Immunoproteomic analysis and identification of possible allergenic proteins in Artemisia annua pollen.

BACKGROUND

Artemisia annua (A. annua) is a wind-pollinated weed and a major allergen responsible for allergic respiratory diseases in Northern China.

METHODS

This study involved the separation of pollen proteins from A. annua utilizing SDS-PAGE and Coomassie Blue staining techniques. The effectiveness of extracting allergens from Artemisia annua pollen (AAP) were confirmed both in vivo and in vitro. A mouse model was established using A. annua pollen (AAP) extracts. In vitro, the interaction between allergenic proteins in the AAP extract and specific IgE antibodies present in patients' sera was analyzed, using IgE-immunoblotting and ELISA methods. Protein bands were subsequently analyzed by mass spectrometry. The recombinant Art an 3 (rArt an 3) protein was obtained via recombination, expression, and purification. Finally, the binding activity of rArt an 3 specific IgE was subsequently examined using Western blot and inhibition ELISA (iELISA).

RESULTS

The electrophoretic profiles of AAP showed band patterns ranging from 10 to 70 kDa, with the most prominet IgE-binding pollen proteins detected at approximately 12 kDa. After stimulation of AAP, the sensitized group of mice exhibited significant allergic symptoms compared to the control group. Mass spectrometry analysis identified 7 proteins, including putative aldehyde oxidase Art an 7, Art v 1-like protein, Art an 3.0101 allergen precursor, Art an 3.0102 allergen precursor, Art an 3.0201 allergen precursor, lipid transfer protein 3, and non-specific lipid-transfer protein. The results of immunoblotting and iELISA showed that rArt an 3 could bind to IgE antibodies in the patient sera, and co-incubation of rArt an 3 with serum, the binding serum significantly inhibited IgE binding to the crude AAP.

CONCLUSIONS

The Art an 3 is a key allergenic protein in AAP with a high IgE sensitization rate in the studied population sample. These findings enhance our understanding of the sensitization components of AAP and its sensitization characteristics within the Chinese population, thereby promoting the development of precise molecular diagnostics and therapeutic interventions.