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) being cloned into a prokaryotic T7 RNA polymerase-regulated expression vector was disclosed. A streptavidin-binding peptide (SBP) sequence fused to a 6His tag is then attac...

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Hauptverfasser:	FULTON R. ELAINE, NAGATA LESLIE P, HU WEIGANG, ALVI AZHAR Z
Format:	Patent
Sprache:	eng
Schlagworte:	BEER BIOCHEMISTRY CHEMISTRY COMPOSITIONS THEREOF CULTURE MEDIA ENZYMOLOGY HUMAN NECESSITIES HYGIENE INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIRCHEMICAL OR PHYSICAL PROPERTIES MEASURING MEDICAL OR VETERINARY SCIENCE METALLURGY MICROBIOLOGY MICROORGANISMS OR ENZYMES MUTATION OR GENETIC ENGINEERING ORGANIC CHEMISTRY PEPTIDES PHYSICS PREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS SPIRITS TESTING VINEGAR WINE
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Zusammenfassung:	A recombinant gene encoding a single-chain variable fragment (scFv) antibody against Venezuelan equine encephalitis virus (VEE) being cloned into a prokaryotic T7 RNA polymerase-regulated expression vector was disclosed. A streptavidin-binding peptide (SBP) sequence fused to a 6His tag is then attached downstream to the scFv gene. The recombinant fusion protein is expressed in bacteria and then purified by immobilized metal affinity chromatography. 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, but also possessed streptavidin-binding activity. This discovery obviates the need for chemical biotinylation of antibodies and the risk associated with antibody denaturation and provides a stable and reproducible reagent for rapid and efficient immunoassay of VEE.