Mukantayev Kanatbek, Tursunov Kanat, Adish Zhansaya, Kanayev Darkhan, Tokhtarova Laura, Nurtleu Malika, Abirbekov Bisultan
Laboratory of Immunochemistry and Immunobiotechnology, National Center for Biotechnology, 010000, Astana, Kazakhstan.
Department of Natural Sciences, L.N. Gumilyov Eurasian National University, 010008, Astana, Kazakhstan.
Vet World. 2024 Sep;17(9):2096-2103. doi: 10.14202/vetworld.2024.2096-2103. Epub 2024 Sep 15.
Bovine interleukin 15 (bIL15) is a potential immunotherapy that can block the spread of bovine leukemia virus (BLV). However, immune checkpoints that maintain body homeostasis may reduce their effectiveness. Thus, an analysis of the effectiveness of bIL15 while blocking negative immune regulators is necessary. We aimed to obtain recombinant bIL15 (rbIL15) and determine its percentage using monoclonal antibodies against bovine cytotoxic T-lymphocyte antigen 4 (CTLA-4) and programmed cell death ligand 1 (PD-L1). To achieve this goal, peripheral blood mononuclear cells (PBMCs) from healthy and BLV+ cattle were treated with bIL15 using a CTLA-4- and PD-L1-blocking algorithm.
The codon-optimized gene was synthesized under conditions using polymerase chain reaction (PCR). The synthesized gene was cloned into pET28 and transformed into electrocompetent BL21 cells; rbIL15 was purified using metal affinity chromatography and analyzed using sodium dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. The expression of the , , and genes was studied using qualitative PCR. An enzyme-linked immunosorbent assay (ELISA) was used to analyze interferon (IFN)-γ production by rbIL15-treated mononuclear cells.
Analysis of rbIL15 using SDS-PAGE and western blotting revealed a specific product weighing 24 kDa. The optimal conditions for rbIL15 induction were 0.2 mM isopropyl-β-D-1-galactopyranoside and 37°C. When rbIL15 was added to PBMCs from healthy cattle, the , , and genes were expressed. ELISA of the culture medium of rbIL15-treated PBMCs revealed IFN-γ production. When PBMCs from healthy cows were treated with rbIL15, CTLA-4, and PD-L1 blockade together, they did not produce more IFN-γ than the rbIL15 group. Using PBMCs from BLV+ cattle, combination treatment increased IFN-γ production.
The biological activity of rbIL15 is characterized by the induction of transcription factors and the production of IFN-γ. Using rbIL15 with CTLA-4 and PD-L1 blockade in PBMCs from healthy and BLV+ cows led to the production of a transcription factor and cytokine. The results demonstrate the possibility of using this method to improve immunity and immunological memory in patients with chronic viral infections.
牛白细胞介素15（bIL15）是一种潜在的免疫疗法，可阻断牛白血病病毒（BLV）的传播。然而，维持机体稳态的免疫检查点可能会降低其有效性。因此，有必要分析bIL15在阻断负性免疫调节因子时的有效性。我们旨在获得重组bIL15（rbIL15），并使用抗牛细胞毒性T淋巴细胞抗原4（CTLA-4）和程序性细胞死亡配体1（PD-L1）的单克隆抗体来测定其比例。为实现这一目标，采用CTLA-4和PD-L1阻断算法，用bIL15处理健康牛和感染BLV牛的外周血单个核细胞（PBMC）。
在特定条件下使用聚合酶链反应（PCR）合成密码子优化的基因。将合成的基因克隆到pET28中，并转化到电感受态BL21细胞中；使用金属亲和层析法纯化rbIL15，并通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和蛋白质印迹法进行分析。使用定性PCR研究相关基因的表达。采用酶联免疫吸附测定（ELISA）分析rbIL15处理的单核细胞产生干扰素（IFN）-γ的情况。
通过SDS-PAGE和蛋白质印迹法分析rbIL_15，发现了一种重24 kDa的特异性产物。rbIL15诱导的最佳条件是0.2 mM异丙基-β-D-1-吡喃半乳糖苷和37°C。当将rbIL15添加到健康牛的PBMC中时，相关基因表达。对rbIL15处理的PBMC培养基进行ELISA检测，发现有IFN-γ产生。当健康奶牛的PBMC同时用rbIL15、CTLA-4和PD-L1阻断剂处理时，它们产生的IFN-γ并不比rbIL15组多。使用感染BLV牛的PBMC，联合治疗可增加IFN-γ的产生。
rbIL15的生物学活性表现为诱导转录因子和产生IFN-γ。在健康牛和感染BLV牛的PBMC中，将rbIL15与CTLA-4和PD-L1阻断剂联合使用可导致转录因子和细胞因子的产生。结果表明，使用这种方法可提高慢性病毒感染患者的免疫力和免疫记忆。
