Genetic Engineering of Streptavidin-Binding Peptide Tagged Single-Chain Variable Fragment Antibody to Venezuelan Equine Encephalitis Virus
| Autor: | Wei-Gang Hu, Azhar Z. Alvi, R. Elaine Fulton, Mavanur R. Suresh, Les P. Nagata |
|---|---|
| Rok vydání: | 2002 |
| Předmět: |
Streptavidin
medicine.drug_class Recombinant Fusion Proteins Blotting Western Molecular Sequence Data Immunology Alphavirus Monoclonal antibody medicine.disease_cause Encephalitis Virus Venezuelan Equine chemistry.chemical_compound Genetics medicine Amino Acid Sequence Immunoglobulin Fragments Expression vector Base Sequence biology biology.organism_classification Molecular biology Fusion protein chemistry Biotinylation SBP-tag Venezuelan equine encephalitis virus Electrophoresis Polyacrylamide Gel Carrier Proteins |
| Zdroj: | Hybridoma and Hybridomics. 21:415-420 |
| ISSN: | 1536-8599 |
| DOI: | 10.1089/153685902321043945 |
| Popis: | A recombinant gene encoding a single-chain variable fragment (scFv) antibody against Venezuelan equine encephalitis virus (VEE) was cloned into a prokaryotic T7 RNA polymerase-regulated expression vector. A streptavidin-binding peptide gene fused to a 6His tag was attached downstream to the scFv gene. The recombinant fusion protein was expressed in bacteria as inclusion bodies that were subsequently solubilized with 8 M urea and renatured by an arginine system. Purification of the fusion protein was achieved by immobilized metal affinity chromatography. Enzyme-linked immunosorbent assay (ELISA) and Western blotting results revealed that the fusion protein not only retained VEE antigen binding and specificity properties similar to those of its parent native monoclonal antibody (MAb), but also possessed streptavidin-binding activity. This experimental approach can eliminate the need for chemical biotinylation of antibodies and the risk associated of antibody denaturation and can provide a stable and reproducible reagent for rapid and efficient immunoassay of VEE when detected by horseradish peroxidase (HRP)-conjugated streptavidin. |
| Databáze: | OpenAIRE |
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