Fusion Molecules of Heat Shock Protein HSPX with Other Antigens of Mycobacterium tuberculosis Show High Potential in Serodiagnosis of Tuberculosis

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.

Enhanced serodiagnostic potential of a fusion molecule consisting of Rv1793, Rv2628 and a truncated Rv2608 of Mycobacterium tuberculosis

“…The double fusion construct Rv1793-Rv2628 carrying NdeI and NheI restriction sites were finally ligated into tnRv2608 recombinant plasmid to generate the final fusion construct TriFu64. The amplified DNA fragments were gel purified, cloned into pTZ57R/T vector and then sub-cloned into a pET28a(+) expression vector, as described previously [10]. The recombinant plasmids were carried by initial cloning host E. coli DH5α cells.…”

“…A fusion construct, tn2FbpC1-tnPstS1 was developed by joining truncated variants of two antigens FbpC and PstS1 with improved sensitivity of 72.2% [9]. To ensure soluble expression of the fusion proteins the heat shock protein, HspX was added and HspX-tnPstS1 showed 57.7% sensitivity [10]. Rv2031c-Tn1Rv1984c-Rv1352 with the combination of three antigens showed enhanced sensitivity of 71.4% [11] and HspX-EspC-CFP7-PPE57 fusion construct developed by joining four different Mtb antigens showed 69% sensitivity [12].…”

Development and Evaluation of a Fusion Polyprotein Based on HspX and Other Antigen Sequences for the Serodiagnosis of Tuberculosis

“…Nine antigens, previously identified as exhibiting TB serodiagnostic potential, would be fused as a polyprotein ( Supplementary Table 5 ). Since HspX was a serodiagnostic antigen with 33.33% sensitivity and 100% specificity ( 18 ), and expression of the fusion molecule HspX with other antigens increased by about 50% as compared with those of the individual antigen, resulting in cheaper production of the fusion antigens ( 31 ), the whole sequence of HspX was retained in the fusion protein to enhance immunogenicity. For the other eight antigens, polypeptides were screened as described in the Materials and Methods and fused as an entirety by glycine- and proline-rich (GPGPGPGPGPG) spacers.…”

Diagnostic performance and problem analysis of commercial tuberculosis antibody detection kits in China

“…If the M.tb antigen selected had a cross-reaction with the sera from a person infected by other bacteria, especially in the environment of non-TB Mycobacteria , it would be easy to obtain false-positive results [ 30 ]. Therefore, it is important to select a number of highly sensitive, strongly specific and complementary antigens to create mixtures or fusions to improve the performance of TB sera diagnosis [ 11 , 31 – 33 ]. Additionally, the preparation method, purity and concentration of the antigen used also affects the quality of the kit [ 22 ].…”