Identification And Function Verification Of Antibacterial Peptide In Strongylocentrotus Intermedius

Antibacterial peptides are widely distributed in living Eukaryotic organisms,with wide antimicrobial spectrum,and not easy to induce bacterial resistance.They play an important role in regulating and enhancing organism immunity,which makes them very promising drug candidates to be developed and approved,having great potential for exploitation.Previous studies of antimicrobial peptides have focused on mammals,with less research in invertebrate animals with natural immunity.This study is conducted with the following experiments based on Strongylocentrotus intermedius.Two gene segments named SiStrongylocin1 and SiStrongylocin2 were screened from the transcriptome of Strongylocentrotus intermedius,the datas we have owned.Then the coding sequences of SiStrongylocin1 and SiStrongylocin2 were separately amplified and isolated by RACE technique.The sequence characteristics were analyzed using various bioinformatics software.This study also analyzed and discussed the expression of SiStrongylocin1 and SiStrongylocin2 in different organizations(coelomic fluid,intestine,peristomial membrane,tube feet,gonad)and different development stages(fertilized egg,eight cells,blastula stage,gastrul stage,prismatic stage,two wrist period,four wrist period)by RT-qPCR.After constructing the recombinants pGEX-4T-1-SiStrongylocin1 and pGEX-4T-1-SiStrongylocin2 with the double digests we transformed them into E.coli competent cells BL21(DE3)pLysS and Transseta,which were induced by IPTG in the following steps.The expression condition were optimized in order to obtain recombinant protein with high yield.Trace double dilution method in 96 Well Cell Culture Plates was applied to measure antimicrobial activities of antibacterial peptides.The major results are as follows: coding sequences of SiStrongylocin1 and SiStrongylocin2 was 252 bp and 249 bp respectively,and the coding sequences were composed of signal peptides,presequences and mature peptides.The amino acid sequences had six highly conserved cysteine residues.The tertiary structure prediction showed that SiStrongylocin1 contained three alpha helices,SiStrongylocin2 contained two simple alpha helices and one beta fold.In evolutionary terms,the antibacterial peptide of Strongylocentrotus intermedius was similar to the antibacterial peptide of Strongylocentrotus droebachiensis and Strongylocentrotus purpuratus.Antibacterial peptides were expressed in different tissues and different developmental stages in Strongylocentrotus droebachiensis.SiStrongylocin1 was expressed highest in intestinal while SiStrongylocin2 was expressed highest in the coelomic fluid.SiStrongylocin1 and SiStrongylocin2 were expressed highest in blastocyst stage.Through sds-page gel electrophoresis,recombinant proteins were detected to be in inclusion body.The optimal expression condition with IPTG as inducers was 28 ?,200 rpm,IPTG 1 mM,8 hours forinduction.The experiment to verify the antibacterial activity of two recombinant proteins showed that antibacterial activity of SiStrongylocin2 was more significant than SiStrongylocin1,while both AMPs inhibit the E.coli bacteria more than Vibrio fortis significantly.Together,this research obtain the CDS of two antibacterial peptides in Strongylocentrotus intermedius and find they have the general characteristics of antibacterial peptides.The expression level of antibacterial peptideds was related to different tissues and different developmental stages closely.Antibacterial activity experiment also prove that the two kinds of recombinant proteins have strong antibacterial activity on E.coli and Vibrio fortis.This study not only provides reference for the development of effective antimicrobial products,but also accumulates experimental datas for understanding sea urchins immune function and the immune mechanism.