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Molecular and Immunological Characterisation of Proteins from Anisakis Pegreffii and Their Immune Stimulatory Effect on the Human Health System

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Molecular and Immunological Characterisation of Proteins from Anisakis Pegreffii and Their Immune Stimulatory Effect on the Human Health System Title Molecular and immunological characterisation of proteins from Anisakis pegreffii and their immune stimulatory effect on the human health system A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) Abdouslam Hassan Asnoussi Alsharif (M.Sc. in Applied Biology & Biotechnology) School of Applied Sciences College of Science Engineering and Health RMIT University June 2015 Declaration I certify that except where due acknowledgement has been made, the work is that of the author alone; the work has not been submitted previously, in whole or in part, to qualify for any other academic award; the content of the thesis/project is the result of work which has been carried out since the official commencement date of the approved research program; any editorial work, paid or unpaid, carried out by a third party is acknowledged; and, ethics procedures and guidelines have been followed. Abdouslam Hassan Asnoussi Alsharif 27/06/2015 i ACKNOWLEDGMENTS I would like to express my sincere thanks and deepest gratitude to my supervisors Professor Andreas Lopata, Professor Peter M. Smooker and Professor Robin Gasser for their guidance, constant support, advice and encouragement throughout the course of my study. I gratefully acknowledge the financial support provided by Ministry of Higher Education in Libya- Scholarship Program, without which my PhD studies would not be possible. I thank Luke Norbury, Natalie Kikidopoulos, Aya Taki, Ibukun Aibinu, Eltaher Elshgmani, Khaled Allemailem, Mohamed Said, Abdulatif Mansur and members of the biotechnology laboratory for their support and encouragement all the way. I am not able to mention everybody’s name here but I hold all members of the biotechnology laboratory dear to my heart and I thank you all for your assistance and friendship. An honorable mention to my wife (Fatma) for being a great companion with whom I shared my frantic moments, unrealistic ideas, tears, fears and triumphant times in Australia over the past 5 years, thank you darlyn! My family is truly a treasure and can never be replaced. During my journey, my little princesses (girls), (Fatema and Moona), and princes (sons), (Hassan and Eyad) came to this world making our life a lot happier and full of fun (thank you for sharing the love and fun). Last but definitely not least, thanks from the bottom of my heart to my parents and my brothers and sisters for their love, patience and support over the many years. I dedicate this thesis to my parents, my family and my kids to thank them for all their love, encouragement and support during the pursuit of achieving my aspirations. Although I and my parents were separated by distance, their presence prevailed through their thoughts and prayers for me. My love and respect will remain strong and high for them, I hope I have made them proud! Abdouslam Alsharif ii Table of Contents Title ...................................................................................................................................................... 1 ACKNOWLEDGMENTS .......................................................................................................................... ii List of Figures ..................................................................................................................................... xii List of Tables ...................................................................................................................................... xiv Published article from this research work…………………………………………………………………………………….xvi Abbreviations ................................................................................................................................... xvii Abstract ............................................................................................................................................. xix Chapter one: Introduction .................................................................................................................. 1 Chapter two: Literature review ........................................................................................................... 5 2.1 Allergy........................................................................................................................................ 6 2.2 Food allergy ............................................................................................................................... 6 2.2.1 Changes in the Prevalence of Food Allergies................................................................................ 9 2.3 Mechanism of IgE mediated-allergic reaction ........................................................................ 10 2.4 Allergens .................................................................................................................................. 10 2.4.1 Allergen History ....................................................................................................................................... 11 2.4.2 Allergen Nomenclature .......................................................................................................................... 11 2.4.3 Allergen Structure (Isoallergens and isoforms) ............................................................................. 12 2.4.4 Types of allergens .................................................................................................................................... 13 2.4.4.1 Proteases .................................................................................................................... 13 2.5 Allergens in the Different Seafood Groups ............................................................................. 14 2.5.1 Seafood Allergens ................................................................................................................................... 14 2.5.2 Studies of Fish Allergens ...................................................................................................................... 17 2.5.3 Studies of Crustacean Allergens ......................................................................................................... 18 2.5.4 Studies of Mollusk Allergens............................................................................................................... 20 2.5.5 Studies of parasite Anisakis Allergens .............................................................................................. 22 2.6 Tropomyosin ........................................................................................................................... 23 2.6.1 Tropomyosin as Allergens in A. simplex and other Invertebrate Groups ............................... 24 2.7 Cysteine proteinase inhibitors (CPIs) ...................................................................................... 26 2.7.1 Cysteine-protease inhibitors as Allergens in A. simplex and other Invertebrate Groups .. 28 2.8 Allergen cross-reactions .......................................................................................................... 28 2.8.1 Antigenic and allergenic cross-reactions between A. simplex (s.s.) and other Anisakis nematodes ............................................................................................................................................................. 29 2.8.2 Antigenic and allergenic cross-reactions between A. simplex (s.s.) and anisakid nematodes ............................................................................................................................................................. 29 iii 2.8.3 Antigenic and allergenic cross-reactions between A. simplex (s.s.) and other groups of nematodes ............................................................................................................................................................. 30 2.8.4 Antigenic and allergenic cross-reactions between A. simplex (s.s.) and non-nematode environmental, food allergens or transmissible agents ........................................................................... 31 2.9 The Life Cycle of Anisakis species ............................................................................................ 31 2.10 Genetics of parasite infections .............................................................................................. 33 2.10.1 Significance of parasites and studying genetic variation .......................................................... 33 2.10.2 DNA regions used for studying genetic variation in parasites ................................................ 34 2.10.2.1 Ribosomal DNA (rDNA) ........................................................................................ 35 2.10.2.2 Mitochondrial DNA ................................................................................................ 36 2.10.3 Genetic variation of rDNA on the internal transcribed spacers (ITS) of nematodes ....... 37 2.10.4 Morphological and molecular approaches ..................................................................................... 37 2.10.4.1 Morphology-based approaches................................................................................ 37 2.10.4.2 Molecular methods for studying genetic variation in parasites ............................... 38 2.10.4.2.1 Protein-based approaches ................................................................................. 38 2.10.4.2.2 DNA-based approaches ...................................................................................
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