Trans-complementation of polyhedrin by a stably transformed Sf9 insect cell line allows occ− baculovirus occlusion and larval per os infectivity
Oral infection of insect larvae with baculovirus is an advantageous methodology for producing high levels of recombinant proteins and for achieving plague control. However, many recombinant baculoviruses express a foreign protein in lieu of the polyhedrin and hence do not form occlusion bodies (occ−...
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| Format: | Artículo |
| Language: | Inglés |
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Elsevier
2019
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| Online Access: | https://www.sciencedirect.com/science/article/pii/S0168165609004842 http://hdl.handle.net/20.500.12123/5110 https://doi.org/10.1016/j.jbiotec.2009.10.015 |
| _version_ | 1855483557758107648 |
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| author | López, María Gabriela Alfonso, Victoria Carrillo, Elisa Cristina Taboga, Oscar Alberto |
| author_browse | Alfonso, Victoria Carrillo, Elisa Cristina López, María Gabriela Taboga, Oscar Alberto |
| author_facet | López, María Gabriela Alfonso, Victoria Carrillo, Elisa Cristina Taboga, Oscar Alberto |
| author_sort | López, María Gabriela |
| collection | INTA Digital |
| description | Oral infection of insect larvae with baculovirus is an advantageous methodology for producing high levels of recombinant proteins and for achieving plague control. However, many recombinant baculoviruses express a foreign protein in lieu of the polyhedrin and hence do not form occlusion bodies (occ−), resulting in extremely reduced per os infectivity in larvae. To overcome this limitation, stably transformed insect cell lines expressing polyhedrin capable of occluding occ− recombinant baculovirus by trans-complementation were developed to obtain oral inoculum for insect larvae infection. First, the optimum regulatory region of polyhedrin promoter was determined utilizing chloramphenicol acetyl transferase (CAT) as the reporter gene. After infection with occ− baculovirus, the higher expression levels of CAT were achieved when a region of 2735 bp that contained sequences known to have transcriptional enhancer functions were present upstream the polyhedrin coding sequence. This regulatory region was selected to drive polyhedrin expression in insect cell lines. Transfection of Sf9 cells with plasmid carrying polyhedrin gene and stable cell lines established by selection with blasticidin showed polyhedrin expression and, moreover, crystalline polyhedron-like structures were visualized by optic microscopy. Oral infectivity was demonstrated by fluorescence detection in Rachiplusia nu larvae infected with occluded AcGFPpolh− baculovirus obtained using the system presented here. |
| format | Artículo |
| id | INTA5110 |
| institution | Instituto Nacional de Tecnología Agropecuaria (INTA -Argentina) |
| language | Inglés |
| publishDate | 2019 |
| publishDateRange | 2019 |
| publishDateSort | 2019 |
| publisher | Elsevier |
| publisherStr | Elsevier |
| record_format | dspace |
| spelling | INTA51102019-05-14T13:48:33Z Trans-complementation of polyhedrin by a stably transformed Sf9 insect cell line allows occ− baculovirus occlusion and larval per os infectivity López, María Gabriela Alfonso, Victoria Carrillo, Elisa Cristina Taboga, Oscar Alberto Biotecnología Insecta Baculovirus Genética Biotechnology Genetics Polihedrina Cuerpos de Oclusión Polyhedrin Occlusion Bodies Oral infection of insect larvae with baculovirus is an advantageous methodology for producing high levels of recombinant proteins and for achieving plague control. However, many recombinant baculoviruses express a foreign protein in lieu of the polyhedrin and hence do not form occlusion bodies (occ−), resulting in extremely reduced per os infectivity in larvae. To overcome this limitation, stably transformed insect cell lines expressing polyhedrin capable of occluding occ− recombinant baculovirus by trans-complementation were developed to obtain oral inoculum for insect larvae infection. First, the optimum regulatory region of polyhedrin promoter was determined utilizing chloramphenicol acetyl transferase (CAT) as the reporter gene. After infection with occ− baculovirus, the higher expression levels of CAT were achieved when a region of 2735 bp that contained sequences known to have transcriptional enhancer functions were present upstream the polyhedrin coding sequence. This regulatory region was selected to drive polyhedrin expression in insect cell lines. Transfection of Sf9 cells with plasmid carrying polyhedrin gene and stable cell lines established by selection with blasticidin showed polyhedrin expression and, moreover, crystalline polyhedron-like structures were visualized by optic microscopy. Oral infectivity was demonstrated by fluorescence detection in Rachiplusia nu larvae infected with occluded AcGFPpolh− baculovirus obtained using the system presented here. Instituto de Biotecnología Fil: Lopez, Maria Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Alfonso, Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Carrillo, Elisa Cristina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Taboga, Oscar Alberto. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina 2019-05-14T13:45:07Z 2019-05-14T13:45:07Z 2010-01-15 info:ar-repo/semantics/artículo info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion https://www.sciencedirect.com/science/article/pii/S0168165609004842 http://hdl.handle.net/20.500.12123/5110 0168-1656 https://doi.org/10.1016/j.jbiotec.2009.10.015 eng info:eu-repo/semantics/restrictedAccess application/pdf Elsevier Journal of Biotechnology 145 (2) : 199-205 (January 2010) |
| spellingShingle | Biotecnología Insecta Baculovirus Genética Biotechnology Genetics Polihedrina Cuerpos de Oclusión Polyhedrin Occlusion Bodies López, María Gabriela Alfonso, Victoria Carrillo, Elisa Cristina Taboga, Oscar Alberto Trans-complementation of polyhedrin by a stably transformed Sf9 insect cell line allows occ− baculovirus occlusion and larval per os infectivity |
| title | Trans-complementation of polyhedrin by a stably transformed Sf9 insect cell line allows occ− baculovirus occlusion and larval per os infectivity |
| title_full | Trans-complementation of polyhedrin by a stably transformed Sf9 insect cell line allows occ− baculovirus occlusion and larval per os infectivity |
| title_fullStr | Trans-complementation of polyhedrin by a stably transformed Sf9 insect cell line allows occ− baculovirus occlusion and larval per os infectivity |
| title_full_unstemmed | Trans-complementation of polyhedrin by a stably transformed Sf9 insect cell line allows occ− baculovirus occlusion and larval per os infectivity |
| title_short | Trans-complementation of polyhedrin by a stably transformed Sf9 insect cell line allows occ− baculovirus occlusion and larval per os infectivity |
| title_sort | trans complementation of polyhedrin by a stably transformed sf9 insect cell line allows occ baculovirus occlusion and larval per os infectivity |
| topic | Biotecnología Insecta Baculovirus Genética Biotechnology Genetics Polihedrina Cuerpos de Oclusión Polyhedrin Occlusion Bodies |
| url | https://www.sciencedirect.com/science/article/pii/S0168165609004842 http://hdl.handle.net/20.500.12123/5110 https://doi.org/10.1016/j.jbiotec.2009.10.015 |
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