Design and evaluation of a novel multi-epitope antigen for evaluate the diagnostic immunity responses against Leishmania infantum infection

Abstract

Background Leishmania infantum is the causative agent of visceral leishmaniasis in the Mediterranean region. The diagnosis of complex visceral leishmaniasis and delays in the diagnosis of the infection are associated with the death of patients. Proper diagnosis of infection is an important measure in controlling and preventing the disease. However, studies have shown that the accuracy of antigens used in current diagnostic tests is insufficient, for this reason, researchers are trying to identify multi-epitope antigens as diagnostic markers to increase the specificity and sensitivity of diagnostic tests. In this study, the design and expression of Leishmania infantum multi-epitope antigens were carried out in two parts of the structure design using bioinformatics tools and the laboratory part for the production of the recombinant protein. Materials and Methods The aim of this study was to design and computationally analyze and express Leishmania infantum multi-epitope antigens. In this study, nine antigenic proteins (CPB, H1, KMP11, GP63, HASPB, A2, K39, LACK, and PSA) were selected. Bioinformatics analyzes such as prediction of immune cell epitopes, design of recombinant structure, antigenicity, allergenicity, evaluation of physicochemical properties, solubility, prediction of secondary structure and tertiary structure, refinement and validation of 3D model structure and finally in silico cloning optimization of protein construct were performed. After synthesis of the designed recombinant gene fusion sequence in pUC57 cloning vector, its subcloning was performed in pET26b prokaryotic expression vector using BamHI/ HindIII restriction enzymes. The expression of recombinant multi-epitope antigen was performed in E. coli B (BL21) strain using IPTG inducer and confirmed by SDS-PAGE and western blotting techniques. Results The results of computational analysis showed that the complete structure, which is suitable for immunogenicity and is non-allergenic, was successfully cloned into pET-26b and expressed as a complete protein. Conclusion Finally, the protein was approved. Based on the expression of recombinant proteins and bioinformatics analysis, this structure can be studied in mouse models and its safety can be evaluated.