Study On Allergen Of Alternaria Alternata

Alternaria Alternata is one kind of common and important inhalant allergens. It can induce allergic asthma and allergic rhinitis. High-quality extraction of alternaria alternata is very important for specific diagnosis and therapy. In this study, alternaria alternata was cultured, identified, extracted, analyzed and fingerprinted by two-dimensional electrophoresis.The research was expressed in five parts to facilitate discussionPart 1: Clinical feature of patients who are sensitive to alternaria alternata is studied and standard pooled serum is collectedIn order to study the clinical feature of the patients who are sensitive to alternaria alternata, fifty-five patients were studied and their serum was collected from august 2005 to may 2007, aged 6～75. Among fifty-five patients, 43 (78.2%) were suffering from allergic asthma, 30 (54.5%) were from allergic rhinitis, 18 (33%) were from allergic rhinitis with allergic asthma.In order to study the allergen of alternaria alternata, of fifty-five serum, twenty were collected for individual serum and pooled sera.Part 2: Alternaria alternata culturation and identificationThe aim of this study was to culture and identify alternaria alternata. Petri dish was used to get alternaria alternata colonies from air. Alternaria alternata was cultured and appraised by morphology and molecular technology.Fungus that cultured for seven days, its colony show brown and black, the spores was ovoid. Its large subunit (26S) ribosomal RNA gene was amplified by PCR, sequenced by sequencer and blasted by genebank, the similarity with Alternaria alternata was over 99%.The fungus that was cultured in this study was identified to be alternaira alternata.Part 3: Research on extract allergen of alternaria alternata based on different extraction method In order to find the best method to extract the allergen ofalternaria alternata, four different extracting procedures with burnisher, liquid nitrogen, burnisher plus ultrosonic and with liquid nitrogen plus ultrosonic separately were tested and compared. Extracts was analyzed by bradford, SDS-PAGE and RAST inhibition test.Results: Among four different extractions, the extraction with liquid nitrogen plus ultrosonic had the highest protein recovery, the largest number of protein bands and the lowest protein in fifty percent inhibition rates.Part 4: SDS-PAGE immunoblotIn order to study the allergen of the extraction ofalternairia alternata, we used SDS-PAGE immunoblot to analyze the extraction obtained by liquid nitrogen plus ultrosonic method.Results: Seven protein bands which bind to Alternaria alternata-specific IgE were shown by SDS-PAGE immunoblot, molecular weight of them were 92 KD, 87KD, 67KD, 53KD, 45KD, 29KD, 22KD; of seven allergens, three were bound by more than half of sera as chief allergen which were 92KD, 53KD, 22KD.Part 5: The extraction of alternaria alternata was analyzed by two-dimensional electrophoresis (2-DE)To develop an identification method to the allergen of alernaria alternata. Protein dots of alternaria alternata were detected by 2-DE.Results: 1450±35 protein spots were identified by the Two-dimensional electrophoresis (2-DE) and six bioactivity proteins were found by 2-DE immunoblot. In our study, fingerprinting based on 2-DE was used to identify alternaria alternata for its high specificity, but to further study is necessary.