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Isolation, cDNA cloning and expression of Lig v 1, the major allergen from privet pollen
E Batanero1, M A Gonzalez De La Peña, M Villalba
1Departamento de Bioquímica y Biología Molecular, Facultad de Química, Universidad Complutense, Madrid, Spain.
Background:
An olive allergen-like protein has been detected in privet pollen. This protein could be involved in the allergenic cross-reactivity described for privet and olive tree pollen extracts.
Objective:
Isolation and characterization of natural Lig v 1. Cloning and expression of its cDNA in order to assess its structural similarity with the olive allergen.
Methods:
Current chromatographic methods were used to isolate the privet counterpart of Ole e 1. A pool of sera from subjects allergic to olive tree pollen was used to immunodetect the protein in the elution profiles. Ole e 1-specific polyclonal antibody and allergic sera were used in immunoblotting assays of the isolated protein. Polymerase chain reaction amplification of the first strand cDNA synthesized from the privet pollen total RNA was carried out to prepare a full-length fragment encoding Lig v 1. After nucleotide sequencing, expression of one clone was performed in Escherichia coli, under the form of a fusion protein with glutathione S-transferase. The IgE binding capability of the recombinant protein was also analysed.
Results:
The major allergen from privet pollen. Lig v 1, was purified to homogeneity by two gel filtration chromatographies and one reverse-phase high-performance liquid chromatography. Its amino acid composition and N-terminal amino acid sequence were determined. Two different clones encoding Lig v 1 were sequenced. Strong sequence similarity between Lig v 1 and Ole e 1 was observed, the identity being 85 and 96%. One of the sequenced clones was expressed and the recombinant product exhibited IgG and IgE binding activities against both anti-Ole e 1 polyclonal antibodies and olive-allergic sera.
Conclusion:
Privet pollen contains a protein structurally and immunologically related to the major allergen of olive pollen. The similarity exhibited by these proteins could explain the cross-reactivity observed between the two pollen extracts. Since these allergens are highly polymorphic, the expression of an immunologically active recombinant Lig v 1 will permit the preparation of well defined molecules for both research and clinical purposes.