ISOLATION, CDNA CLONING AND EXPRESSION OF LIG-V-1, THE MAJOR ALLERGENFROM PRIVET POLLEN

Background An olive allergen-like protein has been detected in privet pollen. This protein could be involved in the allergenic cross-reactiv ity described for privet and olive tree pollen extracts. Objective Iso lation and characterization of natural Lig v 1. Cloning and expression of its cDNA in order to assess its structural similarity with the oli ve allergen. Methods Current chromatographic methods were used to isol ate the privet counterpart of Ole e 1. A pool of sera from subjects al lergic to olive tree pollen was used to immunodetect the protein in th e elution profiles. Ole e 1-specific polyclonal antibody and allergic sera were used in immunoblotting assays of the isolated protein. Polym erase chain reaction amplification of the first strand cDNA synthesize d from the privet pollen total RNA was carried out to prepare a full-l ength fragment encoding Lig v 1. After nucleotide sequencing, expressi on of one clone was performed in Escherichia coli, under the form of a fusion protein with glutathione S-transferase. The IgE binding capabi lity of the recombinant protein was also analysed. Results The major a llergen from privet pollen, Lig v 1, was purified to homogeneity by tw o gel filtration chromatographies and one reverse-phase high-performan ce liquid chromatography. Its amino acid composition and N-terminal am ino 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 an d olive-allergic sera. Conclusion Privet pollen contains a protein str ucturally and immunologically related to the major allergen of olive p ollen. The similarity exhibited by these proteins could explain the cr oss-reactivity observed between the two pollen extracts. Since these a llergens 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.