Designing and generating a single-chain fragment variable (scFv) antibody against IL2Rα (CD25): An in silico and in vitro study

Objective(s): 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 antibodybased therapy. This study aimed to find the most suitable linker peptide to construct a functional antiCD25 singlechain fragment variable (scFv) by bioinformatics studies and its production in a bacterial expression system. Materials and Methods: 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 antiCD25 scFvs with (Gly4Ser)3 linker were constructed and cloned into pET22b(+). Then, recombinant plasmids were transformed into Escherichia coli Bl21 (DE3) for expression using IPTG and lactose as inducers. AntiCD25 scFv was purified from the periplasm and detected by SDSPAGE and Western blot. Afterward, functionality was evaluated using ELISA. Results: In silico analysis showed that the model containing (Gly4Ser)3 as a linker has more stability compared with other linkers. The results of SDSPAGE, Western blot, and ELISA confirmed the accuracy of antiCD25 scFv production and its ability to bind to the human CD25. Conclusion: Conclusively, our work provides a theoretical and experimental basis for production of an antiCD25 scFv, which may be applied for various malignant disorders and autoimmune diseases.