Detection of recombinant human lactoferrin and lysozyme produced in a bitransgenic cow
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 prod...
| Main Authors: | , , , , , , , , , |
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| Format: | info:ar-repo/semantics/artículo |
| Language: | Inglés |
| Published: |
2018
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| Subjects: | |
| Online Access: | http://hdl.handle.net/20.500.12123/2318 https://doi.org/10.3168/jds.2016-11173 |
| _version_ | 1855034941391241216 |
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| author | 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_browse | Aller Atucha, Juan Florencio Calvo, Miguel Gonzalez, Vega Hozbor, Federico Andres Kaiser, German Gustavo Mucci, Nicolas Crescencio Mutto, Adrián Angel Parrón, José Antonio Pérez, María Dolores Sánchez, Lourdes |
| 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_sort | Kaiser, German Gustavo |
| collection | INTA Digital |
| 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. |
| format | info:ar-repo/semantics/artículo |
| id | INTA2318 |
| institution | Instituto Nacional de Tecnología Agropecuaria (INTA -Argentina) |
| language | Inglés |
| publishDate | 2018 |
| publishDateRange | 2018 |
| publishDateSort | 2018 |
| record_format | dspace |
| spelling | INTA23182019-03-26T12:02:20Z Detection of recombinant human lactoferrin and lysozyme produced in a bitransgenic cow 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 Lactoferrinas Lisozima Vaca Animales Transgénicos ELISA Leche Milk Transgenic Animals Cows Lysozyme Lactoferrin 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 2018-05-03T17:02:29Z 2018-05-03T17:02:29Z 2017-03 info:ar-repo/semantics/artículo info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion http://hdl.handle.net/20.500.12123/2318 0022-0302 https://doi.org/10.3168/jds.2016-11173 eng info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by-nc-sa/4.0/ Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0) application/pdf Journal of dairy science 100 (3) : 1605–1617. (March 2017) |
| spellingShingle | Lactoferrinas Lisozima Vaca Animales Transgénicos ELISA Leche Milk Transgenic Animals Cows Lysozyme Lactoferrin 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 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 |
| 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_short | 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 |
| topic | Lactoferrinas Lisozima Vaca Animales Transgénicos ELISA Leche Milk Transgenic Animals Cows Lysozyme Lactoferrin |
| url | http://hdl.handle.net/20.500.12123/2318 https://doi.org/10.3168/jds.2016-11173 |
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