CLONING AND IMMUNE FUNCTIONAL ANALYSIS OF THE X-TYPE LECTIN GENE (OMITLN) FROM ONYCHOSTOMA MACROLEPIS

Intelectin is a novel glycan-binding lectin in the innate immune response of fish, which can bind bacterial surface carbohydrates to agglutinate bacteria. To study the role of intelectin in the innate immunity of Onychostoma macrolepis during bacterial infection, the Onychostoma macrolepis intelectin (OmITLN) was identified in liver transcriptome database and cloned. RT-qPCR was used to analyze the expression of OmITLN in different tissues and after Aeromonas hydrophila infection. OmITLN recombinant protein was successfully obtained by prokaryotic expression and the binding ability of OmITLN to different bacteria was detected by ELISA and fluorescent labeling. The result showed that the sequence was 960 bp in full length, containing 945 bp open reading frame and encoding 315 amino acids. Protein functional domain analysis showed that OmITLN encoded an N-terminal fibrinogen associated domain (FReD) and a C-terminal Intelectin-specific domain. The homology comparison and the phylogenetic tree showed that the OmITLN protein had a higher homology with the other Cyprinidae, such as Ctenopharyngodon idella, Hypophthalmichthys nobilis, Megalobrama amblycephala and Carassius auratus. The qRT-PCR results indicated that OmITLN was expressed in the all examined tissues including liver, muscle, spleen, gonad, intestine, brain and gill in Onychostoma macrolepis, and it was expressed at the highest level in liver and spleen. Compared with the control group, OmITLN was significantly up-regulated and then down-regulated after stimulation with Aeromonas hydrophila. In the liver, the expression of OmITLN reached its maximum at 12 hours post-infection (hpi) and was gradually down-regulated over time. In spleen, OmITLN was reached the highest value at 6hpi and then gradually returned to the initial level after 24hpi. OmITLN recombinant protein was successfully obtained and authenticated by SDS-PAGE and Western Blotting. OmITLN can agglutinated all tested bacteria, including three gram-positive bacteria (Escherichia coli, Vibrio parahaemolyticus and Aeromonas hydrophila) and three gram-negative bacteria (Streptococcus agalactiae, Bacillus cereus and Staphylococcus aureus) with Ca²⁺. OmITLN showed the best binding activity to D-lactose, followed by xylose, D-galactose and sucrose, with the lowest expression in D-glucose, but showed no binding activity to mycose, D-fructose and D-maltose. The binding activity of OmITLN to PGN which is gram-positive bacteria recognition structure was higher than LPS which is gram-negative bacteria recognition structure. As an important pattern recognition receptor, OmITLN is involved in the immune defense of Onychostoma macrolepis against bacteria.