牛淋巴细胞活化基因3的生物信息学分析及其多克隆抗体制备

As an important immunomolecule molecule, lymphocyte activation gene 3 (LAG3) is a potential molecule connecting immunosuppression and neuroregulation, and its functional study is of great significance for revealing the mechanism of stress-induced neuro-immune imbalance. This study predicted the physicochemical properties, structural domains, and modification sites of bovine (Bos taurus) LAG3 protein. The coding sequence of the LAG3 extracellular domain was amplified from bovine peripheral blood lymphocyte cDNA by gene cloning technology. The prokaryotic expression vector pET-28a-LAG3 was constructed and transformed into Escherichia coli BL21 (DE3) to induce the expression of recombinant protein. The inclusion bodu-form recombinant protein purified by nickel column affinity chromatography was used as the immunogen to immunizing BALB/c mice (Mus musculus) to obtain polyclonal antibodies. Indirect ELISA was emplyed to detect antibody titers, Western blot was used to analyze antibody specificity. The results showed that the coding sequence of bovine LAG3 gene was 1 551 bp in length, encoding 516 amino acids. The protein molecular formula was C₂₅₅₉H₃₉₇₄N₇₂₈O₇₁₂S₁₁, with a relative molecular mass of 56.68 kD. It was a hydrophilic, non-lipid-soluble, unstable alkaline protein containing 1 signal peptide, 1 transmenbrance region, 48 potential posphorylation sites, 3 N-glycosylation sites and 19 antigenic epitopes. The concentration was 1.03 mg/mL after purification, and the prepared polyclonal antibody could bind to the total protein of bovine peripheral blood lymphocytes, showing a single band with a size of 45 kD. The highest antibody titer reached 1∶1 048 576. In conclusion, this study characterized the structural features of bovine LAG3 and successfully prepared a high-titer, high-specificity mouse anti-bovine LAG3 polyclonal antibody. These findings provide key tools for in-depth research on expression patterns, subcellular localization, and immunoregulatory mechanisms of LAG3 in bovine stress responses.