Molecular cloning and expression of a C-type lectin-like protein from orange-spotted grouper Epinephelus coioides.
A C-type lectin-like protein (Ec-CTLP) was cloned from the grouper Epinephelus coioides. The full-length cDNA of Ec-CTLP was composed of 905 bp with a 522 bp open reading frame that encodes a 174-residue protein. The putative amino acid sequence of Ec-CTLP contains a signal peptide of 19 residues at the N-terminus and a CLECT domain from Cys43 to Arg169 and a conserved imperfect WND (Trp-Asn-Asp) motif. The homologous identity of deduced amino acid sequences is from 32 to 42% with other fishes. The expression of Ec-CTLP was differently upregulated in E. coioides spleen (germline stem) cells after being challenged at 16 and 4° C. Intracellular localization revealed that Ec-CTLP was distributed only in the cytoplasm. Recombinant Ec-CTLP (rEc-CTLP) was expressed in Escherichia coli BL21 (DE3) and purified for mouse Mus musculus anti-Ec-CTLP serum preparation. The rEc-CTLP fusion protein does not possess haemagglutinating activity, but improves survival from frozen bacteria. The survival of bacteria (including gram-negative E. coli and gram-positive Staphylococcus aureus) was positively correlated with the concentration of the rEc-CTLP. These findings can provide clues to help understand the probable C-type lectin in marine fish innate immunity.
State Key Laboratory Breeding Base for Sustainable Exploitation of Tropical Biotic Resources, College of Marine Science, Hainan University, Haikou, 570228, P.R. China.
H
H
Ji
J
J
Wei
S
S
Wei
Y
Y
Yan
Y
Y
Huang
X
X
Huang
S
S
Zhou
Y
Y
Zhou
Q
Q
Qin
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D029941:Fish Proteins / Q000235:genetics / Q000378:metabolism*; D037181:Lectins, C-Type / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010473:Perciformes / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D059467:Transcriptome
D018076:DNA, Complementary; D029941:Fish Proteins; D037181:Lectins, C-Type
bacteria freezing-survival test; grouper spleen cells; haemagglutinating activity; intracellular localization