|Authors: ||B. Morgun, S. Deshmukh, A. Richter, V. Stepanyuk, K. Kalai, G. Nagy, N. Lukacs|
|Keywords: ||recombinant antibody, plantibody, scFv, double-stranded RNA, virus replication, Nicotiana tabacum|
Antibody expression and immunomodulation in plants are modern molecular techniques for producing pharmaceuticals and for interfering with cellular metabolism or pathogen infectivity.
Large-scale production of antibodies in transgenic plants is possible and offers many advantages.
The achievement of immunomodulation, however, is still a challenge as it is still difficult to express correctly folded active antibodies or antibody fragments in a particular cell compartment, especially in the cytoplasm.
In our group, mouse monoclonal antibodies, K1, J2 and P6, which specifically recognize double-stranded RNA (dsRNA) and clones of their respective cDNAs, had been produced in an earlier project.
Our goal is to use these antibodies to modulate the biological activity of dsRNA in plants, in particular by influencing virus replication through the stabilization of double-stranded replication intermediates.
The aim of this research was to establish strategies for the expression of correctly assembled single-chain antibody fragments (scFv) in various, pre-determined compartments of the plant.
By adding different polypeptide targeting and stabilizing signals, we aimed to target these scFv derivatives to obtain either cytoplasmic expression, anchoring on the cytoplasmic side of the plasma membrane, secretion to the apoplast or ER-residence in transgenic N. tabacum plants.
Very high levels of protein expression were observed in case of all scFv when they were made ER-resident by adding the N-terminal signal sequence and the C-terminal ER-retention signal KDEL. Without the KDEL-signal, i.e. when scFv were targeted to the apoplast, neither J2- nor P6-scFv reached detectable concentrations.
Large differences between each of the scFvs were observed in the case of cytoplasmic expression: scFv P6 was easily detectable on Western-blots, while for scFv J2 and K1 only high levels of mRNA, but no protein, could be detected.
Only one antibody derivative, the ER-resident J2-scFv, was found to have an effect on the replication of potato virus Y (PVY).
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