A new cysteine protease allergen from Ambrosia trifida pollen: proforms and mature forms.

Giant ragweed (Ambrosia trifida) pollen is closely associated with respiratory allergy in late summer and autumn, and the prevalence of giant ragweed pollen allergy progressively increases. Compared with short ragweed (Ambrosia artemisiifolia), allergenic components from giant ragweed pollen are poorly investigated. To promote component-resolved diagnosis and treatment for giant ragweed pollen allergy, it becomes necessary to identify and characterize unknown allergens from giant ragweed pollen. In the present study, we identified and characterized a new cysteine-protease (CP) allergen from giant ragweed pollen, named as Amb t CP. The cloned Amb t CP gene encoded 387 amino acids. Recombinant Amb t CP (rAmb t CP) and natural Amb t CP (nAmb t CP) were purified by high-affinity Ni2+ resin and immunoaffinity chromatography respectively. During refolding, purified rAmb t CP could autocatalytically converted to its mature forms displaying a higher enzymatic activity. Moreover, the autocatalytic conversion of proforms to mature forms of nAmb t CP could cause their amount to change in giant ragweed pollen extracts. Then, the allergenicity of Amb t CP was characterized: 23 (33.8%) of 68 Chinese patients with ragweed pollen allergy showed positive IgE binding to nAmb t CP by enzyme-linked immunosorbent assay (ELISA); the result of subsequent ELISA showed that IgE-binding activity of proforms and mature forms of rAmb t CP was different, with positive rate of 39.1% (9/23) and 47.8% (11/23) respectively; Amb t CP showed IgE cross-reactivity with the CP components from short ragweed, Artemisia annua and Artemisia sieversiana pollen. Our findings will help to promote component-resolved diagnosis and treatment for giant ragweed pollen allergy, standardize allergen products and individualize allergen-specific immunotherapy.

Expression, purification and epitope analysis of Pla a 2 allergen from Platanus acerifolia pollen.

Platanus acerifolia is one of the major sources of outdoor allergens to humans, and can induce allergic asthma, rhinitis, dermatitis and other allergic diseases. Pla a 2 is a polygalacturonase and represents the major allergen identified in P. acerifolia pollen. The aim of the present study was to express and purify Pla a 2, and to predict B and T cell epitopes of Pla a 2. The gene encoding Pla a 2 was cloned into the pET28a vector and subsequently transfected into ArcticExpress™ (DE3) Escherichia coli cells; purified Pla a 2 was analyzed by western blot analysis. The results of the present study revealed that the Pla a 2 allergen has the ability to bind immunoglobulin E within the sera of patients allergic to P. acerifolia pollen. In addition, the B cell epitopes of Pla a 2 were predicted using the DNAStar Protean system, Bioinformatics Predicted Antigenic Peptides and BepiPred 1.0 software; T cell epitopes were predicted using NetMHCIIpan ­3.0 and ­2.2. In total, eight B cell epitopes (15­24, 60­66, 78­86, 109­124, 232­240, 260­269, 298­306 and 315­322) and five T cell epitopes (62­67, 86­91, 125­132, 217­222 and 343­350) were predicted in the present study. These findings may be used to improve allergen immunotherapies and reduce the frequency of pollen­associated allergic reactions.

Expression and purification of a major allergen, Pla a 1, from Platanus acerifolia pollen and the preparation of its monoclonal antibody.

Platanus acerifolia pollen is considered an important source of airborne allergens in numerous cities. Pla a 1 is a major allergen from P. acerifolia pollen. The present study aimed to express and purify Pla a 1, and to prepare its monoclonal antibody. In the present study, the Pla a 1 gene was subcloned into a pET­28a vector and transformed into the ArcticExpress™ (DE3) RP Escherichia coli host strain. The purified Pla a 1 was then used to immunize BALB/c mice. When serum detection was positive, spleen cells were isolated from the mice and fused with SP2/0 myeloma cells at a ratio of 10:1. Hybridoma cells were screened by indirect ELISA and limiting dilution. Positive cells were used to induce the formation of antibody­containing ascites fluid, and the antibodies were purified using protein A­agarose. The results of the present study demonstrated that recombinant Pla a 1 was successfully expressed and purified, and exhibited positive immunoglobulin E­binding to serum from patients allergic to P. acerifolia. A total of 11 hybridomas that steadily secreted anti­Pla a 1 antibody were obtained and an immunoblotting analysis indicated that all of these monoclonal antibodies specifically recognized the Pla a 1 protein. These results suggested that specific anti­Pla a 1 antibodies may be obtained, which can be used for the rapid detection of Pla a 1 allergens and in the preparation of vaccines against P. acerifolia pollen.

Expression, purification and epitope analysis of Pla a 3 allergen from Platanus acerifolia pollen.

Platanus acerifolia (P. acerifolia) is an important cause of pollinosis in cities. The use of allergen extracts on patients with allergic diseases is the most commonly applied method to attempt to treat pollinosis. Pla a 3, a non­specific lipid transfer protein, is a major allergen present in P. acerifolia pollen extracts. In the present study, the Pla a 3 gene was sub­cloned into a pSUMO­Mut vector using Stu I and Xho I sites and transformed into the Arctic Express™ (DE3) RP E. coli host strain. The purified Pla a 3 allergen was analyzed by western blotting and the results revealed that the Pla a 3 allergen has the ability to bind IgE in the P. acerifolia pollen of allergic patients' sera. Moreover, the authors predicted the potential B cell epitopes of the Pla a 3 allergen using the DNAStar Protean system, the Bioinformatics Predicted Antigenic Peptides system and the BepiPred 1.0 server. In addition, the T cell epitopes were predicted by the SYFPEITHI database and the NetMHCII­2.2 server. As a result, two B cell epitopes (35­45 and 81­86) and four potential T cell epitopes including 2­15, 45­50, 55­61 and 67­73 were predicted in the present study. The current results can be used to contribute to allergen immunotherapies and useful in peptide­based vaccine designs of pollen allergy.