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Development of a Replication-Defective Baculovirus Platform Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2020 Development of a replication-defective baculovirus platform Scott Ryan Ackerman Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Recommended Citation Ackerman, Scott Ryan, "Development of a replication-defective baculovirus platform" (2020). Graduate Theses and Dissertations. 18266. https://lib.dr.iastate.edu/etd/18266 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Development of a replication-defective baculovirus platform by Scott Ackerman A thesis submitted to the graduate faculty in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Major: Immunobiology Program of Study Committee: Brad Blitvich, Co-major Professor Eric M. Vaughn, Co-major Professor Jesse Hostetter The student author, whose presentation of the scholarship herein was approved by the program of study committee, is solely responsible for the content of this thesis. The Graduate College will ensure this thesis is globally accessible and will not permit alterations after a degree is conferred. Iowa State University Ames, Iowa 2020 Copyright © Scott Ackerman, 2020. All rights reserved. ii TABLE OF CONTENTS Page LIST OF FIGURES ....................................................................................................................... iv LIST OF TABLES ...........................................................................................................................v ACKNOWLEDGMENTS ............................................................................................................. vi ABSTRACT .................................................................................................................................. vii CHAPTER 1. INTRODUCTION ....................................................................................................1 Background ................................................................................................................................ 1 Objectives .................................................................................................................................. 1 Thesis Organization ................................................................................................................... 2 CHAPTER 2. LITERATURE REVIEW .........................................................................................3 A Brief History of Vaccines ...................................................................................................... 3 Porcine Epidemic Diarrhea Virus as a Vaccine Target ............................................................. 4 Baculovirus Expression Vector Systems ................................................................................... 5 Utility of the Baculovirus Platform and Potential Concerns ..................................................... 8 Plasmid-based Protein Expression in Insect Cells ................................................................... 10 Lambda Red Recombineering ................................................................................................. 11 Baculovirus Envelope Protein gp64 ........................................................................................ 12 Baculovirus Display of Envelope Glycoproteins .................................................................... 13 CHAPTER 3. DEVELOPING A REPLICATION-DEFECTIVE BACULOVIRUS PLATFORM ..................................................................................................................................15 Introduction ............................................................................................................................. 15 Materials and Methods ............................................................................................................ 15 Design of Expression vectors for gp64 Constitutive Expression in Insect Cells ............... 15 Design and Construction of a Red Recombination Vector for the AcMNPV gp64 Locus .................................................................................................................................. 17 Recombination to Remove gp64 from AcMNPV .............................................................. 18 Alternate Recombination to Replace gp64 with PEDV Spike Protein .............................. 19 Flow Cytometry Analysis of gp64 Expression by Transfected Insect Cells ...................... 19 Immunofluorescence-based Staining ................................................................................. 20 Sucrose Gradient Fractionation .......................................................................................... 20 SDS-PAGE and Western Blotting ...................................................................................... 21 qPCR of gp64 and Blasticidin Resistance Gene Integration Rates in Sf9 Cells ................ 21 DNA Purification ............................................................................................................... 21 Immunoprecipitation .......................................................................................................... 22 Next Generation Sequencing for Confirmation of Gene Deletion .......................................... 22 Results and Discussion ............................................................................................................ 23 Preparation of gp64-Complementing Insect Cell Line ....................................................... 23 Deletion of gp64 from AcMNPV ....................................................................................... 25 iii Recombination to Replace gp64 with PEDV Spike Protein .............................................. 26 Reduced Viral Titers in gp64-deleted Baculovirus Grown in gp64-Complementing Cells .................................................................................................................................... 27 Expression of PEDV Spike in Standard Sf9 Cells without gp64 ....................................... 30 CHAPTER 4. CONCLUSIONS ....................................................................................................32 REFERENCES ..............................................................................................................................34 APPENDIX. TABLES AND FIGURES .......................................................................................37 iv LIST OF FIGURES Page Figure 1. Map of the pIB V5/His plasmid. ................................................................................... 39 Figure 2. Relative location of polh and gp64 promoter regions of the AcNPV bacmid............... 40 Figure 3. A visual representation of two-step Red recombination ............................................... 41 Figure 4. A visual representation of alternate recombination method .......................................... 41 Figure 5. Western blot of transfection supernatants showing expression of gp64. ...................... 42 Figure 6. Flow cytometric evaluation of gp64 expression with anti-gp64 mAb Acv5................. 43 Figure 7. gp64 expression in stably transfected Sf9 cells without Blasticidin maintenance. ....... 44 Figure 8. Transfection of standard Sf9 cells and gp64-complementing Sf9 Op64 #2 cells with gp64-deleted bacmid. ....................................................................................... 45 Figure 9. Immunofluorescence-based staining of Replication-defective Baculovirus Constructs in Sf9 and Sf9 Op64 #2 Cells. ................................................................ 46 Figure 10. Western blot of PEDV Spike Expression. ................................................................... 47 Figure 11. gp64 Modified Baculovirus Titers............................................................................... 48 Figure 12. Differing Infectivity of gp64 Deleted Baculovirus in Sf9 Op64 Cells vs Sf9 Ac64 Cells. ............................................................................................................... 48 Figure 13. Sucrose gradient fractions of BaculoG/Δgp64 PEDVS BD. ....................................... 49 Figure 14. Western Blots of BaculoG/Δgp64 PEDVS BD Immunoprecipitation. ....................... 50 v LIST OF TABLES Page Table 1. Antibodies for Immunofluorescence-based staining ...................................................... 37 Table 2. Antibodies for Western Blot ........................................................................................... 37 Table 3. qPCR Primer/Probe Sequences ....................................................................................... 38 Table 4. Insect Cell Expression Plasmids with Alternate Promoters............................................ 38 vi ACKNOWLEDGMENTS I would like to thank my co-major professors Dr. Eric Vaughn and Dr. Brad Blitvich, along with committee member Dr. Jesse Hostetter for their instruction and guidance throughout the course of my research and coursework. I would also like to thank my colleagues at Boehringer-Ingelheim Animal Health for their input and guidance in my development
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