Resumo

Inflammatory bowel diseases are characterized by chronic intestinal inflammation that leads to severe destruction of the intestine mucosa and whose exact etiology is still unknown. Therefore, the understanding of its etiology as well as the development of new medicines is an important step for the treatment of such diseases. In this context, the development of Lactococcus lactis strains capable of delivering a vector, pValac::dts, one of which codifies the cytokine IL-4 of Mus musculus would represent a new strategy for the elaboration of a more effective alternative therapy against Crohn’s Disease. Thus, so far, the goals of this study were the construction of the therapeutic plasmid pValac::dts::IL-4, the in vitro evaluation of its functionality and verification of the rIL-4 protein production. To this end, the IL-4 ORF of M. musculus – which was amplified from a synthetic vector – was, firstly, cloned into the commercial plasmid pCR^TM-Blunt in order to be, then, subcloned into the eukaryotic expression vector pValac::dts, being the final construction obtained in Escherichia coli TG1. Also, to evaluate, in vitro, the functionality of the therapeutic plasmid and verify, in vitro, the production of the recombinant protein, Flp-In^TM-CHO (Chinese Hamster Ovary) cells, which were transfected with the vector pValac::dts::IL-4 by the use of the Lipofectamine® 2000 system, were subjected to fluorescence confocal microscopy and flow cytometry. Thereby, the construction of a functional eukaryotic expression plasmid containing the IL-4 ORF opens perspectives for producing recombinant L. lactis strains – invasive or not – able to deliver the vector in question to mammalian cells, what leads to the development of a more efficacious alternate therapy against Crohn’s Disease. This strategy, in turn, takes advantage of mucosal immunity for modulating the gastrointestinal tract immune responses, which constitutes a highly innovative and promising approach for the control and the prevention of one of the main inflammatory bowel diseases.