Laboratório de Vacinologia, Núcleo de Biotecnologia, Centro de Desenvolvimento Tecnológico, Universidade Federal de Pelotas, Pelotas, RS, Brazil.
Faculdade de Medicina, Universidade Federal Do Rio Grande, Rio Grande, RS, Brazil.
Mol Biol Rep. 2024 Sep 25;51(1):1012. doi: 10.1007/s11033-024-09940-5.
The development of therapies and vaccines for various diseases often necessitates the analysis of cellular immunity. However, unlike other rodents, the limited availability of reagents for Syrian hamsters restricts immunological analysis, particularly in the determination of serum effector molecules such as cytokines. In this study, we aim to produce and characterize the cytokines IFN-γ, TGF-β, IL-6, IL-10, and TNF-α from Syrian hamsters in recombinant form and to generate polyclonal antibodies against them in rats.
Cytokine transcript sequences were cloned into expression vectors in E. coli. Recombinant proteins were produced, purified through affinity chromatography, and characterized by Western blot using an anti-6xHis monoclonal antibody. Rats were immunized with the recombinant proteins to generate polyclonal antibodies (pAbs). These pAbs were characterized by Western blot and titrated by indirect ELISA. The recombinant cytokines rTNF-α, rIL-10, rIFN-γ, rTGF-β, and rIL-6 were produced and specifically recognized at their expected molecular weights of 22.3 kDa, 19.8 kDa, 18.9 kDa, 11.8 kDa, and 22.9 kDa. pAbs were produced and demonstrated the ability to specifically recognize their target proteins with titers of 409,600 (rIL-10), 204,800 (rTNF-α), 102,400 (rIL-10), 51,200 (rTGF-β), and 25,600 (rIFN-ɣ).
The reagents produced represent a starting point for developing immunoassays to detect hamster cytokines, facilitating the analysis of cellular immunity in this biomodel.
各种疾病的疗法和疫苗的开发通常需要分析细胞免疫。然而，与其他啮齿动物不同，叙利亚仓鼠的试剂供应有限，限制了免疫分析，特别是在确定血清效应分子（如细胞因子）方面。在这项研究中，我们旨在以重组形式产生和表征叙利亚仓鼠的细胞因子 IFN-γ、TGF-β、IL-6、IL-10 和 TNF-α，并在大鼠中针对它们产生多克隆抗体。
细胞因子转录序列被克隆到大肠杆菌中的表达载体中。通过亲和层析生产重组蛋白，并通过 Western blot 用抗 6xHis 单克隆抗体进行鉴定。用重组蛋白免疫大鼠以产生多克隆抗体 (pAb)。通过 Western blot 鉴定这些 pAb，并通过间接 ELISA 滴定。产生了重组细胞因子 rTNF-α、rIL-10、rIFN-γ、rTGF-β 和 rIL-6，并特异性地在预期的 22.3 kDa、19.8 kDa、18.9 kDa、11.8 kDa 和 22.9 kDa 分子量处被识别。产生了 pAb，并证明了它们以 409,600（rIL-10）、204,800（rTNF-α）、102,400（rIL-10）、51,200（rTGF-β）和 25,600（rIFN-γ）的滴度特异性识别其靶蛋白的能力。
所产生的试剂为开发检测仓鼠细胞因子的免疫分析提供了起点，便于在这种生物模型中分析细胞免疫。
