SCREENING AND IDENTIFICATION OF INTERACTING PROTEINS OF LECT2 IN CHANNA ARGUS

Leukocyte-derived chemotaxin-2 (LECT2) is a multifunctional immunoregulator that plays a key role in immune regulation, cell growth, differentiation, self-damage repair, and tumorigenesis. We investigated the ligand protein regulated by snakehead LECT2 (CaLECT2) in the antibacterial immunity process. The sequence of the CDs region of the CaLECT2 gene was obtained through homology search and gene cloning technology. The immune-enhancing effect of CaLECT2 on snakehead was assessed through in vivo injection tests. Interacting proteins of CaLECT2 were analyzed using His-Pull down combined with mass spectrometry and Co-IP. The mRNA expression of CaLECT2 significantly increased in the spleen, kidney, and liver of snakehead fish after infection with Nocardia seriolae. The CaLECT2 recombinant protein was effectively expressed and purified using the pET-32a prokaryotic expression system. Western Blot analysis confirmed that the anti-rCaLECT2 polyclonal antibody specifically identified the recombinant CaLECT2 protein. Injection of the rCaLECT2 protein into snakehead fish significantly increased the number of macrophages in the spleen. Pull down assays combined with mass spectrometry identified 253 potential interacting proteins, including Fetuin-like protein, Serotransferrin, and CD209. Co-IP verification confirmed an interaction between CaLECT2 and CD209. This study lays the groundwork for further exploration into the roles of ligand proteins regulated by CaLECT2.