Generation and characterization of single-chain antibody fragments specific against transmembrane envelope glycoprotein gp46 of maedi-visna virus.
A single-chain antibody fragments (scFv) was developed directed against transmembrane envelope glycoprotein gp46 of the virus maedi-visna, by the application of the antibody phage display library. To get specific scFv binders, the library was panned against the biotinylated peptide of 20 amino acids corresponding to the principal immunodominant domain of gp46 protein. The number of positively binding scFvs was evaluated by scFv-phage ELISA, BstN1 fingerprinting and DNA sequencing. The scFvs were expressed in soluble form and purified by immobilized metal affinity chromatography (IMAC) with a yield of 2-2.5 mg/l. Two scFvs have shown to recognize gp46 and gp150 proteins in Western blot analysis. The scFvs also recognized the virus in infected cells as shown by immunofluorescence assay. The affinity of the obtained antibody fragments to gp46 peptide was measured by surface plasmon resonance, and the resulting K(A) was in the 10(6)-10(7)lmol(-1) range. The application of characterized scFvs for expression as intrabodies in intracellular immunization against virus maedi-visna infection and for the diagnosis of this virus is discussed.
Blazek
Dalibor
D
Institute of Microbiology and Immunology, Faculty of Veterinary Medicine, Veterinary and Pharmaceutical University Brno, Palackého 1/3, 612 42 Brno, Czech Republic. dalizek@hotmail.com
Celer
Vladimir
V
Navrátilová
Iva
I
Skládal
Petr
P
D000595:Amino Acid Sequence; D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology*; D000918:Antibody Specificity; D002460:Cell Line; D016172:DNA Fingerprinting / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000276:immunology*; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D008969:Molecular Sequence Data; D019151:Peptide Library; D017422:Sequence Analysis, DNA; D012756:Sheep; D020349:Surface Plasmon Resonance; D014759:Viral Envelope Proteins / Q000276:immunology*; D014790:Visna-maedi virus / Q000276:immunology*
D000914:Antibodies, Viral; D007128:Immunoglobulin Fragments; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D014759:Viral Envelope Proteins