Genetic engineering of streptavidin-binding peptide tagged single-chain variable fragment antibody to Venezuelan equine encephalitis virus.

This research shows the successful genetic engineering of a fusion protein that attaches to the Venezuelan equine encephalitis virus and makes it easier to detect. The new protein also binds to a molecule called streptavidin, which simplifies the identification process and minimizes the risk associated with previous methods of detection.

Objective of the Study

• The primary aim of this research was to create a recombinant fusion protein comprised of a single-chain variable fragment (scFv) antibody against the Venezuelan equine encephalitis virus (VEE) and a streptavidin-binding peptide gene tagged with a 6His tag. This genetic engineering offers an improved and more stable technique for detecting the virus.

Research Methodology

• The researchers cloned the gene encoding the scFv antibody against VEE into a prokaryotic T7 RNA polymerase-regulated expression vector, a type of plasmid used for the efficient production of proteins in bacteria.
• A streptavidin-binding peptide gene attached to a 6His tag was fused downstream of the scFv gene to produce the fusion protein.
• This fusion protein was then expressed in bacteria as inclusion bodies, which were then solubilized with 8 M urea and reformed using an arginine system. This is a method to refold denatured proteins into their active conformations.
• The fusion protein was then isolated through immobilized metal affinity chromatography, a process that exploits the affinity of the 6His tag to nickel ions immobilized on a matrix to purify the protein.

Findings and Conclusion

• The researchers tested the fusion protein using enzyme-linked immunosorbent assay (ELISA) and Western blotting. The results showed that the fusion protein maintained ability to bind to the VEE antigen and had the same specificity as the original monoclonal antibody.
• Additionally, the protein exhibited streptavidin-binding activity. This is significant since streptavidin commonly binds to a small molecule called biotin. Therefore the results indicate that using this fusion protein could eliminate the need for chemical biotinylation of antibodies.
• The researchers concluded that their new method provides a more stable and reproducible reagent for rapid and efficient immunoassay of VEE, since detection can be done by horseradish peroxidase (HRP)-conjugated streptavidin which is a common method used to amplify the signal in immunoassays.