RESUMEN
In our previous study, an ompP2 mutant of a Haemophilus parasuis SC096 strain showed sensitivity to serum bactericidal activity. In this study, we inactivated two gal genes, galU and galE, and these mutants were found to be serum sensitive to porcine sera. Furthermore, the galE mutant exhibited greater sensitivity than the galU mutant in serum sensitivity assays. Biofilm formation ability was also investigated. The galU mutant is unable to form biofilms, while more biofilm mass was produced by the galE mutant compared with SC096. Lack of expression of GalU protein by the galU mutant increased its tendency to autoagglutinate. The results indicated that the galU plays a role in autoagglutination and biofilm formation, while galE may affect the biofilm production indirectly. Both genes are significant for serum resistance in the H. parasuis SC096 strain.
Asunto(s)
Biopelículas/crecimiento & desarrollo , Haemophilus parasuis/fisiología , UDPglucosa 4-Epimerasa/genética , UTP-Glucosa-1-Fosfato Uridililtransferasa/genética , Animales , Actividad Bactericida de la Sangre , Galactosemias , Haemophilus parasuis/enzimología , Haemophilus parasuis/genética , Haemophilus parasuis/inmunología , Mutación , Determinación de Anticuerpos Séricos Bactericidas , Porcinos , UDPglucosa 4-Epimerasa/inmunología , UTP-Glucosa-1-Fosfato Uridililtransferasa/inmunologíaRESUMEN
The study was aimed to generate monoclonal antibodies (mAbs) against homo sapiens UDP-glucose pyrophosphorylase 2 (UGP2). Normal human liver tissues homogenized, and cytosolic proteins isolated by centrifugation were used to immunize BALB/c mice to generate mAbs by hybridoma technique. The mAbs were identified by ELISA, Western blot, and immunohistochemistry assay. The antibody specificity was confirmed by Uni-ZAP expression library screening. The results indicated that one hybridoma BAD062 secreting specific mAb against UGP2 was established. The Ig subclass of this mAb was IgG(2b) (kappa), and it could be used in ELISA, Western blot, immunohistochemistry assay. The antigen recognized by BAD062 mAb was localized in the hepatocyte cytoplasm, with molecular weight of 56 kD in the cytosolic proteins of human liver tissue. The BAD062 mAb was further confirmed by immunoscreening of Uni-ZAP XR liver cDNA expression library. It is concluded that a hybridoma cell line stably secretes specific mAb against UGP2. This mAb reacted with UGP2 in ELISA, Western blot, immunohistochemistry assay, and would be very useful for the UGP2 studies.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , UTP-Glucosa-1-Fosfato Uridililtransferasa/inmunología , Animales , Anticuerpos Monoclonales/análisis , Especificidad de Anticuerpos , Secuencia de Bases , Humanos , Hibridomas/metabolismo , Hígado/metabolismo , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia MolecularRESUMEN
Based on a recently published unusual ase of food allergy in a latex-allergic patients, the present study identifies Hev b UDPGP as a novel allergen in natural rubber latex able to cause latex-fruit allergy syndrome and as a novel, potential pan-allergen in vegetable foods.