Transient expression of a human tissue transglutaminase (htTG) specific antibody in Nicotiana benthamiana

SCABONE, CAMILA MARIA; VECCHI, BRUNO; PETRUCCELLI, SILVANA

Porto

Congreso; Fourth International Conference- Plant Based Vaccines and Antibodies- PBVA 2011; 2011

Numerous efforts have been made over the last decades to develop and optimize plant expression platforms for the large-scale production of both industrial and clinically useful proteins. We are focus on two proteins with potential applications in diagnostic of celiac disease pathogenesis: the autoantigen htTG and a monoclonal antibody 2G3 specific to htTG. These two proteins are difficult to produce in other systems. Total mRNA was isolated from 2G3 hybridoma cells, then a cDNA was obtained and the genes encoding the variable domains (HV and LV) of the mAb 2G3 were amplified using a set of mouse V-specific primers. The PCR products were cloned into the expression vector pComb3X producing scFv or Fab versions and recognition to htTG was evaluated by Phage ELISA. Recombinant full length immunoglobulin genes (a-htTG-Ig) were generated using overlapping extension PCR to fuse the genes encoding the 2G3 light and heavy variable domains to mouse kappa and gamma 1 constant region domains, respectively. In order to develop new strategies to target full length antibody to different subcellular compartments, a red fluorescent protein fused to two different vacuolar sorting signals (VSS) were introduced at the  C-terminal ends of the  2G3 antibody light and heavy chains (LC or HC-mRFP-VSS). The obtained fusions were cloned into the pGWB2 binary vector, introduced into Agrobacterium tumefaciens and temporal expression in N. benthamiana. Different combination of theses constructs and also secretory controls were analyzed 3-4 days after agroinfiltration. Dependent of the combination tags/chains used a-htTG-Ig was found in the endoplasmic reticulum, apoplastic space or central vacuole. The delivery of a-htTG-Ig -RFP-VSS to vacuoles proved their benefits by increasing antibody expression up to 3-fold. The antigen-binding activity of a-htTG-Ig-RFP was confirmed by enzyme-linked immunosorbent assay.