RESUMO
BACKGROUND: Rational design of synthetic phage-displayed libraries requires the identification of the most appropriate positions for randomization using defined amino acid sets to recapitulate the natural occurrence. The present study uses position-specific scoring matrixes (PSSMs) for identifying and randomizing Camelidae nanobody (VHH) CDR3. The functionality of a synthetic VHH repertoire designed by this method was tested for discovering new VHH binders to recombinant coagulation factor VII (rfVII). METHODS: Based on PSSM analysis, the CDR3 of cAbBCII10 VHH framework was identified, and a set of amino acids for the substitution of each PSSM-CDR3 position was defined. Using the Rosetta design SwiftLib tool, the final repertoire was back-translated to a degenerate nucleotide sequence. A synthetic phage-displayed library was constructed based on this repertoire and screened for anti-rfVII binders. RESULTS: A synthetic phage-displayed VHH library with 1 × 108 variants was constructed. Three VHH binders to rfVII were isolated from this library with estimated dissociation constants (KD) of 1 × 10-8 M, 5.8 × 10-8 M and 2.6 × 10-7 M. CONCLUSION: PSSM analysis is a simple and efficient way to design synthetic phage-displayed libraries.
Assuntos
Biologia Computacional , Biblioteca de Peptídeos , Anticorpos de Domínio Único , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/imunologia , Animais , Camelidae/genética , Camelidae/imunologia , Fator VII/genética , Fator VII/química , Fator VII/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Sequência de AminoácidosRESUMO
The single variable domains of camelid heavy-chain only antibodies, known as nanobodies, have taken a long journey since their discovery in 1989 until the first nanobody-based drug's entrance to the market in 2022. On account of their unique properties, nanobodies have been successfully used for diagnosis and therapy against various diseases or conditions. Although research on the application of recombinant antibodies has focused on human medicine, the development of nanobodies has paved the way for incorporating recombinant antibody production in favour of veterinary medicine. Currently, despite many efforts in developing these biomolecules with diversified applications, significant opportunities exist for exploiting these highly versatile and cost-effective antibodies in veterinary medicine. The present study attempts to identify existing gaps and shed light on paths for future research by presenting an updated review on camelid nanobodies with potential applications in veterinary medicine.
Assuntos
Camelidae , Anticorpos de Domínio Único , Medicina Veterinária , Animais , Anticorpos de Domínio Único/imunologia , Camelidae/imunologia , Medicina Veterinária/métodosRESUMO
Mammalian oocyte activation is a critical process occurring post-gamete fusion, marked by a sequence of cellular events initiated by an upsurge in intracellular Ca2+. This surge in calcium orchestrates the activation/deactivation of specific kinases, leading to the subsequent inactivation of MPF and MAPK activities, alongside PKC activation. Despite various attempts to induce artificial activation using distinct chemical compounds as Ca2+ inducers and/or Ca2+-independent agents, the outcomes have proven suboptimal. Notably, incomplete suppression of MPF and MAPK activities persists, necessitating a combination of different agents for enhanced efficiency. Moreover, the inherent specificity of activation methods for each species precludes straightforward extrapolation between them. Consequently, optimization of protocols for each species and for each technique, such as PA, ICSI, and SCNT, is required. Despite recent strides in camelid biotechnologies, the field has seen little advancement in chemical activation methods. Only a limited number of chemical agents have been explored, and the effects of many remain unknown. In ICSI, despite obtaining blastocysts with different chemical compounds that induce Ca2+ and calcium-independent increases, viable offspring have not been obtained. However, SCNT has exhibited varying outcomes, successfully yielding viable offspring with a reduced number of chemical activators. This article comprehensively reviews the current understanding of the physiological activation of oocytes and the molecular mechanisms underlying chemical activation in mammals. The aim is to transfer and apply this knowledge to camelid reproductive biotechnologies, with emphasis on chemical activation in PA, ICSI, and SCNT.
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Oócitos , Animais , Oócitos/fisiologia , Oócitos/efeitos dos fármacos , Feminino , Camelidae , Técnicas de Transferência Nuclear/veterináriaRESUMO
Nanobody (Nb) is a novel type of antibody discovered in the serum of Camelidae. It is characterized by its small size, high specificity, stability, and ease of preparation. Nanobodies exhibit the ability to identify hidden epitopes and have diverse applications across various fields. This review aims to introduce three key stages in the screening and optimization of nanobodies, including nanobody library construction, in vitro surface display, and affinity maturation. We provide a brief description of preparation and characteristics of natural, immunological, and semi-synthetic/synthetic libraries. Additionally, we systematically explain eight in vitro display methods, including phage display, yeast display, bacterial display, ribosome display/mRNA display, and eukaryotic cell display. Furthermore, we discuss the application of yeast two-hybrid system high-throughput sequencing and mass spectrometry identification. A thorough analysis of their advantages and limitations is presented in this protocols. Finally, we summarize the platforms for in vitro or computer-aided affinity maturation techniques aimed at enhancing the functional stability of nanobodies. Consequently, this review provides a comprehensive approach to the integrated utilization of various technologies for the rapid development of stable, reliable, and specific nanobody-based drugs or diagnostic agents.
Assuntos
Anticorpos de Domínio Único , Animais , Anticorpos de Domínio Único/genética , Camelidae , Clonagem Molecular , Epitopos , Saccharomyces cerevisiae/genéticaRESUMO
Infectious diseases continue to pose significant global health challenges. In addition to the enduring burdens of ailments like malaria and HIV, the emergence of nosocomial outbreaks driven by antibiotic-resistant pathogens underscores the ongoing threats. Furthermore, recent infectious disease crises, exemplified by the Ebola and SARS-CoV-2 outbreaks, have intensified the pursuit of more effective and efficient diagnostic and therapeutic solutions. Among the promising options, antibodies have garnered significant attention due to their favorable structural characteristics and versatile applications. Notably, nanobodies (Nbs), the smallest functional single-domain antibodies of heavy-chain only antibodies produced by camelids, exhibit remarkable capabilities in stable antigen binding. They offer unique advantages such as ease of expression and modification and enhanced stability, as well as improved hydrophilicity compared to conventional antibody fragments (antigen-binding fragments (Fab) or single-chain variable fragments (scFv)) that can aggregate due to their low solubility. Nanobodies directly target antigen epitopes or can be engineered into multivalent Nbs and Nb-fusion proteins, expanding their therapeutic potential. This review is dedicated to charting the progress in Nb research, particularly those derived from camelids, and highlighting their diverse applications in treating infectious diseases, spanning both human and animal contexts.
Assuntos
Camelidae , Anticorpos de Domínio Único , Animais , Anticorpos de Domínio Único/imunologia , Anticorpos de Domínio Único/uso terapêutico , Humanos , Camelidae/imunologia , Doenças Transmissíveis/imunologia , Doenças Transmissíveis/terapia , Camelídeos Americanos/imunologia , COVID-19/imunologia , COVID-19/terapiaRESUMO
Camelid immunoglobulins represent a unique facet of antibody biology, challenging conventional understandings of antibody diversification. IgG2 and IgG3 in particular are composed solely of heavy chains and exhibit a reduced molecular weight (90 kDa); their elongated complementarity determining region (CDR) loops play a pivotal role in their functioning, delving deep into enzyme active sites with precision. Serum therapy stands as the primary venom-specific treatment for snakebite envenomation, harnessing purified antibodies available in diverse forms such as whole IgG, monovalent fragment antibody (Fab), or divalent fragment antibody F (ab')2. This investigation looks into the intricacies of IgGs derived from camelid serum previously immunized with crotamine and crotoxin, toxins predominantly in Crotalus durissus venom, exploring their recognition capacity, specificity, and cross-reactivity to snake venoms and its toxins. Initially, IgG purification employed affinity chromatography via protein A and G columns to segregate conventional antibodies (IgG1) from heavy chain antibodies (IgG2 and IgG3) of camelid isotypes sourced from Lama glama serum. Subsequent electrophoretic analysis (SDS-PAGE) revealed distinct bands corresponding to molecular weight profiles of IgG's fractions representing isotypes in Lama glama serum. ELISA cross-reactivity assays demonstrated all three IgG isotypes' ability to recognize the tested venoms. Notably, IgG1 exhibited the lowest interactivity in analyses involving bothropic and crotalic venoms. However, IgG2 and IgG3 displayed notable cross-reactivity, particularly with crotalic venoms and toxins, albeit with exceptions such as PLA2-CB, showing reduced reactivity, and C. atrox, where IgGs exhibited insignificant reactivity. In Western blot assays, IgG2 and IgG3 exhibited recognition of proteins within molecular weight (≈15 kDa) of C. d. collilineatus to C. d. terrificus, with some interaction observed even with bothropic proteins despite lower reactivity. These findings underscore the potential of camelid heavy-chain antibodies, suggesting Lama glama IgGs as prospective candidates for a novel class of serum therapies. However, further investigations are imperative to ascertain their suitability for serum therapy applications.
Assuntos
Antivenenos , Imunoglobulina G , Animais , Antivenenos/imunologia , Imunoglobulina G/imunologia , Crotalus/imunologia , Venenos de Crotalídeos/imunologia , Reações Cruzadas , Camelídeos Americanos/imunologia , Crotoxina/imunologia , Camelidae/imunologiaRESUMO
Abstract The llama (Lama glama) is the largest domesticated animal species from South America and is today found worldwide. Andean peoples have used the llama for millennia for meat, wool, packing, spiritual etc. In order to know the history of the llama, we must learn about the peoples that have known the animal and the ways those relationships have changed over time. While also considering closely related species, including alpaca, guanaco, and vicuña, this article posits three eras of llama/human entanglements: the era of domestication in pre-Columbian Andean sites; the era of dispersal and co-mingling, from 1530s to the 1890s; and finally popular fads and global appeal.
Resumo A lhama (Lama glama) é a maior espécie animal domesticada da América do Sul e atualmente é encontrada em todo o mundo. Os povos andinos a utilizam há milênios para transporte de carga, obtenção de lã e carne, uso espiritual etc. Para conhecer sua história, precisamos aprender sobre os povos que conhecem e se relacionam com esse animal, e como esses relacionamentos se transformaram ao longo do tempo. O artigo considera ainda outras espécies relacionadas, incluindo alpaca, guanaco e vicunha, e apresenta três eras da interação lhamas/humanos: a da domesticação em sítios andinos pré-colombianos; a da dispersão e mistura, dos anos 1530 à década de 1890; e, finalmente, modismos e interesse global.