Abstract
Variable individual response against the antigens of Mycobacterium tuberculosis necessitates detection of multiple antibodies for enhancing reliability of serodiagnosis of tuberculosis. Fusion molecules consisting of two or more antigens showing high sensitivity would be helpful in achieving this objective. Antigens of M. tuberculosis HSPX and PE35 were expressed in a soluble form whereas tnPstS1 and FbpC1 were expressed as inclusion bodies at 37°C. Heat shock protein HSPX when attached to the N-termini of the antigens PE35, tnPstS1 and FbpC1, all the fusion molecules were expressed at high levels in E. coli in a soluble form. ELISA analysis of the plasma samples of TB patients against HSPX-tnPstS1 showed 57.7% sensitivity which is nearly the same as the expected combined value obtained after deducting the number of plasma samples (32) containing the antibodies against both the individual antigens. Likewise, the 54.4% sensitivity of HSPX-PE35 was nearly the same as that expected from the combined values of the contributing antigens. Structural analysis of all the fusion molecules by CD spectroscopy showed that α-helical and β-sheet contents were found close to those obtained through molecular modeling. Molecular modeling studies of HSPX-tnPstS1 and HSPX-PE35 support the analytical results as most of the epitopes of the contributing antigens were found to be available for binding to the corresponding antibodies. Using these fusion molecules in combination with other antigenic molecules should reduce the number of antigenic proteins required for a more reliable and economical serodiagnosis of tuberculosis. Also, HSPX seems to have potential application in soluble expression of heterologous proteins in E. coli.
Highlights
Tuberculosis (TB) represents an ongoing threat to global health, with the current epidemic fuelled by HIV-coinfection and an increasing incidence of drug-resistant strains of M. tuberculosis
His-Heat shock protein X (HSPX) and His-PE35 were expressed in soluble form whereas HistnPstS1 was expressed in insoluble form at 37°C as well as at 18°C as reported previously [28]
His-FbpC1 was expressed in insoluble format 37°C, but at 25°C it was expressed in a soluble form [29]
Summary
Tuberculosis (TB) represents an ongoing threat to global health, with the current epidemic fuelled by HIV-coinfection and an increasing incidence of drug-resistant strains of M. tuberculosis. In 2014, World Health Organization (WHO) reported 9.0 million cases of TB and 1.5 million deaths all over the world [1]. The standard of care for TB diagnosis recommended by WHO is (i) sputum smear microscopy for all cases, and (ii) expansion of the use of culture to diagnose all bacteriologically positive cases [2]. The sensitivity is much lower among HIV-positive patients than among HIV-negative patients [3]. Culture is the most sensitive of currently available tests, but require long growth time and in 10–20% of cases the bacillus is not successfully cultured [4]
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