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 Prof 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 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 plantbased recombinant antigens of African Swine Fever virus for subunit vaccine development” and project code: VINIF.2020.DA22 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 .i ACKNOWLEDGEMENTS ii TABLE OF CONTENT iii LIST OF ABBREVIATION vi LIST OF TABLES vii LIST OF FIGURES viii ABSTRACT ix PART I INTRODUCTION PART LITERATURE REVIEW 2.1 General review .3 2.2 African swine fever virus .4 2.2.1 Molecular biology of virus 2.2.2 Transmission and dangerous effect of the ASFV 2.3 Many attempts for virus prevention .8 2.4 Gene EP 402R encoded for CD2v protein 12 2.5 The transient expression for production of subunit vaccines in plants and potential for their application 15 PART III MATERIALS AND METHODS 17 3.1 Materials .17 3.1.1 Vectors and genes .17 3.1.2 Primers 17 3.1.3 Media 18 3.1.4 Antibiotics 18 iii 3.1.5 Commercial kits 18 3.1.6 Bacteria and plant .18 3.1.7 Antibody .18 3.1.8 Chemicals agents and technique machines 18 3.2 Methods 19 3.2.1 Amplification of the EP402R gene encoding the CD2V protein of the ASFV virus 19 3.2.2 DNA purification method 20 3.2.3 Plasmid extraction method 20 3.2.4 Digestion method 20 3.2.5 Ligation method 21 3.2.6 Transformation into E.coli XLI Blue by heat shock method 21 3.2.7 Transformation into AGL1 by electroporation method 22 3.2.8 Colony PCR method 22 3.2.9 Transient expression of recombinant protein in the leaf of N benthamiana by Agro-Infiltration methods 23 3.2.10 Test the transient expression of CD2V protein by Western blot 23 PART IV RESULTS AND DISCUSSION 25 4.1 Results 25 4.1.1 Amplification of the EP402R gene encoding the CD2V protein of the ASFV virus 25 4.1.2 Construction of the cloning vectors that contain the gene encoding for CD2V protein of ASFV 26 4.1.3 Construction of the expression vectors that contain the gene encoding CD2V protein of ASFV 29 4.1.4 Transformation into A tumefaciens AGLI 33 iv 4.1.5 Transient expression of recombinant protein in N benthamiana by AgroInfiltration methods 35 4.1.6 Evaluation the transient expression of CD2V protein by Western blot .37 4.2 Discussion 40 PART V CONCLUSION AND SUGGESTION 43 5.1 Conclusion 43 5.2 Suggestion 43 REFERENCES 44 APPENDIX 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 11 Table 3.1 The list of primers for amplification of targeting region 17 Table 3.2 PCR components and process .19 Table 3.3 Components for DNA digestion for cloning construction 21 Table 3.4 Components for DNA ligation for cloning construction 21 Table 3.5 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 (B) Electron microscopy image of ASFV virions emerging from infected cells Figure 2.2 2D Schematic representation of ASFV CD2v (https://www.nationalhogfarmer.com/) 13 Figure 4.1 The diagram depicts a general strategies of CD2v protein construction and expression process in plants 25 Figure 4.2 PCR products of CD2v structures ≈ 1083 bp, ≈ 1063 bp, ≈ 550 bp, ≈ 300 bp, respectively 26 Figure 4.3 Schematic representation of cloning vector construction 26 Figure 4.4 The electrophoresis results of colony PCR demonstrated successful plasmid transfer into XLI Blue in the cloning construction process 27 Figure 4.5 The results of double digestion with BamHI and PspOMI to check for the presence of the desired gene CD2v in the cloning plasmid .29 Figure 4.6 Schematic representation of expression vector construction 29 Figure 4.7 Single digestion with restriction enzyme HindIII of different CD2v constructs .31 Figure 4.8 The electrophoresis results of colony PCR demonstrated successful plasmids transfer into XLI Blue in the expression construction process 32 Figure 4.9 The results of single digestion with NotI to check for the presence of the desired gene CD2v .33 Figure 4.10 The electrophoresis results of colony PCR demonstrated successful plasmid transfer into AGLI 35 Figure 4.11 Picture depicting the physical state of N benthamiana transformed by AGLI which contains transgenic plasmids CD2v of constructs 36 Figure 4.12 Western-Blot results with crude extract were electrophoresis on SDS PAGE gel and detected by Cmyc antibodies 37 viii cassettes pCB301 CD2v PII has created a 3-molecule CD2v protein linked together, however when separated by β-mercaptoethanol- SDS-PAGE, the structural space of this protein was partially or completely broken The results determined by western blot showed that the strip size is about kDa corresponding to monomer and dimer size Full-length of CD2v: expected 89 kDa Sequence: MIILIFSFNKTIILDSNITNDNNDINGVSWNFFNNSFNTLATCGKAGNFCECSNY STSIYNITNNCSLTIFPHNDVFDTTYQVVWNQIINYTIKLLTPATPPNITYNCTNF LITCKKNNGTNTNIYLNINDTFVKYTNESILEYNWNNSNINNFTATCIINNTISTS NETTLINCTYLTLSSNYFYTFFKLYYIPLSIIIGITISILLISIITFLSLRKRKKHVEEI ESPPPESNEEEQCQHDDTTSIHEPSPREPLLPKPYSRYQYNTPIYYMRPSTQPLN PFPLPKPCPPPKPCPPPKPCPPPKPCPSAESYSPPKPLPSIPLLPNIPPLSTQNISLIH VDRIIGPKRMKQIEDKIEEILSKIYHIENEIARIKKLIAAAITTIITEQKLISEEDLN GSKDEL CD2v without signal peptide: 80 kDa Sequence: SFNKTIILDSNITNDNNDINGVSWNFFNNSFNTLATCGKAGNFCECSNYSTSIY NITNNCSLTIFPHNDVFDTTYQVVWNQIINYTIKLLTPATPPNITYNCTNFLITCK KNNGTNTNIYLNINDTFVKYTNESILEYNWNNSNINNFTATCIINNTISTSNETT LINCTYLTLSSNYFYTFFKLYYIPLSIIIGITISILLISIITFLSLRKRKKHVEEIESPPP ESNEEEQCQHDDTTSIHEPSPREPLLPKPYSRYQYNTPIYYMRPSTQPLNPFPLP KPCPPPKPCPPPKPCPPPKPCPSAESYSPPKPLPSIPLLPNIPPLSTQNISLIHVDRII GPKRMKQIEDKIEEILSKIYHIENEIARIKKLIAAAITTIITEQKLISEEDLNGSKDE L CD2v cytoplasmic domain: 26 kDa Sequence: SFNKTIILDSNITNDNNDINGVSWNFFNNSFNTLATCGKAGNFCECSNYSTSIY NITNNCSLTIFPHNDVFDTTYQVVWNQIINYTIKLLTPATPPNITYNCTNFLITCK KNNGTNTNIYLNINDTFVKYTNESILEYNWNNSNINNFTATCIINNTISTSNETT 38 LINCTYLTLSGPKRMKQIEDKIEEILSKIYHIENEIARIKKLIAAAITTIITEQKLISE EDLNGSKDEL CD2v extracellular domain: 21 kDa Sequence: KRKKHVEEIESPPPESNEEEQCQHDDTTSIHEPSPREPLLPKPYSRYQYNTPIYY MRPSTQPLNPFPLPKPCPPPKPCPPPKPCPPPKPCPSAESYSPPKPLPSIPLLPNIPP LSTQNISLIHVDRIIGPKRMKQIEDKIEEILSKIYHIENEIARIKKLIAAAITTIITEQ KLISEEDLNGSKDEL The full-length of CD2v showed no sign and the cause of the non-expression can be explained by the region SP and TM, causing the gene segment to be attached to the membrane of the endoplasmic reticulum, hence the gene cannot enter the nucleus and transcription in the cytoplasm to obtain the full form protein of CD2v antigen CD2v may either be retained within membrane compartments as it is to the cell surface, or may be retrieved to these compartments from the cell surface (Kay-Fedorov, Penelope & Goatley et al., 2004) CD2v without SP, after omitting the signal peptide, showed very weak expression, but the protein weight was still about 45 kDa, therefore it was still as expected, but this construct had the appearance of a band nearly 35 kDa, similar to the CD2v extracellular domain, these data indicated that the CD2v protein was cleaved within the luminal domain and that this occurs in the endoplasmic reticulum or Golgi compartments (Lynnette C Goatley and Linda K Dixon, 2011) CD2v cytoplasmic domain showed weak yet quite as expected expression band, size about 26 kDa, this part was C-terminal and it is non-glycosylation CD2v extracellular domain was predicted to be at the N-terminal and being glycosylated, resulting in a higher band than predicted by about 36 kDa compared to the expected protein band of about 21 kDa According to Lynnette C Goatley and Linda K Dixon, N-terminal glycosylated fragment was 63 kDa in the vero cell system, compared to our result in a plant system, about 36 kDa This can be explained by using different components in recombinant plasmid as well as glycosylation position and other factors 39 As a result, the truncated CD2v proteins (extracellular and cytoplasmic domain) were expressed in N.benthamiana under the control of the CaMV 35S promoter by Agro-infiltration However, the full CD2v protein was not expressed and the truncated CD2v protein without signal peptide was slightly expressed The results demonstrated that the partial CD2v protein was successful expressed in plant 4.2 Discussion Novel aspects of vaccine innovation could be taken into consideration Currently, there is no vaccine to prevent African swine fever, while the outbreak has been strong since the end of 2019, destroying millions of pigs, causing great economic damage to farmers and export needles In our country, failing to meet the demand for pork of the entire population, the high price of pork, difficulties in re-herding of pigs due to the lack of clean breeds forced the Government, the Ministry of Industry and Trade and the Ministry of Agriculture Industry and Rural Development joined in and had to increase the import of frozen pork from many countries around the world such as Germany, Poland, Brazil, the United States and Russia According to the Ministry of Agriculture and Rural Development, as of March 15, imported pork and pork products reached nearly 25,300 tons, up 205% over the same period in 2019 (https://thanhtra.com.vn / economy / thi-truong /) Therefore, the research and development of vaccines for African swine fever caused by ASFV virus is an urgent requirement and should be done as soon as possible for the safe development of the pigs ensuring the economy and the needs of the people and the country The production direction of subunit vaccines on plant expression systems is being commercially developed in many countries such as Canada, Japan, the United States, and South Korea and has been conducting effective research at the Institute of Biotechnology, Vietnam Academy of Science and Technology to prevent influenza A-/-H5N1, A-/-H7N9, PRRS, acute diarrhea in PEDV pigs The process of creating subunit vaccines derived from plants has many advantages because of its safety for the environment as well as humans; simple process, easy to increase production scale, very fast production time after only 4-6 weeks can produce a large amount of vaccines to respond promptly when there is a risk of pandemic 40 outbreak and price will also be lower than other vaccine manufacturing technologies However, the production of ASFV plant-based African swine fever subunit vaccine ASFV has not yet published any research in the world and in Vietnam From the above practical and up-to-date reasons this project has been proposed to at present Effect level considered as one of the most crucial factors of vaccine invention The African swine fever is caused by the ASFV virus, which has seriously damaged the economy of our country from the end of 2019 up to now Currently, a commercial vaccine for African swine fever is not available, so there is a risk of an outbreak at any time Therefore, the research to find a vaccine to prevent African swine fever will have a great impact on the socio-economic development of the country, helping to stabilize the farmers' mentality herd, meeting the demand for fresh pork of the people, helping to stabilize pork prices across the country; reducing the import of too much frozen pork from abroad At the same time, the project to develop subunit vaccines against African swine fever with plant origin will further contribute to the development of Science and Technology in the country a new, fast, effective method of vaccine production effective and safe to fight ASFV virus Furthermore, since the vaccines produced in crops are subunit vaccines, it fully meets the DIVA (Differentiating Infected from Vaccinated Animals) standard allowing for easier disease monitoring than the use of inactivated vaccine There have been a number of studies showing that expression in plants is often hindered by transcriptional and post-transcriptional gene silencing There are at least three modes of gene silencing in plants, whose roles have been identified including defense against viruses, regulation of gene expression, and condensation of chromosomes to heterochromatin To overcome this, more than 30 proteins that inhibit the gene silencing of plant viruses were found Therefore, the simultaneous expression of genes encoding silencing-suppressing proteins and target genes in plant cells will increase the expression of the target protein In this study, however, we used the support of the Hc-Pro protein that inhibits the RNA silencing mechanism in plants by inhibiting the breakdown of mRNAs and doublestranded RNAs thereby reducing the amount of siRNA or inducing Failure of the cleavage function of the Dicer and RISC cleavage enzymes leads to the second non41 expression due to the antigen structure of CD2v containing specific sequence regions, particularly the transmembrane region and signal peptide sequences The signal peptide sequence gives the antigen protein a target for the protein to function, the CD2v protein is involved in cell-cell adhesion, virulence enhancement, and immune response modulation Its possible role in the pathogenesis of ASFV infection, further role in tissue tropism, immune evasion, and enhancing virus replication The transmembrane region causes the protein to be stored in the Endoplasmic Reticulum membrane, so that transmission function of the virus also contributes to suppressing the expression of the protein in another plant system The suspicion of the second case has led us to choose to optimize the gene segments of CD2v, which is to cut the above sequences one by one, to get positive results and to express successfully instead of leaving it alone, gene sites regulate the CD2v protein Thus, the removal of the SP structure resulted in weak expression of the protein in the plant system Next, we decided to cut both SP and TM, resulting in fairly strong protein expression in the plant system The preliminary conclusion showed that SP and TM had a significant effect on antigen protein expression in another system, in this case the plant system 42 PART V CONCLUSION AND SUGGESTION 5.1 Conclusion This is the first study about expression of full and truncated CD2v proteins in plant using agroinfiltration After completing experiments, we conclude as following: The DNA sequence encoding CD2v protein of ASFV strain isolated in Vietnam was collected, codon optimized and successfully artificial synthesized Plant based expression vectors pCB301 harboring expression cassettes containing full CD2v protein or truncated CD2v protein (without signal peptide, extracellular, cytoplasmic domain) were successfully constructed The resulting expression vectors pCB301 was successfully transformed in N.benthamiana by agroinfiltration Truncated CD2v protein (extracellular, cytoplasmic domain) was strongly expressed in N benthamiana Besides, truncated CD2v protein without signal peptide was slightly expressed in N benthamiana, while full CD2v protein was not expressed 5.2 Suggestion Further experiments will be required to construct plant expression vector containing the truncated CD2v protein without signal peptide and transmembrane region In addition, the expression of truncated CD2v protein (without signal peptide) in N.benthamiana will be optimized via optimization of factors such as bacterial concentration, age of plants, number of days after transformation… CD2v proteins will be purified for structural and bio-functional characterization; and evaluation its 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Poueymirou W, MacDonald D, Yancopoulos GD, Sleeman MA, Murphy AJ, Skokos D Combination cancer immunotherapy targeting PD-1 and GITR can rescue CD8+ T cell dysfunction and maintain memory phenotype Sci Immunol 2018 Nov 2;3(29):eaat7061 doi: 10.1126/sciimmunol.aat7061 PMID: 30389797 48 APPENDIX pRTRA CD2v full-length PII pCB301 CD2v full-length PII Recombinant plasmid construction of CD2v full-length pRTRA CD2v without signal peptide PII pCB301 CD2v without signal peptide PII Recombinant plasmid construction of CD2v without signal peptide pRTRA CD2v cytoplasmic domain PII pCB 301 CD2v cytoplasmic domain PII Recombinant plasmid construction of CD2v cytoplasmic domain pRTRA CD2v extracellular domain PII pCB301 CD2v extracellular domain PII Recombinant plasmid construction of CD2v extracellular domain