Identification and characterization of natural PR-1 protein as major allergen from Humulus japonicus pollen.

BACKGROUND: The Humulus japonicus pollen is one of the most common allergenic pollens in China. However, little is unveiled regarding the allergenic components in Humulus japonicus pollen. Our study aimed to purify and identify the pathogenesis-related 1 (PR-1) protein from Humulus japonicus pollen, and to characterize the molecular and immunochemical properties of this novel allergen. METHODS: The natural PR-1 protein (named as Hum j PR-1) was purified from Humulus japonicus pollen extracts with a combined strategy of chromatography, and identified by mass spectrometry. The coding sequence of Hum j PR-1 was confirmed by cDNA cloning. The recombinant Hum j PR-1 was expressed and purified from Escherichia coli. The allergenicity was assessed by immunoblot, enzyme-linked immunosorbent assay (ELISA), inhibition ELISA, and basophil activation test using Humulus japonicus allergic patients' whole blood. The physicochemical properties and 3-dimensional structure of it were comprehensively characterized by in silico methods. RESULTS: The allergenicity analysis revealed that 76.6 % (23/30) of the Humulus japonicus pollen allergic patients displayed specific IgE recognition of the natural Hum j PR-1. The cDNA sequence of Hum j PR-1 had a 516-bp open reading frame encoding 171 amino acids. Physicochemical analysis indicated that Hum j PR-1 was a stable and relatively thermostable protein. Hum j PR-1 shared a similar 3-dimensional folding pattern with other homologous allergens, which was a unique αßα sandwich structure containing 4 α-helices and 6 antiparallel ß-sheets, encompassing 4 conserved CAP domain. CONCLUSION: The natural PR-1 was firstly purified and characterized as a major allergenic allergen in Humulus japonicus pollen. These findings would contribute to developing diagnostic and therapeutic strategies for Humulus japonicus pollinosis.

The development of a candidate of desensitization vaccines against Der f 2 nearly without IgE-binding activity.

Considering the important role of Der f 2 in house dust mites mediating allergic diseases and allergic adverse effects during allergen-specific immunotherapy (AIT), we intend to develop a candidate of desensitization vaccines against Der f 2 without allergenicity. According to the reported immunoglobulin E (IgE)-binding B and T cell epitopes of Der f 2, four candidates of desensitization vaccines against Der f 2 were developed. Recombinant wild-type Der f 2 (rWt-Der f 2) preserved conformational and linear IgE-binding B epitopes. rWt-Der f 2 linearized by reduction and alkylation reactions (rWt-Der f 2 (red/alk)) and recombinant modified-type Der f 2 (rMt-Der f 2) were developed via destroying conformational and linear IgE-binding B epitopes respectively. rMt-Der f 2 linearized by reduction and alkylation reactions (rMt-Der f 2 (red/alk)) was developed by destroying conformational and linear IgE-binding B epitopes. T cell epitopes of 4 candidates were preserved. The change of their IgE-binding activity was determined by enzyme linked immunosorbent assay (ELISA), western blot and inhibition ELISA. Compared with rWt-Der f 2, the IgE-binding activity of rWt-Der f 2 (red/alk), rMt-Der f 2 and rMt-Der f 2 (red/alk) all decreased, which was consistent with the result of western blot. The IgE-binding activity of rMt-Der f 2 and rMt-Der f 2 (red/alk) was not significantly different (P = 0.0863 > 0.05), which was comparable to that of their corresponding negative controls (P = 0.3488 and 0.4459, both > 0.05). The result of inhibition ELISA also showed that their IgE-binding activity decreased, and rMt-Der f 2 (red/alk) was the lowest. Conclusively, we developed the candidate of desensitization vaccines against Der f 2, rMt-Der f 2 or rMt-Der f 2 (red/alk), nearly without allergenicity, which would potentially prevent HDM allergic patients from allergic adverse effects caused by AIT.