Made available in DSpace on 2016-04-28T20:16:24Z (GMT). No. of bitstreams: 1 2007-Catia Marques da Costa.pdf: 899456 bytes, checksum: b8d00f84cefd48cf338e385b27121da0 (MD5) Previous issue date: 2007-02-28 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / Anaplasma marginale (Ricktsialles: Anaplasmataceae) is a ricketsial hemoparasite responsible for causing great economic losses in cattle from tropical and subtropical regions. The objectives of this work were to produce the recombinant membrane proteins AM254, VirB9 and VirB10 and to evaluate its possible antigenicity. The genes am254, virB9 and virB10 were submitted to various experiments and were , linked to pET47 (b) plasmid. After the certification of the recombinant plasmid (pET-47-am254, pET-47-virB9 and pET-47- virB10), it was transformed into E. coli Rosetta cells for expression. The recombinant proteins produced, were analyzed by Western blot assay where the reaction of the anti-histidin monoclonal antibody against rAM254 (47kDa), rVirB9 (31kDa) and rVirB10 (60kDa) was observed. After the confirmation of the production of recombinant proteins the indirect Enzyme-Linked Immnunosorbent Assay (ELISA) was standardized with sera previously confirmed by PCR and ELISA (rMSP1 and rMSP5). The averages of the optic densities (OD) of the positive sera were of 1.339; 1.288 and 1.240 and of the negative sera of 0.470, 0.324 and 0.414 for AM254, VirB9 and VirB10 respectively with significant difference to the level of 5% between positives and negatives for each recombinant protein. The study demonstrated that the proteins rAM 254, rVirB9 and rVirB10 of A. marginale are recognized by sera of bovines immunized by different Brazilian isolates of the ricketsia (homologous and heterologous), showing the antigenicity potential of these proteins. / Anaplasma marginale (Ricktsialles: Anaplasmataceae) ? uma riquetsia intraeritroc?tica, respons?vel por ocasionar grandes perdas econ?micas na pecu?ria bovina das regi?es tropical e sub-tropical. O objetivo desse trabalho foi produzir as prote?nas de membrana recombinantes AM254, VirB9 e VirB10 e avaliar sua poss?vel antigenicidade. Os genes am254, virB9 e virB10 foram submetidos a v?rios procedimentos experimentais at? serem ligados ao plasm?deo pET-47b(+). Ap?s a certifica??o do plasm?deo recombinante (pET-47bam254, pET-47b-virB9 e pET-47bvirB10) este foi transformado em E. coli Rosetta para express?o. As prote?nas recombinantes produzidas foram analisadas pelo ensaio Western blot onde foi observada a rea??o do anticorpo monoclonal anti-histidina contra rVirB9 (31kDa), rVirB10 (60kDa) e rAM254 (47kDa). Ap?s a confirma??o da produ??o das prote?nas recombinantes realizou-se a padroniza??o do ensaio de imunoadsor??o enzim?tica (ELISA) indireto com soros oriundos de sangue total previamente confirmados por PCR; os mesmos soros tamb?m foram testados por ELISA (rMSP1 e rMSP5). As m?dias das densidades ?pticas (DO) dos soros positivos foram de 1,339; 1,288 e 1,240 e dos soros negativos de 0,470, 0,324 e 0,414 para AM254, VirB9 e VirB10 respectivamente com diferen?a significativa ao n?vel de 5% entre positivos e negativos para cada prote?na recombinante. O estudo demonstrou que as prote?nas rAM 254, rVirB9 e rVirB10 de A. marginale s?o reconhecidas por soros de bovinos imunizados com diferentes isolados brasileiros da riqu?tsia (hom?logo e heter?logos), revelando o potencial antig?nico dessas prote?nas.