Sequência completa e caracterização do plasmídeo crípico pVCM1 isolado de Salmonella enterica

• Autor(a) principal: PENIDO, Ana Flávia Batista
• Data de Publicação: 2009
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: Repositório Institucional da UFG
• Texto Completo: http://repositorio.bc.ufg.br/tede/handle/tde/1297
• topic: 1. Plasmídeo; 2. Salmonella entérica 3. Replicação via círculo-rolante; 1. Plasmídeo; 2. Replicação via círculo-rolante; 3. Salmonella; 1. Plasmid; 2. Rolling-circle replication; 3. Salmonella; CNPQ::CIENCIAS BIOLOGICAS::BIOLOGIA GERAL

Samonella spp are Gram negatives bactérias belonging to Enterobacteriaceae family. S. enterica comprise about 2.500 sorovars. These sorovars can infect a broad range, including poultry, cattle, swins and humans, and are agent causative of salmonellosis an important public health issue worldwide. Small multicopy plasmids are frequently isolated from Gram negatives and Gram positives bacterias. In Salmonella, low molecular weight plasmids are found on 10% of Salmonella strains and their biological functions are unknown. However, many plasmids in Salmonella control important properties, such as, virulence factors, heavy metals and antibiotics resistance, and utilizations of alternative carbon sources. The pVCM1 plasmid was extracted from one strain of Samonella enteretidis isolated from broilers carcass. The strains were grown in liquid or solid Luria-Bertani broth at 37 °C. The plasmid was purified, separated on 1% agarose electrophoresis and visualized by ethidium bromide staining for analysis. Plasmid was digested with EcoRI enzyme and subcloned in the pUC18 vector.The plasmidal stability was evaluated, inoculating E. coli cells transformated with pVCM1 plasmid (cloned in pUC18) in liquid Luria-bertani broth supplemented with ampicillin. The pVCM1 was stable after 240 generations. The total DNA sequence of plasmid pVCM1 has 1981 pb. Genbank search resulted that pVCM1 showed 99% of identity with pB and 92% with pJ, which were isolated from Salmonella enteretidis. Only one ORF founded in pVCM1 showed significative similarity with others proteins of GenBank. The protein encoded by this ORF showed homology to Rep proteins of plasmids that replicates by rolling-circle mechanism. The pVCM1 posses three impotents elements: rep gene, single strand origin (SSO) and inverted repeat sequences. Such elements are importants for the rolling circle replication, suggesting that pVCM1 use this mechanism. The rep gene was amplified and cloned in the pGEMT-easy vector, but the heterologous expression of Rep protein wasn t gotten successfully.