Navabi Parnian, Ganjalikhany Mohamad Reza, Jafari Sepideh, Dehbashi Moein, Ganjalikhani-Hakemi Mazdak
Department of Immunology, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.
Department of Cell and Molecular Biology, Faculty of Biological Science and Technology, University of Isfahan, Isfahan, Iran.
Iran J Basic Med Sci. 2021 Mar;24(3):360-368. doi: 10.22038/ijbms.2021.51709.11728.
IL-2Rα plays a critical role in maintaining immune function. However, expression and secretion of CD25 in various malignant disorders and autoimmune diseases are now well established. Thus, CD25 is considered an important target candidate for antibody-based therapy. This study aimed to find the most suitable linker peptide to construct a functional anti-CD25 single-chain fragment variable (scFv) by bioinformatics studies and its production in a bacterial expression system.
Here, the 3D structures of the scFvs with different linkers were predicted and molecular dynamics simulation was performed to compare their structures and dynamics. Then, interactions between five models of scFv and human CD25 were calculated via molecular docking. According to MD and docking results, the anti-CD25 scFvs with (Gly4Ser)3 linker were constructed and cloned into pET-22b(+). Then, recombinant plasmids were transformed into Bl21 (DE3) for expression using IPTG and lactose as inducers. Anti-CD25 scFv was purified from the periplasm and detected by SDS-PAGE and Western blot. Afterward, functionality was evaluated using ELISA.
analysis showed that the model containing (Gly4Ser)3 as a linker has more stability compared with other linkers. The results of SDS-PAGE, Western blot, and ELISA confirmed the accuracy of anti-CD25 scFv production and its ability to bind to the human CD25.
Conclusively, our work provides a theoretical and experimental basis for production of an anti-CD25 scFv, which may be applied for various malignant disorders and autoimmune diseases.
白细胞介素-2受体α（IL-2Rα）在维持免疫功能中起关键作用。然而，CD25在各种恶性疾病和自身免疫性疾病中的表达及分泌情况现已明确。因此，CD25被认为是基于抗体治疗的重要候选靶点。本研究旨在通过生物信息学研究找到构建功能性抗CD25单链可变片段（scFv）的最合适连接肽，并在细菌表达系统中生产该片段。
在此，预测了带有不同连接肽的scFv的三维结构，并进行分子动力学模拟以比较其结构和动力学。然后，通过分子对接计算了scFv的五种模型与人类CD25之间的相互作用。根据分子动力学和对接结果，构建了带有（Gly4Ser）3连接肽的抗CD25 scFv，并将其克隆到pET-22b(+)中。然后，将重组质粒转化到BL21(DE3)中，使用异丙基-β-D-硫代半乳糖苷（IPTG）和乳糖作为诱导剂进行表达。从周质中纯化抗CD25 scFv，并通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和蛋白质免疫印迹法（Western blot）进行检测。之后，使用酶联免疫吸附测定（ELISA）评估其功能。
分析表明，与其他连接肽相比，含有（Gly4Ser）3作为连接肽的模型具有更高的稳定性。SDS-PAGE、Western blot和ELISA的结果证实了抗CD25 scFv生产的准确性及其与人CD25结合的能力。
总之，我们的工作为抗CD25 scFv的生产提供了理论和实验基础，其可能应用于各种恶性疾病和自身免疫性疾病。
