title: Design and selection of an intrabody library produced de-novo for the non-structural protein NSP5 of rotavirus.
abstract: Intracellular antibodies or intrabodies have great potential in protein knockout strategies for intracellular antigens. We applied the Intracellular Antibody Capture Technology for the direct selection in yeast of a mouse scFv library (V(L)-V(H) format) constructed from animals immunised with recombinant non-structural protein NSP5 of Rotavirus. We selected five different intracellular antibodies (ICAbs), which specifically recognize Delta2, an NSP5 deletion mutant used as bait. The anti-NSP5 ICAbs were well expressed both in yeast and mammalian cells as cytoplasmic or nuclear-tagged forms. By immunofluorescence and co-immunoprecipitation assays we characterised the intracellular interaction of the five anti-NSP5 ICAbs with the co-expressed antigens.
authors.affiliation: International Centre for Genetic Engineering and Biotechnology, Padriciano 99, 34012 Trieste, Italy.
authors.forename: Fulvia
authors.initials: F
authors.lastname: Vascotto
authors.forename: Michela
authors.initials: M
authors.lastname: Visintin
authors.forename: Antonino
authors.initials: A
authors.lastname: Cattaneo
authors.forename: Oscar R
authors.initials: OR
authors.lastname: Burrone
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D000918:Antibody Specificity; D000956:Antigens, Viral / Q000276:immunology; D002460:Cell Line; D015723:Gene Library; D005822:Genetic Vectors / Q000235:genetics; D051379:Mice; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D013154:Spleen / Q000276:immunology / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000276:immunology*
chemical_list: D000906:Antibodies; D000956:Antigens, Viral; C434828:NSP5 protein, rotavirus group A; D017361:Viral Nonstructural Proteins