Identification of outer membrane protein ompR from rickettsia-like organism and induction of immune response in Crassostrea ariakensis.
Rickettsia-like organism (RLO) caused mass mortality of oysters, but little is known about the protective immune response to this microorganism. The present study was undertaken to identify a gene, ompR, encoding an outer membrane protein of rickettsia-like organism from oyster Crassostrea ariakensis. The role of this protein in promoting immune responses was characterized through analyzing the interaction between RLO and oyster. The results indicated: (i) full-length DNA of ompR is 531 bp and encodes 176 amino acid residues. Theoretical isoelectric point and molecular weight for the ompR protein are 9.76 and 19.76 kDa, respectively; (ii) the recombinant ompR was successfully expressed in Escherichia coli BL21 (DE3) cells, and the titre of anti-ompR antibody raised against rabbits was about 1:4100. A specific immunoreactive band was detected when anti-ompR antibody was opposed to the total outer membrane proteins of RLO; (iii) the expression level of TNF-alpha (tumor necrosis factor-alpha) and Myd88 (myeloid differentiation factor 88) in hemocytes was induced by ompR, whereas TGF-beta (transforming growth factor-beta) was not; (iv) in hemocytes monolayers, a rapid and persistent increase in the level of phosphorylated P38 and a large decrease in the level of phosphorylated JNK were induced by ompR, whereas the level of phosphorylated ERK did not change with ompR incubation; (v) the DNA binding activity of NF-kappaB (nuclear factor kappaB) in hemocytes increased after ompR stimulation.
Zhu
Baojian
B
College of Animal Sciences, Zhejiang University, 268 Kaixuan Road, Hangzhou 310029, PR China.
Wu
Xinzhong
X
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D001426:Bacterial Proteins / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification / Q000494:pharmacology; D015153:Blotting, Western; D049896:Crassostrea / Q000187:drug effects / Q000201:enzymology / Q000276:immunology*; D004247:DNA / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D006434:Hemocytes / Q000187:drug effects / Q000201:enzymology; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification / Q000494:pharmacology; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012281:Rickettsia / Q000187:drug effects / Q000276:immunology*; D017422:Sequence Analysis, DNA; D017186:Titrimetry; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D000906:Antibodies; D001426:Bacterial Proteins; D008565:Membrane Proteins; D016328:NF-kappa B; D011994:Recombinant Proteins; D004247:DNA; D048049:Extracellular Signal-Regulated MAP Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases