Molecular Cloning And Expression Of Peptidoglycan Recognition Protein(PGRPs) Gene From Hyriopsis Cumingii

Peptidoglycan recognition protein as a kind of pattern recognition receptors that can recognize peptidoglycan and pathogen which contain peptidoglycan,that play an important role in identifying and regulating function in the innate immune defense system.This study cloned the cDNA sequence of HcPGRP shot,HcPGRP long,HcPGRP S2 gene from Hyriopsis cumingii.Real-time quantitative PCR was used to analyze these genes expression characterization in tissues of hemocytes,hepatopancreas,musle,mantle,gill from H.cumingii,and gill after PBS,PGN and Aeromonas hydrophila infection.Using gene recombination technology to construct the recombinant expression vector(pET30-HcPGRP long and pET30-HcPGRP S2),using e.coli expression system to expression and purify protein.The complete cDNA sequence of HcPGRP short,HcPGRP long and HcPGRP S2 were length in 1493,1200 and 1037bp,consisting of a 5'-terminal untranslated region(UTR)of 127,334 and 287bp,a 3'-terminal UTR of 508,404 and 93bp,containing an open reading frame(ORF)of 858,462 and 657bp,encode 285,154 and 219 amino acids,respectively.These three genes have N-acetylmuramoyl-L-alanine amidase II domain,also known as PGRP domain.The N-terminus of HcPGRP short and HcPGRP S2 had a signal peptide(HcPGRP short:MAALHITTSMSRASVLTAYRLFICFVCARCLLRNVNG;HcPGRP S2:MFQSSFRRLELNCHEPEEITMWLLFLVTLCVLSCEG)and N-glycosylation site(HcPGRP short:40(NPTH)and 92(NYSC);HcPGRP S2:44(NVTL));Both HcPGRP short and HcPGRP long had transmembrane structure domain.That predicted HcPGRP short as secretory of transmembrane proteins;HcPGRP long as transmembrane proteins;HcPGRP S2 as secretory proteins.In the phylogenetic tree,these three genes clustered with the EsPGRP4,that showed the close evolutionary relationship between them.The multiple sequences alignment showed that the amino acids sites which can bind with Zn2+ and maintain amidase activity were partly conserved in HcPGRP short and HcPGRP long but highly conserved in HcPGRP S2;The amino acids sites which can bind with the Dap type of PGN were highly conserved in HcPGRP short and HcPGRP long but partly conserved in HcPGRP S2.Real-time quantitative PCR detected that all these three genes were expressed in different tissues in H.cumingii and had the same trend on expression quantity that from high to low were hepatopancreas,gill,mantle,adductor muscle and hemocytes.The highest level of expression quantity of HcPGRP short,HcPGRP long and HcPGRP S2 mRNA in hepatopancreas was 6.61,11.29 and 4.95 times as the lowest level of expression quantity in hemocytes,respectively.The expression quantity of HcPGRP long in all detected tissues was the lowest in these three genes.After PGN and A.hydrophila stimulation,in addition to the expression of HcPGRP long had no significant difference in hemocytes,the expression of these three genes had significant difference in tissues of hepatopancreas,hemocytes and gill.The expression of these three genes after A.hydrophila stimulation had significant or extremely significant difference higher than PBS stimulation in some point of these three detected tissues,but the expression PGN stimulation had significant difference higher than PBS stimulation only in some tissues.Through plasmid pET-30 to cpmstruct the recombinant expression vector pET30-HcPGRP long and pET30-HcPGRP S2,they existed as insoluble inclusion bodies after induction.The inclusion body of HcPGRP S2 was purified in the form of having activity of soluble recombinant protein by degeneration and renaturation,The inclusion body of HcPGRP long was purified in the form of soluble recombinant protein by degeneration.