Molecular Cloning and Expression of a New Allergen of Acacia farnesiana (Aca f 2).

Inhalation of pollens from different species of Acacia is a common cause of respiratory allergy in tropical areas of the world. Acacia farnesiana is commonly used as street trees in towns and ornamental shade trees in parks and gardens throughout arid and semi-arid regions of Asia. This study aimed to produce and purify the A. farnesiana pollen profilin (Aca f 2) and evaluate its nucleotide sequence homology with profilins of common allergenic plants to predict allergenic cross-reactivity. Thirty-nine patients who were allergic to Acacia pollens were included in the study. Cloning of Acacia profilin-coding sequence was performed by polymerase chain reaction using primers from Acacia pollen RNA. The cDNA of Acacia pollen profilin was then expressed in Escherichia coli using pET-21b(+) vector and purified by metal affinity chromatography. Immunoreactivity of the recombinant Acacia profilin (rAca f 2) was evaluated by specific ELISA, immunoblotting, and inhibition assays. The coding sequence of the Acacia profilin cDNA was recognized as a 399-bp open reading frame encoding 133 amino acid residues. Eighteen patients (18/39, 46.15%) had significant specific IgE levels against Aca f 2. Immunodetection and inhibition assays indicated that purified Aca f 2 might be the same as that in the crude extract. Aca f2, the first allergen from A. farnesiana pollen, was identified as belonging to the family of profilins. The amino acid sequence homology analysis showed high cross-reactivity between Aca f 2 and other profilins from botanically unrelated common allergenic plants.

Pro j 2 is mesquite profilin: molecular characteristics and specific IgE binding activity.

BACKGROUND: Pollens from mesquite (Prosopis juliflora) are potent allergen responsible in causing immediate hypersensitivity reactions in susceptible people in tropical countries. OBJECTIVE: This study aimed to clone, express and purify the mesquite pollen profilin (Pro j 2) as well as evaluating its nucleotide sequence homology in order to predict allergenic cross-reactivity with profilins of common allergenic plants. METHODS: Immunoblotting assay and specific ELISA were applied to determine the immunoreactivity of sera from 35 patients who were allergic to mesquite pollen. The mesquite profilin-coding sequence was cloned into PTZ57R/T vector and amplified. The cDNA of mesquite pollen profilin was then expressed in Escherichia coli using pET-21b (+) vector and puri?ed by one-step Ni2+ a?nity chromatography. IgE binding capacity of the recombinant mesquite profiling (rPro j 2) was analyzed by specific ELISA, immunoblotting, and inhibition assays. RESULTS: cDNA nucleotide sequencing revealed an open reading frame of 399bp encoding for 133 amino acids which belongs to the profilin family. Seventeen patients (17/35, 48.57%) had significant specific IgE level for rPro j 2. Immunodetection and inhibition assays indicated that puri?ed rPro j 2 might be similar as that in the crude extract. CONCLUSION: Pro j 2, as a new allergen from mesquite pollen, was produced in E. coli with an IgE-reactivity similar to that of its natural counterpart. The amino acid sequences homology analysis of mesquite profilin and several profilin molecules from other plants showed high degree of cross-reactivity among plant-derived profilins from unrelated families.

Immunochemical characterization of prosopis juliflora pollen allergens and evaluation of cross-reactivity pattern with the most allergenic pollens in tropical areas.

Allergy to Prosopis juliflora (mesquite) pollen is one of the common causes of respiratory allergy in tropical countries. Mesquite is widely used as street trees in towns and ornamental shade trees in parks and gardens throughout arid and semiarid regions of Iran. The inhalation of mesquite pollen and several species of Amaranthus/Chenopodiaceae family is the most important cause of allergic respiratory symptoms in Khuzestan province. This study was designed to evaluate IgE banding proteins of mesquite pollen extract and its IgE cross-reactivity with other allergenic plants. Twenty patients with allergic symptoms and positive skin prick tests (SPT) for mesquite pollen extract participated in the study. Crude pollen extract was prepared from local mesquite trees and used for the evaluation of allergenic profiles of P. juliflora pollen extract by Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and IgE-immunoblotting. There were several protein bands in mesquite pollen extract using SDS-PAGE with the approximate range of molecular weight of 10-85 kDa. The most frequent IgE reactive bands among the patients' sera were approximately 20 and 66 kDa. However, there were other IgE reactive protein bands among the patients' sera with molecular weights of 10, 15, 35, 45, 55 and 85 kDa. Inhibition experiments revealed high IgE cross-reactivity between mesquite and acacia. There are several IgE-binding proteins in P. juliflora pollen extract. Results of this study indicate that proteins with a molecular weight of 10 to 85 kDa are the major allergens in P. juliflora pollen extract.