Functional Analysis Of The Peptidoglycan Recognition Protein2 (PGRP2) Of Common Carp(Cyprinus Carpio)

Innate immunity is an innate immune protection mechanism in living organisms that protects organisms from invasion by foreign pathogens and initiates related immune signaling pathways.The innate immune system recognizes microorganisms through a number of pattern recognition receptors?PRRs?that are highly conserved in evolution.These PRRs identify specific components of foreign pathogenic microorganisms known as pathogenic associated molecular patterns?PAMPs?In this way,the relevant signal pathway can be activated to activate the natural immune response,so as to effectively remove and eliminate pathogens.Peptidoglycan recognition protein?PGRPs?is a kind of important natural immune receptor family,is widely distributed in many invertebrates and vertebrates in various tissues and organs.Innate immunity is through evolutionary conservative PRRs congenital immune recognition PAMPs to activate the related pathways,and can identify the invasion of pathogenic microorganisms,such as PGRPs,PGRPs can combine and,in some cases,hydrolysis of bacterial cell wall peptidoglycan?PGN?,Because peptidoglycan is a component of almost all bacterial cell walls,PGRPs is particularly important for organisms to resist the invasion of foreign pathogens and initiate the relevant immune response.The ORF length of PGRP2 in carp was 1449 bp by PCR amplification in this experiment,and conservative domain prediction,signal peptide prediction,sequence analysis of different species and evolutionary analysis were analyzed.In this experiment,the expression of PGRP2 at the mRNA level was investigated by extracting RNA from various tissues of common freshwater fish species-carp,which were immunologically stimulated byhydrophila.The results showed that the expression of PGRP2 in liver,head kidney,foregut and midgut was up-regulated at 3h after immune stimulation,and it was significant in liver,while the expression of PGRP2 in spleen and hindgut was significantly up-regulated at 6h after stimulation.After immersion stimulation,PGRP2 gene is generally highly expressed in liver and spleen during the whole stimulation cycle,and the expression of PGRP2gene in liver,head kidney,foregut and midgut is up-regulated 3h after stimulation,and PGRP2 gene in hindgut is up-regulated 6h after stimulation,and down-regulated to varying degrees in the later stage of immune stimulation.Secondly,we also detected the expression of PGRP2 gene in individuals at the early stage of development of carp.It was found to be very low in the fertilized egg period,and the expression level was significantly up-regulated on the second day of hatching,and was significantly reduced on the 14th day of hatching.There was a significant up-regulation in the 21 days of incubation.It may be speculated that the differences in water environment and the incomplete development of the immune system in eggs and young fish lead to the relatively large changes in PGRP2 gene.These results suggest that PGRP2 may play a role in bacterial immunity and early development.In order to further verify the function of carp PGRP2,we expressed PGRP2 in vitro.In this experiment,we constructed the prokaryotic expression vector of carp PGRP2,and then transferred it to expression strain BL21 and successfully induced expression and purification of inclusion body protein.After protein renaturation Functional verification was carried out in bacteriostatic experiments,bacterial binding,bacterial agglutination,and polysaccharide binding.Bacterial inhibition experiments showed that recombinant PGRP2 can inhibit the growth of A.hydrophila and S.aureus,and this inhibition is dependent on the presence of Zn²⁺;in bacterial binding experiments,recombinant PGRP2 can bind to A.hydrophila,K.pneumoniae,E.coli,S.aureus,B.thuringiensis,and B.subtilis at different degrees;Bacterial agglutination experiments show that recombinant PGRP2 has obvious agglutination effect on S.aureus in the presence of Zn²⁺,The agglutination effect of A.hydrophila is weak;in the experiment of scanning electron microscopy,it can be found that the bacterial surface damage of recombinant PGRP2 against S.aureus is stronger than that of A.hydrophila;the binding experiment of recombinant PGRP2 and polysaccharide shows that it is combined with peptide.The binding ability of sugar is stronger than that of lipopolysaccharide.The above experimental results show that carp PGRP2 can bind to bacteria in different degrees,agglutinate and damage the surface of bacteria,and the ability to bind to the surface component of the bacterial peptidoglycan is higher than that of lipopolysaccharide.The effect is more pronounced in the presence of metal ion Zn²⁺,which is related to the Zn²⁺binding site in which the PGRP2 gene itself has amidase activity.Through the above experiments,we conducted the carp PGRP2 gene protein sequence analysis,preliminary build carp bacteria stimulation model analysis expression patterns of different organizations,and to do some research on antibacterial function,preliminary estimate the carp PGRP2 the stimulus against pathogens and antimicrobial mechanisms play an important role in natural natural immunity,provides the application value for fish production and protection,as well as provides a better basis for study of fish innate immune basis.