Bioinformatics tools and experimental analysis combination for production of specific scFv against CD133

Abstract

Prominin-1, or CD133, is a membrane-bound pentaspan protein that has been utilized recently to identify cancer stem cells (CSCs) in a variety of carcinomas. Today, bioinformatics offers a potent tool for studying the structural and functional relationships of fusion proteins. A structure-activity relationship model based on physicochemical characteristics, biological functions of single-chain antibodies, and molecular conformation can be developed by the integration of biomolecular computer models with biological experiments. In the present study, a mice library of single-chain variable fragment (scFv) antibodies was developed by mRNA extracted from mice immunized for the efficient and specific identification of the N-terminal domain of recombinant CD133 (D-EC1). First, a part of sequences of the scFvs library were cloned in the T.vector and sequenced. Then, bioinformatics was used to select the scFvs with high affinity by molecular dynamics simulations and docking. Based on bioinformatics analysis, three scFvs were cloned and expressed. Finally, the ability of the selected scFv was confirmed with the indirect enzyme-linked immunosorbent assay (ELISA) and immunocytochemistry (ICC). ELISA data showed that scFv3 had a greater affinity for the N-terminal of recombinant CD133, and it was selected for the immunocytochemistry (ICC) analysis. The immunocytochemistry experiments confirmed that the obtained scFv could bind to the CD133-expressing HT-29 cells. Our results suggest that using bioinformatics tools could be applied as a new, rapid, and valid method for the design and development of antibodies with improved properties. The selected scFv may be successfully applied in scFv-based diagnostics and therapeutics.

Keywords: Bioinformatics; CD133; Cancer stem cells (CSCs); Single-chain variable fragment (scFv).