Caracterização in silico, clonagem e expressão heteróloga em Escherichia coli de cinco proteínas de Corynebacterium pseudotuberculosis possivelmente implicadas na virulência deste microrganismo

• Ano de defesa: 2014
• Autor(a) principal: Karina Talita de Oliveira Santana
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de Minas Gerais; UFMG
• Palavras-chave em Português: Corynebacterium pseudotuberculosis; Fatores de virulência; Expressão heteróloga; Linfadenite caseosa; Escherichia coli; Interações hospedeiro-patógeno; Genética
• Link de acesso: http://hdl.handle.net/1843/BUOS-9UVNGZ

Corynebaterium pseudotuberculosis is the causative agent of Caseous lymphadenitis (CL), a disease that affects small ruminants and results in decreased productivity and loss in the agribusiness. Different approaches aim the control of this disease by identifying new drugs and developing new vaccines and diagnostic tests. However, there is not yet an satisfactory and efficient method for the control of LC; nevertheless, the sequencing of the genomes of Corynebacterium pseudotuberculosis has enabled the identification of new virulence factors of this microorganism that may contribute to a better understanding of its biology and host-pathogen relationships. In this work, proteins PLD, PknG, SpaC, SodC and NanH were selected as targets of the study based on in silico analysis and literature data mining. PLD is the major virulence factor of C. pseudotuberculosis whereas the others are pointed as potential virulence factors. In silico characterization of these proteins were performed from predictions of physicochemical parameters, signal peptide, conserved domains, assessment of their conservation in eukaryotes and epitopes. In addition, the proteins were also expressed using a heterologous expression system in Escherichia coli and five different strains were tested for expression. By using the strains that showed the highest level of expression of each protein, we assessed the expression kinetics and solubility of the recombinant proteins. The PknG protein was successfully purified after an optimization step of the expression protocol that aimed to obtain the protein in the soluble fraction. Its purification was confirmed by Western blot. Following the purification of the other proteins we will now evaluate them regarding their antigenicity and protection against infection. Since virulence factors are important candidates for drug targets, diagnostic tests or even vaccine targets, obtaining these proteins is important to perform studies that may contribute to the development of a control strategy against LC.