Abstract
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.
Highlights
Dengue virus (DENV) infection is one of the most important human diseases transmitted by arthropods
We purified and characterized the recombinant protein, which presented antigenic potential, with the ability to recognize anti-dengue antibodies in the serum of infected patients with high specificity and sensitivity in enzyme-linked immunosorbent assays (ELISA)
A NS1DENV2 fragment from pUC57_NS1DENV2 was inserted into pCAMBIA3301, forming pCAMBIA3301_NS1DENV2, which was transformed in E. coli TOP10F again and stored
Summary
Dengue virus (DENV) infection is one of the most important human diseases transmitted by arthropods. A quick, accurate and low-cost diagnosis is essential to confirm suspicions of dengue cases, favoring the adequate treatment of infected patients, especially in countries with limited health care resources. Integration and expression in plants is a valid strategy for obtaining large-scale and low-cost heterologous proteins (Schillberg et al, 2019). We purified and characterized the recombinant protein, which presented antigenic potential, with the ability to recognize anti-dengue antibodies in the serum of infected patients with high specificity and sensitivity in enzyme-linked immunosorbent assays (ELISA). The study takes a step forward by using the plant as the key part of a valid strategy for large-scale and efficient production of a protein that will be used in serological tests, since the production of this antigen is still a key point that generates high cost in commercial diagnosis kits
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