ABSTRACT New Viral Vectors for the Expression of Antigens and Antibodies in Plants Zun Liu, Ph.D. Mentor: Christopher M. Kearney, Ph.D. Plants viruses are increasingly being examined as alternative recombinant protein expression systems. Future development of plant virus expression vectors needs to focus on the most important economic hosts, namely cereals and legumes, to develop tools to aid breeding of such hosts and systems for edible vaccine production. Sunn hemp mosaic virus (SHMV) is a tobamovirus, which infects leguminous plants. This work reports on new SHMV-based viral vectors for high yield of target proteins in legumes. In the SHEC vector series, the coat protein gene of SHMV was substituted by a reporter gene. In the SHAC vector series, the coat protein was substituted by a reporter gene and the coat protein gene from another tobamovirus, tobacco mild green mosaic virus (TMGMV). Co-agroinoculation of SHEC/GFP with an RNA silencing suppressor resulted in high levels of local GFP expression by 3 days post inoculation. Co-agroinoculation with SHAC/GFP led to systemic fluorescence in 12-19 dpi. Foxtail mosaic virus (FoMV) is a species of the group Potexvirus, which infects cereal plants. A new viral vector series named FECT was constructed by eliminating the triple gene block and coat protein genes, reducing the viral genome by 29%. Interestingly, agroinoculation of the vector alone results in only slight transient expression, whereas co-inoculation with silencing suppressor genes allows for GFP expression of 40% total soluble protein. Full-sized HC and LC components of an anti- langerin IgG, each carried by a separate FECT vector, expressed and folded into immunologically functional antibody upon co-inoculation. This may prove a useful and environmentally safe vector for both transient expression and perhaps transgenic plants. Mountain cedar (Juniperus ashei) pollen causes severe allergies in Texas and the central USA. Jun a 1 is the dominant allergen protein of mountain cedar pollen and would be a good allergen vaccine candidate. Recombinant Jun a 1 was expressed in Nicotiana benthamiana using an agroinoculation-compatible tobacco mosaic virus vector and isolated in good quantity from the apoplast by vacuum infiltration (100 μg/g leaf material). The recombinant protein samples were characterized. Pectate lyase activity was detected from plant extracts, suggesting the cause of severe necrotic reaction in plants. New Viral Vectors for the Expression of Antigens and Antibodies in Plants by Zun Liu, B.S., M.S. A Dissertation Approved by the Department of Biology ___________________________________ Robert D. Doyle, Ph.D. Chairperson Submitted to the Graduate Faculty of Baylor University in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Approved by the Dissertation Committee ___________________________________ Christopher M. Kearney, Ph.D., Chairperson ___________________________________ Myeongwoo Lee, Ph.D. __________________________________ Mark F. Taylor, Ph.D. ___________________________________ Sang-Chul Nam, Ph.D. ___________________________________ Jiahuan Ding, M.D., Ph.D. Accepted by the Graduate School May 2009 ___________________________________ J. Larry Lyon, Ph.D., Dean Page bearing signatures is kept on file in the Graduate school. Copyright © 2009 by Zun Liu All rights reserved TABLE OF CONTENTS LIST OF FIGURES v LIST OF TABLES vii ACKNOWLEDGMENTS viii CHAPTER ONE 1 Literature Review 1 Plant Virus Expression of Recombinant Proteins 1 DNA Virus Overexpression Vectors 4 RNA Virus Overexpression Vectors 6 Tobacco Mosaic Virus as an Expression Vector 9 PVX-based Expression Systems 13 Other Plant Viruses Used as Expression Vectors 18 New Approaches for Increased Efficacy and Biosafety 25 Virus Induced Gene Silencing (VIGS) 31 Recombinant Antibodies that are Expressed in Plants 33 Post-translational Modification 35 Allergens and Allergy 37 CHAPTER TWO 46 Efficiency Protein Expression in Legumes and Nicotiana from Agroinfection- Compatible Sunn Hemp Mosaic Virus Expression Vectors 46 Abstract 46 Materials and Methods 50 Results 58 Discussion 70 CHAPTER THREE 75 Highly Efficient Suppressor-dependent Protein Expression in Plants with A Foxtail Mosaic Virus Vector 75 Abstract 75 Introduction 76 Methods and Materials 81 Results 91 Discussion 106 CHAPTER FOUR 113 Plant-expressed Recombinant Mountain Cedar Allergen Jun a 1 is Immunogenic and has Pectate Lyase Activity 113 Abstract 113 Introduction 114 Results 124 Discustion 131 APPENDIX 136 Bench Protocol 136 Formaldehyde-Agarose Gel Electrophoresis 136 Quantification of RNA Using Spectrophotometry 137 iii RNA Extraction from Plant Tissue 138 RNA Extraction from Plant Tissue with Tri Reagent 139 Alkaline Lysis Miniprep: Small Scale Isolation of Plasmid DNA 141 DNA Plasmid Maxiprep Protocol 143 Preparing E. coli Electrocompentent Cells 148 Bacterial Transformation by Electroporation 151 Bacterial Transformation by Heat-shock 152 Preparation of electroporation competent Agrobacterium cells 153 Transformation of Agrobacterium Cells by Electroporation 154 Agroinfiltration 155 Histochemical GUS Assay 157 Spectrophotometric GUS Assay. 158 Protein Extraction by Grinding 159 Protein Extraction by Vacuum Infiltration 159 SDS Polyacrylamide Gel Electrophoresis 160 Transfer of Protein from Gel to PVDF Membrane 162 Ponceau Staining (Sensitive Staining of Proteins) 163 Western Blot for the Detection of Jun a 1 163 Alkaline Phosphatase ELISA Protocol 164 Purification of Jun a 1 165 Purification of rJun a 1 167 Enzyme-Linked Immunoassays ELISAs 168 Freezing Cells 169 Beta-hexosaminidase Release Assay 170 Thawing Cells 171 REFERENCES 172 iv LIST OF FIGURES Figure 1. Genome organization of tobacco mosaic virus 10 Figure 2. Genome organization of potato virus X 14 Figure 3. Genome organization of cowpea mosaic virus 20 Figure 4. Genome organization of Potyvirus 22 Figure 5. Schematic diagram of binary plasmid in SHMV study 52 Figure 6. Infection with wild type SHMV via agroinoculation 59 Figure 7. Electron Microscopy of SHMV virion. 60 Figure 8. Effect of gene silencing suppressor on the agroinfection efficiency of SHEC 62 Figure 9. Time course analysis of GFP expression from SHEC74. 63 Figure 10. Fluorescence microscopy of plants infected with SHEC/SHAC:GFP 66 Figure 11. Histochemical GUS assay for plant leaves agroinfected with SHEC:GUS 67 Figure 12. β-Glucuronidase expression level of SHEC:GUS and 35S:GUS 68 Figure 13. Agroinfection induced systemical expression of SHAC vector 70 Figure 14. Schematic diagram of binary plasmids used in FoMV study 86 Figure 15. Effect of gene silencing suppressor on the agroinfection efficiency of FECT 93 Figure 16. Comparing the expression efficiency of FECT22 and FECT40 95 Figure 17. Time course analysis of GFP expression from FECT40/GFP 96 Figure 18. High efficient expression of FECT40 viral vector 98 Figure 19. Quantification analysis of GFP expression 99 Figure 20. Dilution experiment of agroinfection 100 Figure 21. Fluorescence microscopy of monocots agroinfected with FECT40/GFP 102 v Figure 22. Effect of gene silencing suppressor p19 in cis and trans constructs 103 Figure 23. Western blot analysis of full-length recombinant antibody 105 Figure 24. Diagram of binary plasmid pJL36/Jun a 1 and mutant variants 124 Figure 25. Protein analysis of rJun a 1 expression and purification 125 Figure 26. Symptoms of Jun a 1 Expressing Plants 127 Figure 27. Human IgE ELISA assay of native and recombinant Jun a 1. 128 Figure 28: Biological activity of wild type and recombinant Jun a 1 130 vi LIST OF TABLES Table 1. Primers used for plasmid construction in SHMV study. 54 Table 2. Primers used for plasmid construction in FoMV study. 84 vii ACKNOWLEDGMENTS There are many people I would like to thank for who have been encouraging and supporting me to complete my dissertation. I have special thanks to all of my supervisory committee members who have played a significant and critical role in helping me achieving my goal for my degree. I am particularly thankful to my supervisor Professor Chris Kearney for receiving me at the Biology Department and for serving as my academic advisor and guiding me all the way through my doctoral education at Baylor; for all the support, incentive and wise comments, being always very accessible. I am also very thankful for his supervision and guidance and many nice discussions; for all the support as he shared his vast experience and knowledge of viruses. I am thankful for him being so patient to correct my dissertation manuscript and for his help in making my life in US less stressful. A special mention for Dr. Lee, as my dissertation committee member, is justified for his special friendship, care and concern throughout the period of my project, for his readiness to share his knowledge in scientific and non-scientific discussions. There are not enough words to express my appreciation. I am also very grateful to Dr. Ding for his help in introducing me to the newest techniques in molecular biology and biotechnology courses, for his regular advice and his friendship. I cannot be more thankful to Dr. Baker and Dr. Taylor for serving as my dissertation committee members and for their constant concern and encouragement. All of them have always had helpful and and have provided interesting comments about my projects. I would like to acknowledge the research group from UTMB at Galveston, Dr. Randy Goldblum and Dr. Terumi Midoro-Horiuti, for supporting research direction and viii ideas on the mountain cedar