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Molecular cloning, nucleotide sequencing, and characterization of a 27-kDa antigenic protein from Paracoccidioides brasiliensis

A gene encoding a 27-kDa antigenic protein from Paracoccidioides brasiliensis was cloned, sequenced, and characterized. A cDNA library of the mycelial phase was produced and packed in Uni-Zap-XR vector, lambda Zap II synthesis kit (Stratagene, La Jolla, Ca). The screening of the library was carried...

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Institution:	Academia Colombiana De Ciencias Exactas Fisicas Y Naturales ACCEFYN
Main Authors:	McEwen, J.G., Ortiz, B.L., Garcı́a, A.M., Florez, A.M., Botero, S., Restrepo Moreno, Ángela, Elsevier, Nancy Keller
Format:	article
Published:	Academic Press 1996
Subjects:	Clonación molecular Paracoccidioidomicosis Paracoccidioides brasiliensis
Online Access:	https://repositorio.accefyn.org.co/handle/001/1064
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spelling	McEwen, J.G. Ortiz, B.L. Garcı́a, A.M. Florez, A.M. Botero, S. Restrepo Moreno, Ángela Elsevier, Nancy Keller 2021-11-16T16:50:45Z 2021-11-16T16:50:45Z 1996 10871845 https://repositorio.accefyn.org.co/handle/001/1064 10.1006/fgbi.1996.0027 A gene encoding a 27-kDa antigenic protein from Paracoccidioides brasiliensis was cloned, sequenced, and characterized. A cDNA library of the mycelial phase was produced and packed in Uni-Zap-XR vector, lambda Zap II synthesis kit (Stratagene, La Jolla, Ca). The screening of the library was carried out using a pool of sera from paracoccidioidomycosis patients that had proven reactive in serological testing. Among 44,000 immuno-screened clones from the library, 2 were positive (clones 2 and 3). The former was not characterized further. The latter has a 1-kb DNA insert with an open reading frame encoding a protein of 259 amino acids with a predicted molecular mass of 28.6 kDa (27 kDa by SDS-PAGE). This protein corresponds to a 25-kDa protein in antigenic preparations of P. brasiliensis as determined by Western blot analysis. Comparison of the transcribed sequence with different gene banks failed to reveal a high degree of homology with other proteins. The cloned DNA fragment was easily expressed in Escherichia coli without the need of induction by isopropyl-beta-D-thiogalactopyranoside. These findings suggest that the gene encodes a P. brasiliensis-specific protein. Se clonó, secuenció y caracterizó un gen que codifica una proteína antigénica de 27 kDa de Paracoccidioides brasiliensis. Se produjo una biblioteca de ADNc de la fase micelial y se empaquetó en el vector Uni-Zap-XR, kit de síntesis lambda Zap II (Stratagene, La Jolla, Ca). El cribado de la biblioteca se llevó a cabo utilizando un conjunto de sueros de pacientes con paracoccidioidomicosis que habían demostrado ser reactivos en las pruebas serológicas. Entre 44.000 clones de la biblioteca cribados inmunológicamente, 2 fueron positivos (clones 2 y 3). El primero no se caracterizó más. Este último tiene un inserto de ADN de 1 kb con un marco de lectura abierto que codifica una proteína de 259 aminoácidos con una masa molecular predicha de 28,6 kDa (27 kDa por SDS-PAGE). Esta proteína corresponde a una proteína de 25 kDa en preparaciones antigénicas de P. brasiliensis según lo determinado por análisis de transferencia Western. La comparación de la secuencia transcrita con diferentes bancos de genes no reveló un alto grado de homología con otras proteínas. El fragmento de ADN clonado se expresó fácilmente en Escherichia coli sin necesidad de inducción por isopropil-beta-D-tiogalactopiranósido. Estos hallazgos sugieren que el gen codifica una proteína específica de P. brasiliensis. eng Academic Press http://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.1075.4278&rep=rep1&type=pdf 20 2 Molecular cloning, nucleotide sequencing, and characterization of a 27-kDa antigenic protein from Paracoccidioides brasiliensis article info:eu-repo/semantics/article Artículo info:eu-repo/semantics/openAccess Acceso abierto Clonación molecular Paracoccidioidomicosis Paracoccidioides brasiliensis Fungal Genetics and Biology 125 131 McEwen, J.G.; Ortiz, B.L.; Garcı́a, A.M.; Florez, A.M.; Botero, S. & Restrepo, A. (1996). Molecular cloning, nucleotide sequencing, and characterization of a 27-kDa antigenic protein from Paracoccidioides brasiliensis. Fungal Genetics and Biology, 20(2), 125-131
institution	Academia Colombiana De Ciencias Exactas Fisicas Y Naturales ACCEFYN
collection	d_repositorio.accefyn.org.co-DSPACE
title	Molecular cloning, nucleotide sequencing, and characterization of a 27-kDa antigenic protein from Paracoccidioides brasiliensis
spellingShingle	Molecular cloning, nucleotide sequencing, and characterization of a 27-kDa antigenic protein from Paracoccidioides brasiliensis McEwen, J.G. Ortiz, B.L. Garcı́a, A.M. Florez, A.M. Botero, S. Restrepo Moreno, Ángela McEwen, J.G. Ortiz, B.L. Garcı́a, A.M. Florez, A.M. Botero, S. Restrepo Moreno, Ángela Elsevier, Nancy Keller Clonación molecular Paracoccidioidomicosis Paracoccidioides brasiliensis
title_short	Molecular cloning, nucleotide sequencing, and characterization of a 27-kDa antigenic protein from Paracoccidioides brasiliensis
title_full	Molecular cloning, nucleotide sequencing, and characterization of a 27-kDa antigenic protein from Paracoccidioides brasiliensis
title_fullStr	Molecular cloning, nucleotide sequencing, and characterization of a 27-kDa antigenic protein from Paracoccidioides brasiliensis
title_full_unstemmed	Molecular cloning, nucleotide sequencing, and characterization of a 27-kDa antigenic protein from Paracoccidioides brasiliensis
title_sort	molecular cloning, nucleotide sequencing, and characterization of a 27-kda antigenic protein from paracoccidioides brasiliensis
author	McEwen, J.G. Ortiz, B.L. Garcı́a, A.M. Florez, A.M. Botero, S. Restrepo Moreno, Ángela McEwen, J.G. Ortiz, B.L. Garcı́a, A.M. Florez, A.M. Botero, S. Restrepo Moreno, Ángela Elsevier, Nancy Keller
author_facet	McEwen, J.G. Ortiz, B.L. Garcı́a, A.M. Florez, A.M. Botero, S. Restrepo Moreno, Ángela McEwen, J.G. Ortiz, B.L. Garcı́a, A.M. Florez, A.M. Botero, S. Restrepo Moreno, Ángela Elsevier, Nancy Keller
building	Repositorio digital
topic	Clonación molecular Paracoccidioidomicosis Paracoccidioides brasiliensis
topic_facet	Clonación molecular Paracoccidioidomicosis Paracoccidioides brasiliensis
publishDate	1996
publisher	Academic Press
format	article
description	A gene encoding a 27-kDa antigenic protein from Paracoccidioides brasiliensis was cloned, sequenced, and characterized. A cDNA library of the mycelial phase was produced and packed in Uni-Zap-XR vector, lambda Zap II synthesis kit (Stratagene, La Jolla, Ca). The screening of the library was carried out using a pool of sera from paracoccidioidomycosis patients that had proven reactive in serological testing. Among 44,000 immuno-screened clones from the library, 2 were positive (clones 2 and 3). The former was not characterized further. The latter has a 1-kb DNA insert with an open reading frame encoding a protein of 259 amino acids with a predicted molecular mass of 28.6 kDa (27 kDa by SDS-PAGE). This protein corresponds to a 25-kDa protein in antigenic preparations of P. brasiliensis as determined by Western blot analysis. Comparison of the transcribed sequence with different gene banks failed to reveal a high degree of homology with other proteins. The cloned DNA fragment was easily expressed in Escherichia coli without the need of induction by isopropyl-beta-D-thiogalactopyranoside. These findings suggest that the gene encodes a P. brasiliensis-specific protein. Se clonó, secuenció y caracterizó un gen que codifica una proteína antigénica de 27 kDa de Paracoccidioides brasiliensis. Se produjo una biblioteca de ADNc de la fase micelial y se empaquetó en el vector Uni-Zap-XR, kit de síntesis lambda Zap II (Stratagene, La Jolla, Ca). El cribado de la biblioteca se llevó a cabo utilizando un conjunto de sueros de pacientes con paracoccidioidomicosis que habían demostrado ser reactivos en las pruebas serológicas. Entre 44.000 clones de la biblioteca cribados inmunológicamente, 2 fueron positivos (clones 2 y 3). El primero no se caracterizó más. Este último tiene un inserto de ADN de 1 kb con un marco de lectura abierto que codifica una proteína de 259 aminoácidos con una masa molecular predicha de 28,6 kDa (27 kDa por SDS-PAGE). Esta proteína corresponde a una proteína de 25 kDa en preparaciones antigénicas de P. brasiliensis según lo determinado por análisis de transferencia Western. La comparación de la secuencia transcrita con diferentes bancos de genes no reveló un alto grado de homología con otras proteínas. El fragmento de ADN clonado se expresó fácilmente en Escherichia coli sin necesidad de inducción por isopropil-beta-D-tiogalactopiranósido. Estos hallazgos sugieren que el gen codifica una proteína específica de P. brasiliensis.
issn	10871845
url	https://repositorio.accefyn.org.co/handle/001/1064
url_str_mv	https://repositorio.accefyn.org.co/handle/001/1064
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score	11.260413

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