Cloning, recombinant expression and validation by microarray of potentially allergenic food proteins

Author(s)

Gabriela Justamante Händel Schmitz

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ)
• Defense date: 2019-03-12

Examining board members

João Roberto Oliveira do Nascimento; Fabio Fernandes Morato Castro; Fernando Luiz Affonso Fonseca; Helder Takashi Imoto Nakaya

• Advisor: João Roberto Oliveira do Nascimento

Abstract

The majority of allergic reactions to foods is IgE-mediated, which can be detected for the diagnosis of food allergy. However, purified allergens are necessary to produce different kinds of allergy tests, including microarray, which is a useful tool for simultaneous analysis, as well as for the identification of cross-reactivity. In this respect, it is essential to expand the platform of allergens to include them on microarrays. Nowadays, some foods that are object of interest in the clinical area in Brazil and it is necessary a further evaluation about their potential allergens, since there is a limited information about them, are: pineapple, papaya, cassava and mango. Therefore, the aim of this study was cloning, expressing and purifying potentially allergenic proteins of important Brazilian foods. After confirmed by immunological tests, these proteins were used in microarray production and validation by assays with sera from allergic patients to the selected foods. Achieving this goal, matching potentially allergenic proteins were selected, which were identified by comparison among taxonomically closer species (in silico) and 2D Western Blotting coupled with Mass Spectrometry. Nineteen proteins: 4 from pineapple, 5 from papaya, 6 from cassava and 4 from mango were expressed in Pichia pastoris, purified and printed on a microarray. After incubating those proteins with sera from allergic patients to the selected foods, 18 proteins were detected as potentially allergenic. In addition, cross-reactivity was observed among the proteins from the studied foods, and also regarding to the latex and other fruits. (AU)

• FAPESP's process: 16/01308-7 - Cloning, recombinant expression and validation by microarray of potentially allergenic food proteins.
• Grantee: Gabriela Justamante Handel Schmitz
• Support Opportunities: Scholarships in Brazil - Doctorate