RESUMO
BACKGROUND: As a complex molecule requiring post-translational processing, it has been difficult to produce the Der p 1 major allergen from the Dermatophagoides pteronyssinus house dust mite in a recombinant form. OBJECTIVE: Here, we tested whether transgenic tobacco plants are suitable to express Der p 1, either as a wild-type molecule or as variants lacking N-glycosylation sites (Gly(-)) and/or cysteine protease activity (Enz(-)). Methods Using Agrobacterium tumefaciens-based transformation, pro Der p 1 molecules bearing mutations within either the N-glycosylation sites (N34Q, N150Q) and/or the cysteine protease-active site (C132V) were expressed in tobacco plants. After purification by ion exchange chromatography, allergens were characterized using immunoblotting, circular dichroism (CD), as well as basophil and T lymphocyte stimulation assays. RESULTS: Four forms of recombinant Der p 1 (i.e. wild-type Gly(+)/Enz(+), as well as Gly(-)/Enz(+), Gly(+)/Enz(-) or Gly(-)/Enz(-) variants) were successfully expressed in tobacco leaves as pro Der p 1 molecules. Spontaneous cleavage of the pro-peptide was observed in tobacco leaf extracts for all forms of recombinant Der p 1 (r Der p 1). CD confirmed that all r Der p 1 molecules, with the exception of the Gly(-)/Enz(-) variant, exhibited secondary structures comparable to the natural protein. A cysteine protease activity was associated only with the Gly(+)/Enz(+) form. All these molecules exhibit a profile similar to natural Der p 1 with respect to IgE immunoreactivity, basophil activation and T cell recognition. CONCLUSION: A tobacco plant expression system allows the production of various forms of mature Der p 1, which could be used for diagnostic or immunotherapeutic purposes.
Assuntos
Antígenos de Dermatophagoides/biossíntese , Clonagem Molecular , Nicotiana/genética , Antígenos de Dermatophagoides/genética , Antígenos de Dermatophagoides/imunologia , Proteínas de Artrópodes , Basófilos/imunologia , Basófilos/metabolismo , Linhagem Celular , Cisteína Endopeptidases , Humanos , Diester Fosfórico Hidrolases/imunologia , Diester Fosfórico Hidrolases/metabolismo , Folhas de Planta/genética , Plantas Geneticamente Modificadas , Pirofosfatases/imunologia , Pirofosfatases/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
A new method for the tear IgE measurement Stallerdiag-IgE is presented. For this assay, the tears are collected on a strip of filter paper introduced into the conjunctival cul-de-sac. Then the IgE are measured by a simple enzymo-immunological assay. Tear IgE were determined in a normal population and in three groups of patients with keratoconjunctivitis. In the normal population, the tear IgE level is 0.37±0.66 kIU/1. The IgE level observed in the patients with vernal keratoconjunctivitis (19.6±21.5 kIU/1) or chronic allergic keratoconjunctivitis (11.1±16.2 kIU/1) is significantly different (p<0.0005) from the IgE level measured in the patients suffering from non-allergic keratoconjunctivitis (3.4±10.4 kIU/1). By comparison with a non-allergic group, the sensitivity is 80%, the specificity is 89% giving an overall efficiency of 83%.
