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Background: Pollinosis from Amaranthus retroflexus pollen is a common cause of respiratory allergy in Iran with a high positive rate (68.8%) among Iranian allergic patients. The aim of the present study was to evaluate the allergenicity of the A. retroflexus pollen profilin., Methods: Using sera from twelve patients allergic to A. retroflexus pollen, IgE-binding proteins from the A. retroflexus pollen extract was identified by immunoblotting. The cDNA of A. retroflexus pollen profilin was amplified, then cloned into the pET-21b (+) vector, expressed in Escherichia coli, and finally purified by metal affinity chromatography. The IgE-binding capacity of the recombinant protein was then analyzed by the ELISA, immunoblotting, and inhibition assays, as well as by the skin prick test (SPT)., Results: Immunoblotting results indicated a 14.6kDa protein with IgE-reactivity to 33% (4/12) among A. retroflexus pollen-allergic patients. Nucleotide sequencing of the cDNA revealed an open reading frame of 399 bp encoding for 133 amino acid residues which was belonged to the profilin family and designated as Ama r 2. A recombinant Ama r 2 (rAma r 2) was then produced in E. coli as a soluble protein which showed a strong IgE-reactivity via ELISA confirmed by the SPT. Inhibition experiments revealed high IgE cross-reactivities with the profilins from other plants., Conclusions: The profilin from the A. retroflexus pollen, Ama r 2, was firstly identified as an allergen. Moreover, rAma r 2 was produced in E. coli as a soluble immunoreactive protein with an IgE-reactivity similar to that of its natural counterpart.

Salsola kali pollen is a common cause of pollinosis during summer and early fall in desert and semi-desert regions. The aim of this study was the identification and characterization of Sal k 3, a new allergen from S. kali pollen. S. kali pollen extract was fractionated by SDS-PAGE and the allergenic profile was determined by IgE-immunoblotting using twelve S. kali allergic patients. Protein identification was carried out by the means of mass spectrometry. Using degenerated primers, two DNA fragments encoding N- and C-terminal domain of Sal k 3 were amplified by PCR, then cloned into the PTZ57R/T vector and sequenced. The open reading frame of Sal k 3 fragments were subcloned in the pET-32b(+) vector, expressed in E. coli, and purified by Ni2+ affinity chromatography. The IgE-binding capacity of rSal k 3 fragments was then studied by IgE-immunoblotting, inhibition assays, and skin prick tests. A 45-kDa allergen was identified as a fragment of the cobalamin-independent methionine synthase (MetE) by mass spectrometry and was detected in the sera of 8/12 (66.6%) of S. kali allergic patients. Moreover, inhibition assays demonstrated that the purified rSal k 3 fragments were similar to their counterparts in the crude extract. Sal k 3 represents a new allergen of S. kali pollen and seems to be an important allergenic compound in S. kali pollen.

The importance of Amaranthus retroflexus pollen in causing respiratory allergy has been well ascertained in many countries including Iran with a high positive rate (69%) among Iranian allergic patients. The aim of the present study is to identify the allergenic properties of A. retroflexus pollen. Sixteen patients with allergy to A. retroflexus pollen were selected for the study. The antigenic and allergenic profiles of the A. retroflexus pollen extract as well as pollen extracts from other species of the Amaranthaceae/Chenopodiaceae family, including Chenopodium album, Kochia scoparia, and Salsola kali, were evaluated by ELISA, immunoblotting, and immunoblot inhibition assays. The resolved protein fractions on SDS-PAGE ranged from 10-85 kDa. Several allergenic components (MW 85, 45, 39, 18, 15, and 10 kDa) of the A. retroflexus pollen extract were recognized by using patients' sera by specific antibody of IgE class using ELISA and immunoblot assays. The IgE reactivity of the A. retroflexus pollen extract was partially inhibited by all three pollen extracts tested. The inhibition by the S. kali pollen extract was more than those by other pollen extracts. Moreover, the wheal diameters by the A. retroflexus pollen extract were highly correlated with those by C. album, K. scoparia and S. kali pollen extracts. In conclusion, three proteins with apparent MWs of 39, 45, and 66 kDa are suggested as the common allergenic components among the four pollens from the Amaranthaceae/Chenopodiaceae family. It appears that there are some common (similar) epitopes among the four common allergenic pollens.

Background: The inhalation of Salsola kali pollen is an important cause of pollinosis during summer and early fall throughout desert and semi-desert areas. Sal k 1 has been previously reported as a major allergen of S. kali pollen. In this study, we produced the recombinant Sal k 1 and also its low IgE-binding mutant form. We further compared the IgE binding ability of these two recombinant molecules., Methods: The recombinant Sal k 1 and its low IgE-binding variant, obtained by three amino acid exchanges (R(142)-->S, P(143)-->A, D(144)-->V), were cloned and expressed in E. coli, as proteins fused with thioredoxin and His-tags, and then purified by Ni2+ affinity chromatography. The IgE-binding capacity of the wild-type and mutated rSal k 1 was compared using immunoblotting, ELISA and inhibition assays by ten sera from S. kali allergic patients. Moreover, in vivo IgE-reactivity was investigated by the skin prick test., Results: Both the recombinant and the mutated form of Sal k 1 were expressed in E. coli at a relatively high amount and soluble form. All sera recognized rSal k 1 via immunoassay analysis. In addition, inhibition assays demonstrated that the purified rSal k 1 was similar to its counterpart in the crude extract. The mutated rSal k 1 exhibited a reduced IgE-binding capacity against wild-type rSal k 1., Conclusions: This study demonstrates that purified rSal k 1 is comprised of IgE-epitopes similar to that of its natural counterpart and that the mutated variant showed a reduced IgE-binding capacity based on in vitro assays and in vivo provocation testing.

The aim of this study was to investigate a new allergen of Salsola kali, Sal k 4, and to investigate the predictive value of the conserved conformational regions in cross-reactivity with other plant-derived profilins. The Sal k 4-coding sequence was cloned, expressed, and purified by one-step Ni2+ affinity chromatography to recover high-purity target protein. We assessed cross-reactivity and predicted conserved conformational regions among rSal k 4 and other plant-derived profilins. Immunodetection and inhibition assays using 30 individual sera from S. kali allergic patients indicated that purified rSal k 4 might be the same as that in the crude extract. The results of inhibition assays among rSal k 4 and other plant-derived profilins were in accordance with the homology of the predicted conserved conformational regions. Amino acid sequence homology analysis showed that a high degree of IgE cross-reactivity among plant-derived profilins might depend on the predicted conserved conformational regions.