title: Functional expression in bacteria and plants of an scFv antibody fragment against tospoviruses.
abstract: BACKGROUND
  Recombinant antibodies expressed in plants ('plantibodies'), directed against crucial antigens and addressed to the right cell compartment, may be able to protect against viral diseases. Moreover, antibody fragments produced in bacteria or plants may provide low cost reagents for immunodiagnosis.
  
  
  OBJECTIVE
  In an attempt to develop genetic immunisation against tomato spotted wilt tospovirus (TSWV), we engineered an scFv fragment starting from a monoclonal antibody (mAb) able to recognise an epitope of the glycoprotein G1 conserved among a large number of tospoviruses. After establishing functional expression in bacteria, we aimed to drive expression of this molecule in the secretory pathway of plants.
  
  
  METHODS
  An antibody phage display expression system was used to isolate the correct VH and VL binding regions from the hybridoma secreting the original mAb. To assess functional expression in plant, we first used an epichromosomal expression vector derived from potato virus X (PVX). In this vector the scFv gene was cloned to produce a cytosolic or a secretory protein. For secretion, the signal sequence derived from the polygalacturonase-inhibiting protein (PGIP) of Phaseolus vulgaris was used. Subsequently, the gene encoding the secretory scFv, was used to transform Nicotiana benthamiana plants.
  
  
  RESULTS
  High expression levels of fully active molecule were obtained in Escherichia coli. The engineered molecule retained the binding specificity and dissociation rate constant (k(off)) of the cognate monoclonal antibody. Both PVX-infected and transformed plants expressed fully functional scFv molecules in the secretory pathway.
  
  
  CONCLUSIONS
  This engineered scFv may be valuable for inexpensive diagnosis, for studying the role of the glycoproteins in virus transmission and, possibly, for a 'plantibody'-mediated resistance to tospoviruses.
authors.affiliation: ENEA, Dipartimento Innovazione, C.R. Casaccia, Rome, Italy.
authors.forename: R
authors.initials: R
authors.lastname: Franconi
authors.forename: P
authors.initials: P
authors.lastname: Roggero
authors.forename: P
authors.initials: P
authors.lastname: Pirazzi
authors.forename: F J
authors.initials: FJ
authors.lastname: Arias
authors.forename: A
authors.initials: A
authors.lastname: Desiderio
authors.forename: O
authors.initials: O
authors.lastname: Bitti
authors.forename: D
authors.initials: D
authors.lastname: Pashkoulov
authors.forename: B
authors.initials: B
authors.lastname: Mattei
authors.forename: L
authors.initials: L
authors.lastname: Bracci
authors.forename: V
authors.initials: V
authors.lastname: Masenga
authors.forename: R G
authors.initials: RG
authors.lastname: Milne
authors.forename: E
authors.initials: E
authors.lastname: Benvenuto
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D000914:Antibodies, Viral / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics / Q000276:immunology; D007128:Immunoglobulin Fragments / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D008969:Molecular Sequence Data; D010947:Plants, Toxic; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology; D014026:Nicotiana / Q000235:genetics / Q000276:immunology; D017865:Tospovirus / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins; C055966:immunoglobulin Fv