Expression of the rice hoja blanca virus (RHBV) non-structural protein 3 (NS3) in Escherichia coli and its in situ localization in RHBV-infected rice tissues
The non-structural NS3 protein gene from the rice hoja blanca virus (RHBV) was fused to the glutathione- S-transferase carboxilic end and expressed in Escherichia coli strain JM83. Large quantities of fusion protein were produced in insoluble form. The fusion protein was fractionated in SDS-PAGE and...
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Universidad de Costa Rica
2004-09-01
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| Series: | Revista de Biología Tropical |
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| Online Access: | http://www.scielo.sa.cr/scielo.php?script=sci_arttext&pid=S0034-77442004000300037 |
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| author | Miguel Muñoz Isela Bolaños Griselda Arrieta-Espinoza Ana M Espinoza |
| author_facet | Miguel Muñoz Isela Bolaños Griselda Arrieta-Espinoza Ana M Espinoza |
| author_sort | Miguel Muñoz |
| collection | DOAJ |
| description | The non-structural NS3 protein gene from the rice hoja blanca virus (RHBV) was fused to the glutathione- S-transferase carboxilic end and expressed in Escherichia coli strain JM83. Large quantities of fusion protein were produced in insoluble form. The fusion protein was fractionated in SDS-PAGE and purified by electroelution, polyclonal antibodies were raised in rabbit and the antiserum was absorbed with bacterial crude extract. A band of similar size as that of NS3 protein was observed in Western blots using extracts from RHBV-infected rice plants. Immunoelectron microscopy with colloidal gold-labeled antibodies against NS3 protein and the viral nucleocapsid protein revealed in situ accumulation of NS3 protein in the cytoplasm but not in the viral inclusion bodies, vacuoles or chloroplasts of RHBV-infected plants, following the same pattern of distribution as the RHBV nucleocapsid protein. Rev. Biol. Trop. 52(3): 765-775. Epub 2004 Dic 15.<br>El gen que codifica por la proteína no estructural NS3 del virus de la hoja blanca de arroz (RHBV) se fusionó al extremo carboxilo del gen de la glutationa-S-transferasa y se expresó en la cepa JM83 de Escherichia coli. Se obtuvieron altas concentraciones de la proteína de fusion (GST-NS3) en forma insoluble. La proteína de fusión se fraccionó en geles de SDS-PAGE, se purificó por electroelución, y se utilizó para producir anticuerpos policlonales en conejo . El antisuero producido se absorbió con extractos crudos de E. coli. Extractos crudos de plantas de arroz sanas e infectadas con el RHBV se evaluaron por Western blots detectándose una banda de peso molecular similar al estimado para la proteína NS3 (23KDa) en las plantas infectadas con el virus. Los tejidos provenientes de plantas infectadas con el RHBV se analizaron por medio de microscopia inmunoelectrónica con oro colloidal marcado con anticuerpos contra la proteína NS3 y la nucleoproteína viral N. Se observó una acumulación in situ de la proteína NS3 en el citoplasma, pero no se detectó en los cuerpos de inclusion, vacuolas o cloroplastos. Se demostró que la proteína NS3 sigue el mismo patron de distribución que el de la nucleoproteína viral N del RHBV. |
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| spelling | doaj.art-d182a808ac66458b80ffc93507c91dd32024-08-02T21:54:59ZengUniversidad de Costa RicaRevista de Biología Tropical0034-77442215-20752004-09-01523765775Expression of the rice hoja blanca virus (RHBV) non-structural protein 3 (NS3) in Escherichia coli and its in situ localization in RHBV-infected rice tissuesMiguel MuñozIsela BolañosGriselda Arrieta-EspinozaAna M EspinozaThe non-structural NS3 protein gene from the rice hoja blanca virus (RHBV) was fused to the glutathione- S-transferase carboxilic end and expressed in Escherichia coli strain JM83. Large quantities of fusion protein were produced in insoluble form. The fusion protein was fractionated in SDS-PAGE and purified by electroelution, polyclonal antibodies were raised in rabbit and the antiserum was absorbed with bacterial crude extract. A band of similar size as that of NS3 protein was observed in Western blots using extracts from RHBV-infected rice plants. Immunoelectron microscopy with colloidal gold-labeled antibodies against NS3 protein and the viral nucleocapsid protein revealed in situ accumulation of NS3 protein in the cytoplasm but not in the viral inclusion bodies, vacuoles or chloroplasts of RHBV-infected plants, following the same pattern of distribution as the RHBV nucleocapsid protein. Rev. Biol. Trop. 52(3): 765-775. Epub 2004 Dic 15.<br>El gen que codifica por la proteína no estructural NS3 del virus de la hoja blanca de arroz (RHBV) se fusionó al extremo carboxilo del gen de la glutationa-S-transferasa y se expresó en la cepa JM83 de Escherichia coli. Se obtuvieron altas concentraciones de la proteína de fusion (GST-NS3) en forma insoluble. La proteína de fusión se fraccionó en geles de SDS-PAGE, se purificó por electroelución, y se utilizó para producir anticuerpos policlonales en conejo . El antisuero producido se absorbió con extractos crudos de E. coli. Extractos crudos de plantas de arroz sanas e infectadas con el RHBV se evaluaron por Western blots detectándose una banda de peso molecular similar al estimado para la proteína NS3 (23KDa) en las plantas infectadas con el virus. Los tejidos provenientes de plantas infectadas con el RHBV se analizaron por medio de microscopia inmunoelectrónica con oro colloidal marcado con anticuerpos contra la proteína NS3 y la nucleoproteína viral N. Se observó una acumulación in situ de la proteína NS3 en el citoplasma, pero no se detectó en los cuerpos de inclusion, vacuolas o cloroplastos. Se demostró que la proteína NS3 sigue el mismo patron de distribución que el de la nucleoproteína viral N del RHBV.http://www.scielo.sa.cr/scielo.php?script=sci_arttext&pid=S0034-77442004000300037Virus hoja blancaarrozRHBVNS3anticuerpos policlonalesinmunoelectro microscopíaRice hoja blanca virusRHBVNS3polyclonal antibodiesimmunoelectron microscopy |
| spellingShingle | Miguel Muñoz Isela Bolaños Griselda Arrieta-Espinoza Ana M Espinoza Expression of the rice hoja blanca virus (RHBV) non-structural protein 3 (NS3) in Escherichia coli and its in situ localization in RHBV-infected rice tissues Revista de Biología Tropical Virus hoja blanca arroz RHBV NS3 anticuerpos policlonales inmunoelectro microscopía Rice hoja blanca virus RHBV NS3 polyclonal antibodies immunoelectron microscopy |
| title | Expression of the rice hoja blanca virus (RHBV) non-structural protein 3 (NS3) in Escherichia coli and its in situ localization in RHBV-infected rice tissues |
| title_full | Expression of the rice hoja blanca virus (RHBV) non-structural protein 3 (NS3) in Escherichia coli and its in situ localization in RHBV-infected rice tissues |
| title_fullStr | Expression of the rice hoja blanca virus (RHBV) non-structural protein 3 (NS3) in Escherichia coli and its in situ localization in RHBV-infected rice tissues |
| title_full_unstemmed | Expression of the rice hoja blanca virus (RHBV) non-structural protein 3 (NS3) in Escherichia coli and its in situ localization in RHBV-infected rice tissues |
| title_short | Expression of the rice hoja blanca virus (RHBV) non-structural protein 3 (NS3) in Escherichia coli and its in situ localization in RHBV-infected rice tissues |
| title_sort | expression of the rice hoja blanca virus rhbv non structural protein 3 ns3 in escherichia coli and its in situ localization in rhbv infected rice tissues |
| topic | Virus hoja blanca arroz RHBV NS3 anticuerpos policlonales inmunoelectro microscopía Rice hoja blanca virus RHBV NS3 polyclonal antibodies immunoelectron microscopy |
| url | http://www.scielo.sa.cr/scielo.php?script=sci_arttext&pid=S0034-77442004000300037 |
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