Cloning and expression of functional single-chain Fv antibodies directed against NIa and coat proteins of potato virus Y.
Three single-chain variable fragment (scFv) antibodies recognizing the nuclear inclusion a (NIa) and capsid proteins of potato virus Y were obtained from two mouse derived hybridoma clones secreting, respectively, an anti-NIa (22-1) and an anti-coat protein (136-13) monoclonal antibodies. The first monoclonal antibody was able to inhibit in vitro the PVY polyprotein cleavage by blocking the NIa protease activity. The amplified scFv cDNAs were first inserted into the TOPO vector and then sequenced. Several recombinant E. coli clones carrying the accurate scFv sequences were selected and the corresponding cDNAs were subcloned in pHEN phagemid and transferred in E. coli strain. The expressed scFv fragments showed an antibody activity that recognized the viral target proteins in infected tissues. Their activity was comparable to the parental monoclonal antibodies.
Rouis
Souad
S
Centre de Biotechnologie de Sfax, BP K, 3018 Sfax, Tunisia.
Lafaye
Pierre
P
Jaoua-Aydi
Leila
L
Sghaier
Zidani
Z
Ayadi
Hammadi
H
Gargouri-Bouzid
Radhia
R
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology*; D036022:Capsid Proteins / Q000276:immunology; D003001:Cloning, Molecular; D018076:DNA, Complementary; D010450:Endopeptidases; D004926:Escherichia coli / Q000235:genetics; D019084:Fluorescent Antibody Technique, Indirect; D006825:Hybridomas; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology*; D008969:Molecular Sequence Data; D017800:Potyvirus / Q000276:immunology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D017422:Sequence Analysis, DNA; D011198:Solanum tuberosum / Q000821:virology; D014764:Viral Proteins / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D036022:Capsid Proteins; D018076:DNA, Complementary; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins; D014764:Viral Proteins; D010450:Endopeptidases; C059992:nuclear inclusion protein a, mosaic viruses