A Murine Model Of Mango Fruit Allergy And The Immunological Identification Of Mango Chitinase

Background: The incidence of allergic diseases has been increasing in the past decades in parallel with global industrialization.Mango is an important source of fruit-derived allergens.Consumption of mango or even contact with processed mango food products can cause serious allergies,including dermatitis,oral allergy syndrome and asthma,etc.Up to now,most research publications on mango only focus on case reports,reactivity or crossreactivity of patient's serum IgE with mango crude extract protein.The understanding of protein allergens and their sensitization mechanisms of mango fruit are still lacking.Objective:(1)To explore the sensitization mechanism of mango fruit allergy by establishing a murine model.(2)To identify a new allergen from mango and explore its allergenicity,epitopes and structure.Methods:1)A murine model of mango fruit allergy.Six female Balb/c mice of each group,6-8weeks,were sensitized on days 0,3,7,with an intraperitoneal injection of 150 ?l saline containing 20 ?g MCEP and 2 mg Alum,and challenged on days 14,16,18,20,with nasal drop of 20 ?l saline containing 50 ?g MCEP.Normal control mice were sensitized and challenged with saline.Mice were sacrificed on day 21.Mouse serum,bronchoalveolar lavage fluid(BALF)and spleen cell culture supernatants were collected for the detection of specific antibodies(IgE,IgG1),cytokines(IL-4,IFN-?,IL-5,TSLP,TNF-?,IL-17A).Histopathological changes in the lungs were also visualized to evaluate the model.2)Cloning,expression and immunological identification of a new major allergen from mango.Western blot and Mass spectrometry were used to identify a new IgE-reactive protein band.Deduced coding gene of the band was synthesized and inserted into a recombinant plasmid and expressed as a fusion protein.The epitopes and immunogenicity of the protein were assayed by sera of mango allergic patients or mice.Results:1)A murine model of mango fruit allergy.In comparison with the control mice,the mice in the model group had aggravated inflammation in the lungs and elevated secretion of bronchial mucus;more eosinophils in the BALF were induced(p<0.001).cytokines IL-4(Th2 type)and IL-17A(Th17 type)as well as TSLP were augmented(p<0.01,p<0.01 and p<0.001,respectively);the expression of a chemokine mMCP-1 was enhanced(p<0.05);significant higher level of IgE antibody was observed(p<0.05);Similarly,much more IL-4 and TNF-? were detected in the supernatant of spleen cells(p<0.01),while the level of Th1 type cytokine IFN-? was damped(p<0.001).2)Cloning,expression and immunological identification of a new major allergen from mango.Sequence of the newly found IgE-reactive band revealed that it is a chitinase from mango(GenBank: ACD69683.1).The chitinase was fused with tag and expressed in E.coli and purified by affinity column or the ion exchange column.Western blot studies showed it reacted with 76% sera of 13 mango allergic patients.We therefore name this major allergen as Man i 2 in accordance with the international allergen nomenclature.Dot blot studies showed the 8-22 aa of mango chitinase was the main linear IgE binding epitope.Conclusion:(1)A murine model of mango fruit allergy was successfully established,confirming that type I hypersensitivity could be induced by MCEP in mice.(2)A new major mango allergen was identified as a chitinase and named Man i 2.