Expression characteristic and functional identification of CD209v-1 gene on Oreochromis niloticus

【Objective】This paper clarified the correlation between the differentiated group antigen 209（CD209）variant CD209v-1 and the immune function of tilapia，exploring the mechanism of CD209 and its family members in the fish immune system，and laying a foundation for the prevention and cure of tilapia bacterial disease and the research and deve-lopment of new vaccines.【Method】The open reading frame（ORF）of the Oreochromis niloticus CD209v-1 gene （OnCD209v-1）was amplified by PCR. Bioinformatics analysis was performed using online softwares including BerryPsite，InterProScan，TMHMM 2.0，ExPASy，SignalP 4.1 and SWISS-MODEL. OnCD209v-1 gene expression in various tissues of O. niloticus was detected by real-time fluorescence quantitative PCR. Subsequently，the extracellular segment of OnCD209v-1 gene was cloned to construct pET-OnCD209v-1 recombinant prokaryotic expression plasmid. After prokaryotic-induced expression，the recognition，binding，and agglutination function of rOnCD209v-1 protein on a variety of aquatic animal pathogenic bacteria were explored by bacterial binding assay and pooled test in vitro.【Result】The ORF of OnCD209v-1 gene was 780 bp and encoded 259 amino acid residues；the relative molecular weight of the encoding protein of OnCD209v-1 gene was 29.424 kD. The theoretical isoelectric point（pI）was 6.54，and there was a conserved C-type lectin（CTL）domain between amino acids 113 and 246. The OnCD209v-1 amino acid sequence had the highest similarity to Simochromis diagramma CD209 amino acid sequence（86.49%）. The phylogenetic evolutionary tree based on the CD209 amino acid sequence similarity also showed that the O. niloticus was clustered with S. diagramma，and was most closely related to S. diagramma. OnCD209v-1 gene existed in all analyzed tissues，with higher relative expression in the liver and intestine especially，followed by blood. The rOnCD209v-1 protein obtained by prokaryotic-induced expression recognized and bound a variety of pathogenic bacteria and had agglutination activity in Aeromonas hydrophila，Escherichia coli，Streptococcus agalactiae，Vibrio parahemolyticus and Staphylococcus aureus in the presence of Ca²⁺.【Conclusion】The OnCD209v-1 gene is a variant of tilapia CD209 gene，with a conserved CTL domain，and is widely expressed in tilapia tissues；rOnCD209v-1 obtained by prokaryotic-induced expression can recognize and bind common pathogenic bacteria，such as A. hydrophila，E. coli，Vibrio harveyi，S. agalactiae V. parahaemolyticus and S. aureus， with agglutination activity in a variety of pathogens in the presence of Ca²⁺. The OnCD209v-1 gene participates as an important immune molecule in the immune process of tilapia’ s response to bacterial infection.