RESUMO
We prepared single-chain immunoglobulin Fv fragments (scFv) SLH10 specific for the HepG2 cell line after biopanning from a large human-naive phage display library (Griffin. 1 Library). The three-dimensional (3D) structure of SLH10 was modelled by the Insight II molecule simulation software.The structure was refined using the molecular dynamics method.The structures with the least steric clashes and lowest energy were determined finally. The optimized structures of heavy (VH) and light (VL) variable chains of SLH10 scFv were obtained.Then SLH10 bivalent single-chain Fv (BsFv) was constructed that would be suitable for high-affinity targeting.SLH10 BsFv was generated by linking scFvs together and identified by sequencing. Its expression products were confirmed by western blot analysis.The relative molecular masses of scFv and BsFv were approximately 30 kDa and 60 kDa,respectively. Flow cytometry revealed that SLH10 BsFv bound the selected cell lines with greater signal intensity than the parental scFv. The improved antigen binding of SLH10 BsFv may be useful for immunodiagnostics or targeted gene therapy for liver cancer.
Assuntos
Anticorpos , Linhagem Celular , Hepatócitos/imunologia , Humanos , Fragmentos de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas , Cadeias Leves de Imunoglobulina , Modelos Biológicos , Modelos Moleculares , Biblioteca de Peptídeos , Ligação Proteica , Conformação Proteica , Homologia de Sequência de AminoácidosRESUMO
We describe the expression of an anti-Z-DNA single chain variable region antibody fragment (scFv) on a filamentous phage surface. Four vectors for phage display were constructed. Two of them are able to display multiple copies of the antibody fragment, and the others can be used to make monovalent libraries. The vectors use different promoter/leader sequences to direct the expression of the fused proteins. All were able to promote the assembly of fusion virion particles. In this paper we also show the affinity selection (biopanning) of those phage-antibodies based on the capacity of their products to recognize the antigen. We used biotinylated Z-DNA and the selection was performed in a solution phase fashion. The data presented here indicate that these vectors can be further used to construct anti-nucleic acid antibody fragment libraries that can be used to study the basis of nucleic acid-protein interaction and its role in autoimmunity mechanisms.