Molecular Cloning And Function Analysis Of A Class ? Scavenger Receptor From Octopus Ocellatus

As a kind of pattern recognition receptor(PRR),scavenger receptor could re cognize the “self” and “non-self” components and participate in the immune mec hanism of the organism against external stimulation.Octopus ocellatus is a coast al economic species with high medicinal and nutritional value.Like other inverte brates,it mainly dependents on the innate immune system to resist the external s timulation of invading microorganisms.We studied the gene structure and functio n of a class I scavenger receptor from O.ocellatus to provide support for the re search of scavenger receptor in the immune recognition and molecular response mechanisms of the innate immunity.In this study,a scavenger receptor(OoSR-I)was cloned from the cDNA lib rary of O.ocellatus and the full-length sequence was obtained by RACE(Rapid Amplification of c DNA Ends).The structure and phylogenetic characteristics of Oo SR-I were analyzed and predicted by bioinformatics.The mRNA expression p atterns of OoSR-I,both in tissues and in hemocytes after Listonella anguillaruma nd Micrococcus luteus stimulation,were then detected by real-time fluorescence q uantitative PCR.The prokaryotic expression system was constructed to obtain rec ombinant OoSR-I protein.Enzyme-linked immunosorbent assay(ELISA)was used to detect the binding ability of OoSR-I recombinant protein(rOoSR-I)and ligand s.It was founded that the recombinant protein could recognize a variety of ligan ds.The OoSR-I gene was identified by indirect immunofluorescene.The rOoSR-I was not located on the cell surface of hemocytes and it indicated that OoSR-I w as a secretory and soluble scavenger receptor.The OoSR-I gene played an impor tant role in the innate immune system of O.ocellatus as pattern recognition rece ptors.(1)The full-length cDNA of OoSR-I was 1006 bp,containing a 5' terminal un-translated region(UTR)of 104 bp,3' UTR of 152 bp with a poly(A)tail of 19 bp.The open reading frame(ORF)was 750 bp encoding a polypeptide of 249 amino acids with a predicted molecular weight of 27.76 KDa and theoretical isoelectric point of 6.05.It contained a signal peptide,two repeats SR domain and five N-glycosylation sites.Analysis of multiple sequence alignment,construction of phylogenetic tree and prediction of tertiary structure revealed that OoSR-I and were similar to class I scavenger receptor members in amino acid sequence and structure,and it was one of class I scavenger receptor family members.(2)The expression of Oo SR-I m RNA in various tissues and in hemocytes a fter L.anguillarum and M.Luteus stimulation was detected by real-time fluoresc ence quantitative PCR(RT-PCR).The results showed that OoSR-I was widely ex pressed in a range of tissues including gonad,mantle,stomach,muscle,gill,hep atopancreas,systemic heart and hemocytes with the highest level in hemocytes a nd the lowest expression in the gonad.The expression of Oo SR-I m RNA was u p-regulated once to 4.34-fold of blank group and reached the peak at 12 h post L.anguillarum.While it increased two times at 12 h and 48 h post M.Luteus whic h was 2.92 and 37.7-fold of blank group,respectively.High expression of OoSR-I gene after stimulation indicated that OoSR-I played a crucial role in the immu ne defense of O.ocellatus.(3)The recombinant protein of Oo SR-I gene was obtained,the anti-rOoSR-I polyclonal antibody was prepared by immunizing mouse,and then analyzed by Western Blot.The anti-rOoSR-I polyclonal antibody could specially bind to rOoS R-I and employed for ELISA.The ability of rOoSR-I binding to ligands was det ected by ELISA with LPS,PGN,glucan,zymosan,mannan,LDL,Ox-LDL and Ac-LDL as ligands.The results showed that the binding activity of the rOoSR-I to these ligands dependented on Ca2+.The OoSR-I encoding protein was identifie d by indirect immunofluorescene,and the results showed that OoSR-I was notloc ated on the cell surface of hemocytes,indicating OoSR-I was a secretory and so luble scavenger receptor.In summary,a OoSR-I gene was cloned from O.ocellatus and indentified a s class I scavenger receptor.It's expression could be induced by L.anguillarum and M.Luteus stimulation,and the recombinant protein could bind a variety of ligands,indicating that Oo SR-I gene was a pattern recognition receptor which par ticipated in the immune mechanism of the organism against invading microorgani sms.Oo SR-I encoding protein was not located on the cell surface,it was a secr etory or soluble scavenger receptor and played an important role in the immune defense process.This research could provide theoretical support for the immune defense mechanism of marine invertebrates.