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Obtención de la proteína verde fluorescente recombinante y su anticuerpo policlonal Igy
Obtaining the recombinant green fluorescent protein and its IgY polyclonal antibody
DOI:
https://doi.org/10.15446/rev.colomb.biote.v25n1.91675Palabras clave:
Anticuerpos policlonales, Biotecnología, Proteínas recombinantes, Proteína Verde Fluorescente Mejorada (EGFP) (es)Polyclonal antibodies, Biotechnology, Recombinant proteins, Enhanced Green Fluorescent Protein (EGFP) (en)
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La Proteína Verde Fluorescente (Green Fluorescent Protein, GFP) es ampliamente utilizada en ensayos in vivo e in vitro. Se han generado múltiples variantes de esta proteína para diversificar sus características, como la GFP-enhancer (EGFP) que emite una señal de fluorescencia 35 veces mayor en comparación con la proteína silvestre, siendo implementada como proteína fusión en estudios de localización y estabilidad estructural, entre otros. La detección de esta proteína y sus variantes puede ser directa o indirecta, mediante el uso de anticuerpos anti-GFP. Aunque el uso de GFP es generalizado y de evidente utilidad en investigación y en docencia, los insumos para su estudio exhiben un alto costo dado que deben ser importados, constituyendo un recurso limitado en Colombia. El presente trabajo reporta la clonación y expresión de la proteína recombinante 6xHisEGFP, cuya purificación se completó a partir de la fracción soluble e insoluble del sistema heterólogo Escherichia coli mediante cromatografía de afinidad a metales inmovilizados y electroforesis preparativa, respectivamente. La proteína purificada se implementó como antígeno para la producción de anticuerpos policlonales aviares (IgY) contra la EGFP, los cuales se obtuvieron desde los huevos colectados y el suero de las sangrías de las gallinas inmunizadas. En este sentido, la estrategia metodológica planteada constituye un avance en el desarrollo de un sistema biotecnológico para la producción nacional de herramientas moleculares como los anticuerpos policlonales aviares a bajo costo.
Green Fluorescent Protein (GFP) is widely used in in vivo and in vitro assays. Multiple variants of this protein have been generated to diversify its characteristics, such as the enhancer GFP (EGFP) that emits a 35-fold higher fluorescence signal compared to the wild-type protein, being implemented as a fusion reporter in localization and structural stability studies, among others. Detection of this protein can be direct or indirect, fusing anti-GFP antibodies. Although the use of GFP is generalized and of evident utility in research and teaching, the molecular tools for its study exhibit a high cost since they must be imported, constituting a limited resource in Colombia. This work reports the cloning and expression of the recombinant protein 6xHisEGFP, which purification was completed from the soluble and insoluble fraction of the heterologous Escherichia coli system by immobilized metal affinity chromatography and preparative SDS-PAGE, respectively. The purified protein was implemented as an antigen to produce avian polyclonal antibodies (IgY) against EGFP, which were obtained from collected eggs and blood serum from immunized hens. In this sense, the proposed methodological strategy constitutes an advance in the development of a biotechnological system for the national production of molecular tools such as avian polyclonal antibodies at low-cost.
Referencias
Abdelwhab, E. M., Grund, C., Aly, M. M., Beer, M., Harder, T. C., & Hafez, H. M. (2016). Benefits and limits of egg yolk vs. serum samples for avian influenza virus serosurveillance. Avian Diseases, 60(2), 496-499. DOI: https://doi.org/10.1637/11207-060115-ResNote
Amro, W. A., Al-Qaisi, W., & Al-Razem, F. (2018). Production and purification of IgY antibodies from chicken egg yolk. Journal of Genetic Engineering and Biotechnology, 16(1), 99-103. DOI: https://doi.org/10.1016/j.jgeb.2017.10.003
Ausubel F, Brent R, Kingston RE, et al. (2003). Current Protocols in Molecular Biology. John Wiley and Sons Inc, ringbou edition.
Chalfie, M., Tu, Y., Euskirchen, G., Ward, W., & Prasher, D. (1994). Green fluorescent protein as a marker for gene expression. Science, 263(5148), 802–805. DOI: https://doi.org/10.1126/science.8303295
Ching, K. H., Collarini, E. J., Abdiche, Y. N., Bedinger, D., Pedersen, D., Izquierdo, S., ... & Harriman, W. D. (2018). Chickens with humanized immunoglobulin genes generate antibodies with high affinity and broad epitope coverage to conserved targets. In Mabs, 10, 71-80. DOI: https://doi.org/10.1080/19420862.2017.1386825
Contreras, L. E., Neme, R., & Ramírez, M. H. (2015). Identification and functional evaluation of Leishmania braziliensis Nicotinamide Mononucleotide Adenylyltransferase. Protein expression and purification, 115, 26-33. DOI: https://doi.org/10.1016/j.pep.2015.08.022
Criste, A., Urcan, A. C., & Corcionivoschi, N. (2019). Avian IgY antibodies, ancestors of mammalian antibodies–production and application. Rom. Biotechnol. Lett., 25, 41-49. DOI: https://doi.org/10.25083/rbl/25.2/1311.1319
Crowley, E. L., & Rafferty, S. P. (2019). Review of lactose-driven auto-induction expression of isotope-labelled proteins. Protein Expression and Purification, 157, 70-85. DOI: https://doi.org/10.1016/j.pep.2019.01.007
Hammond, J. B., & Kruger, N. J. (1988). Chapter 2- The Bradford Method for Protein Quantitation. In J. M. Walker (Ed.), New Protein Techniques. Methods in Molecular Biology. Vol 3 (Issue I, pp. 25–32). Humana Press. DOI: https://doi.org/10.1385/0-89603-126-8:25
Heim, R., Cubitt, A. B., & Tsien, R. Y. (1995). Improved green fluorescence. Nature, 373(6516), 663–664. DOI: https://doi.org/10.1038/373663b0
Gambotto, A., Dworacki, G., Cicinnati, V., Kenniston, T., Steitz, J., Tüting, T., ... & DeLeo, A. B. (2000). Immunogenicity of enhanced green fluorescent protein (EGFP) in BALB/c mice: identification of an H2-Kd-restricted CTL epitope. Gene therapy, 7(23), 2036-2040. DOI: https://doi.org/10.1038/sj.gt.3301335
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259): 680-685. DOI: https://doi.org/10.1038/227680a0
Leiva, C. L., Gallardo, M. J., Casanova, N., Terzolo, H., & Chacana, P. (2020). IgY-technology (egg yolk antibodies) in human medicine: a review of patents and clinical trials. International Immunopharmacology, 81, 106269. DOI: https://doi.org/10.1016/j.intimp.2020.106269
Liu ZQ, Mahmood T, Yang PC. (2014). Western blot: Technique, theory and trouble shooting. N Am J Med Sci., 6(3), 160. DOI: https://doi.org/10.4103/1947-2714.128482
McRae, S. R., Brown, C. L., & Bushell, G. R. (2005). Rapid purification of EGFP, EYFP, and ECFP with high yield and purity. Protein expression and purification, 41(1), 121-127. DOI: https://doi.org/10.1016/j.pep.2004.12.030
Montini, M. P. O., Fernandes, E. V., Ferraro, A. C. N. D. S., Almeida, M. A., da Silva, F. C., & Venancio, E. J. (2018). Effects of inoculation route and dose on production and avidity of IgY antibodies. Food and Agricultural Immunology, 29(1), 306-315. DOI: https://doi.org/10.1080/09540105.2017.1376036
Moreno-González PA, Díaz GJ, Ramírez-Hernández MH. (2013) Producción y purificación de anticuerpos aviares (IgYs) a partir de cuerpos de inclusión de una proteína recombinante central en el metabolismo del NAD+. Rev. Colomb. Quim.,42 (2),12-20.
Müller, S., Schubert, A., Zajac, J., Dyck, T., & Oelkrug, C. (2015). IgY antibodies in human nutrition for disease prevention. Nutrition journal, 14(1), 109. DOI: https://doi.org/10.1186/s12937-015-0067-3
Palmer, I., & Wingfield, P. T. (2012). Preparation and extraction of insoluble (inclusion‐body) proteins from Escherichia coli. Current protocols in protein science, 70(1), 6-3. DOI: https://doi.org/10.1002/0471140864.ps0603s70
Park, W. J., You, S. H., Choi, H. A., Chu, Y. J., & Kim, G. J. (2015). Over-expression of recombinant proteins with N-terminal His-tag via subcellular uneven distribution in Escherichia coli. Acta Biochim Biophys Sin, 47(7), 488-495. DOI: https://doi.org/10.1093/abbs/gmv036
Patterson GH, Knobel SM, Sharif WD, Kain SR, Piston DW. (1997) Use of the green fluorescent protein and its mutants in quantitative fluorescence microscopy. Biophysical journal. 73(5):2782-2790. doi:10.1016/S0006-3495(97)78307-3 DOI: https://doi.org/10.1016/S0006-3495(97)78307-3
Pauly, D., Chacana, P. A., Calzado, E. G., Brembs, B., & Schade, R. (2011). IgY technology: extraction of chicken antibodies from egg yolk by polyethylene glycol (PEG) precipitation. JoVE (Journal of Visualized Experiments), (51), e3084. DOI: https://doi.org/10.3791/3084
Pereira, E. P. V., van Tilburg, M. F., Florean, E. O. P. T., & Guedes, M. I. F. (2019). Egg yolk antibodies (IgY) and their applications in human and veterinary health: A review. International immunopharmacology, 73, 293-303. DOI: https://doi.org/10.1016/j.intimp.2019.05.015
Ponomarenko JV, Bui H, Li W, Fusseder N, Bourne PE, Sette A, Peters B. (2008). ElliPro: a new structure-based tool for the prediction of antibody epitopes. BMC Bioinformatics 9:514. DOI: https://doi.org/10.1186/1471-2105-9-514
Remington, SJ. (2011) Green fluorescent protein: a perspective. Protein Science, 20(9), 1509-1519. DOI: https://doi.org/10.1002/pro.684
Sá e Silva, M., & Swayne, D. E. (2012). Serum and egg yolk antibody detection in chickens infected with low pathogenicity avian influenza virus. Avian Diseases, 56(3), 601-604. DOI: https://doi.org/10.1637/10087-022312-ResNote.1
Sarkar, P., & Chattopadhyay, A. (2018). GFP fluorescence: A few lesser-known nuggets that make it work. Journal of Biosciences, 43(3), 421–430. DOI: https://doi.org/10.1007/s12038-018-9779-9
Sheng, Y. J., Ni, H. J., Zhang, H. Y., Li, Y. H., Wen, K., & Wang, Z. H. (2015). Production of chicken yolk IgY to sulfamethazine: comparison with rabbit antiserum IgG. Food and Agricultural Immunology, 26(3), 305-316. DOI: https://doi.org/10.1080/09540105.2014.914468
Shimomura, O., Johnson, F. H., & Saiga, Y. (1962). Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. Journal of cellular and comparative physiology, 59(3), 223-239. DOI: https://doi.org/10.1002/jcp.1030590302
Singh, A., Upadhyay, V., Upadhyay, A.K. et al. (2015). Protein recovery from inclusion bodies of Escherichia coli using mild solubilization process. Microbial Cell Factories, 14, 41. https://doi.org/10.1186/s12934-015-0222-8 DOI: https://doi.org/10.1186/s12934-015-0222-8
Sochor, M. A. (2014). Allostery and applications of the lac repressor. Publicly Accessible Penn Dissertations, 1448.
Villamil-Silva, S. E., Ortiz-Joya, L. J., Contreras-Rodríguez, L. E., Díaz, G. J., & Ramírez-Hernández, M. H. (2021). Identificación de una triparedoxina peroxidasa citoplasmática en Leishmania braziliensis. Revista Colombiana de Química, 50(2), 3-14. DOI: https://doi.org/10.15446/rev.colomb.quim.v50n2.91721
Yang, F., Moss, L. G., & Phillips, G. N. (1996). The molecular structure of green fluorescent protein. Nature biotechnology, 14(10), 1246. DOI: https://doi.org/10.1038/nbt1096-1246
Zacharias, D. A., & Tsien, R. Y. (2006). Molecular biology and mutation of green fluorescent protein. Methods of biochemical analysis, 47, 83-120. DOI: https://doi.org/10.1002/0471739499.ch5
Zhang, G., Gurtu, V., & Kain, S. R. (1996). An enhanced green fluorescent protein allows sensitive detection of gene transfer in mammalian cells. Biochemical and biophysical research communications, 227(3), 707-711. DOI: https://doi.org/10.1006/bbrc.1996.1573
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