Design and selection of an intrabody library produced de-novo for the non-structural protein NSP5 of rotavirus.
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.
Vascotto
Fulvia
F
International Centre for Genetic Engineering and Biotechnology, Padriciano 99, 34012 Trieste, Italy.
Visintin
Michela
M
Cattaneo
Antonino
A
Burrone
Oscar R
OR
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*
D000906:Antibodies; D000956:Antigens, Viral; C434828:NSP5 protein, rotavirus group A; D017361:Viral Nonstructural Proteins