Expressão heteróloga da protease Rv 2467 de Mycobacterium tuberculosis

• Autor(a) principal: Andrade, Rayanny Gomes de
• Data de Publicação: 2018
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: Repositório Institucional da UFG
• Texto Completo: http://repositorio.bc.ufg.br/tede/handle/tede/8693
• topic: Mycobacterium tuberculosis; Virulência; Infecção; Protease; Virulence; Iinfection; MICROBIOLOGIA::MICROBIOLOGIA APLICADA

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