Genetic engineering of streptavidin-binding peptide tagged single-chain variable fragment antibody to Venezuelan equine encephalitis virus.
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.
Hu
Wei-Gang
WG
Chemical Biological Defence Section, Defence Research & Development Canada-Suffield, Box 4000, Station Main, Medicine Hat, Alberta, Canada T1A 8K6.
Alvi
Azhar Z
AZ
Fulton
R Elaine
RE
Suresh
Mavanur R
MR
Nagata
Les P
LP
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004666:Encephalitis Virus, Venezuelan Equine / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D007128:Immunoglobulin Fragments / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D019809:Streptavidin / Q000378:metabolism
D002352:Carrier Proteins; D007128:Immunoglobulin Fragments; D011993:Recombinant Fusion Proteins; C452700:streptavidin-binding peptide; D019809:Streptavidin