RESUMO
Tobacco seeds can be used as a cost effective system for production of recombinant vaccines. Avian influenza is an important respiratory pathogen that causes a high degree of mortality and becomes a serious threat for the poultry industry. A safe vaccine against avian flu produced at low cost could help to prevent future outbreaks. We have genetically engineered tobacco plants to express extracellular domain of hemagglutinin protein from H5N1 avian influenza virus as an inexpensive alternative for production purposes. Two regulatory sequences of seed storage protein genes from Phaseolus vulgaris L. were used to direct the expression, yielding 3.0 mg of the viral antigen per g of seeds. The production and stability of seed-produced recombinant HA protein was characterized by different molecular techniques. The aqueous extract of tobacco seed proteins was used for subcutaneous immunization of chickens, which developed antibodies that inhibited the agglutination of erythrocytes after the second application of the antigen. The feasibility of using tobacco seeds as a vaccine carrier is discussed.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vacinas contra Influenza/farmacologia , Nicotiana/metabolismo , Sementes/genética , Testes de Aglutinação , Animais , Galinhas/virologia , Cromatografia Líquida de Alta Pressão , Regulação da Expressão Gênica de Plantas , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vacinas contra Influenza/genética , Vacinas contra Influenza/imunologia , Influenza Aviária/prevenção & controle , Phaseolus/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Polissacarídeos/análise , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Sementes/metabolismo , Nicotiana/genéticaRESUMO
A broad variety of foreign genes can be expressed in transgenic plants, which offer the opportunity for large-scale production of pharmaceutical proteins, such as therapeutic antibodies. Nimotuzumab is a humanized anti-epidermal growth factor receptor (EGFR) recombinant IgG1 antibody approved in different countries for the treatment of head and neck squamous cell carcinoma, paediatric and adult glioma, and nasopharyngeal and oesophageal cancers. Because the antitumour mechanism of nimotuzumab is mainly attributed to its ability to interrupt the signal transduction cascade triggered by EGF/EGFR interaction, we have hypothesized that an aglycosylated form of this antibody, produced by mutating the N(297) position in the IgG(1) Fc region gene, would have similar biochemical and biological properties as the mammalian-cell-produced glycosylated counterpart. In this paper, we report the production and characterization of an aglycosylated form of nimotuzumab in transgenic tobacco plants. The comparison of the plantibody and nimotuzumab in terms of recognition of human EGFR, effect on tyrosine phosphorylation and proliferation in cells in response to EGF, competition with radiolabelled EGF for EGFR, affinity measurements of Fab fragments, pharmacokinetic and biodistribution behaviours in rats and antitumour effects in nude mice bearing human A431 tumours showed that both antibody forms have very similar in vitro and in vivo properties. Our results support the idea that the production of aglycosylated forms of some therapeutic antibodies in transgenic plants is a feasible approach when facing scaling strategies for anticancer immunoglobulins.
Assuntos
Anticorpos Monoclonais Humanizados/biossíntese , Anticorpos Monoclonais Humanizados/farmacologia , Receptores ErbB/antagonistas & inibidores , Imunoglobulinas/biossíntese , Nicotiana/genética , Nicotiana/metabolismo , Planticorpos/farmacologia , Animais , Antineoplásicos/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Camundongos Nus , Agricultura Molecular/métodos , Fosforilação/efeitos dos fármacos , Planticorpos/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Ratos , Ratos Wistar , Proteínas Recombinantes , Tirosina/metabolismoRESUMO
Agrobacterium tumefaciens technology is the battle horse for tomato genetic transformation. However, tomato varieties with low regeneration capacity are very difficult to transform. In the past, tomato transformation through Agrobacterium infection was focused on varieties capable of high regeneration yield, while successful transformation of low regenerable cultivars has not been reported. The genotype response to tissue culture conditions is believed to drive the frequency of regeneration of transgenic plant, whereas the capacity for cell proliferation could determine the transformation efficiency through this technology. The Campbell-28 cultivar is an example of constraints arising from a high morphogenetic potential with low conversion compared to normal plants. In the present work the roles that contribute to improved transgenic plant recovery from this recalcitrant variety were explored for factors like Agrobacterium concentration and antibiotics for bacterial removal and transformant selection. Analysis of the efficiency from independent transformation experiments revealed a more than twofold increase of transformant regeneration after selection on ammonium glufosinate compared to kanamycin selection, showing a transformation efficiency of 21.5%.