1 research outputs found
Comparaci贸n de metodolog铆as para la purificaci贸n de prote铆nas de membrana externa de聽Brucella abortus.
Las prote铆nas de la membrana externa de la Brucella abortus, Cepa 19 y RB-51 se purificaron por dos m茅todos, el primero basado en la extracci贸n secuencial por tratamientos sucesivos con detergentes y lisozima y el segundo, en la formaci贸n del saco de peptidoglican con sus prote铆nas asociadas, utilizando dodecil sulfato de so-dio (SOS) a 50掳C y extracci贸n directa de las prote铆nas de la membrana externa con 50 mM de MgCl2 en SDS-23 Mercaptoetanol a 37掳. Se utiliz贸 la sonicacion para lograr la ruptura bacterial y su efecto se comprob贸 por microscopia electr贸nica. Las prote铆nas obtenidas se caracterizaron por electroforesis en geles de poliacrilamida. El m茅todo de extracci贸n por detergentes ani貌nicos d茅biles y dipolares y tratamiento con lisozima, comprob贸 ser superior en calidad, selectividad y concentraci贸n de las prote铆nas de inter茅s. Los pesos moleculares obtenidos para las prote铆nas denominadas porinas van de 37 a 41 kilo Daltons (k0), asi mismo se detecta una prote铆na de bajo peso molecular (14kI3). Se detecto reacci贸n especifica de las prote铆nas en sueros controles de bovinos con infecci贸n natural de B. abortus por to t茅cnica de inmunotransferencia en papel de nitrocelulosa.Outer membrane proteins of Bruce/la abortus strain 19 and strain RB-51 were purified using two methods. One based on sequential detergent extraction plus iysosyme treatment and the other obtaining peptidoglican saculae associated proteins
by sodium dodecil sulfate (SDS) treatment at 50掳C, and external protein extraction
with SDS-23 mercaptoetanol, 50 mM MgCl2 at 37掳C. Sonication was used in order
to disrupt the cells and its effect was monitored by electron microscopy. The proteins were characterized by polycrilamide gel electrophoresis. Detergent and lysozyme treatment demonstrated superiority over SDS extraction regarding selectivity,
antigenicity and yield. The proteins obtained from strain RB-51 had less LPS contamination. The molecular weights of the porin proteins obtained ranged between
37 and 41 kD, and a 14 kD protein was also detected. Specific reaction of the purified proteins was detected by western-blotting with positive bovine control serums.Ganado de leche-Ganader铆a lech