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Comparing Influenza Virus Hemagglutinin (HA) Expression in Three Different Baculovirus Expression Systems

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Comparing Influenza Virus Hemagglutinin (HA) Expression in Three Different Baculovirus Expression Systems Comparing influenza virus hemagglutinin (HA) expression in three different baculovirus expression systems by Alexandra Elliott A thesis presented to The University of Guelph In partial fulfilment of requirements for the degree of Master of Science in Molecular and Cellular Biology Guelph, ON, Canada ©Alexandra Elliott, August, 2012 ABSTRACT COMPARING INFLUENZA VIRUS HEMAGGLUTININ (HA) EXPRESSION IN THREE DIFFERENT BACULOVIRUS EXPRESSION SYSTEMS Alexandra Elliott Advisor: University of Guelph, 2012 Professor P.J. Krell Co-advisor: Dr. Éva Nagy In this study, the expression of HA, a key immunogenic protein of influenza viruses, in insect cells was compared using three baculovirus expression strategies: protein over-expression, surface (GP64) display, and capsid (VP39) display. Further, a recombinant virus expressing NA, another immunogenic influenza virus protein, was generated and fused to an HA epitope-tag. Western immunoblot using various antibodies, including those against HA, demonstrated the expression of HA and NA for all recombinant viruses. HA showed stronger expression when fused to the C-terminus of VP39 than the N-terminus, but unlike other expression methods, there was no observable cleavage of HA in VP39-displayed viruses. Cells infected with only over- expressed and surfaced-displayed HA were biologically active, and capable of hemadsorption and hemagglutination of chicken red blood cells. These results suggest that GP64 display or over-expression are the most efficacious modes of HA-expression for use as antigen to detect anti-HA antibodies in poultry. ACKNOWLEDGEMENTS My gratitude goes to my co-advisors, Dr. Peter Krell and Dr. Éva Nagy for giving me the opportunity to complete my graduate research in their laboratories. I would also like to thank you for your consistent support, advice, and for thoroughly enriching my graduate experience. I would also like to acknowledge Dr. Sarah Wootton, the third member of my committee, for contributing ideas, offering guidance and supporting me throughout this research. A number of materials were received from colleagues, enabling this research to take place. Thank you to Dr. Gary Blissard at Cornell University in Ithaca NY, for supplying us with the monoclonal anti-GP64 antibody. Thank you to Dr. Congyi Zheng and Qingzhen Liu (Wuhan University, Wuhan, China) for supplying us with the H5, N1 and M2 cDNA clones. Thank-you to Dr. Robert Webster at the St. Jude’s Childrens’ Research Hospital in Memphis, TN for supplying the monoclonal anti-HA antibodies. Thank you to Dr. Rob Kotin for supplying us with the anti-VP39 antibody. Thank-you to Dan-hui Yang, a previous post-doctoral fellow in the Krell and Nagy labs for supplying us with the FAdV-HA viruses and serum from vaccinated chickens. Thank you to OGS, OMAFRA and NSERC for funding this research. The members of the Krell lab have been indispensible to the completion of this research. Thank you to Jeffrey Hodgson for training me in the lab and for your continued support and plentiful guidance throughout this research. Thank you to James Ackford, an undergraduate project student for your contribution to the NA over-expression work. Thank you to Yang, Dave, Mike, J.D. and Guozhong for your friendship, advice and encouragement. Without the support of my family and friends, the completion of this Masters would not have been possible. Thank you to my parents, Anne and Morris, brother Cameron, sister Lauren, and brother-in-law Ned for your love and support. Finally, I would like to thank my partner and best friend Nathan for your continued emotional support throughout the past two years. It is such a comfort to know that no matter what, you are always in my corner. iii TABLE OF CONTENTS Title Page ........................................................................................................................................ i Abstract .......................................................................................................................................... ii Acknowledgements ....................................................................................................................... iii Table of Contents .......................................................................................................................... iv List of Figures .............................................................................................................................. vii List of Tables ................................................................................................................................ ix List of Abbreviations ......................................................................................................................x List of Virus and Protein Abbreviations ...................................................................................... xii Chapter 1: Review of the Literature .................................................................................................1 1.1 Influenza and influenza virus ...........................................................................................1 1.2 Hemagglutinin and neuraminidase ...................................................................................5 1.3 Evolution of influenza viruses and avian influenza virus ................................................8 1.4 HPAI control strategies in humans ................................................................................11 1.5 HPAI control strategies in poultry ..................................................................................14 1.6 Baculoviruses as expression vectors ..............................................................................15 1.7 Baculovirus expression, overexpression and display .....................................................18 1.8 Applications of baculoviral foreign gene expression to influenza virus research .........23 1.9 Research objectives and experimental design ................................................................27 Chapter 2: Materials and Methods .................................................................................................30 2.1 Cells and virus ...............................................................................................................30 2.1.1 Insect cell culture ...............................................................................................30 2.1.2 Influenza virus, HA and NA clones ...................................................................30 2.2.General DNA manipulation ............................................................................................30 2.2.1 Bacterial cultures and plasmid isolation ...........................................................30 2.2.2 Restriction digestions and PCR..........................................................................31 2.2.3 Plasmid construction and cell transformation ...................................................32 2.3 Generation of recombinant baculovirus constructs ......................................................32 2.3.1 Removal of histidine (6-His) tag from pFastbacB ............................................32 2.3.2 Generation of ΔHispFastBacBHAFLAG .............................................................35 2.3.3 Generation of ΔHispFastBacBHA:VP39FLAG and ΔHispFastBacBVP39:HAFLAG (VP39 fusion constructs) .............................................................................................35 2.3.4 Generation of ΔHispFastBacBHA:GP64FLAG ...................................................37 2.3.5 Generation of ΔHisPFastbacBNAHA .................................................................38 iv 2.4 Construction, amplification and titration of recombinant baculoviruses .......................38 2.4.1 Generation of recombinant bacmids using the Bac-toBac system ....................38 2.4.2 Purification of bacmid DNA ..............................................................................39 2.4.3 Transfection, amplification and titration of recombinant viruses ......................40 2.5 Infections and concentration of recombinant budded virions .......................................41 2.5.1 Infection of insect cells with recombinant virus ..............................................41 2.5.2 Virus growth curve.............................................................................................42 2.5.3 Recovery of infected cells and general protein sample preparation ..................42 2.5.4 Concentration of recombinant budded virions ...................................................43 2.5.5 Quantification of total protein (Bradford assay) ................................................43 2.6 General SDS-PAGE and Western Immunoblot ............................................................44 2.6.1 SDS-PAGE.........................................................................................................44 2.6.2 Western Immunoblot..........................................................................................44 2.6.3 Coomassie blue staining to confirm equal loading ............................................45 2.7 Assessment of HA activity in insect cells ......................................................................46 2.7.1 Collection and preparation of red blood cells ...................................................46 2.7.2 Hemagglutination assays ...................................................................................46 2.7.3 Hemadsorption assays
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