RESUMO
Dengue fever is endemic in more than 120 countries, which account for 3.9 billion people at risk of infection worldwide. The absence of a vaccine with effective protection against the four serotypes of this virus makes differential molecular diagnosis the key step for the correct treatment of the disease. Rapid and efficient diagnosis prevents progression to a more severe stage of this disease. Currently, the limiting factor in the manufacture of dengue (DENV) diagnostic kits is the lack of large-scale production of the non-structural 1 (NS1) protein (antigen) to be used in the capture of antibodies from the blood serum of infected patients. In this work, we use plant biotechnology and genetic engineering as tools for the study of protein production for research and commercial purposes. Gene transfer, integration and expression in plants is a valid strategy for obtaining large-scale and low-cost heterologous protein production. The authors produced NS1 protein of the dengue virus serotype 2 (NS1DENV2) in the Arabidopsis thaliana plant. Transgenic plants obtained by genetic transformation expressed the recombinant protein that was purified and characterized for diagnostic use. The yield was 203 µg of the recombinant protein per gram of fresh leaf. By in situ immunolocalization, transgenic protein was observed within the plant tissue, located in aggregates bodies. These antigens showed high sensitivity and specificity to both IgM (84.29% and 91.43%, respectively) and IgG (83.08% and 87.69%, respectively). The study goes a step further to validate the use of plants as a strategy for obtaining large-scale and efficient protein production to be used in dengue virus diagnostic tests.
RESUMO
The expression of recombinant proteins in plants is a valuable alternative to bioreactors using mammalian cell systems. Ease of scaling, and their inability to host human pathogens, enhance the use of plants to generate complex therapeutic products such as monoclonal antibodies. However, stably transformed plants expressing antibodies normally have a poor accumulation of these proteins that probably arise from the negative positional effects of their flanking chromatin. The induction of boundaries between the transgenes and the surrounding DNA using matrix attachment regions (MAR) and insulator elements may minimize these effects. With the PHB-01 antibody as a model, we demonstrated that the insertion of DNA elements, the TM2 (MAR) and M4 insulator, flanking the transcriptional cassettes that encode the light and heavy chains of the PHB-01 antibody, increased the protein accumulation that remained stable in the first plant progeny. The M4 insulator had a stronger effect than the TM2, with over a twofold increase compared to the standard construction. This effect was probably associated with an enhancer-promoter interference. Moreover, transgenic plants harboring two transcriptional units encoding for the PHB-01 heavy chain combined with both TM2 and M4 elements enhanced the accumulation of the antibody. In summary, the M4 combined with a double transcriptional unit of the heavy chain may be a suitable strategy for potentiating PHB-01 production in tobacco plants.