乌鳢白细胞衍生趋化因子LECT2互作蛋白的筛选与鉴定

SCREENING AND IDENTIFICATION OF INTERACTING PROTEINS OF LECT2 IN CHANNA ARGUS

  • 摘要: 为了解乌鳢(Channa argus)白细胞衍生趋化因子2 (CaLECT2)在抗菌免疫过程中所调控的配体蛋白, 克隆获得CaLECT2基因, 并对其表达特征进行了分析。利用体内注射试验分析了CaLECT2对乌鳢机体的免疫增强作用。利用His-pull down联合质谱、Co-IP分析了CaLECT2的互作蛋白。结果显示, 在感染鰤诺卡氏菌的乌鳢脾脏、肾脏和肝脏中, CaLECT2 mRNA的表达量显著上调。利用原核表达系统表达纯化获得了CaLECT2重组蛋白。通过Western Blot证明rCaLECT2抗血清可以特异性识别CaLECT2重组蛋白。向乌鳢体内注射CaLECT2蛋白可以显著提高脾脏内巨噬细胞数量。Pull down联合质谱鉴定得到Fetuin-like protein、Serotransferrin和CD209等253个潜在的互作蛋白。Co-IP结果进一步确认了CaLECT2与CD209存在互作关系。研究为深入探讨CaLECT2所调控的配体蛋白的功能奠定了前期基础。

     

    Abstract: 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.

     

/

返回文章
返回