INTRODUCTION
LITERATURE REVIEW
Animal diseases pose significant threats to health, food safety, the economy, and the environment African swine fever (ASF) is a particularly devastating virus affecting pigs and wild suids, with no available vaccine or effective treatment, leading to mortality rates as high as 100% Since its introduction in Georgia in 2007, ASF has rapidly spread to neighboring countries in Europe and Asia, raising alarms, especially in China, which houses half of the world's pig population and reported its first case in 2018 By May 2019, the disease had been reported in over 59 countries globally In Vietnam, ASF was officially announced on February 19, 2019, with outbreaks first identified in two northern provinces By September 2019, the virus had spread to 63 provinces and cities, resulting in the culling of over 4.4 million pigs.
In a study by Le Van Phan et al (2019), African Swine Fever (ASF) was first identified in East Africa in the early 1900s and subsequently spread to Kenya in the 1920s, Europe and South America in the 1960s, Georgia in 2007, and China in 2018 In January 2019, an outbreak occurred at a family-owned pig farm in Hung Yen Province, Vietnam, located approximately 50 km from Hanoi and 250 km from the China border, where 20 sows were housed Initial symptoms included marked redness, conjunctivitis, and hemorrhagic diarrhea in one piglet and one sow, while breeding gilts exhibited anorexia, cyanosis, and high fever By February 2019, the mortality rate at the farm exceeded 50%, prompting Le Van Phan and colleagues to collect organ samples Collaborating with the Vietnam National University of Agriculture, they identified the virus's complete genome, which was subsequently cataloged in Genbank under several accession numbers.
4 respectively Also, the phylogenetic analysis of the major capsid protein genome was constructed (Phan et al., 2019)
Therefore, ASF prevention strategies are important recently to avoid economic losses worldwide The preventive and control measurements should be recommended and developed via intercontinent collaboration
African swine fever is caused by the African Swine Fever Virus (ASFV), the sole member of the Asfarviridae family This virus shares characteristics with Poxvirus and Iridovirus, as they are all cytoplasmic DNA viruses The structure of ASFV includes an internal core, an internal lipid membrane, an icosahedral capsid, and an outer lipid envelope (Alonso et al., 2018).
ASFV is a large, cytoplasmic, double-stranded DNA, length approximate 170-
African Swine Fever Virus (ASFV) has a genome of 190 Kbs, encoding around 160 proteins, and primarily replicates in the mononuclear phagocyte system, particularly in monocytes and macrophages Other cell types can also become infected as the disease progresses The replication of ASFV in target cells is crucial for the infection process and significantly influences the virus's pathogenesis (Yolanda Revilla et al., 2017) ASFV virions are approximately 200 nm in size and exhibit icosahedral structures composed of concentric layers, including an internal core, core shell, inner membrane, capsid, and an external envelope in extracellular virions.
The structure of the extracellular African Swine Fever Virus (ASFV) and its virion egress from infected cells is illustrated in Figure 2.1 Part (A) presents an electron microscopy image of the extracellular ASFV particle, while part (B) shows ASFV virions emerging from infected cells, as reviewed by Salas and Andres in 2013.
The inner envelope, adjacent to the core shell, appears as a single lipid membrane under electron microscopy and is derived from the endoplasmic reticulum (ER), containing viral proteins such as p17/D117Lp, p54/E183Lp, E248Rp, and p12/O61Rp (Salas and Andres, 2013) The capsid, the outermost layer of intracellular virions, is primarily composed of the p72/B646Lp and E120Rp, B438Lp proteins that form the capsomers During the budding process, the outer envelope of extracellular viral particles is acquired from the cellular plasma membrane, incorporating essential proteins like CD2v/EP402Rp, p12/O61Rp, and p24 The core shell includes cleavage products of the polyprotein, such as pp220/CP2485Lp, p150, p37, p34, p14, pp62/CP530Rp, p35, p15, and S273Rp Additionally, the inner core houses the genome along with proteins and enzymes, including RNA polymerase, necessary for initiating viral infection.
The ASFV virus particle, known as CD2v, is a homologue of the cellular CD2 protein and plays a crucial role in mediating hemadsorption to infected cells (Salas and Andres, 2013) Morphogenesis of ASFV occurs in specialized cytoplasmic regions called viral factories, which form near the nucleus and the microtubule organization center These viral factories are characterized by the exclusion of host proteins and are encased in endoplasmic reticulum membranes and vimentin boxes (Heath et al., 2001).
The urgent need for research and development of vaccines against African swine fever is underscored by the virus's complex nature and large genome, which currently prevent the creation of an effective vaccine worldwide.
2.2.2 Transmission and dangerous effect of the ASFV
The Ornithodoros tick serves as the primary vector for transmitting the African swine fever virus from wild boars to domestic pigs This virus predominantly targets pig macrophages, leading to severe outbreaks among both domesticated pigs and wild boars The transmission occurs via the respiratory and digestive tracts, as well as through direct or indirect contact with contaminated items, including barns, vehicles, tools, clothing, and leftover food containing infected pork (OIE, 2013).
Understanding virus evasion and transmission strategies, along with cellular mechanisms such as infection and replication, is essential for developing future vaccines Recent advancements in ASFV virology involve the use of proteomic tools, including two-dimensional gel electrophoresis and mass spectrometry, to analyze ASFV-infected cells and virus particles A study by Alejo et al (2018) focused on the proteome of highly purified extracellular virions from the Vero-adapted strain BA71V, leading to the creation of a comprehensive atlas of the ASFV particle through immunoelectron microscopy Additionally, Keòler et al (2018) investigated the expression of ASFV genes in three mammalian cell lines—WSL-HP from wild boar, human HEK 293, and Vero cells—after infection with a recombinant derivative of the OURT88/3 strain These studies demonstrated that mass spectrometry could confirm the presence of corresponding proteins for most predicted ASFV open reading frames in mammalian cell cultures.
Recent research has elucidated the mechanism of African Swine Fever Virus (ASFV) entry into host cells, which involves outer envelope disruption, capsid disassembly, and inner envelope fusion, allowing the viral core to be released from endosomes According to Elena G Sánchez et al (2017), ASFV enters cells through receptor-mediated endocytosis, a process that is energy-dependent and influenced by vacuolar pH and temperature Additionally, studies by Galindo et al (2015) indicate that viral entry is clathrin-dependent and requires dynamin Following internalization, ASFV navigates the endolysosomal system, with the capsid and inner envelope initially found in early endosomes or macropinosomes, co-localizing with EEA1 and Rab5, and later associating with markers of late endosomes and lysosomes, such as Rab7 and Lamp1.
ASFV uncoating begins with the removal of outer capsid layers, followed by the fusion of the inner membrane with endosomes, which releases the nucleocapsid into the cytosol Early studies indicate that ASFV entry can occur as quickly as 30 minutes, with nearly 60% of virions internalized The entry process is rapid, with viral particles detectable inside endosomes as early as 1 to 15 minutes post-infection.
African Swine Fever Virus (ASFV) can be transmitted directly among domestic pigs, resulting in mortality rates nearing 100% Key clinical signs of the disease include high fever, haemorrhagic lesions, cyanosis, anorexia, and ataxia In acute and subacute forms of ASF, various organs exhibit severe vascular changes, such as renal petechiae, diffuse haemorrhage in lymph nodes, pulmonary oedema, disseminated intravascular coagulation, and thrombocytopenia (Ning Jia et al., 2017).
African swine fever virus (ASFV) is a highly contagious and transboundary viral disease affecting swine, with no commercially available prophylactic vaccine (Teshale Teklue et al., 2019) Recent studies have highlighted the role of MGFs as determinants of macrophage host range, promoting the survival of infected cells while suppressing type I IFN expression Notably, Reis et al (2016) reported that MGF-deleted ASFV derived from the virulent strain Benin 97/1 exhibited reduced virulence.
In domestic pigs, a protective response is induced against viral invasions, primarily through the elimination of infected cells via apoptosis However, the African Swine Fever Virus (ASFV) has evolved mechanisms to counteract this process by expressing proteins that compete with and sequester pro-apoptotic proteins Notably, ASFV encodes several anti-apoptotic proteins, including A179L, A224L, EP153R, and DP71L, which help modulate host immune responses and promote the survival of infected cells Despite these findings, the key features of immune evasion mechanisms remain incompletely understood and warrant further investigation.
2.3 Many attempts for virus prevention
MATERIALS AND METHODS
Materials
Cloning vector: pRTRA_CaMV35S-H5-GCN4pII-cmyc-his-KDEL (Phan HT et al., 2013)
Expression vector: pCB301 (Xiang et al., 1996)
The CD2v sequence of the ASFV strain isolated in Vietnam in 2019, as reported by Le et al (2019), is accessible in the National Center for Biotechnology Information (NCBI) under the accession number MK757459.1 This sequence was tobacco codon optimized and synthesized by Genewiz (USA), and is referred to as pUC54-CD2v.
Table 3.1The list of primers for amplification of targeting region
Enzyme: BamHI, PspOMI, HindIII, NotI (Thermo Scientific)
LB agar: Components in 1L: Peptone (10g), Yeast extract (5g), NaCl (5g), Agar (15g)
LB liquid: Components in 1L: Peptone (10g), Yeast extract (5g), NaCl (5g)
Kanamycin (50 mg/l), Rifampicin (50 mg/l), Carbenicillin (50 mg/l)
GenJET Gel Extraction Kit (Thermo scientific), Kit GeneJET PCR Purification (Thermo Scientific), Master mix for PCR (Thermo Scientific)
Escherichia coli XLI Blue, Agrobacterium tumefaciens AGLI and Nicotiana benthamiana was grown in the greenhouse at 21 °C, 16 hours light per day (department of Applied DNA technology, Institution of Biotechnology
The anti-c-myc monoclonal antibody, clone 9E10, specifically targets the region between amino acids 408 and 239 of the proto-oncogene p62, as established by Evan et al in 1985 This antibody was utilized in Western blotting to identify recombinant proteins that feature the c-myc tag.
Secondary antibody: Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP (Invitrogen) was used for Western blot
3.1.8 Chemicals agents and technique machines
Chemical agents: Peptone (biobasic Canada), TAE buffer: Merck, Alcohol (Merck), GeneRuler 1 kb DNA Ladder (Thermo Scientific™), Solution 1 (Glucozo 2M,
25mM Tris HCL pH=8 1M, EDTA 10mM 0.5M, Water H2O), solution 2 (NaOH 0.2N, SDS 1% and Water H2O), solution 3 (CH3COOK 5M, CH3COOH 11.5% and Water
The laboratory is equipped with advanced machinery, including the Sorvall Legend micro 21R and Eppendorf Centrifuge 5415R for efficient sample processing For PCR applications, the Eppendorf Mastercycler x50s is utilized, alongside equipment from Applied Biosystems (Germany) Additional tools include an oven from China, a Culture Lamina Flow Cabinet from N-biotech, and a precision scale from Switzerland pH measurements are conducted using instruments from the USA, while magnetic stirring is also sourced from the USA Pipetting systems are provided by Eppendorf and iKIA, complemented by UV lighting from Vilber Lourmat (France) and an incubation shaker from IKA (Germany).
KS 260 basic), Freezer Alaska Vietnam and Thermo scientific, Fume Hoods LFS (French), Orbital shaker incubator MRC (USA), and others…
Methods
3.2.1 Amplification of the EP402R gene encoding the CD2V protein of the ASFV virus
The EP402R gene, which encodes the CD2v protein, was amplified in full and partially through PCR using various primer pairs, resulting in four distinct CD2v products The details of the PCR contents are provided in Table 3.2.
Table 3.2PCR components and process
5x HF buffer 10 àl dNTP 1 àl
PCR processes to amplify targeting CD2v products were carried out as follow:
The Kit GeneJET PCR Purification method (Thermo Scientific) involves several key steps for DNA purification First, DNA samples are mixed with a binding buffer in a 1:1 ratio and incubated at 55°C The gel is then transferred to a purification tube and centrifuged at 13,000 rpm for 1 minute at 4°C, after which the bottom solution is discarded Next, 700 µl of 70% ethanol is added to the purification tube, followed by another centrifugation at the same speed and temperature, with the solution again removed This ethanol wash is repeated once more The purified tube is then transferred to a 1.5 ml Eppendorf tube, and 30 µl of H2O is added A final centrifugation at 13,000 rpm for 1 minute at 4°C is performed, and the resulting tube is retained as it contains the purified DNA.
Bacteria were cultured overnight in 5 ml of LB medium supplemented with 5 µl of Carbe The following day, 4 ml of the bacterial suspension was centrifuged at 13,000 rpm for 1 minute Subsequently, 200 µl of Solution I and 400 µl of Solution II were added to the residue.
300 àl Solution III (invert gently in each solution adding step) Then we centrifuge at
Centrifuge the solution at 13,000 rpm for 30 minutes at 4 °C, then collect 600 µL or 700 µL of the supernatant Next, add an equal volume of isopropanol to the tube and refrigerate at -20 °C for 20 minutes Afterward, centrifuge again at 13,000 rpm for 30 minutes at 4 °C Add 1 mL of 70% ethanol, gently invert the tube, and centrifuge at 13,000 rpm for 10 minutes at 4 °C, discarding the supernatant while retaining the pellet Allow the pellet to dry in a culture box until it becomes transparent, which should take about 5-10 minutes Finally, add 30 µL of RNase + H2O and incubate at 37 °C for 15 minutes.
The extracted plasmid underwent a digestion process in order to create the same sticky position in both genes and vectors Enzymes BamHI and PspOMI are used in the
The cloning construction process involves the use of HindIII for expression construction, with Buffer Tango 10x utilized for the enzymes BamHI and PspOMI Additionally, Buffer R is employed for HindIII and Buffer O for NotI, following the guidelines provided by Thermo Scientific The general components of the digestion process are outlined in Table 3.3.
Table 3.3Components for DNA digestion for cloning construction
After the digestion of genes and vectors to create sticky ends, a ligation process is performed to assemble a complete recombinant vector The necessary components for this ligation are outlined in Table 3.4.
Table 3.4Components for DNA ligation for cloning construction
3.2.6 Transformation into E.coli XLI Blue by heat shock method
To begin the transformation process, competent E coli cells are retrieved from the -80°C cabinet and thawed on ice for 5 minutes Subsequently, 15 µL of recombinant plasmid, obtained after the ligation process, is added to a tube containing 50 µL of the transformable cells, which is then kept on ice for another 5 minutes Finally, the tube is placed in the microwave at a power setting of 150W for 1 minute.
30 seconds Afterwards we keep the tube on ice for 5 minutes In the next stage, we add
500 àL fresh liquid LB to this tube and incubate for recovery at 37 0 C, in 30 minutes to
1 hour, then we spill around 100-200 àl of bacterial solution onto the LB plate with the addition of Carbenicillin (50 mg/l) and incubate dishes at 37 °C overnight
3.2.7 Transformation into AGL1 by electroporation method
To transform A.tumefaciens competent cells, begin by thawing them on ice for 5 minutes Next, add 5 µl of recombinant plasmid to the competent cells and keep the mixture on ice for another 5 minutes Transfer the solution to a cooled cuvette and maintain it on ice for an additional 5 minutes before applying an electric pulse at 2.5 kV, 25 µF, and 200 Ω After pulsing, add 500 µl of recovery medium to the cuvette and transfer the entire solution to a 2 ml tube, incubating at 28 °C for 2 hours Finally, spread 100-200 µl of the bacterial suspension onto an LB dish containing selective antibiotics: Kanamycin (50 mg/l), Carbenicillin (50 mg/l), and Rifampicin (50 mg/l), and incubate at 28 °C for 2 days.
Colony PCR for selecting the desirable colony carried gene cassette by using the appropriate primers as follow table 2 The colony PCR components are depicted in the Table 3.5:
Table 3.5PCR components and process for colony PCR of cloning construction
Master Mix Dream tag 7.5àl
Process are expressed as following:
3.2.9 Transient expression of recombinant protein in the leaf of N benthamiana by Agro-Infiltration methods
A.tumefaciens containing the shuttle vectors for the expression of recombinant
HcPro and CD2v proteins are independently pre-cultivated in 5 ml of LB medium with kanamycin, carbenicillin, and rifampicin at 28 °C for 16-18 hours with shaking at 200 rpm Following this, 200 ml of fresh LB medium containing the same antibiotics is added to the preculture for inoculation.
Bacteria were collected after 24 hours and resuspended in an infiltration buffer containing 10 mM 2–(N–morpholino) ethanesulfonic acid (MES) and 10 mM MgSO4 at pH 5.6 The A tumefaciens suspension was mixed and diluted in a 1:1 ratio to achieve a final optical density of OD600 = 1 This suspension was then used to infiltrate 5–6 week old N benthamiana plants under vacuum conditions for 1.5 minutes at 27 inches and 0 atm Following infiltration, the plants were placed in a greenhouse with hydroponic cultivation at temperatures of 21-23 °C and exposed to 16 hours of light daily Leaf samples were collected for five days post-infiltration and stored at −80 °C for subsequent protein extraction.
3.2.10 Test the transient expression of CD2V protein by Western blot
Leaf samples were processed using a Mixer Mill MM 300 (Retsch, Haan, Germany) and dissolved in an SDS sample buffer containing 50 mM Tris-HCl (pH 6.8), 2% SDS, 0.1% Bromophenol blue, and 10% glycerol The samples were denatured at 95 °C for 10 minutes and then centrifuged at 13,000 rpm for 30 minutes at 4 °C Following this, 10-30 µg of protein was separated using SDS-PAGE electrophoresis with a 30% polyacrylamide gel and transferred to a nitrocellulose membrane using a Fast film transfer machine blotter (Thermo Scientific) at 40V for 2 hours The membrane was subsequently blocked with a 5% (w/v) fat-free milk powder solution in PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4).
The membrane is first incubated at room temperature for 2 hours with a 1:200 dilution of Cmyc primary antibody Following this, it is treated with a 1:2000 dilution of anti-mouse IgG secondary antibody for an additional 2 hours at room temperature Specific signals are then detected by incubating the membrane with 3,3-diaminobenzidine (DAB).
24 Thermo Scientific Pierce) dissolved in 0.05 M Tris–HCl and 0.04% hydrogen peroxide for 10 min in the dark (Adapted from HoTT et al., 2020)
RESULTS AND DISCUSSION
Results
Figure 4.1 The diagram depicts a general strategies of CD2v protein construction and expression process in plants
4.1.1 Amplification of the EP402R gene encoding the CD2V protein of the ASFV virus
The EP402R gene segment first encodes the antigen CD2v protein as double strand DNA, and consists of 4 parts: signal peptide (1-19), extracellular domain (20-
The study focused on the CD2v gene, which consists of a transmembrane region and a cytoplasmic domain Using PCR amplification, four distinct products of varying sizes were obtained from the CD2v gene segment, with the full-length CD2v measuring approximately 195 to 375 base pairs.
The CD2v gene is 1083 base pairs (bp) in size, while the CD2v variant without the signal peptide (SP) measures approximately 1063 bp The cytoplasmic domain of CD2v is around 550 bp, and the extracellular domain is about 300 bp The electrophoresis results are illustrated in Figures 4.2 below.
Figure 4.2 PCR products of full and a part of gene encoding for CD2v protein
A: Full length of CD2v, B: CD2v cytoplasmic domain, CD2v extracellular domain and CD2v without signal peptide, M: Maker 1kb (Thermo Scientific)
The DNA sequences encoding CD2v proteins were successfully amplified by PCR, resulting in clear and specific bands that were separated on agarose gel The purified PCR products were then prepared for the next step.
4.1.2 Construction of the cloning vectors that contain the gene encoding for
All 4 different products of CD2v in the session 1were inserted in a cloning vector based on the diagram shown in Figure 4.3:
Figure 4.3Schematic representation of cloning vector construction
The cloning vector pRTRA PII, along with four different PCR products, was double digested using BamHI and PspOMI enzymes to create compatible ends for ligation The resulting recombinant plasmids were transformed into E coli XLI Blue via heat shock and screened through colony PCR to identify colonies containing the desired gene cassette Five bacterial colonies were selected for PCR analysis to confirm the correct strain, followed by electrophoresis to visualize the results and assess transformation efficiency, ultimately restoring the colonies with the expected gene.
Figure 4.4 The electrophoresis results of colony PCR demonstrated successful plasmid transfer into XLI Blue in the cloning construction process
A: Full length of CD2v, B: CD2v cytoplasmic domain, CD2v extracellular domain and CD2v without signal peptide, M: Maker 1kb (Thermo Scientific)
The expected bands of CD2v in full-length, CD2v without SP, CD2v cytoplasmic domain and CD2v extracellular domain were: ≈ 1500 bp, ≈ 1400 bp, ≈ 1000 bp ≈ 750 bp, respectively (Figure 4.4)
After PCR colony, we performed a double digestion with 2 enzymes: BamHI and
The re-examination of PspOMI yielded results displayed through electrophoresis on a 1% agarose gel The expected bands included the vector frame pRTRA PII at approximately 3363 bp, the full-length CD2v at around 1083 bp, CD2v without the signal peptide at about 1063 bp, the cytoplasmic domain of CD2v at roughly 550 bp, and the extracellular domain at approximately 300 bp Additionally, colony PCR results confirmed the successful insertion of all four DNA sequences encoding the CD2v protein into the cloning vectors pRTRA.
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
A: Full length of CD2v, B: CD2v cytoplasmic domain, CD2v extracellular domain and CD2v without signal peptide, M: Maker 1kb (Thermo Scientific)
4.1.3 Construction of the expression vectors that contain the gene encoding
Figure 4.6 Schematic representation of expression vector construction
We utilized pCB301 as a binary vector to house plant-expression cassettes after creating a cloning vector for amplifying recombinant plasmids The construction of the expression vector is illustrated in Figure 4.6 We cultured colonies containing the plasmid pRTRA PII, which included various constructs: CD2v full-length, CD2v without SP, CD2v cytoplasmic domain, and CD2v extracellular domain Following the plasmid extraction method, we extracted the plasmids of these four constructs Each plasmid was then digested with the enzyme HindIII to generate compatible ends, and the digestion products were analyzed using electrophoresis on a 1.5% agarose gel.
After digestion with HindIII, the expected band size for the frame vector pRTRA PII is approximately 2600 bp The constructs CD2v full-length, CD2v without SP, CD2v cytoplasmic domain, and CD2v extracellular domain yielded sizes of approximately 2100 bp, 2000 bp, 1500 bp, and 1400 bp, respectively Figure 4.7 illustrates that the bands for these four constructs align with the expected sizes.
Figure 4.7 Single digestion with restriction enzyme HindIII of 4 different CD2v constructs
A: Full length of CD2v, B: CD2v cytoplasmic domain, CD2v extracellular domain and CD2v without signal peptide, M: Maker 1kb (Thermo Scientific)
The cassettes for each construct—CD2v full-length (≈ 2100 bp), CD2v without SP (≈ 2000 bp), CD2v cytoplasmic domain (≈ 1500 bp), and CD2v extracellular domain (≈ 1400 bp)—were extracted from the gel, purified, and then ligated with the expression vector pCB 301 Following this, the recombinant plasmids were transformed into XLI Blue via heat shock Colony PCR was conducted to verify the presence of the desired gene segments, with expected band sizes of approximately 1500 bp for CD2v full-length, 1400 bp for CD2v without SP, 1000 bp for CD2v cytoplasmic domain, and 750 bp for CD2v extracellular domain The results, as shown in Figure 4.8, confirmed the successful amplification of the bands for each construct.
Figure 4.8 The electrophoresis results of colony PCR demonstrated successful plasmids transfer into XLI Blue in the expression construction process
A: Full length of CD2v, B: CD2v cytoplasmic domain, CD2v extracellular domain and CD2v without signal peptide, M: Maker 1kb (Thermo Scientific)
In the next step, plasmids pCB301 contain desirable cassette of 4 constructs were undergo single digested with enzyme NotI for confirmation The results were
Figure 4.9 illustrates the expected band of the pCB301 frame at approximately 5561 bp The constructs of CD2v show varying lengths: the full-length CD2v is around 2043 bp, CD2v without the signal peptide (SP) is approximately 2000 bp, the CD2v cytoplasmic domain measures about 1500 bp, and the CD2v extracellular domain is roughly 1400 bp.
Figure 4.9 The results of single digestion with NotI to check for the presence of the desired gene CD2v
A: Full length of CD2v, B: CD2v cytoplasmic domain, CD2v extracellular domain and CD2v without signal peptide, M: Maker 1kb (Thermo Scientific)
In summary, this step successfully transformed constructs with DNA sequences encoding full-length CD2v, CD2v without the signal peptide, the cytoplasmic domain of CD2v, and the extracellular domain of CD2v into the expression vector pCB301, resulting in recombinant plasmids that are ready for transformation into AGLI.
E.coli carried the recombinant plasmid pCB 301 CD2v PII each of 4 constructs:
CD2v full-length, CD2v without SP, CD2v cytoplasmic domain, and CD2v extracellular domain were cultured in liquid LB medium, followed by the extraction and purification of DNA plasmids using a DNA purification method The electroporation technique was employed to transform these plasmids into A tumefaciens AGLI After a two-day incubation period, colony PCR was conducted to verify the presence of the desired genes in AGLI.
Expected band of 4 constructs above mentioned would be ≈ 1500 bp, ≈ 1400 bp,
The gel analysis revealed bands at approximately 1000 bp and 750 bp, confirming our expectations As illustrated in Figure 4.10, the recombinant plasmid containing the gene for the CD2v protein was successfully transformed.
A tumefaciens and well prepared for transformation into N benthamiana
Figure 4.10 The electrophoresis results of colony PCR demonstrated successful plasmid transfer into AGLI
A: Full length of CD2v, B: CD2v cytoplasmic domain, CD2v extracellular domain and CD2v without signal peptide, M: Maker 1kb (Thermo Scientific)
4.1.5 Transient expression of recombinant protein in N benthamiana by Agro- Infiltration methods
Five days post-transformation of A tumefaciens with recombinant plasmid into N benthamiana, leaf samples were collected to assess CD2v protein expression Although the leaves appeared green, signs of wilting were observed due to the plant's natural immune response, which targets foreign gene sequences in the infected areas Despite this, the foreign protein remained expressed on the leaves, indicating that this was the optimal time for harvesting and detecting the presence of CD2v protein, as illustrated in Figure 4.11 below.
Figure 4.11 Picture depicting the physical state of N benthamiana transformed by
AGLI which contains transgenic plasmids CD2v of 4 constructs
A: CD2v full-length, B: CD2v without signal peptide, C: CD2v cytoplasmic domain, D: CD2v extracellular domain
4.1.6 Evaluation the transient expression of CD2V protein by Western blot
After a 5-day collection period, the leaves are crushed and denatured We then extracted 10-30 µg of protein, which was separated using SDS-PAGE electrophoresis with 30% polyacrylamide The proteins were subsequently transferred to a Nitrocellulose membrane using a Fast Film Transfer Machine (Thermo Scientific) The expression of CD2v proteins was successfully detected via Western blotting with a c-myc antibody, as illustrated in Figure 4.12.
Figure 4.12 Western-Blot results with crude extract were electrophoresis on SDS -
PAGE gel and detected by Cmyc antibodies
(A) Full: Full length of CD2v (B) W/o SP: CD2v without signal peptide (C) Cyto:
Cytoplasmic domain of CD2v, Extra: Extracellular domain of CD2v, M: marker protein and
In the detection by Western Blot system using c myc-specific antibodies, the c myc-tagging facilitates the identification of the recombinant protein based on the antigen-antibody specific hybrid reaction
The full-length CD2v protein did not exhibit membrane presence, suggesting it was not expressed in N benthamiana for unknown reasons In contrast, CD2v lacking the signal peptide showed weak expression in N benthamiana, while the cytoplasmic domain of CD2v demonstrated stronger expression Notably, the extracellular domain of CD2v exhibited the highest expression levels due to glycosylation, significantly outperforming the other constructs despite equal protein loading.
Discussion
The urgent need for vaccine innovation is highlighted by the ongoing African swine fever outbreak, which has devastated millions of pigs since late 2019, leading to significant economic losses for farmers and increased pork prices In response to the pork supply crisis, the government and relevant ministries have ramped up imports of frozen pork from countries like Germany, Poland, Brazil, the United States, and Russia, with imports reaching approximately 25,300 tons by mid-March 2023, a 205% increase from 2019 Consequently, developing a vaccine against the ASFV virus is critical for ensuring the safe growth of the pig population and meeting national economic needs Many countries, including Canada, Japan, the United States, and South Korea, are advancing research on subunit vaccines using plant expression systems, with effective studies also being conducted at the Institute of Biotechnology in Vietnam to combat various pig diseases.
Subunit vaccines derived from plants offer significant advantages, including enhanced safety for both humans and the environment The production process is straightforward and can be easily scaled up, allowing for rapid vaccine development Within just 4-6 weeks, a substantial quantity of vaccines can be produced, enabling a swift response to potential pandemic threats.
The production of a plant-based subunit vaccine for African swine fever virus (ASFV) is a promising approach, as it is expected to be more cost-effective than traditional vaccine manufacturing technologies Despite the urgency of addressing ASF outbreaks, there is currently no published research on this vaccine in Vietnam or globally Given these practical considerations, this project has been proposed as a timely initiative.
The effectiveness level is a critical factor in vaccine development, particularly for African swine fever (ASF), caused by the ASFV virus, which has severely impacted the economy since late 2019 With no commercial vaccine currently available, the risk of outbreaks remains high Research into a vaccine is essential for socio-economic stability, supporting farmers' mental well-being, ensuring a steady supply of fresh pork, stabilizing pork prices, and reducing reliance on imported frozen pork The development of plant-origin subunit vaccines against ASF will advance the country's scientific and technological capabilities, offering a rapid, effective, and safe production method These subunit vaccines meet the DIVA (Differentiating Infected from Vaccinated Animals) standard, facilitating easier disease monitoring compared to inactivated vaccines However, gene expression in plants can be hindered by transcriptional and post-transcriptional gene silencing, which serves various roles, including viral defense and gene regulation To address this challenge, over 30 proteins that inhibit plant virus gene silencing have been identified.
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, we utilized the Hc-Pro protein, which inhibits RNA silencing in plants by preventing the degradation of mRNAs and double-stranded RNAs This action reduces the levels of siRNA and disrupts the cleavage function of the Dicer and RISC enzymes, ultimately leading to a failure in the RNA silencing process.
The CD2v antigen structure, particularly its transmembrane region and signal peptide sequences, plays a crucial role in cell-cell adhesion, virulence enhancement, and immune response modulation The signal peptide directs the protein's functionality, while the transmembrane region facilitates its storage in the Endoplasmic Reticulum membrane, impacting virus transmission and protein expression in plant systems To optimize CD2v gene segments, we systematically removed specific sequences, leading to improved protein expression Our findings indicate that the removal of the signal peptide and transmembrane regions significantly enhances antigen protein expression in plant systems, highlighting their importance in the overall expression process.
CONCLUSION AND SUGGESTION
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
The truncated CD2v protein, specifically its extracellular and cytoplasmic domains, exhibited strong expression in N benthamiana In contrast, the truncated CD2v protein lacking the signal peptide showed only slight expression, while the full CD2v protein was not expressed at all.
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 immunogenicity in animals.
In a study published in December 2019, Abid et al explored the generation and immunogenicity of a recombinant pseudorabies virus that co-expresses the E2 protein of the classical swine fever virus and the capsid protein of porcine circovirus type 2 This research utilized a fosmid library platform, contributing valuable insights into vaccine development for swine diseases The findings are documented in the journal Pathogens, highlighting the potential for improved immunization strategies in the swine industry.
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A study by Sunwoo et al (2019) published in the journal Vaccine examined the effectiveness of a DNA-protein vaccination strategy against the African swine fever virus, specifically the Armenia 2007 strain The findings revealed that this vaccination approach did not provide protection when challenged with the virus For more details, refer to the article at https://doi.org/10.3390/vaccines7010012.
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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