Detection of recombinant human lactoferrin and lysozyme produced in a bitransgenic cow
- Autores
- Kaiser, German Gustavo; Mucci, Nicolas Crescencio; Gonzalez, Vega; Sánchez, Lourdes; Parrón, José Antonio; Pérez, María Dolores; Calvo, Miguel; Aller Atucha, Juan Florencio; Hozbor, Federico Andres; Mutto, Adrián Angel
- Año de publicación
- 2017
- Idioma
- inglés
- Tipo de recurso
- artículo
- Estado
- versión publicada
- Descripción
- Lactoferrin and lysozyme are 2 glycoproteins with great antimicrobial activity, being part of the nonspecific defensive system of human milk, though their use in commercial products is difficult because human milk is a limited source. Therefore, many investigations have been carried out to produce those proteins in biological systems, such as bacteria, yeasts, or plants. Mammals seem to be more suitable as expression systems for human proteins, however, especially for those that are glycosylated. In the present study, we developed a bicistronic commercial vector containing a goat β-casein promoter and an internal ribosome entry site fragment between the human lactoferrin and human lysozyme genes to allow the introduction of both genes into bovine adult fibroblasts in a single transfection. Embryos were obtained by somatic cell nuclear transfer, and, after 6 transferences to recipients, 3 pregnancies and 1 viable bitransgenic calf were obtained. The presence of the vector was confirmed by fluorescent in situ hybridization of skin cells. At 13 mo of life and after artificial induction of lactation, both recombinant proteins were found in the colostrum and milk of the bitransgenic calf. Human lactoferrin concentration in the colostrum was 0.0098 mg/mL and that in milk was 0.011 mg/mL; human lysozyme concentration in the colostrum was 0.0022 mg/mL and that in milk was 0.0024 mg/mL. The molar concentration of both human proteins revealed no differences in protein production of the internal ribosome entry site upstream and downstream protein. The enzymatic activity of lysozyme in the transgenic milk was comparable to that of human milk, being 6 and 10 times higher than that of bovine lysozyme presentin milk. This work represents an important step to obtain multiple proteins or enhance single protein production by using animal pharming and fewer regulatory and antibiotic-resistant foreign sequences, allowing the design of humanized milk with added biological value for newborn nutrition and development. Transgenic animals can offer a unique opportunity to the dairy industry, providing starting materials suitable to develop specific products with high added value. Key words: bitransgenic cow, human lactoferrin, ELISA, human lysozyme.
Fil: Kaiser, German Gustavo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Grupo de Biotecnología de la Reproducción; Argentina
Fil: Mucci, Nicolas Crescencio. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Grupo de Biotecnología de la Reproducción; Argentina
Fil: Gonzalez, Vega. Universidad de Zaragoza. Facultad de Veterinaria. Tecnología de los Alimentos; España
Fil: Sánchez, Lourdes. Universidad de Zaragoza. Facultad de Veterinaria. Tecnología de los Alimentos; España
Fil: Parrón, José Antonio. Universidad de Zaragoza. Facultad de Veterinaria. Tecnología de los Alimentos; España
Fil: Pérez, María Dolores. Universidad de Zaragoza. Facultad de Veterinaria. Tecnología de los Alimentos; España
Fil: Calvo, Miguel. Universidad de Zaragoza. Facultad de Veterinaria. Tecnología de los Alimentos; España
Fil: Aller Atucha, Juan Florencio. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Grupo de Biotecnología de la Reproducción; Argentina
Fil: Hozbor, Federico Andres. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Grupo de Biotecnología de la Reproducción; Argentina
Fil: Mutto, Adrián Angel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina - Fuente
- Journal of dairy science 100 (3) : 1605–1617. (March 2017)
- Materia
-
Lactoferrinas
Lisozima
Vaca
Animales Transgénicos
ELISA
Leche
Milk
Transgenic Animals
Cows
Lysozyme
Lactoferrin - Nivel de accesibilidad
- acceso abierto
- Condiciones de uso
- http://creativecommons.org/licenses/by-nc-sa/4.0/
- Repositorio
.jpg)
- Institución
- Instituto Nacional de Tecnología Agropecuaria
- OAI Identificador
- oai:localhost:20.500.12123/2318
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Detection of recombinant human lactoferrin and lysozyme produced in a bitransgenic cowKaiser, German GustavoMucci, Nicolas CrescencioGonzalez, VegaSánchez, LourdesParrón, José AntonioPérez, María DoloresCalvo, MiguelAller Atucha, Juan FlorencioHozbor, Federico AndresMutto, Adrián AngelLactoferrinasLisozimaVacaAnimales TransgénicosELISALecheMilkTransgenic AnimalsCowsLysozymeLactoferrinLactoferrin and lysozyme are 2 glycoproteins with great antimicrobial activity, being part of the nonspecific defensive system of human milk, though their use in commercial products is difficult because human milk is a limited source. Therefore, many investigations have been carried out to produce those proteins in biological systems, such as bacteria, yeasts, or plants. Mammals seem to be more suitable as expression systems for human proteins, however, especially for those that are glycosylated. In the present study, we developed a bicistronic commercial vector containing a goat β-casein promoter and an internal ribosome entry site fragment between the human lactoferrin and human lysozyme genes to allow the introduction of both genes into bovine adult fibroblasts in a single transfection. Embryos were obtained by somatic cell nuclear transfer, and, after 6 transferences to recipients, 3 pregnancies and 1 viable bitransgenic calf were obtained. The presence of the vector was confirmed by fluorescent in situ hybridization of skin cells. At 13 mo of life and after artificial induction of lactation, both recombinant proteins were found in the colostrum and milk of the bitransgenic calf. Human lactoferrin concentration in the colostrum was 0.0098 mg/mL and that in milk was 0.011 mg/mL; human lysozyme concentration in the colostrum was 0.0022 mg/mL and that in milk was 0.0024 mg/mL. The molar concentration of both human proteins revealed no differences in protein production of the internal ribosome entry site upstream and downstream protein. The enzymatic activity of lysozyme in the transgenic milk was comparable to that of human milk, being 6 and 10 times higher than that of bovine lysozyme presentin milk. This work represents an important step to obtain multiple proteins or enhance single protein production by using animal pharming and fewer regulatory and antibiotic-resistant foreign sequences, allowing the design of humanized milk with added biological value for newborn nutrition and development. Transgenic animals can offer a unique opportunity to the dairy industry, providing starting materials suitable to develop specific products with high added value. Key words: bitransgenic cow, human lactoferrin, ELISA, human lysozyme.Fil: Kaiser, German Gustavo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Grupo de Biotecnología de la Reproducción; ArgentinaFil: Mucci, Nicolas Crescencio. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Grupo de Biotecnología de la Reproducción; ArgentinaFil: Gonzalez, Vega. Universidad de Zaragoza. Facultad de Veterinaria. Tecnología de los Alimentos; EspañaFil: Sánchez, Lourdes. Universidad de Zaragoza. Facultad de Veterinaria. Tecnología de los Alimentos; EspañaFil: Parrón, José Antonio. Universidad de Zaragoza. Facultad de Veterinaria. Tecnología de los Alimentos; EspañaFil: Pérez, María Dolores. Universidad de Zaragoza. Facultad de Veterinaria. Tecnología de los Alimentos; EspañaFil: Calvo, Miguel. Universidad de Zaragoza. Facultad de Veterinaria. Tecnología de los Alimentos; EspañaFil: Aller Atucha, Juan Florencio. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Grupo de Biotecnología de la Reproducción; ArgentinaFil: Hozbor, Federico Andres. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Grupo de Biotecnología de la Reproducción; ArgentinaFil: Mutto, Adrián Angel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina2018-05-03T17:02:29Z2018-05-03T17:02:29Z2017-03info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfhttp://hdl.handle.net/20.500.12123/23180022-0302https://doi.org/10.3168/jds.2016-11173Journal of dairy science 100 (3) : 1605–1617. 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| dc.title.none.fl_str_mv |
Detection of recombinant human lactoferrin and lysozyme produced in a bitransgenic cow |
| title |
Detection of recombinant human lactoferrin and lysozyme produced in a bitransgenic cow |
| spellingShingle |
Detection of recombinant human lactoferrin and lysozyme produced in a bitransgenic cow Kaiser, German Gustavo Lactoferrinas Lisozima Vaca Animales Transgénicos ELISA Leche Milk Transgenic Animals Cows Lysozyme Lactoferrin |
| title_short |
Detection of recombinant human lactoferrin and lysozyme produced in a bitransgenic cow |
| title_full |
Detection of recombinant human lactoferrin and lysozyme produced in a bitransgenic cow |
| title_fullStr |
Detection of recombinant human lactoferrin and lysozyme produced in a bitransgenic cow |
| title_full_unstemmed |
Detection of recombinant human lactoferrin and lysozyme produced in a bitransgenic cow |
| title_sort |
Detection of recombinant human lactoferrin and lysozyme produced in a bitransgenic cow |
| dc.creator.none.fl_str_mv |
Kaiser, German Gustavo Mucci, Nicolas Crescencio Gonzalez, Vega Sánchez, Lourdes Parrón, José Antonio Pérez, María Dolores Calvo, Miguel Aller Atucha, Juan Florencio Hozbor, Federico Andres Mutto, Adrián Angel |
| author |
Kaiser, German Gustavo |
| author_facet |
Kaiser, German Gustavo Mucci, Nicolas Crescencio Gonzalez, Vega Sánchez, Lourdes Parrón, José Antonio Pérez, María Dolores Calvo, Miguel Aller Atucha, Juan Florencio Hozbor, Federico Andres Mutto, Adrián Angel |
| author_role |
author |
| author2 |
Mucci, Nicolas Crescencio Gonzalez, Vega Sánchez, Lourdes Parrón, José Antonio Pérez, María Dolores Calvo, Miguel Aller Atucha, Juan Florencio Hozbor, Federico Andres Mutto, Adrián Angel |
| author2_role |
author author author author author author author author author |
| dc.subject.none.fl_str_mv |
Lactoferrinas Lisozima Vaca Animales Transgénicos ELISA Leche Milk Transgenic Animals Cows Lysozyme Lactoferrin |
| topic |
Lactoferrinas Lisozima Vaca Animales Transgénicos ELISA Leche Milk Transgenic Animals Cows Lysozyme Lactoferrin |
| dc.description.none.fl_txt_mv |
Lactoferrin and lysozyme are 2 glycoproteins with great antimicrobial activity, being part of the nonspecific defensive system of human milk, though their use in commercial products is difficult because human milk is a limited source. Therefore, many investigations have been carried out to produce those proteins in biological systems, such as bacteria, yeasts, or plants. Mammals seem to be more suitable as expression systems for human proteins, however, especially for those that are glycosylated. In the present study, we developed a bicistronic commercial vector containing a goat β-casein promoter and an internal ribosome entry site fragment between the human lactoferrin and human lysozyme genes to allow the introduction of both genes into bovine adult fibroblasts in a single transfection. Embryos were obtained by somatic cell nuclear transfer, and, after 6 transferences to recipients, 3 pregnancies and 1 viable bitransgenic calf were obtained. The presence of the vector was confirmed by fluorescent in situ hybridization of skin cells. At 13 mo of life and after artificial induction of lactation, both recombinant proteins were found in the colostrum and milk of the bitransgenic calf. Human lactoferrin concentration in the colostrum was 0.0098 mg/mL and that in milk was 0.011 mg/mL; human lysozyme concentration in the colostrum was 0.0022 mg/mL and that in milk was 0.0024 mg/mL. The molar concentration of both human proteins revealed no differences in protein production of the internal ribosome entry site upstream and downstream protein. The enzymatic activity of lysozyme in the transgenic milk was comparable to that of human milk, being 6 and 10 times higher than that of bovine lysozyme presentin milk. This work represents an important step to obtain multiple proteins or enhance single protein production by using animal pharming and fewer regulatory and antibiotic-resistant foreign sequences, allowing the design of humanized milk with added biological value for newborn nutrition and development. Transgenic animals can offer a unique opportunity to the dairy industry, providing starting materials suitable to develop specific products with high added value. Key words: bitransgenic cow, human lactoferrin, ELISA, human lysozyme. Fil: Kaiser, German Gustavo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Grupo de Biotecnología de la Reproducción; Argentina Fil: Mucci, Nicolas Crescencio. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Grupo de Biotecnología de la Reproducción; Argentina Fil: Gonzalez, Vega. Universidad de Zaragoza. Facultad de Veterinaria. Tecnología de los Alimentos; España Fil: Sánchez, Lourdes. Universidad de Zaragoza. Facultad de Veterinaria. Tecnología de los Alimentos; España Fil: Parrón, José Antonio. Universidad de Zaragoza. Facultad de Veterinaria. Tecnología de los Alimentos; España Fil: Pérez, María Dolores. Universidad de Zaragoza. Facultad de Veterinaria. Tecnología de los Alimentos; España Fil: Calvo, Miguel. Universidad de Zaragoza. Facultad de Veterinaria. Tecnología de los Alimentos; España Fil: Aller Atucha, Juan Florencio. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Grupo de Biotecnología de la Reproducción; Argentina Fil: Hozbor, Federico Andres. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Grupo de Biotecnología de la Reproducción; Argentina Fil: Mutto, Adrián Angel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina |
| description |
Lactoferrin and lysozyme are 2 glycoproteins with great antimicrobial activity, being part of the nonspecific defensive system of human milk, though their use in commercial products is difficult because human milk is a limited source. Therefore, many investigations have been carried out to produce those proteins in biological systems, such as bacteria, yeasts, or plants. Mammals seem to be more suitable as expression systems for human proteins, however, especially for those that are glycosylated. In the present study, we developed a bicistronic commercial vector containing a goat β-casein promoter and an internal ribosome entry site fragment between the human lactoferrin and human lysozyme genes to allow the introduction of both genes into bovine adult fibroblasts in a single transfection. Embryos were obtained by somatic cell nuclear transfer, and, after 6 transferences to recipients, 3 pregnancies and 1 viable bitransgenic calf were obtained. The presence of the vector was confirmed by fluorescent in situ hybridization of skin cells. At 13 mo of life and after artificial induction of lactation, both recombinant proteins were found in the colostrum and milk of the bitransgenic calf. Human lactoferrin concentration in the colostrum was 0.0098 mg/mL and that in milk was 0.011 mg/mL; human lysozyme concentration in the colostrum was 0.0022 mg/mL and that in milk was 0.0024 mg/mL. The molar concentration of both human proteins revealed no differences in protein production of the internal ribosome entry site upstream and downstream protein. The enzymatic activity of lysozyme in the transgenic milk was comparable to that of human milk, being 6 and 10 times higher than that of bovine lysozyme presentin milk. This work represents an important step to obtain multiple proteins or enhance single protein production by using animal pharming and fewer regulatory and antibiotic-resistant foreign sequences, allowing the design of humanized milk with added biological value for newborn nutrition and development. Transgenic animals can offer a unique opportunity to the dairy industry, providing starting materials suitable to develop specific products with high added value. Key words: bitransgenic cow, human lactoferrin, ELISA, human lysozyme. |
| publishDate |
2017 |
| dc.date.none.fl_str_mv |
2017-03 2018-05-03T17:02:29Z 2018-05-03T17:02:29Z |
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article |
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publishedVersion |
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http://hdl.handle.net/20.500.12123/2318 0022-0302 https://doi.org/10.3168/jds.2016-11173 |
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http://hdl.handle.net/20.500.12123/2318 https://doi.org/10.3168/jds.2016-11173 |
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0022-0302 |
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eng |
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