Method for obtaining a recombinant proteins and its utilization in the assay for for the african swine pest virus (ASPV)

Filing Date: 1990-11-14 -- Priority Data: ES P 8904300 (1989-12-20)The object of the method is the detection of the african swine pest virus by an immuno enzymatic assay wherein the major protein of the virus (p72) is used as antigen, said protein being obtained by expression of the gene which encodes it. The gene was identified by hybridization with oligonucleotide probes derived from structural information of the purified protein. Once its sequence of nucleotides was determined, the gene was inserted in the expression vector pAR3038, to obtain the recombinant plasmid pS72a. Said plasmid was used to transform the strain of E.coli BL21(DE3) which, after induction with IPTG, allowed the high yield production of the recombinant protein p72. The protein was purified by high resolution liquid chromatography and was directly used in antigen-antibody assays with animal serum samples in order to diagnose the disease.Peer reviewe

Método para la obtención de una proteina recombinante y su utilización en la detección del virus de la peste porcina africana (VPPA)

Traducción de Patente Europea E90916994 (fecha de solicitud, 14/11/1990).-- Prioridad: ES198912208904300.-- Titulares: Consejo Universidad de Oviedo, Superior de Investigaciones Científicas (CSIC).El objetivo del método es la detección del virus de la peste porcina africana mediante una prueba inmuno enzimática donde la principal proteina del virus (p72) es utilizado como antígeno, dicha proteina es obtenida por manifestación del gene que la codifica. El gene se identifica por hibridación con ensayos oligonucleótidos derivados de la información estructural de la proteína purificada. Una vez que la secuencia de nucleótidos ha sido determinada, el gene es insertado en el vector de formulación par3038 al objeto de obtener el asociante del plasma ps72a. Dicho plásmido ha sido utilizado en la transformación de la cepa e.coli bl21(de3) que, tras la inducción con iptg, favorece el alto rendimiento de producción de proteína asociante p72. La proteina ha sido purificada por una elevada solución líquida cromatográfica habiéndose sido empleada directamente en ensayos antigeneñanticuerpo con muestras de suero animal a fin de diagnosticar la enfermedad.Peer reviewe

Proteomic profiling of adipose tissue from Zmpste24(-/-) Mice, a model of Lipodystrophy and premature aging, reveals major changes in mitochondrial function and vimentin processing

Lipodystrophy is a major disease involving severe alterations of adipose tissue distribution and metabolism. Mutations in genes encoding the nuclear envelope protein lamin A or its processing enzyme, the metalloproteinase Zmpste24, cause diverse human progeroid syndromes that are commonly characterized by a selective loss of adipose tissue. Similarly to humans, mice deficient in Zmpste24 accumulate prelamin A and display phenotypic features of accelerated aging, including lipodystrophy. Herein, we report the proteome and phosphoproteome of adipose tissue as well as serum metabolome in lipodystrophy by using Zmpste24(-/-) mice as experimental model. We show that Zmpste24 deficiency enhanced lipolysis, fatty acid biogenesis and β-oxidation as well as decreased fatty acid re-esterification, thus pointing to an increased partitioning of fatty acid toward β-oxidation and away from storage that likely underlies the observed size reduction of Zmpste24-null adipocytes. Besides the mitochondrial proteins related to lipid metabolism, other protein networks related to mitochondrial function, including those involved in tricarboxylic acid cycle and oxidative phosphorylation, were up-regulated in Zmpste24(-/-) mice. These results, together with the observation of an increased mitochondrial response to oxidative stress, support the relationship between defective prelamin A processing and mitochondrial dysfunction and highlight the relevance of oxidative damage in lipoatrophy and aging. We also show that absence of Zmpste24 profoundly alters the processing of the cytoskeletal protein vimentin and identify a novel protein dysregulated in lipodystrophy, High-Mobility Group Box-1 Protein. Finally, we found several lipid derivates with important roles in energy balance, such as Lysophosphatidylcholine or 2-arachidonoylglycerol, to be dysregulated in Zmpste24(-/-) serum. Together, our findings in Zmpste24(-/-) mice may be useful to unveil the mechanisms underlying adipose tissue dysfunction and its overall contribution to body homeostasis in progeria and other lipodystrophy syndromes as well as to develop novel strategies to prevent or ameliorate these diseases