidoglycan recognition protein in the hepatopancreas stimulated by Vibrio parahaemolyticus were observed by immunofluorescence technique.Then the biological activities of the recombinant protein were studied,including the amidase activity,agglutination activity and bacteriostatic activity.The main research results were as follows:1.Bioinformatics analysis of the SCPGRP gene familyEight ScPGRP genes were identified from the genome of S.constricta.They were distributed on all four chromosomes.The predicted molecular weight of their proteins ranged from 14.2-25.0 k Da,thus the ScPGRPs were classified to PGRP-S.The PGRP/amidase-2domain exists in all ScPGRPs.In addition,the presence of signal peptides was predicted in ScPGRP-S2,ScPGRP-S3 and Sc PRP-S6,while a transmembrane structure only existed in ScPGRP-S6.The PGRP gene family of S.constricta has a high homology with shellfish,and according to the clustering results of PGRP protein,this protein family can be divided into four subgroups.The tertiary structure analysis indicated that,except for ScPGRP-S5,S7 and S8,the other six proteins had conserved tertiary structures and disulfide bonds.2.Spatial and temporal expression profilesqRT-PCR results showed that ScPGRP-S1,ScPGRP-S2,ScPGRP-S3,ScPGRP-S4 and ScPGRP-S6 were all expressed in the six tissues.Specifically,the high expression patterns of ScPGRP-S1 and ScPGRP-S4 were found in mantle,adductor muscle and foot,while those of ScPGRP-S2,ScPGRP-S3 and ScPGRP-S6 were observed in hepatopancreas.Additionally,after V.parahaemolyticus challenge,compared with the m RNA expression level of ScPGRPs at 0 h,a significant up-regulation expression was found in ScPGRP-S2,ScPGRP-S3 and ScPGRP-S6,and the peak appeared at 24 h,24 h,and 12 h,respectively（P<0.01）,while only the m RNA temporal expression of ScPGRP-S2 and ScPGRP-S3 showed significant increase compared to 0h post Staphylococcus aureus challenge.Notably,Gram-negative V.parahaemolyticus induced higher expression compared with Gram-positive S.aureus.In addition,the m RNA transcripts of ScPGRP-S2,ScPGRP-S3 and ScPGRP-S6 in the hepatopancreases were all up-regulated after injection of PGN and LPS,and the expression levels of ScPGRP-S2 and ScPGRP-S6 were up-regulated twice.3.Prokaryotic expression and immunofluorescence analysisThe recombinant expression vectors of ScPGRP-S2,ScPGRP-S3 and ScPGRP-S6 were constructed and the recombinant proteins were successfully induced and expressed.Solubility analysis showed that expressed fusion proteins of ScPGRP-S6 were expressed in both supernatant and inclusion bodies,while those of ScPGRP-S2 and ScPGRP-S3 only existed in the form of inclusion body.What’s more,afterward,the recombinant protein of ScPGRP-S6with His-tag was purified using Ni-NTA agarose was purified and the corresponding antibody was synthesized.Subsequently,the result of immunofluorescence detection reflected that the positive signal was obviously after V.parahaemolyticus challenge.5.Amidase activity,agglutination ability and antibacterial activityAmidase activity assay showed that the recombinant ScPGRP-S6 protein possessed PGN degradation activity with the presence of Zn²⁺.The recombinant ScPGRP-S6 protein also has bacterial agglutination ability,which can agglutinate V.parahaemolyticus and S.aureus.In addition,the recombinant protein could strongly suppress the growth of Bacillus subtilis,Escherichia coli and S.aureus in the presence of Zn²⁺during the whole period,and the inhibition zone experiment verified that the recombinant ScPGRP-S6 protein had obvious bactericidal effect on V.parahaemolyticus and S.aureus.