Bioinformatics Analysis of Bovine Lymphocyte Activation Gene 3 and Preparation of Its Polyclonal Antibody
HUANG Tian-Tian1,*, WANG Pu-Yang2,*, ZHANG Guang-Xian1, ZANG Ran1, YUAN Fei-Fan1, WANG Gan-Feng1, LI Xian1, CHEN Shu-Lin1, LI Run-Sheng3,**, CONG Ri-Hua1,**
1 College of Veterinary Medicine, Northwest A&F University, Yangling 712199, China; 2 Shaanxi Provincial Feed Work Station, Xi'an 710003, China; 3 Jockey Club College of Veterinary Medicine and Life Sciences, City University of Hong Kong, Jiulong, 999077, China
Abstract：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.
[1] 宋小珍, 付戴波, 瞿明仁, 等. 2012. 热应激对肉牛血清内分泌激素含量、抗氧化酶活性及生理生化指标的影响[J]. 动物营养学报, 24(12): 2485-2490. (Sun X Z, Fu D B, Zhai M R, et al.2012. Effects of heat stress on serum endocrine hormone content, antioxidant enzyme activity and physiological and biochemical indexes in beef cattle[J]. Chinese Journal of Animal Nutrition, 24(12): 2485-2490.) [2] 王晓娜, 李静, 刘仰知, 等. 2018. 抗应激添加剂对运输应激前后肉牛血清激素及细胞因子的影响[J]. 沈阳农业大学学报, 49(2): 214-219. (Wang X N, Li J, Liu Y Z, et al.2018. Effects of anti-stress additives on serum hormones and cytokines in beef cattle before and after transport stress[J]. Journal of Shenyang Agricultural University, 49(2): 214-219.) [3] Aggarwal V, Workman C J, Vignali D A A.2023. LAG-3 as the third checkpoint inhibitor[J]. Nature Immunology, 24(9): 1415-1422. [4] Andrews L P, Cillo A R, Karapetyan L, et al.2022. Molecular pathways and mechanisms of Lag3 in cancer therapy[J]. Clinical Cancer Research, 28(23): 5030-5039. [5] Besedovsky H, Sorkin E.1977. Network of immune-neuroendocrine interactions[J]. Clinical Experimental Immunology, 27(1): 1-12. [6] Borcel E, Perez-Alvarez L, Herrero A I, et al.2008. Chronic stress in adulthood followed by intermittent stress impairs spatial memory and the survival of newborn hippocampal cells in aging animals: Prevention by FGL, a peptide mimetic of neural cell adhesion molecule[J]. Behavioural Pharmacology, 19(1): 41-49. [7] Buisson S, Triebel F.2005. LAG-3(CD223) reduces macrophage and dendritic cell differentiation from monocyte precursors[J]. Immunology, 114(3): 369-374. [8] Carvajal M A, Alaniz A J, Gutierrez-Gomez C, et al.2021. Increasing importance of heat stress for cattle farming under future global climate scenarios[J]. Science of the Total Environment, 801: 149661. [9] Casati C, Camisaschi C, Rini F, et al.2006. Soluble human LAG-3 molecule amplifies the in vitro generation of type 1 tumor-specific immunity[J]. Cancer Research, 66(8): 4450-4460. [10] Chaudhary O, Trotta D, Wang K C, et al.2022. Patients with HIV-associated cancers have evidence of increased T cell dysfunction and exhaustion prior to cancer diagnosis[J]. Journal for ImmunoTherapy of Cancer, 10(4): e004564. [11] Chrousos G P, Gold P W.1992. The concepts of stress and stress system disorders. Overview of physical and behavioral homeostasis[J]. JAMA, 267(9): 1244-1252. [12] Day H E W, Curran E J, Watson S J, et al.1999. Distinct neurochemical populations in the rat central nucleus of the amygdala and bed nucleus of the stria terminalis: Evidence for their selective activation by interleukin-1beta[J]. Journal of Comparative Neurology, 413(1): 113-128. [13] Graydon C G, Mohideen S, Fowke K R.2020. LAG3's enigmatic mechanism of action[J]. Frontiers in Immunology, 11: 615317. [14] Hung A L, Garzon-Muvdi T, Lim M.2017. Biomarkers and immunotherapeutic targets in glioblastoma[J]. World Neurosurgery, 102: 494-506. [15] Kalkman H O, Feuerbach D.2016. Antidepressant therapies inhibit inflammation and microglial M1-polarization[J]. Pharmacology Therapeutics, 163: 82-93. [16] Kim D, Kim G, Yu R, et al.2024. Inhibitory co-receptor Lag3 supports Foxp3+ regulatory T cell function by restraining Myc-dependent metabolic programming[J]. Immunity, 57(11): 2634-2650. [17] Kokkosis A G, Madeira M M, Hage Z, et al.2024. Chronic psychosocial stress triggers microglial-/macrophage-induced inflammatory responses leading to neuronal dysfunction and depressive-related behavior[J]. Glia, 72(1): 111-132. [18] Long L, Zhang X, Chen F, et al.2018. The promising immune checkpoint LAG-3: From tumor microenvironment to cancer immunotherapy[J]. Genes Cancer, 9(5-6): 176-189. [19] Maruhashi T, Sugiura D, Okazaki I M, et al.2020. LAG-3: From molecular functions to clinical applications[J]. Journal for ImmunoTherapy of Cancer, 8(2): e001014. [20] Maruhashi T, Sugiura D, Okazaki I M, et al.2022. Binding of LAG-3 to stable peptide-MHC class Ⅱ limits T cell function and suppresses autoimmunity and anti-cancer immunity[J]. Immunity, 55(5): 912-924. [21] Ming Q Q, Celias D P, Wu C, et al.2022. LAG3 ectodomain structure reveals functional interfaces for ligand and antibody recognition[J]. Nature Immunology, 23(7): 1031-1041. [22] Rimmerman N, Verdiger H, Goldenberg H, et al.2022. Microglia and their LAG3 checkpoint underlie the antidepressant and neurogenesis-enhancing effects of electroconvulsive stimulation[J]. Molecular Psychiatry, 27(2): 1120-1135. [23] Selye H.1998. A syndrome produced by diverse nocuous agents (Reprinted from Nature, vol 138, pg 32, 1936)[J]. Journal of Neuropsychiatry Clinical Neurosciences, 10(2): 230-231. [24] Triebel F.2003. LAG-3: A regulator of T-cell and DC responses and its use in therapeutic vaccination[J]. Trends in Immunology, 24(12): 619-622. [25] Veinante P, Stoeckel M E, Freund-Mercier M J.1997. GABA- and peptide-immunoreactivities co-localize in the rat central extended amygdala[J]. NeuroReport, 8(13): 2985-2989. [26] Wang H, Kaur G, Sankin A I, et al.2019. Immune checkpoint blockade and CAR-T cell therapy in hematologic malignancies[J]. Journal of Hematology Oncology, 12: 59. [27] Wang J, Sanmamed M F, Datar I, et al.2019. Fibrinogen-like protein 1 is a major immune inhibitory ligand of LAG-3[J]. Cell, 176(1-2): 334-347. [28] Zhang B, Sun J Q, Yuan Y S, et al.2023. Proximity-enabled covalent binding of IL-2 to IL-2 Ralpha selectively activates regulatory T cells and suppresses autoimmunity[J]. Signal Transduction and Targeted Therapy, 8(1): 28.
