Expressão e purificação da outer membrane protein A (OmpA) de Rickettsia rickettsii expressa em sistema bacteriano

• Autor(a) principal: Garrefa, Isadora de Cássia
• Data de Publicação: 2023
• Tipo de documento: Trabalho de conclusão de curso
• Idioma: por
• Título da fonte: Repositório Institucional da UFU
• Texto Completo: https://repositorio.ufu.br/handle/123456789/38669
• topic: Ni-NTA agarose; Ni-NTA agarose; MBP-OmpA; MBP-OmpA; ELISA; Rocky Mountain spotted fever; CNPQ::CIENCIAS BIOLOGICAS::MICROBIOLOGIA; CNPQ::CIENCIAS BIOLOGICAS::PARASITOLOGIA; CNPQ::CIENCIAS BIOLOGICAS::GENETICA::GENETICA MOLECULAR E DE MICROORGANISMOS

The infection caused by Rickettsia rickettsii is the main zoonosis transmitted by ticks and has a high mortality rate in humans in Brazil. Currently, the gold standard assay for diagnosing infection with both R. rickettsii and other Rickettsia species, especially those that are part of the Rocky Mountain spotted fever group, is indirect immunofluorescence, which has some disadvantages such as subjectivity and the way of obtaining the antigen, which is done by growing strains of different species of Rickettsia in mammalian cells. In a previous study, truncated Outer membrane protein A (OmpA) from R. rickettsii strain Brazil was expressed in Escherichia coli as a fusion protein with maltose-binding protein (MBP-OmpA) and used in Enzyme-linked immunosorbent assay (ELISA) for testing serum samples from capybaras, opossums, and horses. However, the test showed good reactivity only with sera from opossums, possibly because MBP-OmpA was not subjected to a purification process. Thus, the main objective of this project was to carry out the processes of expression and purification of MBP-OmpA through the 6xHis tail fused to protein. Since the protein formed aggregates during bacterial cell lysis, the precipitate was solubilized with buffer containing 8 M urea and purification was performed under denaturing conditions. With the use of the Ni-NTA spin column, the MBP-OmpA was not observed in electrophoresis. However, with Ni-NTA agarose, a protein band was observed, but with a molecular mass apparently a little higher than expected. Fractions obtained during purification were tested in ELISA, using anti-6xHis monoclonal antibody, and the wells with greater reactivity correspond to the fractions in which the protein bands were more intense on electrophoresis, indicating that these bands probably represent MBP-OmpA. Pools will be carried out with these fractions and the preparation will be evaluated, in future stages, in ELISA using sera from capybaras, opossums and horses.