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Construction of auxotrophic marker in Mycobacterium bovis BCG, knockout strain for the DPPD and proteomic study of tuberculin

By Sibele BORSUK

Abstract

Mycobacterium bovis BCG tem o potencial para ser um vetor efetivo para vacinas recombinantes multivalentes. No entanto, existem dois problemas quanto a sua utiliza????o como vetor vacinal. O primeiro ?? a presen??a de genes que conferem resist??ncia a antibi??ticos nos vetores utilizados para transforma????o gen??tica. O segundo ?? a limita????o de uso de BCG em animais, principalmente por comprometer o teste de tuberculina, utilizado como diagn??stico de tuberculose, o qual se baseia em rea????o de hipersensibilidade ao PPD (Derivado Prot??ico Purificado). Neste trabalho desenvolvemos e avaliamos a complementa????o auxotr??fica como novo marcador de sele????o, fizemos a caracteriza????o das prote??nas componentes de amostras de PPD avi??rio e bovino e desenvolvemos um mutante de BCG por recombina????o hom??loga. Para o uso de complementa????o auxotr??fica como marcador de sele????o, uma cepa de BCG auxotr??fica para o amino??cido leucina foi constru??da por knockout do gene leuD por recombina????o hom??loga. A express??o do gene leuD em um plasm??dio atuou como marcador de sele????o nas cepas auxotr??ficas de M. bovis BCG leuD e M. smegmatis mc2144. A sele????o por complementa????o de BCG auxotr??fica se mostrou equivalente ?? sele????o por resist??ncia a antibi??tico, com a vantagem adicional de proporcionar maior estabilidade do vetor plasmidial, j?? que a press??o seletiva ?? mantida mesmo durante multiplica????o da bact??ria in vivo. A identifica????o das prote??nas que comp??em o PPD foi feita por espectrometria de massa utilizando-se LCMS/ MS (cromatografia l??quida associada ?? espectrometria de massa em tandem). Foram identificadas 147 prote??nas entre 5 amostras de PPD (2 PPD bovino e 3 PPD avi??rio). O PPD bovino teve um n??mero maior de prote??nas comparado ao PPD avi??rio. Foi identificado um grupo de 28 prote??nas presentes em PPD bovino, mas ausentes em PPD avi??rio. Al??m disso, 5 prote??nas encontradas no PPD est??o ausentes em M. bovis BCG. Estes s??o de 9 especial interesse, pois poder??o vir a contribuir para o desenvolvimento de um teste de diagn??stico mais espec??fico, e possivelmente capaz de diferenciar indiv??duo vacinado com BCG e infectado com o bacilo da tuberculose. Um mutante de M. bovis BCG Pasteur foi constru??do. O gene Mb0092 (dppd) foi alvo de inativa????o g??nica por recombina????o hom??loga. Seq????ncias que flanqueiam o gene alvo foram clonadas em um vetor suicida. Duplo crossover foi selecionado utilizando sacB. O gen??tipo mutante foi determinado por PCR e por Southern blot. Esta cepa poder?? ser utilizada como vacina em animais, quando o diagn??stico for feito com DPPD recombinante. Os resultados obtidos apresentam alternativas para os problemas envolvidos quanto ?? utiliza????o de M. bovis BCG como vacina recombinante. O sistema de sele????o por complementa????o auxotr??fica foi est??vel, e pode ser empregado na express??o de ant??genos heter??logos em BCG. A identifica????o dos principais componentes prot??icos do PPD e o desenvolvimento da cepa mutante de BCG possibilitam o desenvolvimento de testes diagn??sticos diferencias, permitindo a utiliza????o de BCG como vacina tamb??m em animas.Mycobacterium bovis BCG has the potential to be an effective live vector for multivalent vaccines. However, there are two problems regarding the utilization of recombinant BCG as vaccine. The first one is that most mycobacterial cloning vectors rely on antibiotic resistance gene as selectable marker, which is used for genetic transformation. The second one is the limited use of BCG in animals because it interferes in the tuberculosis diagnosis by tuberculin skin test, which elicits delayed type hypersensitivity to the purified protein derivative (PPD). In this work we developed and evaluated the use of auxotrophic complementation as a new selectable marker, characterized the proteins that are present in the bovine and avium PPD and developed a knockout BCG strain by homologous recombination. To test the auxotrophic complementation as selectable marker, an auxotrophic BCG strain for the amino acid leucine was constructed by knocking out the leuD gene by homologous recombination. Expression of leuD on a plasmid acted as a selectable marker in the auxotrophic M. bovis BCG leuD and M. smegmatis mc2144. The auxotrophic complementation selection was similar to selection by antibiotic resistance, but with the advantage of promoting stability of the plasmid. The new system was highly stable even during in vivo BCG growth. The identification of proteins from PPD was archived by LC-MS/MS (Liquid Chromatography/Mass Spectrometry/Mass Spectrometry). A total of 147 proteins among five PPD samples (2 bovine PPD and 3 avium PPD) were identified. The bovine PPD had a considerable higher number of proteins comparing to the avium PPD. We identifying a group of 28 proteins present only in bovine PPD and a group of five proteins deleted in M. bovis BCG vaccinal strain. These two groups are of special interest as they can be used in tests with improved specificity, and potentially able to differentiate vaccinated and infected individuals. A mutant BCG strain with the DPPD antigen deleted was constructed. The Mb0092 coding sequence was knocked out by homologous recombination. The 11 sequences flanking the target gene were cloned into a suicide vector. Double crossovers were selected using sacB. The knockout genotype was determined by PCR and by Southern blot. This mutant BCG strain can be useful in animal vaccination as it will not interfere in the tuberculosis diagnostic test, when performed using recombinant DPPD. The results show alternatives for the problems related to the use of M. bovis BCG as a recombinant vaccine. The auxotrophic complementation system was highly stable, efficient and it is suitable for expressing heterologous antigens in BCG. The identification of proteins present in PPD preparations and the mutant BCG obtained provide the possibility for the development of differential diagnostic test, thus allowing the use of BCG as vaccine also in animals

Topics: BCG recombinante, complementa????o auxotr??fica, caracteriza????o PPD, recombinant BCG, auxotrophic complementation, characterization PPD, CNPQ::CIENCIAS BIOLOGICAS::IMUNOLOGIA
Publisher: Universidade Federal de Pelotas
Year: 2008
OAI identifier: oai:agregador.ibict.br.RI_UFPEL:oai:repositorio.ufpel.edu.br:123456789/1257
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