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

Document Type : Original Article

Authors

1 Department of Immunology, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

2 Department of Cell and Molecular Biology, Faculty of Biological Science and Technology, University of Isfahan, Isfahan, Iran

3 Division of Genetics, Department of Cell and Molecular Biology and Microbiology, Faculty of Biological Sciences and Technologies, University of Isfahan, Isfahan, Iran

Abstract

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 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.
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 anti-CD25 scFvs with (Gly4Ser)3 linker were constructed and cloned into pET-22b(+). Then, recombinant plasmids were transformed into Escherichia coli 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.
Results: In silico 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.
Conclusion: 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.

Keywords

References

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Iranian Journal of Basic Medical Sciences
Volume 24, Issue 3
March 2021
Pages 360-368

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