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Identification, Characterization, and Epitope Mapping of Tree Nut Allergens Jason M Florida State University Libraries Electronic Theses, Treatises and Dissertations The Graduate School 2006 Identification, Characterization, and Epitope Mapping of Tree Nut Allergens Jason M. Robotham Follow this and additional works at the FSU Digital Library. For more information, please contact [email protected] THE FLORIDA STATE UNIVERSITY COLLEGE OF ARTS AND SCIENCES IDENTIFICATION, CHARACTERIZATION, AND EPITOPE MAPPING OF TREE NUT ALLERGENS By JASON M. ROBOTHAM A Dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy Degree Awarded: Spring Semester, 2006 Copyright © 2006 Jason Robotham All Rights Reserved The members of the Committee approve the Dissertation of Jason M. Robotham defended on January 27, 2006. __________________________ Kenneth H. Roux Professor Directing Dissertation __________________________ Shridhar K. Sathe Outside Committee Member __________________________ Peter G. Fajer Committee Member __________________________ Thomas C.S. Keller III Committee Member __________________________ Kenneth A. Taylor Committee Member Approved: __________________________ Timothy S. Moerland, Chair, Department of Biological Science __________________________ Joseph Travis, Dean, College of Arts and Sciences The Office of Graduate Studies has verified and approved the above named committee members ii ACKNOWLEDGEMENTS I would like to thank Kenneth Roux for being a patient advisor, mentor, and most importantly a good friend over the course of my dissertation work. I would like to thank my committee members Shridhar Sathe, Thomas Keller, Peter Fajer, and Kenneth Taylor for their assistance in my development as a scientist. Dr. Suzanne Teuber made much of my research possible through a fruitful collaboration. I am also grateful to Drs. Kirsten Beyer and Hugh Sampson for their generosity in supplying our lab with sera and general advice and Dr. Rudolf Jung for supplying me with the soybean glycinin gene when it seemed like nobody else would. I’d like to thank all the former and current lab technicians, graduate students, and postdoctoral fellows from the Roux lab. In particular, I’d like to thank Shirley Roux for helping me to adjust to graduate school early on, Pallavi Tawde for our many thought- filled conversations regarding our work together, Ping Zhu for demonstrating a work ethic that I still strive to achieve, Fang Wang for helping introduce me to Molecular Biology, and both Dillon Fritz and Henry Grise for their support as scientist and friends. I’d like to thank all the support personnel that made the completion of this project possible, including the Analytical lab, the Sequencing lab, the Cloning lab, and the KLB facilities. In particular, I’d like to thank Margaret Seavy for her patience and hard work throughout my many efforts at protein purification and Rani Dhanarajan for teaching me nearly everything I know about the technical aspects of Molecular biology and cell culturing. I’d also like to thank my fellow graduate students throughout the department for their friendship and for creating both a work and non-work community of which I am proud to say I was a part. Finally, a special thanks goes out to my family. I could never have succeeded in graduate school without the advice and support of my older brother and closest friend, Claude Robotham. Similarly, I could have never succeeded in life without the unconditional love and support of my parents, Maria and Kenrick Robotham. How they managed to put two sons through a combined 20 years of higher education is beyond me and if I could love them more I would, but it is not possible. iii TABLE OF CONTENTS List of Tables ................................................................................................ Page vi List of Figures ................................................................................................ Page vii Abstract ...................................................................................................... Page viii 1. FOOD HYPERSENSITIVITY AND TREE NUT ALLERGY...................... Page 1 2. IDENTIFICATION AND CHARACTERIZATION OF IMPORTANT ALLERGENS IN THE ENGLISH WALNUT AND CASHEW TREE NUT BELONGING TO THE 2S ALBUMIN AND 11S GLOBULIN FAMILIES Introduction ................................................................................................ Page 4 Methods ................................................................................................ Page 5 Results ............................................................................................... Page 12 Discussion ............................................................................................... Page 18 3. LINEAR IGE-EPITOPE MAPPING OF ENGLISH WALNUT AND CASHEW NUT 2S ALBUMIN AND 11S GLOBULIN ALLERGENS Introduction ................................................................................................ Page 22 Methods ................................................................................................ Page 23 Results ................................................................................................ Page 26 Discussion ............................................................................................... Page 40 4. IDENTIFICATION OF A CASHEW 11S GLOBULIN CONFORMATIONAL EPITOPE Introduction ................................................................................................ Page 42 Methods ................................................................................................ Page 44 iv Results ............................................................................................... Page 50 Discussion ............................................................................................... Page 57 5. HOMOLOGY MODELING OF VARIOUS 11S GLOBULIN ALLERGENS Introduction ................................................................................................ Page 61 Methods ................................................................................................ Page 62 Results ............................................................................................... Page 62 Discussion ............................................................................................... Page 69 CONCLUSION ................................................................................................ Page 72 REFERENCES ................................................................................................ Page 74 BIOGRAPHICAL SKETCH .............................................................................. Page 83 v LIST OF TABLES 2.1 Primers used for subcloning of walnut and cashew 2S albumin and 11S globulin genes into the pMAL-c2-His expression vector........................... Page 7 2.2 Clinical characteristics of cashew and walnut-allergic subjects................. Page 10 2.3 Proteins showing identity and similarity to Ana o2.................................... Page 13 2.4 Proteins showing identity and similarity to Ana o 3................................... Page 13 3.1 Mutational analysis of the Jug r 1 IgE-binding epitope, E1 ....................... Page 28 3.2 Ana o 3 IgE-binding peptides and relative intensity................................... Page 31 3.3 Jug r 4 IgE-binding peptides and relative intensity .................................... Page 33 3.4 Ana o 2 IgE-binding peptides and relative intensity................................... Page 34 4.1 Primers used for the amplification and subcloning of cashew and soybean Page 45 11S globulin genes and constituent subunits .............................................. 4.2 Primers used to amplify Ana o 2 and Gy1 glycinin large and small subunits Page 46 for use in blunt end ligation reactions......................................................... 5.1 Percent identity and similarity of five 11S globulin allergens.................... Page 63 vi LIST OF FIGURES 2.1 Purification of the expressed recombinant Ana o 2-MBP fusion protein... Page 14 2.2 Western immunoblotting and rJug r 1 inhibition blotting of walnut extract in reducing conditions with patient #3 sera ................................................ Page 15 2.3 Western immunoblotting and rJug r 4 inhibition blotting of walnut extract in reducing conditions with patient #3 sera. ............................................... Page 16 2.4 Western immunoblotting and rAna o 3 inhibition blotting of cashew extract in reducing conditions with patient #31 sera. ............................................. Page 17 2.5 Western immunoblotting and rAna o 2 inhibition blotting of cashew extract in reducing conditions with patient #31 sera .............................................. Page 18 3.1 Schematic representation of solid phase overlapping peptide synthesis of Jug r 1 ................................................................................................ Page 27 3.2 IgE binding analysis of Jug r 1 SPOTs ....................................................... Page 27 3.3 Isolation and inhibition of E1-specific IgE................................................. Page 30 3.4 Amino acid sequence and linear epitope comparison of cashew (Ana o 3), walnut (Jug r 1), sesame, and mustard seed (Sin a 1, segment) 2S albumins Page 32 3.5 Mutational analysis of two immunodominant Ana o 2 IgE-binding peptides Page 35 3.6 Comparison of the IgE-binding regions of Jug r 4 and Ana o 2 with various allergenic 11S globulins.............................................................................
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