Protocolos molecular y celular para elaborar y determinar una cepa vacunal de salmonella enterica serotipo enteriditis 82139 y propuesta de tipos de biorreactores para su produccion
Villanueva Alva, Liliana Verónica
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A molecular and Cellular procedures is proposed to obtain alive attenuated no virulent strains from Salmonella entérica Seroype Enteritidis 82139, as a vaccine elaboration model and production scale bioreactor models as well. Initially, a factorial design allowed the mutant strains selection, which facilitated the genetic role of dsbA, dle y dlp genes in the bacterial motility and invasiveness activities. and with a 99% level of confidence for mobility and 95% for intracellular bacterial invasion. It was obtained the single mutant strains 82139 C, 82139 I, 82139 2; the double mutant strains, they are 82139 2C, 82139 2I y 82139 CI, and a triple mutant 82139 2CI. As dle gen expression control strains were used the following : 82139 2CI pSTB1dle y 82139 pSTB1. That strains belong to the Laboratory of Microbiology II the Complutense University the Madrid. To obtain and construct the mutant strains , it was used several genetic engineering tools such as PCR, electrophoresis, electroelution, sequencing, cloning, minipres and maxipres mono and multicopy plasmid extraction, transformation and transduction, fage doses, vector construction and genic complementation and recombination techniques. In addition to production to larger scale preinoculos and bacterial inocula of vaccine strains is proposed the use of bioreactors; for preinoculos type of Biorreactor Loop of internal circulation, flow invested (with aeration system and propeller type agitator) and cutter foam (BLIIHC - 11) and for the production of bacterial inoculum: vaccine strains, it is proposed the model of bioreactor type Loop of internal circulation, without flow reversal Given the fact that the triple mutant y double mutant, in absence of the three TDOR proteins, did not show motility and a limited invasive capacity below a infective doses in vivo, it can proposed Salmonella enteritidis 82139 2CI and 82139 2C as a susceptible resource of being investigated by means of immunologic assays to determine their possible use as straint alive attenuated vacunal no virulent. in that allowed the successful future of the in vivo tests... The Test in vitro motility test was performed in 0,3% motility agar slants by the sting method in badge. The bacterium invasive capacity and motility decreased in the single and double mutants and decreased ostensibly more in the triple mutants 2CI in absence of the TDOR proteins : DsbA, Dlp, y Dle. It was probed that the DsbA protein determined the motility and invasive capacity, and the complementary function of the Dlp to DsbA; Dle shows only specific and limited function TDOR, associated with the fimbrias biosynthesis SEF operon and possibly of some proteins of virulence. Dle doesn't have effect in the mobility. Despite that DsbA, Dlp and Dle have no function in the growth of the bacterium the mutants have sluggish growth time.The Test in vitro the invasive capacity in was carried out by infection in monocapas of cells MDCK and certain at 1, 2 and 4 hours. The in vitro motility and invasiveness tests allowed relating DsbA, Dlp y Dle Tiol periplasmic oxidative path (TDOR) function to the role of virulence factors. Motility assay was determined to 1% statistical significance level and invasiveness to 5% statistical significance level. El Diseño Factorial, el análisis de Varianza ANAVA y Diferencia Mínima Significativa (DMS) permiten también validar los resultados de los ensayos in vitro con coeficientes de Variación de 4.96% obtenidos en los ensayos de movilidad y de 3.93% en los ensayos de capacidad invasiva, comparado con un límite de aceptabilidad del 30%, lo que indican una buena calidad en la obtención de datos, resultados importantes para la determinación de la aplicación de nuevas vacunas que permita disminuir la forma agresiva de invasión en células de mamífero, y del retraso en su capacidad infectiva que se vea afectada su supervivencia y multiplicación de las bacterias invasivas – internalizadas, time required to allow the early immune response of the host cell. The double mutant 82139 2C missing dsbA y dlp genes and the triple mutant 82139 2CI missing dsbA, dlp y dle genes became potencial genetic resources to be used as vaccinal strains; for those genes code for the proteins TDOR: DsbA, Dlp and Dle, in charge of inducing correct folding and/or transport of the proteins of virulence, fimbria and flagella with the right assemblage, translocation and/or insertion of their proteins such as invationins, adhesionins, and flagelins respectively, containing one or more cystein moieties.