Construção e expressão de uma proteína de fusão recombinante, em Escherichia coli, a partir de antígenos imunodominantes do Mycobacterium tuberculosis

• Autor(a) principal: Marques Neto, Lázaro Moreira
• Data de Publicação: 2014
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: Repositório Institucional da UFG
• dARK ID: ark:/38995/0013000002n9v
• Texto Completo: http://repositorio.bc.ufg.br/tede/handle/tede/6191
• topic: Proteína; Fusão; Antígeno imunodominante; Resposta imune; Tuberculose; Protein; Fusion; Immunodominant antigen; Immune response; Tuberculosis; MEDICINA::ANATOMIA PATOLOGICA E PATOLOGIA CLINICA

Tuberculosis (TB) is an ancient plague which affects mankind for thousands of years and remains a serious public health problem, causing illness of over 8 millions and killing more than 1 million people throughout the year. The currently used vaccine, BCG, was developed in 1921 and began to be used in 1924 as a prophylactic agent against tuberculosis, and today is the most widely used vaccine worldwide. The protective potential of BCG is very variable, with rates of protection ranging from 0 to 80%, but it is still used because it protects against the most serious forms of the disease in childhood. Consequently the need for a new vaccine strategy is urgently needed. Subunit protein vaccines and fusion proteins strategy only deal with the components of the organism that best stimulate the immune system rather than using the whole organism system. However, the development of fusion proteins has no defined rules, as approximating artificial peptides from different proteins leads to new and unpredicted intramolecular interactions. In this study we propose the construction of a fusion protein (CDα) composed of antigenic subunits from proteins of Mycobacterium tuberculosis (Ag85c, MPT51 and HspX). The subunits were chosen due to their potential in inducing a Th1-type immune response and especially for its role in activation and induction of various populations of CD8+ T lymphocytes. Based on analyzes in silico we could design primers that allowed the amplification and subsequent fusion of the subunit gene sequences of, and in addition, inserting a hinge sequence of glycine and serine between each subunit that allowed better protein stability. The gene sequence of CDα was inserted into an expression plasmid and transformed into Escherichia coli BL21 and from this it was possible to induce its expression and purify the recombinant protein on a large scale, however, it was not possible to obtain the protein in a soluble form. The protein sequence was confirmed by sequencing the expression plasmid and analysis of purified recombinant protein composition by mass spectrometry MALDI-TOF. The purified protein was used to standardize an immunoassay test to detect human antibodies, and our data show that the CDα is not antigenic for humoral immune response of patients infected with Mtb (latent TB or active TB). Monoclonal antibodies produced by immunization of BALB/c mice generated titers of 1:20,000 against IgG1 and IgG2a, indicating that the protein has immunogenic potential. We conclude that the CDα protein was constructed without any error or mutation, and this was possible due to technical alliance in silico and in vitro, however further studies are needed to purify the protein in soluble form and especially to ascertain their vaccine potential.