Purification , Identification And Mass Spectrum Analysis Of Major Allergen In Periplaneta Americana

Background Allergic diseases are defined as a worldwide important health problem by WHO. The prevalence of allergic diseases in most human populations is steadily increasing and up to 15-30%, especially in children after urbanization. It may be related to the allergen exposure. Inhalation of environmental allergens from pollens,mites,insects and fungi induces IgE antibody responses, which can cause immediate hypersensitivity reactions and may result in the development of allergic diseases such as rhinitis, asthma and atopic dermatitis in genetically susceptible individuals. The cockroach is one of the most important sources of allergen. In south China, Periplaneta Americana is the major domiciliary cockroach and predominant species. The whole body extract of Periplaneta Americana is difficult to standardize and contains a large amount of non-specific proteins, hence it has many disadvantages for the diagnosis and immunotherapy. Therefore, the naturally purified allergen may be valuable for the research of diagnostic reagent and immunotherapeutic vaccine in the allergic diseases. Here we report the purification and identification of major allergens of Periplaneta Americana. Objective To isolate and identify the major allergen of Periplaneta Americana. Methods Male and female adult whole bodies of Periplaneta Americana were extracted in the Coca's solution after defatting. Sequential purification of two steps including DEAE-cellulose 52 ion exchange chromatography and Hiprep Sephacryl? S-200 gel filtration chromatography were carried out to obtain the purified allergen from the crude extract of Periplaneta Americana. It was analysed by sodium dodcyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and identified by western blotting. After in-gel digestion of the purified protein ,the tryptic peptides mapping of major allergen was run by HPLC－ESI－MS/MS. The data was then searched against NCBInr database by using MS/MS Ions search (http://www.matrixscience.com). Results The results showed that crude extract contained 24 proteins by SDS-PAGE. Western blotting analysis indicated that the obtained protein with molecular weight 74kDa is the major allergen of Periplaneta Americana which is able to react with serum specific IgE (sIgE) from 93.8%(15/16) of atopic patients sensitive to cockroach. Also the major allergen was identified by in-gel digestion and HPLC-ESI-MS/MS, the purity and the recovery of target allergen were up to 90% and 26%, respectively. Conclusions The 74kDa protein is the major allergen of Periplaneta Americana. The purification strategy set up for the gaining of the allergen is simple and effective; and HPLC-ESI-MS/MS, to our knowledge, is first adopted for the identification of major allergen from Periplaneta Americana. Present study may help the development of allergen protein chip.