Generation and characterization of single-chain antibody fragments specific against transmembrane envelope glycoprotein gp46 of maedi-visna virus
Language English Country Netherlands Media print
Document type Evaluation Study, Journal Article, Research Support, Non-U.S. Gov't
PubMed
14656464
DOI
10.1016/j.jviromet.2003.09.020
PII: S0166093403002830
Knihovny.cz E-resources
- MeSH
- Cell Line MeSH
- DNA Fingerprinting methods MeSH
- Enzyme-Linked Immunosorbent Assay MeSH
- Immunoglobulin Fragments chemistry immunology MeSH
- Immunoglobulin Light Chains MeSH
- Molecular Sequence Data MeSH
- Sheep MeSH
- Peptide Library * MeSH
- Surface Plasmon Resonance MeSH
- Viral Envelope Proteins immunology MeSH
- Antibodies, Viral immunology MeSH
- Amino Acid Sequence MeSH
- Sequence Analysis, DNA MeSH
- Antibody Specificity MeSH
- Immunoglobulin Variable Region MeSH
- Visna-maedi virus immunology MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Evaluation Study MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Immunoglobulin Fragments MeSH
- Immunoglobulin Light Chains MeSH
- Peptide Library * MeSH
- Viral Envelope Proteins MeSH
- Antibodies, Viral MeSH
- Immunoglobulin Variable Region MeSH
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.
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