A novel allergen Tab y 1 with inhibitory activity of platelet aggregation from salivary glands of horseflies

International Journal of Molecular Medicine

Jin

et al. 2016

Per a 9 is a major allergen of the American cockroach (CR), which has been recognized as an important cause of imunoglobulin E-mediated type I hypersensitivity worldwide. However, it is not neasy to obtain a substantial quantity of this allergen for use in functional studies. In the present study, the Per a 9 gene was cloned and expressed in Escherichia coli (E. coli) systems. It was found that 13/16 (81.3%) of the sera from patients with allergies caused by the American CR reacted to Per a 9, as assessed by enzyme-linked immunosorbent assay, confirming that Per a 9 is a major allergen of CR. The induction of the expression of CD63 and CCR3 in passively sensitized basophils (from sera of patients with allergies caused by the American CR) by approximately 4.2-fold indicated that recombinant Per a 9 was functionally active. Three immunoinformatics tools, including the DNASTAR Protean system, Bioinformatics Predicted Antigenic Peptides (BPAP) system and the BepiPred 1.0 server were used to predict the potential B cell epitopes, while Net-MHCIIpan-2.0 and NetMHCII-2.2 were used to predict the T cell epitopes of Per a 9. As a result, we predicted 11 peptides (23–28, 39–46, 58–64, 91–118, 131–136, 145–154, 159–165, 176–183, 290–299, 309–320 and 338–344) as potential B cell linear epitopes. In T cell prediction, the Per a 9 allergen was predicted to have 5 potential T cell epitope sequences, 119–127, 194–202, 210–218, 239–250 and 279–290. The findings of our study may prove to be useful in the development of peptide-based vaccines to combat CR-induced allergies.

Section: Methodsmentioning

confidence: 99%

Molecular cloning, expression, IgE binding activities and in silico epitope prediction of Per a 9 allergens of the American cockroach

Yang

International Journal of Molecular Medicine

Jin

et al. 2016

“…Twenty control sera were from individuals who had negative horsefly bite and wasp stinging tests. Sera were obtained from 37 subjects with horsefly allergy in our previous study [16]–[17], 17 children (46%) age 6 to 18 years (mean 12.1 years) and 20 adults (54%) age 19–59 years (mean 37.6 years), with immediate allergic reactions after the bites of T. yao . All sera were stored at −80°C.…”

Section: Methodsmentioning

confidence: 99%

Purification and Characterization of Two New Allergens from the Venom of Vespa magnifica

Wei

et al. 2012

PLoS ONE

Due to poor diagnostic facilities and a lack of medical alertness, allergy to Vespa wasps may be underestimated. Few allergens have been identified from Vespa wasps.Possible native allergen proteins were purified from the wasp venoms (WV) (Vespa magnifica Smith) by gel filtration, ion exchange chromatography, respectively. Their sequences were determined by Edman degradation and cDNA cloning. Their allergenicities were assayed by enzyme-linked immunosorbent assay inhibition tests (ELISA-IT), immunoblots, and skin prick tests (SPTs). Their cross allergencities with Tab y 2 and Tab y 5 purified from the horsefly (Tabanus yao Macquart) were also determined. Two native allergens were identified from the WV, respectively. They are a 25-KDa antigen 5 protein (Ag5) (Vesp ma 5) and a 35-KDa hyaluronidase (Vesp ma 2). They represented major allergens in Vespa magnifica by immunoblots and SPTs. ELISA inhibition of pooled sera IgE reactivity to both the WV and the horsefly salivary gland extracts (HSGE) using four purified allergens (Vesp ma 2, Vesp ma 5 and previously purified Tab y 2 and Tab y 5) was significant. Their cross allergenicities were confirmed by ELISA-IT, immunoblots, and SPTs. They represented the cross reactive allergens from wasp and horsefly and proved the so called wasp-horsefly syndrome.

“…The plates were read with a microplate reader at 450 nm (Epoch Etock, BioTek). Two patients (6 and 8) whose sera recognized purified allergens were chosen for ELISA inhibition assay according to previously described method (11) BSA, 0.05% Tween 20 phosphate buffer saline) were pre-incubated with purified allergen or DME (final concentration: 0.0003-30 g/ml) at 37°C for 1 h and then were added to the microtiter plates previously coated with 100 l per well of 20 g/ml DME. The subsequent steps were the same as those for direct ELISA described above.…”

Section: -D Electrophoresis (2-de) and Immunoblotting Analysis With mentioning

confidence: 99%

“…Enzyme-linked Immunosorbent Assay (ELISA) and ELISA Inhibition-Sera IgE antibodies specific for purified allergens were measured by indirect ELISA as described (10,11). Briefly, 96-well microtiter plates (Nunc, Roskilde, Denmark) were coated with 2 g purified allergen in 100 l carborate-biocarborate buffer (15 mM Na 2 CO 3 and 35 mM NaHCO 3 , pH 9.6) overnight at 4°C and then blocked for 30 min at 37°C with 200 l 3% BSA in PBS.…”

Section: -D Electrophoresis (2-de) and Immunoblotting Analysis With mentioning

confidence: 99%

Dermatophagoides farinae Allergens Diversity Identification by Proteomics

Molecular & Cellular Proteomics

Long

et al. 2013

The most important indoor allergens for humans are house dust mites (HDM). Fourteen Dermatophagoides farinae allergens (Der f 1-3, 6, 7, 10, 11, 13-18, and 22) are reported although more than 30 allergens have been estimated in D. farinae. Seventeen allergens belonging to 12 different groups were identified by a procedure of proteomics combined with two-dimensional immunoblotting from D. farina extracts. Their sequences were determined by Edman degradation, mass spectrometry analysis, and cDNA cloning. Their allergenicities were assayed by enzyme-linked immunosorbent assay inhibition tests, immunoblots, basophil activation test, and skin prick tests. Eight of them are the first report as D. farinae allergens. The procedure of using a proteomic approach combined with a purely discovery approach using sera of patients with broad IgE reactivity profiles to mite allergens was an effective method to investigate a more complete repertoire of D. farinae allergens. The identification of eight new D. farinae allergens will be helpful for HDM allergy diagnosis and therapy, especially for patients without response for HDM major allergens. In addition, the current work significantly extendedthe repertoire of D. farinae allergens. Molecular & Cellular