RESUMO
Oral antibodies that interfere with gastrointestinal targets and can be manufactured at scale are needed. Here we show that a single-gene-encoded monomeric immunoglobulin A (IgA)-like antibody, composed of camelid variable single domain antibodies (VHH) fused to IgA Fc (mVHH-IgA), prevents infection by enterotoxigenic Escherichia coli (F4-ETEC) in piglets. The mVHH-IgA can be produced in soybean seeds or secreted from the yeast Pichia pastoris, freeze- or spray-dried and orally delivered within food.
Assuntos
Doenças Transmissíveis/tratamento farmacológico , Gastroenteropatias/tratamento farmacológico , Imunoglobulina A/uso terapêutico , Anticorpos de Domínio Único/uso terapêutico , Administração Oral , Animais , Doenças Transmissíveis/imunologia , Doenças Transmissíveis/microbiologia , Escherichia coli/patogenicidade , Alimentos , Gastroenteropatias/imunologia , Gastroenteropatias/prevenção & controle , Gastroenteropatias/veterinária , Humanos , Imunoglobulina A/imunologia , Anticorpos de Domínio Único/imunologia , SuínosRESUMO
Plant expression systems have proven to be exceptional in producing high-value complex polymeric proteins such as secretory IgAs (SIgAs). However, polymeric protein production requires the expression of multiple genes, which can be transformed as single or multiple T-DNA units to generate stable transgenic plant lines. Here, we evaluated four strategies to stably transform multiple genes and to obtain high expression of all components. Using the in-seed expression of a simplified secretory IgA (sSIgA) as a reference molecule, we conclude that it is better to spread the genes over two T-DNAs than to contain them in a single T-DNA, because of the presence of homologous recombination events and gene silencing. These T-DNAs can be cotransformed to obtain transgenic plants in one transformation step. However, if time permits, more transformants with high production levels of the polymeric protein can be obtained either by sequential transformation or by in-parallel transformation followed by crossing of transformants independently selected for excellent expression of the genes in each T-DNA.
Assuntos
Arabidopsis/genética , DNA Bacteriano/genética , Imunoglobulina A Secretora/biossíntese , Transformação Genética , Animais , Arabidopsis/metabolismo , Inativação Gênica , Vetores Genéticos , Plantas Geneticamente Modificadas , Sementes/genética , SuínosRESUMO
GoldenBraid (GB) is an iterative and standardized DNA assembling system specially designed for Multigene Engineering in Plant Synthetic Biology. GB is based on restriction-ligation reactions using type IIS restriction enzymes. GB comprises a collection of standard DNA pieces named "GB parts" and a set of destination plasmids (pDGBs) that incorporate the multipartite assembly of standardized DNA parts. GB reactions are extremely efficient: two transcriptional units (TUs) can be assembled from several basic GBparts in one T-DNA less than 24 h. Moreover, larger assemblies comprising 4-5 TUs are routinely built in less than 2 working weeks. Here we provide a detailed view of the GB methodology. As a practical example, a Bimolecular Fluorescence Complementation construct comprising four TUs in a 12 kb DNA fragment is presented.
Assuntos
Biotecnologia/métodos , Clonagem Molecular/métodos , Família Multigênica/genética , Nicotiana/genética , Agrobacterium/citologia , Agrobacterium/genética , Sequência de Bases , Técnicas de Cultura de Células , Escherichia coli/citologia , Escherichia coli/genética , Plasmídeos/genética , Reação em Cadeia da Polimerase , Transformação GenéticaRESUMO
Plant synthetic biology aims to apply engineering principles to plant genetic design. One strategic requirement of plant synthetic biology is the adoption of common standardized technologies that facilitate the construction of increasingly complex multigene structures at the DNA level while enabling the exchange of genetic building blocks among plant bioengineers. Here, we describe GoldenBraid 2.0 (GB2.0), a comprehensive technological framework that aims to foster the exchange of standard DNA parts for plant synthetic biology. GB2.0 relies on the use of type IIS restriction enzymes for DNA assembly and proposes a modular cloning schema with positional notation that resembles the grammar of natural languages. Apart from providing an optimized cloning strategy that generates fully exchangeable genetic elements for multigene engineering, the GB2.0 toolkit offers an evergrowing open collection of DNA parts, including a group of functionally tested, premade genetic modules to build frequently used modules like constitutive and inducible expression cassettes, endogenous gene silencing and protein-protein interaction tools, etc. Use of the GB2.0 framework is facilitated by a number of Web resources that include a publicly available database, tutorials, and a software package that provides in silico simulations and laboratory protocols for GB2.0 part domestication and multigene engineering. In short, GB2.0 provides a framework to exchange both information and physical DNA elements among bioengineers to help implement plant synthetic biology projects.
