Parnian Navabi - Department of Immunology, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
Mohamad Reza Ganjalikhany - Department of Cell and Molecular Biology, Faculty of Biological Science and Technology, University of Isfahan, Isfahan, Iran
Sepideh Jafari - Department of Cell and Molecular Biology, Faculty of Biological Science and Technology, University of Isfahan, Isfahan, Iran
Moein Dehbashi - Division of Genetics, Department of Cell and Molecular Biology and Microbiology, Faculty of Biological Sciences and Technologies, University of Isfahan, Isfahan, Iran
Mazdak Ganjalikhani-Hakemi - Department of Immunology, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

Objective(s): IL-۲Rα plays a critical role in maintaining immune function. However, expression and secretion of CD۲۵ in various malignant disorders and autoimmune diseases are now well established. Thus, CD۲۵ 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-CD۲۵ single-chain fragment variable (scFv) by bioinformatics studies and its production in a bacterial expression system. Materials and Methods: Here, the ۳D 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 CD۲۵ were calculated via molecular docking. According to MD and docking results, the anti-CD۲۵ scFvs with (Gly۴Ser)۳ linker were constructed and cloned into pET-۲۲b(+). Then, recombinant plasmids were transformed into Escherichia coli Bl۲۱ (DE۳) for expression using IPTG and lactose as inducers. Anti-CD۲۵ scFv was purified from the periplasm and detected by SDS-PAGE and Western blot. Afterward, functionality was evaluated using ELISA.Results: In silico analysis showed that the model containing (Gly۴Ser)۳ as a linker has more stability compared with other linkers. The results of SDS-PAGE, Western blot, and ELISA confirmed the accuracy of anti-CD۲۵ scFv production and its ability to bind to the human CD۲۵. Conclusion: Conclusively, our work provides a theoretical and experimental basis for production of an anti-CD۲۵ scFv, which may be applied for various malignant disorders and autoimmune diseases.

CD۲۵ Daclizumab IL, ۲Rα Protein engineering Single, chain variable fragment (scFv)