VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY GRADUATION THESIS TITLE: CONSTRUCTING THE CD2v (FROM AFRICAN SWINE FEVER VIRUS) PLANT-BASED VECTORS AND DETECTING OF TRANSIENT EXPRESSION OF CD2v PROTEIN IN NICOTIANA BENTHAMIANA HANOI, 03 /2021 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY GRADUATION THESIS TITLE: CONSTRUCTING THE CD2v (FROM AFRICAN SWINE FEVER VIRUS) PLANT-BASED VECTORS AND DETECTING OF TRANSIENT EXPRESSION OF CD2v PROTEIN IN NICOTIANA BENTHAMIANA Student’s name : Vu Duy Thai Son Department : Biotechnology Supervisor : Assoc. Pham Bich Ngoc Msc. Ho Thi Thuong Co-supervisor : Msc. Nguyen Quoc Trung HANOI, 03 /2021 STATEMENT OF ORIGINAL AUTHORSHIP The work contained in this thesis has not been previously submitted to meet requirements for an award at this or any other education institution.
To the best of my knowledge and belief, the thesis contains no material previously published or written by another person except where due reference is made. Signature: Date: i ACKNOWLEDGEMENTS First and foremost, my sincere thanks goes to the Department of Applied DNA technology- Institution of Biotechnology- Vietnam Academy of Science and Technology and Faculty of Biotechnology- Vietnam National University of Agriculture for giving me a chance to do this thesis and support me all the time during the process. Secondly, I would like to thank my principal supervisor, Assoc/Prof. Pham Bich Ngoc, and co-supervisor Nguyen Quoc Trung for their ongoing support and suggestions through my graduation thesis and most profoundly, Mrs.Ngoc had accepted me from the first time after I decided to leave the previous lab.
Moreover, I extend deep thanks to Mrs. Ho Thi Thuong, who guided me throughout all sectors of my thesis in particular, and so far, my research direction in general. Without her, this project would not have been done. I also wish to express my appreciation and gratitude to my colleagues who, through their ideas, suggestions, and criticisms, all in order to improve my thesis.
This thesis, like any other, would not have been possible without the involvement and support of Ms. Trinh Thai Vy, Ms. Nguyen Thi Tra, Mr. Ngo Hong Duong, they have all earned my deepest gratitude, giving me many guidance to help me from beginner to premature in research aspects and my skills in research almost improved from their support.
I would like to thank all Staff and students at the Faculty of Applied DNA technology, Vietnam Academy of Science and Technology, who have had to put up with me going through all kinds of emotional swings also deserve my thanks. They were all very friendly and helpful to me, I was so lucky to study in a great working environment. This thesis was financially supported by Vingroup Innovation Foundation (VINIF) for project named: “Study on expression and evaluation of immunogenicity of several plant- based recombinant antigens of African Swine Fever virus for subunit vaccine development” and project code: VINIF. Last, but not least, I express my great gratitude for my family and friends for all their help, encouragement through all these years.
ii TABLE OF CONTENT STATEMENT OF ORIGINAL AUTHORSHIP. ii TABLE OF CONTENT. iii LIST OF ABBREVIATION.vi LIST OF TABLES. vii LIST OF FIGURES.
African swine fever virus. Molecular biology of virus. Transmission and dangerous effect of the ASFV. Many attempts for virus prevention.
Gene EP 402R encoded for CD2v protein. The transient expression for production of subunit vaccines in plants and potential for their application. MATERIALS AND METHODS. Vectors and genes.
Bacteria and plant. Chemicals agents and technique machines. Amplification of the EP402R gene encoding the CD2V protein of the ASFV virus. DNA purification method.
Plasmid extraction method .coli XLI Blue by heat shock method. Transformation into AGL1 by electroporation method. Colony PCR method. Transient expression of recombinant protein in the leaf of N.
benthamiana by Agro-Infiltration methods. Test the transient expression of CD2V protein by Western blot. RESULTS AND DISCUSSION. Amplification of the EP402R gene encoding the CD2V protein of the ASFV virus.
Construction of the cloning vectors that contain the gene encoding for CD2V protein of ASFV. Construction of the expression vectors that contain the gene encoding CD2V protein of ASFV. Transient expression of recombinant protein in N. benthamiana by Agro- Infiltration methods.
Evaluation the transient expression of CD2V protein by Western blot. CONCLUSION AND SUGGESTION .47 v LIST OF ABBREVIATION µL microlitre AGLI A.tumefaciens bp Base pair Carbe Carbenicilin CD Cytoplasmic domain DNA Deoxyribonucleic acid dNTP Deoxynucleoside triphosphate dsDNA Double DNA E.coli XLI Blue Escherichia coli XLI Blue strain ED Extracellular domain IgG antibody Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP’ Kana Kanamycin L litter LB Luria-Bertani broth mg Mini gram mins minutes mL milliliter PCR Polymerase Chain Reaction RE Restriction enzyme Rifa Rifampicin rpm revolutions per minute s second SP Signal peptide TAE Tris – acetate – EDTA TM Transmembrane region w/v Weight/volume vi LIST OF TABLES Table 2.1 The summary of several previous researches in relevant protein antigen .1 The list of primers for amplification of targeting region .2 PCR components and process .3 Components for DNA digestion for cloning construction .4 Components for DNA ligation for cloning construction .5 PCR components and process for colony PCR of cloning construction .22 vii LIST OF FIGURES Figure 2.1 Structure of the extracellular ASFV and virion egress from cells. (A) Electron microscopy image of the extracellular ASFV particle. Electron microscopy image of ASFV virions emerging from infected cells.2 2D Schematic representation of ASFV CD2v (https://www.1 The diagram depicts a general strategies of CD2v protein construction and expression process in plants .2 PCR products of 4 CD2v structures ≈ 1083 bp, ≈ 1063 bp, ≈ 550 bp, ≈ 300 bp, respectively .3 Schematic representation of cloning vector construction .4 The electrophoresis results of colony PCR demonstrated successful plasmid transfer into XLI Blue in the cloning construction process .5 The results of double digestion with BamHI and PspOMI to check for the presence of the desired gene CD2v in the cloning plasmid.6 Schematic representation of expression vector construction .7 Single digestion with restriction enzyme HindIII of 4 different CD2v constructs .8 The electrophoresis results of colony PCR demonstrated successful plasmids transfer into XLI Blue in the expression construction process .9 The results of single digestion with NotI to check for the presence of the desired gene CD2v.10 The electrophoresis results of colony PCR demonstrated successful plasmid transfer into AGLI .11 Picture depicting the physical state of N.
benthamiana transformed by AGLI which contains transgenic plasmids CD2v of 4 constructs.12 Western-Blot results with crude extract were electrophoresis on SDS - PAGE gel and detected by Cmyc antibodies. 37 viii ABSTRACT African swine fever (ASF) is one of the most dangerous infectious diseases in pigs with a rapid spread, mortality up to 100%. The research and development of vaccines against African swine fever virus (ASFV) is urgent and needs to be promoted. Due to the complex nature of the virus and the large genome, so far the development of vaccine against African swine fever is challenge and currently, there is no commercially available vaccine against African swine fever.
Recently, a candidate of live attenuated vaccine ASFV-G-ΔI177L produced in mammalian cells induced a strong virus-specific antibody response, and a sterile immunity against the ASFV Georgia isolate, the method to generate and produce the live attenuated vaccine ASFV-G-ΔI177L is quite complicated and expensive when compare to other strategies for vaccine development. Few subunit vaccines showed a patial protection against exposure to virulent strains of ASFV. Identification of the ASFV antigens and epitopes responsible for induction of relevant immune responses is key to develop effective vaccines against ASFV. CD2v is one of the strategically important proteins for the production of vaccine subunit protein.
The process of creating subunit vaccines derived from plants brings many advantages because of its safety for the environment as well as animals and humans; simple process, easy to increase production scale, well- respond promptly when there is a risk of pandemic outbreak whereas the price will also would be lower than traditional vaccine manufacturing technologies. In this research, we collected a DNA sequence that encodes for CD2v protein of ASFV strain isolated in Vietnam, codon optimized commercially in tobacco, then artificial synthesized and inserted in a pUC57 vector. Four different DNA regions that encode for full CD2v protein and truncated CD2v proteins (CD2v without signal peptide, CD2v cytoplasmic domain and CD2v extracellular domain) were amplified by PCR using different specific primers. These PCR products were then inserted in a cloning vector pRTRA-CaMV35S-H5-GCN4pII-cmyc-his-KDEL) via the BamHI and PspOMI sites.
The resulting expression cassette was inserted into the expression vector pCB301 via HindIII cleavage, then transformed into the Agrobacterium tumefaciens. To express the CD2v recombinant proteins in planta, the agroinfiltration protocol was ix performed to transform A. tumefaciens carrying recombinant plasmids into Nicotiana benthamiana. The expression of CD2v proteins in planta were detected by Western blot.
As a result, the expression of full length CD2v protein was not detected. However, the truncated CD2v protein without signal peptide was slightly expressed in Nicotiana benthamiana, while truncated CD2v protein containing cytoplasmic domain and truncated CD2v protein containing extracellular domain was strongly expressed. The above results are considered as the scientific foundation for the production of antigens in plant systems, the potential for developing new systems with low production costs, while in a short time, making great contributions to innovative medicine. INTRODUCTION African swine fever is one of the most dangerous infectious diseases in pigs with a rapid spread, mortality up to 100%.
From 2016 to 5/2019, the disease appeared in over 59 countries around the world, including Vietnam. Currently, there have been many researches to develop vaccines to prevent African swine fever such as DNA vaccines, inactivated vaccines, and live attenuated vaccines. While the inactivated vaccine in ASFV has not been shown to provide protective efficacy, there is little concern (Stone & Hess, 1967). Development of a subunit vaccine for ASFV prevention by expressing recombinant expression of some ASFV antigens has been studied.
ASFV's identification of the antigens and epitopes responsible for stimulating a good immune response is seen as key to the development of an effective subunit vaccine against ASFV. CD2v protein is one of the viral envelope proteins, which plays an important role in viral transmission and replication. CD2v is the ASFV haemagglutinin and has been implicated previously in protective immunity. CD2v/ EP402R proteins may be important for protection against ASFV infection (Burmakina, G et al.
Expression of vaccine subunit protein produced on plant systems is still a relatively novel approach for vaccine innovation. The potential of plant-based vaccine is to bring relatively large benefits compared to other systems such as low cost, lower time consuming in production and promptly respond to the rapid change of the virus. Therefore, for all above reasons, I proposed the research: constructing the CD2v (from African Swine Fever Virus) plant-based vectors and detecting its transient expression in Nicotiana benthamiana. The major aim of the research is to construct the plant-based vectors containing DNA sequences encoding full and truncated CD2v protein of African Swine Fever Virus (ASFV) isolated in Vietnam and examine the transient expression of CD2v protein in Nicotiana benthamiana by Agro-infiltration methods.
The analysis results of this research will be used for evaluation of biological characteristics of ASFV and potentially contribute to protein subunit vaccine production in the future. Several hypotheses and requirements could be taken into considerations to answer for the research. Firstly, the information should be collected including: DNA sequence encoding CD2v protein of ASFV virus strains circulating in Vietnam, codon 1 optimization for protein expression in Nicotiana benthamiana and artificial synthesis. Secondly, the experiment is conducted begin with constructing the plant-based vectors carrying regions encoding ASFV CD2v antigens and constructing Agrobacterium strains carrying corresponding vectors, ending with examining the transient expression of ASFV CD2v protein in Nicotiana benthamiana.
General review Animal diseases are now putting a threat to animal health, food safety, national economy and environment.