Estudo da expressão do transgene sintético scFv-antiBAP1 em Nicotiana benthamiana

Abstract

The accidents caused by snakes of the genus Bothrops represent a serious public health problem in Brazil. The main treatment for these accidents is serotherapy, which relies on the use of equine antibodies. Currently, plants are widely used in the production of recombinant proteins of pharmaceutical interest, serving as an alternative for the treatment of snakebites through the production of antibody fragments (scFv). This study investigated the expression of the scFv-BaP1 fragment in Nicotiana benthamiana to evaluate its potential as an antivenom. The pK7WG2D-scFvBaP1 plasmid, optimized for plant expression, was cloned into Agrobacterium tumefaciens and used for transient and stable transformation of N. benthamiana. Transient expression was achieved by infiltrating leaves with an A. tumefaciens suspension in MMA medium, while stable transformation was established through co-culture with A. tumefaciens. Selection and PCR confirmation of transformed plants resulted in nine genetically modified plants, with a transformation efficiency of 28%. Additionally, callus induction using growth regulators demonstrated variation in recombinant protein production. The biological activity of the scFv-BaP1 fragment was assessed by its ability to recognize and neutralize toxins from Bothrops atrox, Bothrops leucurus, Bothrops moojeni, and Bothrops jararaca. The fibrinogenolytic assay revealed that fibrinogen neutralization was directly proportional to antibody concentration (25 μg, 50 μg, and 100 μg). These findings suggest that the scFv-BaP1 fragment produced in N. benthamiana could represent a promising alternative for the development of antivenoms.