Aplicación del sistema phage display para la producción de anticuerpos monoclonales: Una aproximación al desarrollo de herramientas para la detección de proteínas
ilustraciones, diagramas, gráficas, tablas
- Autores:
-
Riascos Orjuela, Laura Estefanía
- Tipo de recurso:
- Fecha de publicación:
- 2023
- Institución:
- Universidad Nacional de Colombia
- Repositorio:
- Universidad Nacional de Colombia
- Idioma:
- spa
- OAI Identifier:
- oai:repositorio.unal.edu.co:unal/85255
- Palabra clave:
- 570 - Biología::572 - Bioquímica
Anticuerpos Monoclonales
Inmunización
Antibodies, Monoclonal
Immunization
Phage display
librerías de fagos
Spike
SARS-CoV-2
anticuerpos recombinantes
Phage display
phage libraries
Spike
recombinant antibodies
SARS-CoV-2
- Rights
- openAccess
- License
- Atribución-NoComercial 4.0 Internacional
| id |
UNACIONAL2_ffe002e112ba53e381eb119ff3ff2c83 |
|---|---|
| oai_identifier_str |
oai:repositorio.unal.edu.co:unal/85255 |
| network_acronym_str |
UNACIONAL2 |
| network_name_str |
Universidad Nacional de Colombia |
| repository_id_str |
|
| dc.title.spa.fl_str_mv |
Aplicación del sistema phage display para la producción de anticuerpos monoclonales: Una aproximación al desarrollo de herramientas para la detección de proteínas |
| dc.title.translated.eng.fl_str_mv |
Application of the phage display system for the production of monoclonal antibodies: An approach to the development of tools for protein detection |
| title |
Aplicación del sistema phage display para la producción de anticuerpos monoclonales: Una aproximación al desarrollo de herramientas para la detección de proteínas |
| spellingShingle |
Aplicación del sistema phage display para la producción de anticuerpos monoclonales: Una aproximación al desarrollo de herramientas para la detección de proteínas 570 - Biología::572 - Bioquímica Anticuerpos Monoclonales Inmunización Antibodies, Monoclonal Immunization Phage display librerías de fagos Spike SARS-CoV-2 anticuerpos recombinantes Phage display phage libraries Spike recombinant antibodies SARS-CoV-2 |
| title_short |
Aplicación del sistema phage display para la producción de anticuerpos monoclonales: Una aproximación al desarrollo de herramientas para la detección de proteínas |
| title_full |
Aplicación del sistema phage display para la producción de anticuerpos monoclonales: Una aproximación al desarrollo de herramientas para la detección de proteínas |
| title_fullStr |
Aplicación del sistema phage display para la producción de anticuerpos monoclonales: Una aproximación al desarrollo de herramientas para la detección de proteínas |
| title_full_unstemmed |
Aplicación del sistema phage display para la producción de anticuerpos monoclonales: Una aproximación al desarrollo de herramientas para la detección de proteínas |
| title_sort |
Aplicación del sistema phage display para la producción de anticuerpos monoclonales: Una aproximación al desarrollo de herramientas para la detección de proteínas |
| dc.creator.fl_str_mv |
Riascos Orjuela, Laura Estefanía |
| dc.contributor.advisor.none.fl_str_mv |
Ramírez Hernández, María Helena |
| dc.contributor.author.none.fl_str_mv |
Riascos Orjuela, Laura Estefanía |
| dc.contributor.researchgroup.spa.fl_str_mv |
Laboratorio de Investigaciones Básica en Bioquímica - LIBBIQ |
| dc.subject.ddc.spa.fl_str_mv |
570 - Biología::572 - Bioquímica |
| topic |
570 - Biología::572 - Bioquímica Anticuerpos Monoclonales Inmunización Antibodies, Monoclonal Immunization Phage display librerías de fagos Spike SARS-CoV-2 anticuerpos recombinantes Phage display phage libraries Spike recombinant antibodies SARS-CoV-2 |
| dc.subject.decs.spa.fl_str_mv |
Anticuerpos Monoclonales Inmunización |
| dc.subject.decs.eng.fl_str_mv |
Antibodies, Monoclonal Immunization |
| dc.subject.proposal.spa.fl_str_mv |
Phage display librerías de fagos Spike SARS-CoV-2 anticuerpos recombinantes |
| dc.subject.proposal.eng.fl_str_mv |
Phage display phage libraries Spike recombinant antibodies SARS-CoV-2 |
| description |
ilustraciones, diagramas, gráficas, tablas |
| publishDate |
2023 |
| dc.date.issued.none.fl_str_mv |
2023-10-27 |
| dc.date.accessioned.none.fl_str_mv |
2024-01-12T19:44:48Z |
| dc.date.available.none.fl_str_mv |
2024-01-12T19:44:48Z |
| dc.type.spa.fl_str_mv |
Trabajo de grado - Maestría |
| dc.type.driver.spa.fl_str_mv |
info:eu-repo/semantics/masterThesis |
| dc.type.version.spa.fl_str_mv |
info:eu-repo/semantics/acceptedVersion |
| dc.type.content.spa.fl_str_mv |
Text |
| dc.type.redcol.spa.fl_str_mv |
http://purl.org/redcol/resource_type/TM |
| status_str |
acceptedVersion |
| dc.identifier.uri.none.fl_str_mv |
https://repositorio.unal.edu.co/handle/unal/85255 |
| dc.identifier.instname.spa.fl_str_mv |
Universidad Nacional de Colombia |
| dc.identifier.reponame.spa.fl_str_mv |
Repositorio Institucional Universidad Nacional de Colombia |
| dc.identifier.repourl.spa.fl_str_mv |
https://repositorio.unal.edu.co/ |
| url |
https://repositorio.unal.edu.co/handle/unal/85255 https://repositorio.unal.edu.co/ |
| identifier_str_mv |
Universidad Nacional de Colombia Repositorio Institucional Universidad Nacional de Colombia |
| dc.language.iso.spa.fl_str_mv |
spa |
| language |
spa |
| dc.relation.references.spa.fl_str_mv |
Abbas A, Lichtman A, Pillai S. Cellular and Molecular Immunology. Ninth edit. Journal of Chemical Information and Modeling. Philadelphia: ELSEVIER; 2017. Meyers AJ, Grohs BM, Hall JC. Antibody Production in planta [Internet]. Second Edi. Vol. 4, Comprehensive Biotechnology, Second Edition. Elsevier B.V.; 2011. 287–300 p. Available from: http://dx.doi.org/10.1016/B978-0-08-088504-9.00271-3 Singh A, Chaudhary S, Agarwal A, Verma AS. Antibodies: Monoclonal and Polyclonal [Internet]. Animal Biotechnology: Models in Discovery and Translation. Elsevier; 2013. 265–287 p. Available from: http://dx.doi.org/10.1016/B978-0-12-416002-6.00015-8 Schroeder HW, Cavacini L. Structure and function of immunoglobulins. J Allergy Clin Immunol [Internet]. 2010 Feb;125(2):S41–52. Available from: https://linkinghub.elsevier.com/retrieve/pii/S0091674909014651 Beenhouwer DO. Molecular basis of diseases of immunity [Internet]. Second Edi. Molecular Pathology: The Molecular Basis of Human Disease. Elsevier Inc.; 2018. 329–345 p. Available from: https://doi.org/10.1016/B978-0-12-802761-5.00017-1 Mashoof S, Criscitiello MF. Fish immunoglobulins. Biology (Basel). 2016;5(4):1–23. Munhoz LS, Vargas GDÁ, Fischer G, Lima M de, Esteves PA, Ḧbner S de O. Anticorpos IgY aviário: Características e aplicações em imunodiagnóstico. Cienc Rural. 2014;44(1):153–60. Kumar R, Parray HA, Shrivastava T, Sinha S, Luthra K. Phage display antibody libraries: A robust approach for generation of recombinant human monoclonal antibodies. Int J Biol Macromol [Internet]. 2019;135:907–18. Available from: https://doi.org/10.1016/j.ijbiomac.2019.06.006 Smith G. Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science (80- ). 1985;228(4705):1315–1317. Bazan J, Całkosiñski I, Gamian A. Phage displaya powerful technique for immunotherapy: 1. Introduction and potential of therapeutic applications. Hum Vaccines Immunother. 2012;8(12):1817–28. Hentrich C, Ylera F, Frisch C, Haaf A Ten, Knappik A. Monoclonal antibody generation by phage display: History, state-of-the-art, and future [Internet]. Handbook of Immunoassay Technologies: Approaches, Performances, and Applications. Elsevier Inc.; 2018. 47–80 p. Available from: http://dx.doi.org/10.1016/B978-0-12-811762-0.00003-7 Huse WD, Sastry L, Iverson SA, Kang AS, Alting-Mees M, Burton DR, et al. Generation of a large combinatorial library of the immunoglobulin repertoire in phage lambda. 1989. Biotechnology. 1989;24(1984):517–23. Alfaleh MA, Alsaab HO, Mahmoud AB, Alkayyal AA, Jones ML, Mahler SM, et al. Phage Display Derived Monoclonal Antibodies: From Bench to Bedside. Front Immunol. 2020;11(August). Reader RH, Workman RG, Maddison BC, Gough KC. Advances in the Production and Batch Reformatting of Phage Antibody Libraries. Mol Biotechnol [Internet]. 2019;61(11):801–15. Available from: https://doi.org/10.1007/s12033-019-00207-0 Chiu ML, Goulet DR, Teplyakov A, Gilliland GL. Antibody Structure and Function: The Basis for Engineering Therapeutics. Antibodies. 2019;8(4):55. Ercan I, Tufekci KU, Karaca E, Genc S, Genc K. Peptide Derivatives of Erythropoietin in the Treatment of Neuroinflammation and Neurodegeneration [Internet]. 1st ed. Vol. 112, Advances in Protein Chemistry and Structural Biology. Elsevier Inc.; 2018. 309–357 p. Available from: http://dx.doi.org/10.1016/bs.apcsb.2018.01.007 Aitken R. Antibody Phage Display / METHODS IN MOLECULAR BIOLOGY TM. 2009. 238 p. Somasundaram R, Choraria A, Antonysamy M. An approach towards development of monoclonal IgY antibodies against SARS CoV-2 spike protein (S) using phage display method: A review. Int Immunopharmacol [Internet]. 2020 Aug;85(January):106654. Available from: https://linkinghub.elsevier.com/retrieve/pii/S1567576920315010 Ge S, Xu L, Li B, Zhong F, Liu X, Zhang X. Canine Parvovirus is diagnosed and neutralized by chicken IgY-scFv generated against the virus capsid protein. Vet Res [Internet]. 2020;51(1):1–11. Available from: https://doi.org/10.1186/s13567-020-00832-7 Finlay WJJ, Shaw L, Reilly JP, Kane M. Generation of high-affinity chicken single-chain Fv antibody fragments for measurement of the Pseudonitzschia pungens toxin domoic acid. Appl Environ Microbiol. 2006;72(5):3343–9. Park KJ, Park DW, Kim CH, Han BK, Park TS, Han JY, et al. Development and characterization of a recombinant chicken single-chain Fv antibody detecting Eimeria acervulina sporozoite antigen. Biotechnol Lett. 2005;27(5):289–95. Noakes PS, Michaelis LJ. Innate and adaptive immunity [Internet]. Diet, Immunity and Inflammation. Woodhead Publishing Limited; 2013. 3–33 p. Available from: http://dx.doi.org/10.1533/9780857095749.1.3 Pereira EPV, van Tilburg MF, Florean EOPT, Guedes MIF. Egg yolk antibodies (IgY) and their applications in human and veterinary health: A review. Int Immunopharmacol [Internet]. 2019 Aug;73(January):293–303. Available from: https://linkinghub.elsevier.com/retrieve/pii/S1567576919302206 Cabanillas-Bernal O, Dueñas S, Ayala-Avila M, Rucavado A, Escalante T, Licea-Navarro AF. Synthetic libraries of shark vNAR domains with different cysteine numbers within the CDR3. PLoS One. 2019;14(6):1–24. Alkan SS. Monoclonal antibodies: The story of a discovery that revolutionized scienceand medicine. Nat Rev Immunol. 2004;4(2):153–6. Zhao A, Tohidkia MR, Siegel DL, Coukos G, Omidi Y. Phage antibody display libraries: A powerful antibody discovery platform for immunotherapy. Crit Rev Biotechnol. 2016;36(2):276–89. Brüggemann M, Osborn MJ, Ma B, Hayre J, Avis S, Lundstrom B, et al. Human Antibody Production in Transgenic Animals. Arch Immunol Ther Exp (Warsz). 2015;63(2):101–8. Lee W, Syed Atif A, Tan SC, Leow CH. Insights into the chicken IgY with emphasis on the generation and applications of chicken recombinant monoclonal antibodies. J Immunol Methods [Internet]. 2017;447:71–85. Available from: http://dx.doi.org/10.1016/j.jim.2017.05.001 Comor L, Dolinska S, Bhide K, Pulzova L, Jiménez-Munguía I, Bencurova E, et al. Joining the in vitro immunization of alpaca lymphocytes and phage display: Rapid and cost effective pipeline for sdAb synthesis. Microb Cell Fact. 2017;16(1):1–13. Andris-Widhopf J, Rader C, Steinberger P, Fuller R, Barbas CF. Methods for the generation of chicken monoclonal antibody fragments by phage display. J Immunol Methods. 2000;242(1–2):159–81. Contreras Rodríguez LE, Jutinico Shubach LLM, García Castañeda JE, Ramírez Hernández MH. Functional identification and subcellular localization of NAD kinase in the protozoan parasite Giardia intestinalis. Rev Colomb Química [Internet]. 2019 Jan 1;48(1):16–25. Available from: https://revistas.unal.edu.co/index.php/rcolquim/article/view/75273 Ostos Peña DM. Aproximación a la regulación de algunas enzimas involucradas en el metábolismo del NAD+ en Giardia duodenalis. 2019. p. 1–128. Hu B, Guo H, Zhou P, Shi ZL. Characteristics of SARS-CoV-2 and COVID-19. Nat Rev Microbiol [Internet]. 2020;(December). Available from: http://dx.doi.org/10.1038/s41579-020-00459-7 Wu F, Zhao S, Yu B, Chen YM, Wang W, Song ZG, et al. A new coronavirus associated with human respiratory disease in China. Nature. 2020;579(7798):265–9. Zhou P, Yang X Lou, Wang XG, Hu B, Zhang L, Zhang W, et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature [Internet]. 2020;579(7798):270–3. Available from: http://dx.doi.org/10.1038/s41586-020-2012-7 Zhu N, Zhang D, Wang W, Li X, Yang B, Song J, et al. A novel coronavirus from patients with pneumonia in China, 2019. N Engl J Med. 2020;382(8):727–33. Lv Z, Deng Y-Q, Ye Q, Cao L, Sun C-Y, Fan C, et al. Structural basis for neutralization of SARS-CoV-2 and SARS-CoV by a potent therapeutic antibody. Science (80- ). 2020;1509(September):eabc5881. Álvarez-Díaz DA, Franco-Muñoz C, Laiton-Donato K, Usme-Ciro JA, Franco-Sierra ND, Flórez-Sánchez AC, et al. Molecular analysis of several in-house rRT-PCR protocols for SARS-CoV-2 detection in the context of genetic variability of the virus in Colombia. Infect Genet Evol [Internet]. 2020;84(May):104390. Available from: https://doi.org/10.1016/j.meegid.2020.104390 Chan JF, Kok K, Zhu Z, Chu H, To KK-W, Yuan S, et al. Genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting Wuhan. Emerg Microbes Infect [Internet]. 2020 Jan 1;9(1):221–36. Available from: https://www.tandfonline.com/doi/full/10.1080/22221751.2020.1719902 Takahashi H, Iwasaki Y, Watanabe T, Ichinose N, Okada Y, Oiwa A, et al. Case studies of SARS-CoV-2 treated with favipiravir among patients in critical or severe condition. Int J Infect Dis [Internet]. 2020; Available from: https://doi.org/10.1016/j.ijid.2020.08.047 World Health Organization. Weekly Epidemiological Update on COVID-19. 2020;(October). Available from: https://www.who.int/docs/default-source/coronaviruse/situation-reports/20201012-weekly-epi-update-9.pdf Zhou L, Chandrasekaran AR, Punnoose JA, Bonenfant G, Charles S, Levchenko O, et al. Programmable low-cost DNA-based platform for viral RNA detection. 2020;6246:1–15. Grzelak L, Temmam S, Planchais C, Demeret C, Tondeur L, Huon C, et al. A comparison of four serological assays for detecting anti-SARS-CoV-2 antibodies in human serum samples from different populations. Sci Transl Med. 2020;12(559). Dao Thi VL, Herbst K, Boerner K, Meurer M, Kremer LP, Kirrmaier D, et al. A colorimetric RT-LAMP assay and LAMP-sequencing for detecting SARS-CoV-2 RNA in clinical samples. Sci Transl Med. 2020;12(556). Ponomarenko J, Bui HH, Li W, Fusseder N, Bourne PE, Sette A, et al. ElliPro: A new structure-based tool for the prediction of antibody epitopes. BMC Bioinformatics. 2008;9:1–8. Life Technologies. Champion pET SUMO Protein Expression System. 2010;5(January):1833–9. Froger A, Hall JE. Transformation of Plasmid DNA into E . coli Using the Heat Shock Method. 2007;2007. Lessard JC. Molecular cloning, a laboratory manual. Vol. 529, Methods in Enzymology. 2013. 85–98 p. Palmer I, Wingfield PT. Preparation and extraction of insoluble (Inclusion-body) proteins from Escherichia coli. Curr Protoc Protein Sci. 2012;1(SUPPL.70):1–25. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem [Internet]. 1976 May;72(1–2):248–54. Available from: https://linkinghub.elsevier.com/retrieve/pii/0003269776905273 Mahmood T, Yang PC. Western blot: Technique, theory, and trouble shooting. N Am J Med Sci. 2012;4(9):429–34. Howard GC, Kaser MR. Making and Using Antibodies: A Practical Handbook, Second Edition [Internet]. 2013. 458 p. Available from: https://books.google.com/books?id=AfnRBQAAQBAJ&pgis=1 Pauly D, Chacana PA, Calzado EG, Brembs B, Schade R. Igy technology: Extraction of chicken antibodies from egg yolk by polyethylene glycol (PEG) precipitation. J Vis Exp. 2011;i(51):2–7. Thermo Fisher Scientific. TRIzol Reagent User Guide - Pub. no. MAN0001271 - Rev. A.0. User Guid. 2016;15596018(15596026):1–6. Applied Biosystems Ambion. Gel Loading Buffer II (Denaturing PAGE) User Manual. 2008;1–2. Corporation P. RQ1 RNase-Free DNase Product Infomation. 2018;1–2. Thermo Scientific. Thermo Scientific RevertAid Reverse Transcriptase. 2016;(3):3–6. Zhang J, Gao Y, Huang Y, Fan Q, Lu X. Selection of housekeeping genes for quantitative gene expression analysis in yellow-feathered broilers in yellow-feathered broilers. Ital J Anim Sci [Internet]. 2018;0(0):540–6. Available from: https://doi.org/10.1080/1828051X.2017.1365633 Rajput R, Sharma G, Rawat V, Gautam A, Kumar B, Pattnaik B, et al. Diagnostic potential of recombinant scFv antibodies generated against hemagglutinin protein of influenza A virus. Front Immunol. 2015;6(SEP):1–9. Progen. Surface Expression Phagemid Vector pSEX81. Biotechnol Adv [Internet]. 2003;13(1991). Available from: https://www.progen.com/media/downloads/datasheets/PR3005.pdf Progen. Mouse IgG Library Primer Set. 2000;1–3. Thermo Fisher Scientific. MluI User Guide. :1–3. Available from: https://www.thermofisher.com/order/catalog/product/ER0561?SID=srch-hj-ER0561 Thermo Fisher Scientific. NcoI User Guide. :1–3. Available from: https://www.thermofisher.com/order/catalog/product/ER0572?SID=srch-hj-ER0572 FAVORGEN. FavorPrep GEL / PCR Purification Kit Kit Contents : Brief procedure : Specification : Important Notes : PCR Clean-Up Protocol : For purification of PCR products or reaction mixtures. :1–2. Thermo Fisher Scientific. Product information: T4 DNA Ligase. :1–2. Available from: https://www.thermofisher.com/order/catalog/product/EL0014?SID=srch-srp-EL0014 Moradi-Kalbolandi S, Davani D, Golkar M, Habibi-Anbouhi M, Abolhassani M, Shokrgozar MA. Soluble Expression and Characterization of a New scFv Directed to Human CD123. Appl Biochem Biotechnol. 2016;178(7):1390–406. Hust M. Phage Display [Internet]. Hust M, Lim TS, editors. Methods in Molecular Biology. New York, NY: Springer New York; 2018. 331–347 p. (Methods in Molecular Biology; vol. 1701). Available from: http://link.springer.com/10.1007/978-1-4939-7447-4 Thermo Fisher Scientific. Hind III User Guide. :1–3. Available from: https://www.thermofisher.com/order/catalog/product/ER0502?SID=srch-hj-ER0502 Progen. Product datasheet Hyperphage e M13 KO7pIII. 2022;1–3. Available from: https://us.progen.com/Hyperphage-M13-KO7DpIII/PRHYPE-1 Kay BK, Winter J, McCafferty J. Phage Display of Peptides and Proteins, a Laboratory Manual. 1996. 61–62 p. Kim H, Ho M. Current Protocols in Protein Science: Isolation of Antibodies to Heparan Sulfate on Glypicans by Phage Display. Curr Protoc Protrin Sci. 2018;94(1):1–32. Zhang M-Y, Dimitrov DS. Sequential Antigen Panning for Selection of Broadly Cross- Reactive HIV-1-Neutralizing Human Monoclonal Antibodies. Methods Mol Biol. 2009;562:143–54. Li Y, Ma M, Lei Q, Li Y, Ma M, Lei Q, et al. Linear epitope landscape of the SARS-CoV-2 Spike protein constructed from 1 , 051 COVID-19 patients. CellReports [Internet]. 2021;34(13):108915. Available from: https://doi.org/10.1016/j.celrep.2021.108915 Wang H, Wu X, Zhang X, Hou X, Liang T, Wang D, et al. SARS-CoV ‑ 2 Proteome Microarray for Mapping COVID-19 Antibody Interactions at Amino Acid Resolution. 2020; Shang J, Wan Y, Luo C, Ye G, Geng Q, Auerbach A, et al. Cell entry mechanisms of SARS-CoV-2. Proc Natl Acad Sci U S A. 2020;117(21). Tye EXC, Jinks E, Haigh TA, Kaul B, Patel P, Parry HM, et al. Mutations in SARS-CoV-2 spike protein impair epitope-specific CD4 + T cell recognition. 2022;23(December). Chen L, Pang P, Qi H, Yan K, Ren Y, Ma M, et al. Evaluation of Spike Protein Epitopes by Assessing the Dynamics of Humoral Immune Responses in Moderate COVID-19. 2022;13(March):1–14. Poh CM, Carissimo G, Wang B, Amrun SN, Lee CY, Chee RS, et al. Two linear epitopes on the SARS-CoV-2 spike protein that elicit neutralising antibodies in COVID-19 patients. Nat Commun [Internet]. 2020; Available from: http://dx.doi.org/10.1038/s41467-020-16638-2 Krempl C, Schultze B, Laude H. Point Mutations in the S Protein Connect the Sialic Acid Binding Activity with the Enteropathogenicity of Transmissible Gastroenteritis Coronavirus. 1997;71(4):3285–7. Lu G, Wang Q, Gao GF. Bat-to-human : spike features determining ‘ host jump ’ of MERS-CoV , and beyond. 2020;(January). Chi X, Yan R, Zhang J, Zhang G, Zhang Y, Hao M, et al. A neutralizing human antibody binds to the N-terminal domain of the Spike protein of SARS-CoV-2. 2020;655(August):650–5. Xiaojie S, Yu L, Guang Y, Min Q. Neutralizing antibodies targeting SARS-CoV-2 spike protein. 2020;(January). Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7737530/pdf/main.pdf Robichon C, Luo J, Causey TB, Benner JS, Samuelson JC. Engineering Escherichia coli BL21 ( DE3 ) Derivative Strains To Minimize E . coli Protein Contamination after Purification by Immobilized Metal Affinity Chromatography ᰔ †‡. 2011;77(13):4634–46. Mcguire BE, Mela JE, Thompson VC, Cucksey LR, Stevens CE, Mcwhinnie RL, et al. Escherichia coli recombinant expression of SARS ‑ CoV ‑ 2 protein fragments. Microb Cell Fact [Internet]. 2022;1–13. Available from: https://doi.org/10.1186/s12934-022-01753-0 Glycoprotein S-S, Zhang S, Go EP, Ding H, Anang S, Kappes JC, et al. Analysis of Glycosylation and Disul fi de Bonding of Wild-Type. 2022;96(3):1–27. Rosano GL, Ceccarelli EA. Recombinant protein expression in Escherichia coli : advances and challenges. 2014;5(April):1–17. Laura M, Puglisi A, Dal F, Hochkoeppler A. Production in Escherichia coli of recombinant COVID-19 spike protein fragments fused to CRM197. 2020;(January). Rahbar Z, Nazarian S, Dorostkar R, Sotoodehnejadnematalahi F. Recombinant expression of SARS-CoV-2 receptor binding domain ( RBD ) in Escherichia coli and its immunogenicity in mice. 2022; Liu L, Chen T, Zhou L, Sun J, Li Y, Nie M, et al. A Bacterially Expressed SARS-CoV-2 Receptor Binding Domain Fused With Cross-Reacting Material 197 A-Domain Elicits High Level of Neutralizing Antibodies in Mice. 2022;13(April). Gao X, Peng S, Mei S, Liang K, Saleem M, Khan I, et al. Expression and functional identification of recombinant SARS-CoV-2 receptor binding domain ( RBD ) from E . coli system. Prep Biochem Biotechnol [Internet]. 2021;0(0):1–7. Available from: https://doi.org/10.1080/10826068.2021.1941106 Ge S, Wu R, Zhou T, Liu X, Zhu J, Zhang X. Specific anti ‑ SARS ‑ CoV ‑ 2 S1 IgY ‑ scFv is a promising tool for recognition of the virus. AMB Express [Internet]. 2022; Available from: https://doi.org/10.1186/s13568-022-01355-4 Balasubramaniyam A, Ryan E, Brown D, Hamza T, Harrison W, Gan M, et al. Unglycosylated Soluble SARS-CoV-2 Receptor Binding Domain ( RBD ) Produced in E . coli Combined with the Army Liposomal Formulation Containing QS21 ( ALFQ ) Elicits Neutralizing Antibodies against Mismatched Variants. 2023;21. Tungekar AA, Ruddock LW. Production of neutralizing antibody fragment variants in the cytoplasm of E . coli for rapid screening : SARS ‑ CoV ‑ 2 a case study. Sci Rep [Internet]. 2023;1–12. Available from: https://doi.org/10.1038/s41598-023-31369-2 Singh A, Upadhyay V, Upadhyay AK, Singh SM, Panda AK. Protein recovery from inclusion bodies of Escherichia coli using mild solubilization process. 2015;1–10. Kong B, Guo GL. Soluble Expression of Disulfide Bond Containing Proteins FGF15 and FGF19 in the Cytoplasm of Escherichia coli. 2014;9(1):1–8. Bhatwa A, Wang W, Hassan YI, Abraham N, Li X. Challenges Associated With the Formation of Recombinant Protein Inclusion Bodies in Escherichia coli and Strategies to Address Them for Industrial Applications. 2021;9(February):1–18. Sivashanmugam A, Murray V, Cui C, Zhang Y, Wang J, Li Q. Practical protocols for production of very high yields of recombinant proteins using Escherichia coli. 2009;18(1):936–48. Hata S, Kitamura F, Sorimachi H. Efficient expression and purification of recombinant human l -calpain using an Escherichia coli expression system. 2013;753–63. Trabbic-carlson K, Liu LI, Kim B, Chilkoti A. Expression and purification of recombinant proteins from Escherichia coli : Comparison of an elastin-like polypeptide fusion with an oligohistidine fusion. 2004;3274–84. Rienzo L Di, Ruocco G, Desantis F, Grassmann G, Milanetti E. Dynamical changes of SARS-CoV-2 spike variants in the highly immunogenic regions impact the viral antibodies escaping. 2023;(March):1116–29. Chacón ME. Evaluación de un candidato a transportador de NAD+ en el parásito protozoario Trypanosoma cruzi. 2021; Eliana S, Silva V. Exploration of a Nad + Transporter and / or Its Precursors in. 2021; Lin TW, Huang PH, Liao BH, Chao TL, Tsai YM, Chang SC, et al. Tag-Free SARS-CoV-2 Receptor Binding Domain (RBD), but Not C-Terminal Tagged SARS-CoV-2 RBD, Induces a Rapid and Potent Neutralizing Antibody Response. Vaccines. 2022;10(11):1–11. Gefen T, Vaya J, Khatib S, Rapoport I, Lupo M, Barnea E, et al. The effect of haptens on protein-carrier immunogenicity. Immunology. 2015;144(1):116–26. Ertekin Ö, Akçael E, Kocaağa H, Öztürk S. Biological activity of the carrier as a factor in immunogen design for haptens. Molecules. 2018;23(11). Gonzalez-montalban N, Natalello A, Garcı E, Villaverde A, Doglia SM. In Situ Protein Folding and Activation in Bacterial Inclusion Bodies. 2008;100(4):797–802. Ramón A, Señorale-pose M, Marín M. Inclusion bodies : not that bad . . . 2014;5(February):2010–5. Li S, Zhao Q, Wu T, Chen S, Zhang J, Xia N. The development of a recombinant hepatitis E vaccine HEV 239. 2015;11(4):908–14. Sarantos K, Cleo K. European Journal of Pharmaceutical Sciences Analysis of the landscape of biologically-derived pharmaceuticals in Europe : Dominant production systems , molecule types on the rise and approval trends. Eur J Pharm Sci [Internet]. 2013;48(3):428–41. Available from: http://dx.doi.org/10.1016/j.ejps.2012.11.016 Lua LHL, Connors NK, Sainsbury F, Chuan YP, Wibowo N, Middelberg APJ. Bioengineering Virus-Like Particles as Vaccines. 2014;111(3):425–40. Huang X, Wang X, Zhang J, Xia N, Zhao Q. Escherichia coli- derived virus-like particles in vaccine development. npj Vaccines [Internet]. 2017;(October 2016):1–8. Available from: http://dx.doi.org/10.1038/s41541-017-0006-8 Wei M, Zhang X, Yu H, Tang Z, Wang K, Li Z, et al. Bacteria expressed hepatitis E virus capsid proteins maintain virion-like epitopes. Vaccine [Internet]. 2014;1–7. Available from: http://dx.doi.org/10.1016/j.vaccine.2014.02.025 Qiagen. RNA Isolation with TRIzol ( Invitrogen ) and Qiagen RNAeasy. 2013;1–7. Gandhi V, Brien MHO, Yadav S. High-Quality and High-Yield RNA Extraction Method From Whole Human Saliva. 2020; Yan J, Li G, Hu Y, Ou W, Wan Y. Construction of a synthetic phage-displayed Nanobody library with CDR3 regions randomized by trinucleotide cassettes for diagnostic applications. 2014;1–12. Bashir S, Paeshuyse J. Construction of Antibody Phage Libraries and Their Application in Veterinary Immunovirology. 2020;(Figure 1). Lim CC, Woo PCY, Lim TS. Development of a Phage Display Panning Strategy Utilizing Crude Antigens: Isolation of MERS-CoV Nucleoprotein human antibodies. Sci Rep [Internet]. 2019;9(1):1–15. Available from: http://dx.doi.org/10.1038/s41598-019-42628-6 Li J, Xu Y, Wang X, Li Y, Wang L, Li X. Construction and characterization of a highly reactive chicken-derived single-chain variable fragment (scFv) antibody against Staphylococcus aureus developed with the T7 phage display system. Int Immunopharmacol [Internet]. 2016;35:149–54. Available from: http://dx.doi.org/10.1016/j.intimp.2016.02.024 Hu Z, Liu J, Li H, Xing S, Xue S, Zhang J. Generation of a Highly Reactive Chicken-Derived Single-Chain Variable Fragment against Fusarium verticillioides by Phage Display. 2012;7038–56. Yang T, Yang L, Chai W, Li R, Xie J, Niu B. A strategy for high-level expression of a single-chain variable fragment against TNFα by subcloning antibody variable regions from the phage display vector pCANTAB 5E into pBV220. Protein Expr Purif [Internet]. 2011;76(1):109–14. Available from: http://dx.doi.org/10.1016/j.pep.2010.10.006 Zhan Y, Song Y, Ren H, Zeng Q, Yuan Y, Xia L, et al. Preparation of a Single-Chain Antibody against Nucleocapsid Protein of Porcine Deltacoronavirus by Phage. 2022; Lee YC, Leu SJC, Hung HC, Wu HH, Huang IJ, Hsieh WS, et al. A dominant antigenic epitope on SARS-CoV spike protein identified by an avian single-chain variable fragment (scFv)-expressing phage. Vet Immunol Immunopathol. 2007;117(1–2):75–85. Anandakumar S, Boosi KN, Bugatha H, Padmanabhan B, Sadhale PP. Phage displayed short peptides against cells of Candida albicans demonstrate presence of species, morphology and region specific carbohydrate epitopes. PLoS One. 2011;6(2). O’Brien P, Aitken R. Methods in Molecular Biology: antibody Phage Display. Vol. 178. 2002. Pitaksajjakul P, Lekcharoensuk P, Upragarin N, Barbas CF, Ibrahim MS, Ikuta K, et al. Fab MAbs specific to HA of influenza virus with H5N1 neutralizing activity selected from immunized chicken phage library. Biochem Biophys Res Commun [Internet]. 2010;395(4):496–501. Available from: http://dx.doi.org/10.1016/j.bbrc.2010.04.040 |
| dc.rights.spa.fl_str_mv |
Derechos reservados al autor, 2023 |
| dc.rights.coar.fl_str_mv |
http://purl.org/coar/access_right/c_abf2 |
| dc.rights.license.spa.fl_str_mv |
Atribución-NoComercial 4.0 Internacional |
| dc.rights.uri.spa.fl_str_mv |
http://creativecommons.org/licenses/by-nc/4.0/ |
| dc.rights.accessrights.spa.fl_str_mv |
info:eu-repo/semantics/openAccess |
| rights_invalid_str_mv |
Atribución-NoComercial 4.0 Internacional Derechos reservados al autor, 2023 http://creativecommons.org/licenses/by-nc/4.0/ http://purl.org/coar/access_right/c_abf2 |
| eu_rights_str_mv |
openAccess |
| dc.format.extent.spa.fl_str_mv |
xx, 86 páginas |
| dc.format.mimetype.spa.fl_str_mv |
application/pdf |
| dc.publisher.spa.fl_str_mv |
Universidad Nacional de Colombia |
| dc.publisher.program.spa.fl_str_mv |
Bogotá - Ciencias - Maestría en Ciencias - Bioquímica |
| dc.publisher.faculty.spa.fl_str_mv |
Facultad de Ciencias |
| dc.publisher.place.spa.fl_str_mv |
Bogotá, Colombia |
| dc.publisher.branch.spa.fl_str_mv |
Universidad Nacional de Colombia - Sede Bogotá |
| institution |
Universidad Nacional de Colombia |
| bitstream.url.fl_str_mv |
https://repositorio.unal.edu.co/bitstream/unal/85255/1/license.txt https://repositorio.unal.edu.co/bitstream/unal/85255/2/1026291353.2023.pdf |
| bitstream.checksum.fl_str_mv |
eb34b1cf90b7e1103fc9dfd26be24b4a fb21d71bc06f776696b4ba07746d27a3 |
| bitstream.checksumAlgorithm.fl_str_mv |
MD5 MD5 |
| repository.name.fl_str_mv |
Repositorio Institucional Universidad Nacional de Colombia |
| repository.mail.fl_str_mv |
repositorio_nal@unal.edu.co |
| _version_ |
1806886321836261376 |
| spelling |
Atribución-NoComercial 4.0 InternacionalDerechos reservados al autor, 2023http://creativecommons.org/licenses/by-nc/4.0/info:eu-repo/semantics/openAccesshttp://purl.org/coar/access_right/c_abf2Ramírez Hernández, María Helena3cd22161e24a39009214e3120ed6f39bRiascos Orjuela, Laura Estefaníaa3ea0703489648ac0a8a7739285f45d6Laboratorio de Investigaciones Básica en Bioquímica - LIBBIQ2024-01-12T19:44:48Z2024-01-12T19:44:48Z2023-10-27https://repositorio.unal.edu.co/handle/unal/85255Universidad Nacional de ColombiaRepositorio Institucional Universidad Nacional de Colombiahttps://repositorio.unal.edu.co/ilustraciones, diagramas, gráficas, tablasLa tecnología phage display se ha constituido como una alternativa para la producción de anticuerpos recombinantes monoclonales (rmAbs) de alta calidad. En este trabajo, empleando el modelo aviar, se realizó una primera aproximación a la producción local de herramientas de importancia para la investigación científica en Colombia como los rmAbs desde IgYs para la detección de proteínas clínicamente importantes. En este caso, se utilizaron como antígenos los dominios N-terminal (NTD) y el dominio de unión al receptor (RBD) de la proteína Spike (S) de SARS-CoV-2. Estos dominios se expresaron en el sistema heterólogo de E. coli y se purificaron a partir de cuerpos de inclusión. Las proteínas recombinantes obtenidas (6xHis-SUMO-NTD y 6xHis-SUMO-RBD) fueron inoculadas en gallinas Hy-Line Brown siguiendo un esquema de inmunización previamente estandarizado. Durante el proceso de inmunización, se recolectaron huevos y sangrías con el objetivo de evaluar los anticuerpos policlonales (pAbs) allí presentes. Esto, permitió establecer que los IgYs de los últimos sueros de cada animal permitieron la detección de hasta 15,6ng de 6xHis-SUMO-NTD y 7,8ng de 6xHis-SUMO-RBD respectivamente. Los ensayos de especificidad evidenciaron el reconocimiento cruzado de otras proteínas recombinantes que cuentan con las etiquetas 6xHis o 6xHis-SUMO, indicando que en el proceso de inmunización se generaron anticuerpos contra dichas etiquetas. Con base en esto, se estableció que los dominios NTD y RBD expresados pueden funcionar como proteínas transportadoras o carrier de las etiquetas que se desempeñarían como haptenos. Por otro lado, una vez se finalizó el esquema de inmunización, se sacrificaron los animales para obtener los bazos a fin de extraer ARN. Luego, se sintetizó ADN complementario (ADNc) desde el cuál se amplificaron las regiones encargadas de codificar las cadenas variables ligeras (VL) y pesadas (VH) de las IgYs. Estas regiones fueron clonadas en el fásmido pSEX81 que cuenta con la secuencia codificante de una proteína de cobertura (pIII) del bacteriófago M13 a la cual se acoplan VL y VH. De esta forma, se construyeron librerías de un tamaño de 6,25x106 cfu para NTD y de 3,75x106 cfu para RBD. Células E. coli TG1 fueron transformadas por electroporación con los constructos obtenidos (pSEX81-ScFv) para el antígeno 6xHis-SUMO-RBD e infectadas con el hiperfago M13K07ΔPIII. Finalmente, después de llevar a cabo las rondas de biopanning, se obtuvo la librería a partir de la cual se han aislado clones específicos que reconocen a 6xHis-SUMO-RBD que podrán ser caracterizados y empleados en la producción de los rmAbs. (Texto tomado de la fuente)Phage display technology has become an alternative to produce high-quality recombinant monoclonal antibodies (rmAbs). In this work, using the avian model, we made a first approach to the local production of important tools for scientific research in Colombia such as rmAbs from IgYs for the detection of clinically important proteins. In this case, the N-terminal domains (NTD) and receptor-binding domain (RBD) of the SARS-CoV-2 Spike (S) protein were used as antigens. These domains were expressed in the E. coli heterologous system and purified from inclusion bodies. The recombinant proteins obtained (6xHis-SUMO-NTD and 6xHis-SUMO-RBD) were inoculated into Hy-Line Brown hens following a previously standardized immunization scheme. During the immunization process, eggs and sera were collected in order to evaluate the polyclonal antibodies (pAbs) present there. This allowed us to establish that the IgYs from the last collected sera of each animal allowed the detection of up to 15.6ng of 6xHis-SUMO-NTD and 7.8ng of 6xHis-SUMO-RBD respectively. The specificity assays evidenced the cross recognition of other recombinant proteins that have the 6xHis or 6xHis-SUMO tags, indicating that antibodies against these tags were generated in the immunization process. Based on this, it was established that the expressed NTD and RBD domains can function as transporter proteins or carriers of the tags that would act as haptens. On the other hand, once the immunization scheme was completed, the animals were sacrificed to obtain the spleens in order to extract RNA. Then, complementary DNA (cDNA) was synthesized from which the coding regions for the variable light (VL) and heavy (VH) chains of the IgYs were amplified. These regions were cloned in the phasmid pSEX81 that has the coding sequence for a coat protein (pIII) of the M13 bacteriophage to which VL and VH are coupled. In this way, libraries with a size of 6.25x106 cfu for NTD and 3.75x106 cfu for RBD were built. E. coli TG1 cells were transformed by electroporation with the constructs obtained (pSEX81-ScFv) for the 6xHis-SUMO-RBD antigen and infected with the M13K07ΔPIII hyperphage. Finally, after carrying out the rounds of biopanning, a library was obtained from which specific clones that recognize 6xHis-SUMO-RBD have been isolated, which can be characterized and used in the production of rmAbs.MaestríaMagíster en Ciencias - BioquímicaDesarrollo de herramientas biotecnológicasxx, 86 páginasapplication/pdfspaUniversidad Nacional de ColombiaBogotá - Ciencias - Maestría en Ciencias - BioquímicaFacultad de CienciasBogotá, ColombiaUniversidad Nacional de Colombia - Sede Bogotá570 - Biología::572 - BioquímicaAnticuerpos MonoclonalesInmunizaciónAntibodies, MonoclonalImmunizationPhage displaylibrerías de fagosSpikeSARS-CoV-2anticuerpos recombinantesPhage displayphage librariesSpikerecombinant antibodiesSARS-CoV-2Aplicación del sistema phage display para la producción de anticuerpos monoclonales: Una aproximación al desarrollo de herramientas para la detección de proteínasApplication of the phage display system for the production of monoclonal antibodies: An approach to the development of tools for protein detectionTrabajo de grado - Maestríainfo:eu-repo/semantics/masterThesisinfo:eu-repo/semantics/acceptedVersionTexthttp://purl.org/redcol/resource_type/TMAbbas A, Lichtman A, Pillai S. Cellular and Molecular Immunology. Ninth edit. Journal of Chemical Information and Modeling. Philadelphia: ELSEVIER; 2017.Meyers AJ, Grohs BM, Hall JC. Antibody Production in planta [Internet]. Second Edi. Vol. 4, Comprehensive Biotechnology, Second Edition. Elsevier B.V.; 2011. 287–300 p. Available from: http://dx.doi.org/10.1016/B978-0-08-088504-9.00271-3Singh A, Chaudhary S, Agarwal A, Verma AS. Antibodies: Monoclonal and Polyclonal [Internet]. Animal Biotechnology: Models in Discovery and Translation. Elsevier; 2013. 265–287 p. Available from: http://dx.doi.org/10.1016/B978-0-12-416002-6.00015-8Schroeder HW, Cavacini L. Structure and function of immunoglobulins. J Allergy Clin Immunol [Internet]. 2010 Feb;125(2):S41–52. Available from: https://linkinghub.elsevier.com/retrieve/pii/S0091674909014651Beenhouwer DO. Molecular basis of diseases of immunity [Internet]. Second Edi. Molecular Pathology: The Molecular Basis of Human Disease. Elsevier Inc.; 2018. 329–345 p. Available from: https://doi.org/10.1016/B978-0-12-802761-5.00017-1Mashoof S, Criscitiello MF. Fish immunoglobulins. Biology (Basel). 2016;5(4):1–23.Munhoz LS, Vargas GDÁ, Fischer G, Lima M de, Esteves PA, Ḧbner S de O. Anticorpos IgY aviário: Características e aplicações em imunodiagnóstico. Cienc Rural. 2014;44(1):153–60.Kumar R, Parray HA, Shrivastava T, Sinha S, Luthra K. Phage display antibody libraries: A robust approach for generation of recombinant human monoclonal antibodies. Int J Biol Macromol [Internet]. 2019;135:907–18. Available from: https://doi.org/10.1016/j.ijbiomac.2019.06.006Smith G. Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science (80- ). 1985;228(4705):1315–1317.Bazan J, Całkosiñski I, Gamian A. Phage displaya powerful technique for immunotherapy: 1. Introduction and potential of therapeutic applications. Hum Vaccines Immunother. 2012;8(12):1817–28.Hentrich C, Ylera F, Frisch C, Haaf A Ten, Knappik A. Monoclonal antibody generation by phage display: History, state-of-the-art, and future [Internet]. Handbook of Immunoassay Technologies: Approaches, Performances, and Applications. Elsevier Inc.; 2018. 47–80 p. Available from: http://dx.doi.org/10.1016/B978-0-12-811762-0.00003-7Huse WD, Sastry L, Iverson SA, Kang AS, Alting-Mees M, Burton DR, et al. Generation of a large combinatorial library of the immunoglobulin repertoire in phage lambda. 1989. Biotechnology. 1989;24(1984):517–23.Alfaleh MA, Alsaab HO, Mahmoud AB, Alkayyal AA, Jones ML, Mahler SM, et al. Phage Display Derived Monoclonal Antibodies: From Bench to Bedside. Front Immunol. 2020;11(August).Reader RH, Workman RG, Maddison BC, Gough KC. Advances in the Production and Batch Reformatting of Phage Antibody Libraries. Mol Biotechnol [Internet]. 2019;61(11):801–15. Available from: https://doi.org/10.1007/s12033-019-00207-0Chiu ML, Goulet DR, Teplyakov A, Gilliland GL. Antibody Structure and Function: The Basis for Engineering Therapeutics. Antibodies. 2019;8(4):55.Ercan I, Tufekci KU, Karaca E, Genc S, Genc K. Peptide Derivatives of Erythropoietin in the Treatment of Neuroinflammation and Neurodegeneration [Internet]. 1st ed. Vol. 112, Advances in Protein Chemistry and Structural Biology. Elsevier Inc.; 2018. 309–357 p. Available from: http://dx.doi.org/10.1016/bs.apcsb.2018.01.007Aitken R. Antibody Phage Display / METHODS IN MOLECULAR BIOLOGY TM. 2009. 238 p.Somasundaram R, Choraria A, Antonysamy M. An approach towards development of monoclonal IgY antibodies against SARS CoV-2 spike protein (S) using phage display method: A review. Int Immunopharmacol [Internet]. 2020 Aug;85(January):106654. Available from: https://linkinghub.elsevier.com/retrieve/pii/S1567576920315010Ge S, Xu L, Li B, Zhong F, Liu X, Zhang X. Canine Parvovirus is diagnosed and neutralized by chicken IgY-scFv generated against the virus capsid protein. Vet Res [Internet]. 2020;51(1):1–11. Available from: https://doi.org/10.1186/s13567-020-00832-7Finlay WJJ, Shaw L, Reilly JP, Kane M. Generation of high-affinity chicken single-chain Fv antibody fragments for measurement of the Pseudonitzschia pungens toxin domoic acid. Appl Environ Microbiol. 2006;72(5):3343–9.Park KJ, Park DW, Kim CH, Han BK, Park TS, Han JY, et al. Development and characterization of a recombinant chicken single-chain Fv antibody detecting Eimeria acervulina sporozoite antigen. Biotechnol Lett. 2005;27(5):289–95.Noakes PS, Michaelis LJ. Innate and adaptive immunity [Internet]. Diet, Immunity and Inflammation. Woodhead Publishing Limited; 2013. 3–33 p. Available from: http://dx.doi.org/10.1533/9780857095749.1.3Pereira EPV, van Tilburg MF, Florean EOPT, Guedes MIF. Egg yolk antibodies (IgY) and their applications in human and veterinary health: A review. Int Immunopharmacol [Internet]. 2019 Aug;73(January):293–303. Available from: https://linkinghub.elsevier.com/retrieve/pii/S1567576919302206Cabanillas-Bernal O, Dueñas S, Ayala-Avila M, Rucavado A, Escalante T, Licea-Navarro AF. Synthetic libraries of shark vNAR domains with different cysteine numbers within the CDR3. PLoS One. 2019;14(6):1–24.Alkan SS. Monoclonal antibodies: The story of a discovery that revolutionized scienceand medicine. Nat Rev Immunol. 2004;4(2):153–6.Zhao A, Tohidkia MR, Siegel DL, Coukos G, Omidi Y. Phage antibody display libraries: A powerful antibody discovery platform for immunotherapy. Crit Rev Biotechnol. 2016;36(2):276–89.Brüggemann M, Osborn MJ, Ma B, Hayre J, Avis S, Lundstrom B, et al. Human Antibody Production in Transgenic Animals. Arch Immunol Ther Exp (Warsz). 2015;63(2):101–8.Lee W, Syed Atif A, Tan SC, Leow CH. Insights into the chicken IgY with emphasis on the generation and applications of chicken recombinant monoclonal antibodies. J Immunol Methods [Internet]. 2017;447:71–85. Available from: http://dx.doi.org/10.1016/j.jim.2017.05.001Comor L, Dolinska S, Bhide K, Pulzova L, Jiménez-Munguía I, Bencurova E, et al. Joining the in vitro immunization of alpaca lymphocytes and phage display: Rapid and cost effective pipeline for sdAb synthesis. Microb Cell Fact. 2017;16(1):1–13.Andris-Widhopf J, Rader C, Steinberger P, Fuller R, Barbas CF. Methods for the generation of chicken monoclonal antibody fragments by phage display. J Immunol Methods. 2000;242(1–2):159–81.Contreras Rodríguez LE, Jutinico Shubach LLM, García Castañeda JE, Ramírez Hernández MH. Functional identification and subcellular localization of NAD kinase in the protozoan parasite Giardia intestinalis. Rev Colomb Química [Internet]. 2019 Jan 1;48(1):16–25. Available from: https://revistas.unal.edu.co/index.php/rcolquim/article/view/75273Ostos Peña DM. Aproximación a la regulación de algunas enzimas involucradas en el metábolismo del NAD+ en Giardia duodenalis. 2019. p. 1–128.Hu B, Guo H, Zhou P, Shi ZL. Characteristics of SARS-CoV-2 and COVID-19. Nat Rev Microbiol [Internet]. 2020;(December). Available from: http://dx.doi.org/10.1038/s41579-020-00459-7Wu F, Zhao S, Yu B, Chen YM, Wang W, Song ZG, et al. A new coronavirus associated with human respiratory disease in China. Nature. 2020;579(7798):265–9.Zhou P, Yang X Lou, Wang XG, Hu B, Zhang L, Zhang W, et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature [Internet]. 2020;579(7798):270–3. Available from: http://dx.doi.org/10.1038/s41586-020-2012-7Zhu N, Zhang D, Wang W, Li X, Yang B, Song J, et al. A novel coronavirus from patients with pneumonia in China, 2019. N Engl J Med. 2020;382(8):727–33.Lv Z, Deng Y-Q, Ye Q, Cao L, Sun C-Y, Fan C, et al. Structural basis for neutralization of SARS-CoV-2 and SARS-CoV by a potent therapeutic antibody. Science (80- ). 2020;1509(September):eabc5881.Álvarez-Díaz DA, Franco-Muñoz C, Laiton-Donato K, Usme-Ciro JA, Franco-Sierra ND, Flórez-Sánchez AC, et al. Molecular analysis of several in-house rRT-PCR protocols for SARS-CoV-2 detection in the context of genetic variability of the virus in Colombia. Infect Genet Evol [Internet]. 2020;84(May):104390. Available from: https://doi.org/10.1016/j.meegid.2020.104390Chan JF, Kok K, Zhu Z, Chu H, To KK-W, Yuan S, et al. Genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting Wuhan. Emerg Microbes Infect [Internet]. 2020 Jan 1;9(1):221–36. Available from: https://www.tandfonline.com/doi/full/10.1080/22221751.2020.1719902Takahashi H, Iwasaki Y, Watanabe T, Ichinose N, Okada Y, Oiwa A, et al. Case studies of SARS-CoV-2 treated with favipiravir among patients in critical or severe condition. Int J Infect Dis [Internet]. 2020; Available from: https://doi.org/10.1016/j.ijid.2020.08.047World Health Organization. Weekly Epidemiological Update on COVID-19. 2020;(October). Available from: https://www.who.int/docs/default-source/coronaviruse/situation-reports/20201012-weekly-epi-update-9.pdfZhou L, Chandrasekaran AR, Punnoose JA, Bonenfant G, Charles S, Levchenko O, et al. Programmable low-cost DNA-based platform for viral RNA detection. 2020;6246:1–15.Grzelak L, Temmam S, Planchais C, Demeret C, Tondeur L, Huon C, et al. A comparison of four serological assays for detecting anti-SARS-CoV-2 antibodies in human serum samples from different populations. Sci Transl Med. 2020;12(559).Dao Thi VL, Herbst K, Boerner K, Meurer M, Kremer LP, Kirrmaier D, et al. A colorimetric RT-LAMP assay and LAMP-sequencing for detecting SARS-CoV-2 RNA in clinical samples. Sci Transl Med. 2020;12(556).Ponomarenko J, Bui HH, Li W, Fusseder N, Bourne PE, Sette A, et al. ElliPro: A new structure-based tool for the prediction of antibody epitopes. BMC Bioinformatics. 2008;9:1–8.Life Technologies. Champion pET SUMO Protein Expression System. 2010;5(January):1833–9.Froger A, Hall JE. Transformation of Plasmid DNA into E . coli Using the Heat Shock Method. 2007;2007.Lessard JC. Molecular cloning, a laboratory manual. Vol. 529, Methods in Enzymology. 2013. 85–98 p.Palmer I, Wingfield PT. Preparation and extraction of insoluble (Inclusion-body) proteins from Escherichia coli. Curr Protoc Protein Sci. 2012;1(SUPPL.70):1–25.Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem [Internet]. 1976 May;72(1–2):248–54. Available from: https://linkinghub.elsevier.com/retrieve/pii/0003269776905273Mahmood T, Yang PC. Western blot: Technique, theory, and trouble shooting. N Am J Med Sci. 2012;4(9):429–34.Howard GC, Kaser MR. Making and Using Antibodies: A Practical Handbook, Second Edition [Internet]. 2013. 458 p. Available from: https://books.google.com/books?id=AfnRBQAAQBAJ&pgis=1Pauly D, Chacana PA, Calzado EG, Brembs B, Schade R. Igy technology: Extraction of chicken antibodies from egg yolk by polyethylene glycol (PEG) precipitation. J Vis Exp. 2011;i(51):2–7.Thermo Fisher Scientific. TRIzol Reagent User Guide - Pub. no. MAN0001271 - Rev. A.0. User Guid. 2016;15596018(15596026):1–6.Applied Biosystems Ambion. Gel Loading Buffer II (Denaturing PAGE) User Manual. 2008;1–2.Corporation P. RQ1 RNase-Free DNase Product Infomation. 2018;1–2.Thermo Scientific. Thermo Scientific RevertAid Reverse Transcriptase. 2016;(3):3–6.Zhang J, Gao Y, Huang Y, Fan Q, Lu X. Selection of housekeeping genes for quantitative gene expression analysis in yellow-feathered broilers in yellow-feathered broilers. Ital J Anim Sci [Internet]. 2018;0(0):540–6. Available from: https://doi.org/10.1080/1828051X.2017.1365633Rajput R, Sharma G, Rawat V, Gautam A, Kumar B, Pattnaik B, et al. Diagnostic potential of recombinant scFv antibodies generated against hemagglutinin protein of influenza A virus. Front Immunol. 2015;6(SEP):1–9.Progen. Surface Expression Phagemid Vector pSEX81. Biotechnol Adv [Internet]. 2003;13(1991). Available from: https://www.progen.com/media/downloads/datasheets/PR3005.pdfProgen. Mouse IgG Library Primer Set. 2000;1–3.Thermo Fisher Scientific. MluI User Guide. :1–3. Available from: https://www.thermofisher.com/order/catalog/product/ER0561?SID=srch-hj-ER0561Thermo Fisher Scientific. NcoI User Guide. :1–3. Available from: https://www.thermofisher.com/order/catalog/product/ER0572?SID=srch-hj-ER0572FAVORGEN. FavorPrep GEL / PCR Purification Kit Kit Contents : Brief procedure : Specification : Important Notes : PCR Clean-Up Protocol : For purification of PCR products or reaction mixtures. :1–2.Thermo Fisher Scientific. Product information: T4 DNA Ligase. :1–2. Available from: https://www.thermofisher.com/order/catalog/product/EL0014?SID=srch-srp-EL0014Moradi-Kalbolandi S, Davani D, Golkar M, Habibi-Anbouhi M, Abolhassani M, Shokrgozar MA. Soluble Expression and Characterization of a New scFv Directed to Human CD123. Appl Biochem Biotechnol. 2016;178(7):1390–406.Hust M. Phage Display [Internet]. Hust M, Lim TS, editors. Methods in Molecular Biology. New York, NY: Springer New York; 2018. 331–347 p. (Methods in Molecular Biology; vol. 1701). Available from: http://link.springer.com/10.1007/978-1-4939-7447-4Thermo Fisher Scientific. Hind III User Guide. :1–3. Available from: https://www.thermofisher.com/order/catalog/product/ER0502?SID=srch-hj-ER0502Progen. Product datasheet Hyperphage e M13 KO7pIII. 2022;1–3. Available from: https://us.progen.com/Hyperphage-M13-KO7DpIII/PRHYPE-1Kay BK, Winter J, McCafferty J. Phage Display of Peptides and Proteins, a Laboratory Manual. 1996. 61–62 p.Kim H, Ho M. Current Protocols in Protein Science: Isolation of Antibodies to Heparan Sulfate on Glypicans by Phage Display. Curr Protoc Protrin Sci. 2018;94(1):1–32.Zhang M-Y, Dimitrov DS. Sequential Antigen Panning for Selection of Broadly Cross- Reactive HIV-1-Neutralizing Human Monoclonal Antibodies. Methods Mol Biol. 2009;562:143–54.Li Y, Ma M, Lei Q, Li Y, Ma M, Lei Q, et al. Linear epitope landscape of the SARS-CoV-2 Spike protein constructed from 1 , 051 COVID-19 patients. CellReports [Internet]. 2021;34(13):108915. Available from: https://doi.org/10.1016/j.celrep.2021.108915Wang H, Wu X, Zhang X, Hou X, Liang T, Wang D, et al. SARS-CoV ‑ 2 Proteome Microarray for Mapping COVID-19 Antibody Interactions at Amino Acid Resolution. 2020;Shang J, Wan Y, Luo C, Ye G, Geng Q, Auerbach A, et al. Cell entry mechanisms of SARS-CoV-2. Proc Natl Acad Sci U S A. 2020;117(21).Tye EXC, Jinks E, Haigh TA, Kaul B, Patel P, Parry HM, et al. Mutations in SARS-CoV-2 spike protein impair epitope-specific CD4 + T cell recognition. 2022;23(December).Chen L, Pang P, Qi H, Yan K, Ren Y, Ma M, et al. Evaluation of Spike Protein Epitopes by Assessing the Dynamics of Humoral Immune Responses in Moderate COVID-19. 2022;13(March):1–14.Poh CM, Carissimo G, Wang B, Amrun SN, Lee CY, Chee RS, et al. Two linear epitopes on the SARS-CoV-2 spike protein that elicit neutralising antibodies in COVID-19 patients. Nat Commun [Internet]. 2020; Available from: http://dx.doi.org/10.1038/s41467-020-16638-2Krempl C, Schultze B, Laude H. Point Mutations in the S Protein Connect the Sialic Acid Binding Activity with the Enteropathogenicity of Transmissible Gastroenteritis Coronavirus. 1997;71(4):3285–7.Lu G, Wang Q, Gao GF. Bat-to-human : spike features determining ‘ host jump ’ of MERS-CoV , and beyond. 2020;(January).Chi X, Yan R, Zhang J, Zhang G, Zhang Y, Hao M, et al. A neutralizing human antibody binds to the N-terminal domain of the Spike protein of SARS-CoV-2. 2020;655(August):650–5.Xiaojie S, Yu L, Guang Y, Min Q. Neutralizing antibodies targeting SARS-CoV-2 spike protein. 2020;(January). Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7737530/pdf/main.pdfRobichon C, Luo J, Causey TB, Benner JS, Samuelson JC. Engineering Escherichia coli BL21 ( DE3 ) Derivative Strains To Minimize E . coli Protein Contamination after Purification by Immobilized Metal Affinity Chromatography ᰔ †‡. 2011;77(13):4634–46.Mcguire BE, Mela JE, Thompson VC, Cucksey LR, Stevens CE, Mcwhinnie RL, et al. Escherichia coli recombinant expression of SARS ‑ CoV ‑ 2 protein fragments. Microb Cell Fact [Internet]. 2022;1–13. Available from: https://doi.org/10.1186/s12934-022-01753-0Glycoprotein S-S, Zhang S, Go EP, Ding H, Anang S, Kappes JC, et al. Analysis of Glycosylation and Disul fi de Bonding of Wild-Type. 2022;96(3):1–27.Rosano GL, Ceccarelli EA. Recombinant protein expression in Escherichia coli : advances and challenges. 2014;5(April):1–17.Laura M, Puglisi A, Dal F, Hochkoeppler A. Production in Escherichia coli of recombinant COVID-19 spike protein fragments fused to CRM197. 2020;(January).Rahbar Z, Nazarian S, Dorostkar R, Sotoodehnejadnematalahi F. Recombinant expression of SARS-CoV-2 receptor binding domain ( RBD ) in Escherichia coli and its immunogenicity in mice. 2022;Liu L, Chen T, Zhou L, Sun J, Li Y, Nie M, et al. A Bacterially Expressed SARS-CoV-2 Receptor Binding Domain Fused With Cross-Reacting Material 197 A-Domain Elicits High Level of Neutralizing Antibodies in Mice. 2022;13(April).Gao X, Peng S, Mei S, Liang K, Saleem M, Khan I, et al. Expression and functional identification of recombinant SARS-CoV-2 receptor binding domain ( RBD ) from E . coli system. Prep Biochem Biotechnol [Internet]. 2021;0(0):1–7. Available from: https://doi.org/10.1080/10826068.2021.1941106Ge S, Wu R, Zhou T, Liu X, Zhu J, Zhang X. Specific anti ‑ SARS ‑ CoV ‑ 2 S1 IgY ‑ scFv is a promising tool for recognition of the virus. AMB Express [Internet]. 2022; Available from: https://doi.org/10.1186/s13568-022-01355-4Balasubramaniyam A, Ryan E, Brown D, Hamza T, Harrison W, Gan M, et al. Unglycosylated Soluble SARS-CoV-2 Receptor Binding Domain ( RBD ) Produced in E . coli Combined with the Army Liposomal Formulation Containing QS21 ( ALFQ ) Elicits Neutralizing Antibodies against Mismatched Variants. 2023;21.Tungekar AA, Ruddock LW. Production of neutralizing antibody fragment variants in the cytoplasm of E . coli for rapid screening : SARS ‑ CoV ‑ 2 a case study. Sci Rep [Internet]. 2023;1–12. Available from: https://doi.org/10.1038/s41598-023-31369-2Singh A, Upadhyay V, Upadhyay AK, Singh SM, Panda AK. Protein recovery from inclusion bodies of Escherichia coli using mild solubilization process. 2015;1–10.Kong B, Guo GL. Soluble Expression of Disulfide Bond Containing Proteins FGF15 and FGF19 in the Cytoplasm of Escherichia coli. 2014;9(1):1–8.Bhatwa A, Wang W, Hassan YI, Abraham N, Li X. Challenges Associated With the Formation of Recombinant Protein Inclusion Bodies in Escherichia coli and Strategies to Address Them for Industrial Applications. 2021;9(February):1–18.Sivashanmugam A, Murray V, Cui C, Zhang Y, Wang J, Li Q. Practical protocols for production of very high yields of recombinant proteins using Escherichia coli. 2009;18(1):936–48.Hata S, Kitamura F, Sorimachi H. Efficient expression and purification of recombinant human l -calpain using an Escherichia coli expression system. 2013;753–63.Trabbic-carlson K, Liu LI, Kim B, Chilkoti A. Expression and purification of recombinant proteins from Escherichia coli : Comparison of an elastin-like polypeptide fusion with an oligohistidine fusion. 2004;3274–84.Rienzo L Di, Ruocco G, Desantis F, Grassmann G, Milanetti E. Dynamical changes of SARS-CoV-2 spike variants in the highly immunogenic regions impact the viral antibodies escaping. 2023;(March):1116–29.Chacón ME. Evaluación de un candidato a transportador de NAD+ en el parásito protozoario Trypanosoma cruzi. 2021;Eliana S, Silva V. Exploration of a Nad + Transporter and / or Its Precursors in. 2021;Lin TW, Huang PH, Liao BH, Chao TL, Tsai YM, Chang SC, et al. Tag-Free SARS-CoV-2 Receptor Binding Domain (RBD), but Not C-Terminal Tagged SARS-CoV-2 RBD, Induces a Rapid and Potent Neutralizing Antibody Response. Vaccines. 2022;10(11):1–11.Gefen T, Vaya J, Khatib S, Rapoport I, Lupo M, Barnea E, et al. The effect of haptens on protein-carrier immunogenicity. Immunology. 2015;144(1):116–26.Ertekin Ö, Akçael E, Kocaağa H, Öztürk S. Biological activity of the carrier as a factor in immunogen design for haptens. Molecules. 2018;23(11).Gonzalez-montalban N, Natalello A, Garcı E, Villaverde A, Doglia SM. In Situ Protein Folding and Activation in Bacterial Inclusion Bodies. 2008;100(4):797–802.Ramón A, Señorale-pose M, Marín M. Inclusion bodies : not that bad . . . 2014;5(February):2010–5.Li S, Zhao Q, Wu T, Chen S, Zhang J, Xia N. The development of a recombinant hepatitis E vaccine HEV 239. 2015;11(4):908–14.Sarantos K, Cleo K. European Journal of Pharmaceutical Sciences Analysis of the landscape of biologically-derived pharmaceuticals in Europe : Dominant production systems , molecule types on the rise and approval trends. Eur J Pharm Sci [Internet]. 2013;48(3):428–41. Available from: http://dx.doi.org/10.1016/j.ejps.2012.11.016Lua LHL, Connors NK, Sainsbury F, Chuan YP, Wibowo N, Middelberg APJ. Bioengineering Virus-Like Particles as Vaccines. 2014;111(3):425–40.Huang X, Wang X, Zhang J, Xia N, Zhao Q. Escherichia coli- derived virus-like particles in vaccine development. npj Vaccines [Internet]. 2017;(October 2016):1–8. Available from: http://dx.doi.org/10.1038/s41541-017-0006-8Wei M, Zhang X, Yu H, Tang Z, Wang K, Li Z, et al. Bacteria expressed hepatitis E virus capsid proteins maintain virion-like epitopes. Vaccine [Internet]. 2014;1–7. Available from: http://dx.doi.org/10.1016/j.vaccine.2014.02.025Qiagen. RNA Isolation with TRIzol ( Invitrogen ) and Qiagen RNAeasy. 2013;1–7.Gandhi V, Brien MHO, Yadav S. High-Quality and High-Yield RNA Extraction Method From Whole Human Saliva. 2020;Yan J, Li G, Hu Y, Ou W, Wan Y. Construction of a synthetic phage-displayed Nanobody library with CDR3 regions randomized by trinucleotide cassettes for diagnostic applications. 2014;1–12.Bashir S, Paeshuyse J. Construction of Antibody Phage Libraries and Their Application in Veterinary Immunovirology. 2020;(Figure 1).Lim CC, Woo PCY, Lim TS. Development of a Phage Display Panning Strategy Utilizing Crude Antigens: Isolation of MERS-CoV Nucleoprotein human antibodies. Sci Rep [Internet]. 2019;9(1):1–15. Available from: http://dx.doi.org/10.1038/s41598-019-42628-6Li J, Xu Y, Wang X, Li Y, Wang L, Li X. Construction and characterization of a highly reactive chicken-derived single-chain variable fragment (scFv) antibody against Staphylococcus aureus developed with the T7 phage display system. Int Immunopharmacol [Internet]. 2016;35:149–54. Available from: http://dx.doi.org/10.1016/j.intimp.2016.02.024Hu Z, Liu J, Li H, Xing S, Xue S, Zhang J. Generation of a Highly Reactive Chicken-Derived Single-Chain Variable Fragment against Fusarium verticillioides by Phage Display. 2012;7038–56.Yang T, Yang L, Chai W, Li R, Xie J, Niu B. A strategy for high-level expression of a single-chain variable fragment against TNFα by subcloning antibody variable regions from the phage display vector pCANTAB 5E into pBV220. Protein Expr Purif [Internet]. 2011;76(1):109–14. Available from: http://dx.doi.org/10.1016/j.pep.2010.10.006Zhan Y, Song Y, Ren H, Zeng Q, Yuan Y, Xia L, et al. Preparation of a Single-Chain Antibody against Nucleocapsid Protein of Porcine Deltacoronavirus by Phage. 2022;Lee YC, Leu SJC, Hung HC, Wu HH, Huang IJ, Hsieh WS, et al. A dominant antigenic epitope on SARS-CoV spike protein identified by an avian single-chain variable fragment (scFv)-expressing phage. Vet Immunol Immunopathol. 2007;117(1–2):75–85.Anandakumar S, Boosi KN, Bugatha H, Padmanabhan B, Sadhale PP. Phage displayed short peptides against cells of Candida albicans demonstrate presence of species, morphology and region specific carbohydrate epitopes. PLoS One. 2011;6(2).O’Brien P, Aitken R. Methods in Molecular Biology: antibody Phage Display. Vol. 178. 2002.Pitaksajjakul P, Lekcharoensuk P, Upragarin N, Barbas CF, Ibrahim MS, Ikuta K, et al. Fab MAbs specific to HA of influenza virus with H5N1 neutralizing activity selected from immunized chicken phage library. Biochem Biophys Res Commun [Internet]. 2010;395(4):496–501. Available from: http://dx.doi.org/10.1016/j.bbrc.2010.04.040EstudiantesInvestigadoresMaestrosLICENSElicense.txtlicense.txttext/plain; charset=utf-85879https://repositorio.unal.edu.co/bitstream/unal/85255/1/license.txteb34b1cf90b7e1103fc9dfd26be24b4aMD51ORIGINAL1026291353.2023.pdf1026291353.2023.pdfTesis de Maestría en Ciencias - Bioquímicaapplication/pdf4309578https://repositorio.unal.edu.co/bitstream/unal/85255/2/1026291353.2023.pdffb21d71bc06f776696b4ba07746d27a3MD52unal/85255oai:repositorio.unal.edu.co:unal/852552024-01-12 14:48:50.974Repositorio Institucional Universidad Nacional de Colombiarepositorio_nal@unal.edu.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 |