Expressão heteróloga da protease Rv 2467 de Mycobacterium tuberculosis
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2018-06-12
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Universidade Federal de Goiás
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Mycobacterium tuberculosis is the etiological agent of tuberculosis (TB), which is responsible in 2015 for
10.4 million new cases and 1.8 million casualties registered worldwide. To be able to avoid the defense
barriers of the host, the bacteria evolved mechanisms to modulate or change the environment it is in,
thereby, the secretion of bioactive molecules is an efficient strategy to do so. Among the bioactive
molecules produced by M. tuberculosis, proteases have a crucial role to a successful infection, as they are
involved in several important biological processes of the bacteria, such as DNA replication, cell proliferation
and antigen processing. Thus, proteases are good targets for the development of new anti-TB drugs or as
possible vaccine antigens. This work aimed to clone and express the gene Rv2467, a putative protease with
aminopeptidase activity from M. tuberculosis. To achieve this goal, oligonucleotides design were made to
amplify the gene Rv2467 by Polimerase Chain Reaction (PCR), which was cloned in the pGEM-T easy vector.
The clone Rv2467 gene was then transfere to the expression vector pET-28a. The recombinant protein was
expressed from the recombinant plasmid pET-28a/Rv2467 in Escherichia coli BL21 (DE3) pLysS through
induction with IPTG. The recombinant protein, with the expected size of 94KDa, was expressed and
subjected to the purification process by nickel affinity chromatography. The recombinant protein was
partially purified only in its denatured form. The low yield of purified recombinant protein raised the
possibility of histidine tail loss. To verify that, Western Blotting with anti-histidine antibody was made and
it was verified that the antibody did not recognize the target Rv2467 protein, which made it impossible to
acquire the pure protein, not allowing further characterization tests. Additionally, a literature review was
performed about Mycobacterium tuberculosis proteases that are involved in virulence mechanisms to make
a manuscript to be submitted to a scientific journal
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ANDRADE, R. G. Expressão heteróloga da protease Rv 2467 de Mycobacterium tuberculosis. 2018. 90 f. Dissertação (Mestrado em Medicina Tropical e Saúde Publica) - Universidade Federal de Goiás, Goiânia, 2018.