Cloning, Expression and Characterization of Fusion Proteins Based on Peptides of Rv1980c Disrupting Rv3019c Sequence and Evaluation of its Potential Immunoreactivity in Pulmonary Tuberculosis Sera

Abstract

Mycobacterium tuberculosis-specific antigens (Ag) would be of important value in developing immunodiagnostic test for tuberculosis (TB), however human heterogeneous recognition of Ag epitopes may result in specificity variation of the test. In this work, we described the construction of two fusion proteins, based on two peptides from MPT-64 disrupting MT-10 Ag, F1-MT10.3 (1M-40S):CE15 (173G-187A):MT10.3 (41S-96) and F2- MT10.3 (1M-40S):MPT64 (91L-205A):MT10.3 (41S-96), and their potential immunoreactivity in TB sera. These fusion genes were cloned in expression vector, inserted in E. coli, and their proteins were expressed and purified. Using ELISA technique purified fused proteins and single full antigens were evaluated for IgA, IgM and IgG in sera from individuals diagnosed with pulmonary tuberculosis (TB) and controls with other pulmonary disease. The F1 construction generated a new peptide and F2 generate two modified peptides compared with the single full proteins. Testing of the tuberculosis human sera, the constructions were recognized by all antibodies types but the best results was obtained for ELISA-IgA which predominantly recognized the F2 (66.7%) and F1 (58.3%), follow by full single antigens MT10.3 (41.7%) and MPT64 (16.7%) keeping the highest specificity (95.5%), hitherto being unnoticed. Reactivity of IgG-F1 and IgM-F2 showed higher UAC than full MT10.2 and MPT64. The data demonstrated the viability of the constructions and the usefulness of molecule modification for obtains potential immune reactivity improvement, deserving further immunological characterization.