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Inicio Allergologia et Immunopathologia Identification of polcalcin as a novel allergen of Amaranthus retroflexus pollen
Journal Information
Vol. 47. Issue 4.
Pages 357-364 (July - August 2019)
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Vol. 47. Issue 4.
Pages 357-364 (July - August 2019)
Original Article
DOI: 10.1016/j.aller.2018.12.006
Identification of polcalcin as a novel allergen of Amaranthus retroflexus pollen
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M. Vakili Moghaddama,b, M. Fallahpourc, M. Mohammadid, F.S. Rasi Varaeea, K. Mokhtariane, M. Khoshmirsafaa,g, R. Jafarif, N. Shirzadb, R. Falaka,g,
Corresponding author
a Immunology Research Center, Iran University of Medical Sciences, Tehran, Iran
b Department of Immunology, School of Medicine, Shahrekord University of Medical Sciences, Shahrekord, Iran
c Department of Allergy and Clinical Immunology, Iran University of Medical Sciences, Tehran, Iran
d Persian Gulf Marine Biotechnology Research Center, Persian Gulf Biomedical Sciences Research Institute, Bushehr University of Medical Sciences, Bushehr, Iran
e Clinical Biochemistry Research Center, Basic Health Sciences Institute, Shahrekord University of Medical Sciences, Shahrekord, Iran
f Department of Immunology, School of Medicine, Shahroud University of Medical Sciences, Shahroud, Iran
g Department of Immunology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran
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Tables (2)
Table 1. Clinical characteristics, IgE-reactivity and SPT responses of the A. retroflexus allergic patients.
Table 2. Comparison of specific IgE-reactivity of A. retroflexus and recombinant Ama r 3 with different cut-off points of absorbance (450nm) using ELISA test.
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Abstract
Introduction

Amaranthus retroflexus (Redroot Pigweed) is one of the main sources of allergenic pollens in temperate areas. Polcalcin is a well-known panallergen involved in cross-reactivity between different plants. The aim of this study was the molecular cloning and expression of polcalcin, as well as evaluating its IgE-reactivity with A. retroflexus sensitive patients’ sera.

Methods

Allergenic extract was prepared from A. retroflexus pollen and the IgE-reactivity profile was determined by ELISA and immunoblotting using sera from twenty A. retroflexus sensitive patients. Polcalcin-coding sequence was amplified by conventional PCR method and the product was inserted into pET-21b(+) vector. The recombinant protein was expressed in E. coli BL21 and purified by metal affinity chromatography. The IgE-binding capability of the recombinant protein was analyzed by ELISA and immunoblotting assays, and compared with crude extract.

Results

Of 20 skin prick test positive patients, 17 patients were positive in IgE-specific ELISA. Western blotting confirmed that approximately 53% of ELISA positive patients reacted with 10kDa protein in crude extract. The A. retroflexus polcalcin gene, encoding to 80 amino acid residues was cloned and expressed as a soluble protein and designated as Ama r 3. The recombinant polcalcin showed rather identical IgE-reactivity in ELISA and western blotting with 10kDa protein in crude extract. These results were confirmed by inhibition methods, too.

Conclusion

The recombinant form of A. retroflexus polcalcin (Ama r 3) could be easily produced in E. coli in a soluble form and shows rather similar IgE-reactivity with its natural counterpart.

Keywords:
Allergen characterization
Amaranthus retroflexus
Cloning
Polcalcin

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