Evaluation of antigens of Mycobacterium tuberculosis with potential for use in the diagnosis of tuberculosis
Tuberculosis, caused by Mycobacterium tuberculosis, is one of the most seriousinfectious illnesses in the world, being responsible for millions of deaths. Despite theuse of the vaccination with M. bovis BCG, the number of new cases remains high.Immunological methods are hampered by the reaction afforded by BCG. Betterdiagnostic tools should allow detection and treatment of infected patients before theydevelop active disease. Antigens present in M. tuberculosis but absent from the vaccinestrain are needed for this purpose. Therefore, this work has been developed aimed atthe characterization of proteins Rv3402, Rv3404 and Rv3405, contained in RD16,missing only region M. bovis BCG Moreau, the strain vaccine used in Brazil, butpresent in all other strains of BCG and M. Tuberculosis. The genes rv3402, rv3404 andrv3405 were obtained by PCR from M. tuberculosis H37RV and cloned in the vectorpENTR/D/Topo (Gateway System ? Invitrogen) and used to transform Escherichia coliTOP10F. The coding sequence for each gene was then transferred to the expressionvector pDEST17, which allows the fusion of the protein with a tail of histidine,facilitating their purification. After extracted and purified, these plasmids were used totransform E. coli BL21 pLysS. The purification of the protein was performed by affinitychromatography using the ÄKTAPrime system (Amersham Biosciences). BALB/cmice were inoculated with purified proteins and serum obtained from these animalswas used for characterization of the proteins by ELISA and Western blot. It waspossible to demonstrate that the proteins are immunogenic and that they areexpressed by M. tuberculosis. The protein contained in the RD16 and evaluated in thisstudy, were quantified in a concentration of 0.5 mg.mL-1 for Rv3402 and Rv3404 and0.2 mg.mL-1 for Rv3405 and were able to induce a specific and statisticallysignificant humoral immune response in immunized mice, indicating that they can beused for the development of immunodiagnostic tests for tuberculosis, aiming todifferentiate individuals vaccinated and infected with the tuberculosis bacillus.
Advisor:Odir Antônio Dellagostin
School:Universidade Federal de Pelotas
Source Type:Master's Thesis
Keywords:RD16 imunodiagnóstico Tuberculosis immune diagnostic
Date of Publication:12/07/2007