Transient Expression in Cytoplasm and Apoplast of Rotavirus VP6 Protein Fused to Anti-DEC205 Antibody in Nicotiana benthamiana and Nicotiana sylvestris

Abstract

Rotavirus is the most common cause of severe diarrhea in infants and children worldwide and is responsible for about 215,000 deaths annually. Over 85% of these deaths originate in low-income/developing countries in Asia and Africa. Therefore, it is necessary to explore the development of vaccines that avoid the use of "living" viruses and furthermore, vaccines that have viral antigens capable of generating powerful heterotypic responses. Our strategy is based on the expression of the fusion of the anti-DEC205 single-chain variable fragment (scFv) coupled by an OLLAS tag to a viral protein (VP6) of Rotavirus in Nicotiana plants. It was possible to express transiently in N. benthamiana and N. sylvestris a recombinant protein consisting of the single chain variable fragment linked by an OLLAS tag to the VP6 protein. The presence of the recombinant protein, which had a molecular weight of approximately 75 kDa, was confirmed by immunodetection, in both plant species and in both cellular compartments (cytoplasm and apoplast) where it was expressed. In addition, the recombinant protein was modeled, and it was observed that some epitopes of interest are exposed on the surface, which could favor their immunogenic response.

Neutralization and immunoperoxidase to estimate the immunogenicity of recombinant human rotavirus VP6 structural protein in vitro after insertion of the antibodies into cells using electroporation

“…This indicates the need to use different puri cation method, like ion exchange or gel ltration, to minimize the loss of the protein during the puri cation process. E. coli BL21 strain which was used in this study, is the most commonly used expression system for rVP6 protein [27,71], although, several studies explored different expression systems including eukaryotic systems such as insect cells through baculovirus expression vector [72], yeast including Saccharomyces cerevisiae, Pichia pastoris, and Hansenula polymorpha [73,74], mammalian cells [75], viruses such as herpes simplex virus 1 (HSV-1)based vectors [76], and plant cells [77]. The relatively low protein content obtained in this study may indicate the need to use another expression system in future studies as one of the suggested solutions to increase the amount of the expressed protein.…”