Expression and functional characterization of a single chain FV antibody directed against secretions involved in plant nematode infection process
| Autor: | Pierre Abad, Tanja Borst-Vrenssen, Fred J. Gommers, A. Schouten, Marie-Noëlle Rosso, Jan Roosien, Arjen Schots, Richard S. Hussey, Japp Bakker |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 1996 |
| Předmět: |
Signal peptide
KDEL Recombinant Fusion Proteins Molecular Sequence Data Biophysics Antibodies Helminth Immunoglobulin Variable Region chemical and pharmacologic phenomena Biology Protein Sorting Signals Biochemistry Polymerase Chain Reaction medicine Meloidogyne incognita Escherichia coli Animals Life Science Amino Acid Sequence Tylenchoidea Molecular Biology Gene Immunoglobulin Fragments Laboratorium voor Nematologie Hybridomas Base Sequence food and beverages Cell Biology Protoplast medicine.disease biology.organism_classification Molecular biology Recombinant Proteins Transformation (genetics) Nematode infection Immunoglobulin M biology.protein Antibody EPS Laboratory of Nematology Oligopeptides |
| Zdroj: | Biochemical and Biophysical Research Communications 220 (1996) Biochemical and Biophysical Research Communications, 220, 255-263 |
| ISSN: | 0006-291X |
| Popis: | Expression in plants of antibodies directed against proteins essential for pathogenesis could provide an alternative approach to engineer new resistance traits into crops. Salivary secretions of the root-knot nematode Meloidogyne incognita are known to play a key role during this nematode infection process. From a hybridoma cell line producing an IgM monoclonal antibody specific to these secretions, we have constructed a synthetic gene that encodes an antigen-binding single-chain Fv protein (scFv). The scFv gene was created by polymerase chain reaction amplification of variable domain encoding regions from the IgM antibody. The cloned scFv was initially expressed in Escherichia coli as a 33-kDa protein which could be purified to near homogeneity by immobilized metal affinity chromatography. The produced scFv is fully functional since it shows the same specificity towards a crude extract of M. incognita infective larvae as the corresponding parental IgM. Transient expression assays with tobacco leaf protoplasts using different targeting signals resulted in a high intracellular accumulation of scFv, especially when fused to the tetrapeptide KDEL retention signal. Activity analysis and stability characterization of this scFv in tobacco protoplast represent the first step before plant transformation in order to test a new form of resistance to root-knot nematode in plants. |
| Databáze: | OpenAIRE |
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