Identificação e caracterização de peptídeos bioativos através de Phage display usando genoma completo de Corynebacterium pseudotuberculosis

Abstract

The Caseous Lymphadenitis (CL) is a disease caused by Corynebacterium pseudotuberculosis, which affects mainly sheep and goats. This illness is responsible for significant economic losses related to productivity of animals. Currently, there is no satisfactory diagnosis for CL. In this work the tool Phage Display was used that selects peptides using bacteriophages to express random sequences of peptides on their surface. Phage Display has been used successfully in many applications such as mapping and mimicry antigens, vaccine development and immunogenicity tests. The main goal of the study was to perform the selection and identification of mimotopes peptides of secreted and /or excreted and total C. pseudotuberculosis 1002 proteins through Phage Display technique. A commercial librarary of 12 linear peptides (Ph.D.-12 mer - New England Biolabs - NEB) was used. After three cycles of selection, 366 peptides were obtained, including 116 distinct sequences. In silico analysis of the peptides obtained in Phage Display were tested against the predicted proteome of C. pseudotuberculosis 1002. The indentified proteins showed similarity to protein involved in signaling, nutrition, metabolism, bacterial pathogenesis and transcription factors. The reactivity and specificity of isolated clones were tested by ELISA and Spot synthesis. Through immunoassays for both methods, was possible to select 12 peptides among them three were synthesized, bound to two different formulations of liposomes, with a hydrogenated phosphatidylcholine (HPC) and soy phosphatidylcholine (SPC), the three peptide were used for testing and immunization of BALB/c. Immunization with the peptides 1 and 2 was able to induce a high Th1 immune response before infection. After the challenge, we observed a high production of IFN- in animals immunized with either peptide/SPC or peptide/HPC, and the levels of IL-10 were higher in animals immunized with peptide/SPC only. In contrast, control groups of mice exhibited low levels of IFN- and IL-10. The identification of new antigens or C. pseudotuberculosis specific genes can potentially provide new specific biomarkers with antigens for obtaining vaccine candidate antigens and sub-clinical diagnosis tests for more accurate control and eradication of the CL.