Expression of functional recombinant antibody molecules in insect cell expression systems.
Recombinant single-chain variable-fragment molecules (scFv) were constructed from a cell line expressing a monoclonal antibody against African cassava mosaic virus (ACMV) and expressed in Escherichia coli. DNA sequences that encoded the scFv were manipulated to allow scFv expression in insect cell lines. A recombinant baculovirus containing the scFv cDNA was constructed and large amounts of scFv were produced in each of three insect cell lines infected with the baculovirus. However, the scFv were not secreted into the medium by any of the cell lines despite the scFv having been linked to a honeybee melittin leader sequence. The same scFv cDNA construct was introduced into Drosophila DS2 cells and a stable recombinant cell line was obtained that produced scFv that was secreted into the medium. Culture medium containing the scFv was used directly in enzyme-linked immunosorbent assay (ELISA) tests to detect ACMV in plant tissues. Another construct that encoded the Ckappa domain of human IgG was fused to the C-terminus of the scFv that was produced and expressed in Drosophila cells. This scFv derivative also accumulated in the medium and was more active in ELISA than scFv lacking the Ckappa domain.
Reavy
B
B
Scottish Crop Research Institute, Invergowrie, DD2 5DA, United Kingdom. breavy@scri.sari.ac.uk
Ziegler
A
A
Diplexcito
J
J
Macintosh
S M
SM
Torrance
L
L
Mayo
M
M
D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000378:metabolism*; D000914:Antibodies, Viral / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004330:Drosophila / Q000235:genetics / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D017791:Geminiviridae / Q000235:genetics* / Q000276:immunology; D006801:Humans; D015151:Immunoblotting; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000378:metabolism*; D009029:Mosaic Viruses / Q000235:genetics* / Q000276:immunology; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins