Self-cleaving 2A Peptide Research Articles

A versatile 2A peptide-based strategy for ectopic expression and endogenous gene tagging in Trypanosoma cruzi

Nearly all expression vectors currently available for Trypanosoma cruzi were conceived to produce a single primary transcript containing the genes of interest along with those that confer antibiotic resistance. However, since each messenger RNA (mRNA) matures separately, drug selection will only guarantee the expression of those derived from the selectable marker. Therefore, commonly a considerable fraction of the cells recovered after selection with these expression vectors, although resistant do not express the protein of interest. Consequently, in order to counteract this disadvantage, we developed vectors with an alternative arrangement in which the gene of interest and antibiotic resistance are fused sharing the same mRNA. To test this configuration, we included the coding sequence for the green fluorescent protein (mEGFP) linked to the one conferring neomycin resistance (Neo). Additionally, to allow for the production of two independent proteins the sequence for a Thosea asigna virus self-cleaving 2A peptide (T2A) was inserted in-between. Cells obtained with these vectors displayed higher mEGFP expression levels with more homogeneous transgenic parasite populations than those transfected with more conventional independent mRNA-based alternatives. Moreover, as determined by Western blot, 2A mediated fusion protein dissociation occurred with high efficiency in all parasite stages. In addition, these vectors could easily be transformed into endogenous tagging constructs that allowed the insertion, by ends-in homologous recombination, of a hemagglutinin tag (HA) fused to the actin gene. The use of 2A self-cleaving peptides in the context of single mRNA vectors represents an interesting strategy capable of improving ectopic transgene expression in T. cruzi as well as providing a simple alternative to more sophisticated methods, such as the one based on CRISPR/Cas9, for the endogenous labeling of genes.

Generation of bicistronic Dmp1-Cre knock-in mice using a self-cleaving 2A peptide.

The conditional manipulation of genes using the Cre recombinase-locus of crossover in P1 (Cre/loxP) system is an important tool for revealing gene functions and cell lineages in vivo. The outcome of this method is dependent on the performance of Cre-driver mouse strains. In most cases, Cre knock-in mice show better specificity than randomly inserted Cre transgenic mice. However, following knock-in, the expression of the original gene replaced by Cre is lost. We generated a new differentiated osteoblast- and osteocyte-specific Cre knock-in mouse line that carries the viral T2A sequence encoding a 2A self-cleaving peptide at the end of the coding region of the dentin matrix protein 1 (Dmp1) gene accompanied by the Cre gene. We confirmed that Dmp1-T2A-Cre mice showed high Cre expression in osteoblasts, osteocytes, odontoblasts, and periodontal ligament cells and that the 2A self-cleaving peptide efficiently produced both Dmp1 and Cre proteins. Furthermore, unlike the Dmp1 knockout mice, homozygous Dmp1-T2A-Cre mice showed no skeletal abnormalities. Analysis using the Cre reporter strain confirmed differentiated osteoblast- and osteocyte-specific Cre-mediated recombination in the skeleton. Furthermore, recombination was also detected in some nuclei of skeletal muscle cells, spermatocytes, and intestinal cells. 2A-Cre functions effectively in vivo, and Dmp1-T2A-Cre knock-in mice are a useful tool for studying the functioning of various genes in hard tissues.

Encoding of a transgene in-frame with a Newcastle disease virus protein increases transgene expression and stability.

Newcastle disease virus (NDV) has been extensively explored as a vector for vaccine and oncolytic therapeutic development. In conventional NDV-based vectors, the transgene is arranged as a separate transcription unit in the NDV genome. Here, we expressed haemagglutinin protein (HA) of an avian influenza virus using an NDV vector design in which the transgene ORF is encoded in-frame with the ORF of an NDV gene. This arrangement does not increase the number of transcription units in the NDV genome, and imposes a selection pressure against mutations interrupting the transgene ORF. We placed the HA ORF upstream or downstream of N, M, F and HN ORFs of NDV so that both proteins are encoded in-frame and are separated by either a self-cleaving 2A peptide, furin cleavage site or both. Only constructs in which HA was placed downstream of the NDV HN were viable. These constructs expressed the transgene at a higher level compared to the vector encoding the same transgene in the same position in the NDV genome but as a separate transcription unit. Furthermore, the transgene expressed in one ORF with the NDV protein proved to be more stable over multiple passages. Thus, this design may be useful for applications where the stability of the transgene expression is highly important for a recombinant NDV vector.

The Construction and Immunogenicity Analyses of Recombinant Pseudorabies Virus With NADC30-Like Porcine Reproductive and Respiratory Syndrome Virus-Like Particles Co-expression.

Porcine reproductive and respiratory syndrome (PRRS) and pseudorabies (PR) are highly infectious swine diseases and cause significant financial loss in China. The respiratory system and reproductive system are the main target systems. Previous studies showed that the existing PR virus (PRV) and PRRS virus (PRRSV) commercial vaccines could not provide complete protection against PRV variant strains and NADC30-like PRRSV strains in China. In this study, the PRV variant strain XJ and NADC30-like PRRSV strain CHSCDJY-2019 are used as the parent for constructing a recombinant pseudorabies virus (rPRV)-NC56 with gE/gI/TK gene deletion and co-expressing NADC30-like PRRSV GP5 and M protein. The rPRV-NC56 proliferated stably in BHK-21 cells, and it could stably express GP5 and M protein. Due to the introduction of the self-cleaving 2A peptide, GP5 and M protein were able to express independently and form virus-like particles (VLPs) of PRRSV in rPRV-NC56-infected BHK-21 cells. The rPRV-NC56 is safe for use in mice; it can colonize and express the target protein in mouse lungs for a long time. Vaccination with rPRV-NC56 induces PRV and NADC30-like PRRSV specific humoral and cellular immune responses in mice, and protects 100% of mice from virulent PRV XJ strain. Furthermore, the virus-neutralizing antibody (VNA) elicited by rPRV-NC56 showed significantly lower titer against SCNJ-2016 (HP-PRRSV) than that against CHSCDJY-2019 (NADC30-like PRRSV). Thus, rPRV-NC56 appears to be a promising candidate vaccine against NADC30-like PRRSV and PRV for the control and eradication of the variant PRV and NADC30-like PRRSV.

Abstract 106: Efficient In Vivo Homology-Directed Repair Within Cardiomyocytes

CRISPR/Cas9-based genome editing technologies provide powerful tools for genetic manipulation. Delivery of Cas9 and a homology directed repair (HDR) template using adeno-associated virus (AAV; CASAAV-HDR), was recently shown to enable creation of precise genomic edits, even within postmitotic cells. Here we studied CASAAV-HDR in cardiomyocytes. We constructed an AAV9 vector containing a gRNA targeting the ventricle specific Myl2 gene, and a promoterless HDR template that replaces the native Myl2 stop codon with a self-cleaving 2A peptide followed by mScarlet, a red fluorescent protein. When this vector was injected into Cas9 expressing newborn mice, we observed mScarlet expression within a remarkably high fraction of cardiomyocytes, approximately 45%. Expression was ventricle specific, consistent with the Myl2 expression profile. Similarly, when we targeted the atrial specific Myl7 gene, we observed mScarlet expression in ~20% of atrial cardiomyocytes. Amplicon sequencing of Myl2 and Myl7 transcripts showed that the vast majority of transcripts with an insertion were mutation-free, indicating that CASAAV-HDR is precise. Furthermore, CASAAV-HDR efficiency was comparable when AAV was delivered to fetal, neonatal, or mature mice. Next we targeted seven additional loci: Yap1, Tmem43, Nfatc3, Bdh1, Mkl1, Ttn, and Pln, fusing either an HA tag or mScarlet to each. Insertion efficiency varied dramatically between loci, with HDR efficiency generally correlating with target gene expression. TTN-mScarlet and mScarlet-PLN fusion proteins localized to the sarcomere and sarcoplasmic reticulum, respectively, consistent with the localization of the endogenous proteins. Collectively these data indicate that systemic delivery of CASAAV-HDR vectors can achieve efficient, precise, in vivo somatic genome modification that does not require cardiomyocyte proliferation. We successfully used this technology to monitor protein localization and anticipate it will be useful for many other applications, such as precise introduction of mutations to model disease or probe gene function. CASAAV-HDR may also enable efficient, permanent, and precisely targeted delivery of therapeutic transgenes to validated loci.

A novel 2A-peptide-containing plasmid to generate stable Perkinsus marinus cells expressing organelle-targeted genes.

Genetic manipulation techniques for marine protists are not well-established, despite immense efforts. However, Perkinsus marinus is an exception and can be developed as a genetically tractable model organism for related protists. Here, we designed a new plasmid for P.marinus that allows two proteins from a single mRNA to be differently localized using a self-cleaving 2A peptide. This enabled us to establish a stable transfectant expressing a mitochondrially targeted fluorescent protein. The system can be applied to any protein in theory and would make a powerful tool for investigating unique organelles in P.marinus and related dinoflagellates.

CRISPR/Cas9-Mediated Specific Integration of Fat-1 and IGF-1 at the pRosa26 Locus.

Many researchers have focused on knock-in pigs for site-specific integration, but little attention has been given to genetically modified pigs with the targeted integration of multiple recombinant genes. To establish a multigene targeted knock-in editing system, we used the internal ribosome entry site (IRES) and self-cleaving 2A peptide technology to construct a plasmid coexpressing the fatty acid desaturase (Fat-1) and porcine insulin-like growth factor-1 (IGF-1) genes at equal levels. In this study, pigs were genetically modified with multiple genes that were precisely inserted into the pRosa26 locus by using the clustered regularly spaced short palindrome repeat sequence (CRISPR)/CRISPR-related 9 (Cas9) system and somatic cell nuclear transfer technology (SCNT) in combination. Single copies of the Fat-1 and IGF-1 genes were expressed satisfactorily in various tissues of F0-generation pigs. Importantly, gas chromatography analysis revealed a significantly increased n-3 polyunsaturated fatty acid (PUFA) level in these genetically modified pigs, which led to a significant decrease of the n-6 PUFA/n-3 PUFA ratio from 6.982 to 3.122 (*** p < 0.001). In conclusion, the establishment of an editing system for targeted double-gene knock-in in this study provides a reference for the precise integration of multiple foreign genes and lays a foundation for the development of new transgenic pig breeds with multiple excellent phenotypes.

Receptor-ligand supplementation via a self-cleaving 2A peptide-based gene therapy promotes CNS axonal transport with functional recovery.

Gene replacement approaches are leading to a revolution in the treatment of previously debilitating monogenic neurological conditions. However, the application of gene therapy to complex polygenic conditions has been limited. Down-regulation or dysfunction of receptor expression in the disease state or in the presence of excess ligand has been shown to compromise therapeutic efficacy. Here, we offer evidence that combined overexpression of both brain-derived neurotrophic factor and its receptor, tropomyosin receptor kinase B, is more effective in stimulating axonal transport than either receptor administration or ligand administration alone. We also show efficacy in experimental glaucoma and humanized tauopathy models. Simultaneous administration of a ligand and its receptor by a single gene therapy vector overcomes several problems relating to ligand deficiency and receptor down-regulation that may be relevant to multiple neurodegenerative diseases. This approach shows promise as a strategy to target intrinsic mechanisms to improve neuronal function and facilitate repair.

Expanding base editing scope to near-PAMless with engineered CRISPR/Cas9 variants in plants

Expanding base editing scope to near-PAMless with engineered CRISPR/Cas9 variants in plants

Golden Gate Cloning-Compatible DNA Replicon/2A-Mediated Polycistronic Vectors for Plants.

The expression of multiple proteins and high-throughput vector assembly system are highly relevant in the field of plant genetic engineering and synthetic biology. Deployment of the self-cleaving 2A peptide that mediates polycistronic gene expression has been an effective strategy for multigene expression, as it minimizes issues in coordinated transgene regulation and trait staking in plants. However, efficient vector assembly systems optimized for 2A peptide-mediated polycistronic expression are currently unavailable. Furthermore, it is unclear whether protein expression levels are influenced by the transgene position in the polycistronic expression cassette. In this article, we present Golden Gate cloning-compatible modular systems allowing rapid and flexible construction of polycistronic expression vectors applicable for plants. The genetic modules comprised 2A peptides (T2A and P2A)-linked tricistron expression cassette and its acceptor backbones, named pGO-DV1 and pGO-DV2. While both acceptor backbones were binary T-DNA vectors, pGO-DV2 was specially designed to function as a DNA replicon enhancing gene expression levels. Using the Golden Gate cloning, a set of six tricistronic vectors was constructed, whereby three transgenes encoding fluorescent proteins (mCherry, eYFP, and eGFP) were combinatorially placed along the expression cassette in each of the binary vectors. Transient expression of the construct in tobacco leaves revealed that the expression levels of three fluorescent proteins were comparable each other regardless of the gene positions in the tricistronic expression cassette. pGO-DV2-based constructs were able to increase protein expression level by up to 71%, as compared to pGO-DV1-based constructs.

Co-expression of self-cleaved multiple proteins derived from Porcine Reproductive and Respiratory Syndrome Virus by bi-cistronic and tri-cistronic DNA vaccines

Porcine Reproductive and Respiratory Syndrome caused by Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) remains one of the important diseases in swine industry. A vaccine that is safe, effective and also elicit broad immune response against multiple antigens is desirable. In this study, we developed multi-cistronic DNA vaccines capable of co-expressing multiple structural proteins derived from PRRSV. To preserve the structure and function of each antigen protein, we employed self-cleaving 2A peptides to mediate separation of multiple proteins expressed by multi-cistronic genes. Six bi-cistronic genes encoding PRRSV GP5 and M proteins were generated, by which each construct contains different 2A sequences derived from Foot-and-mouth disease virus (F2A), porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A) either with or without furin cleavage site (Fu). Vectored by the mammalian expression plasmid pTH, all six bi-cistronic genes co-expressed the proteins GP5 and M at comparable level. Importantly, all six types of 2A sequences could mediate a complete self-cleavage of the GP5 and M. We next generated tri-cistronic DNA vaccines co-expressing the PRRSV proteins GP5, M and N. All homologous and heterologous combinations of P2A and F2A in tri-cistronic genes yielded a complete self-cleavage of the GP5, M and N proteins. Our study reports a success in co-expression of multiple PRRSV structural proteins in discrete form from a single vaccine and confirms feasibility of developing one single vaccine that provides broad immune responses against PRRSV.

N-Terminal Amino Acids Determine KLF4 Protein Stability in 2A Peptide-Linked Polycistronic Reprogramming Constructs

SummaryA common strategy for multi-protein expression is to link genes by self-cleaving 2A peptide sequences. Yet, little is known how the 2A peptide-derived N-terminal proline or adjacent non-native residues introduced during cDNA cloning affects protein stoichiometry. Polycistronic reprogramming constructs with altered KLF4 protein stoichiometry can influence induced pluripotent stem cell (iPSC) generation. We studied the impact of N-terminal 2A peptide-adjacent residues on the protein stability of two KLF4 isoforms, and assayed their capacity to generate iPSCs. Here, we show that the N-terminal proline remnant of the 2A peptide, alone or in combination with leucine, introduced during polycistronic cloning, destabilizes KLF4 resulting in increased protein degradation, which hinders reprogramming. Interestingly, the addition of charged and hydrophilic amino acids, such as glutamate or lysine stabilizes KLF4, enhancing reprogramming phenotypes. These findings raise awareness that N-terminal modification with 2A peptide-derived proline or additional cloning conventions may affect protein stability within polycistronic constructs.

Production and Application of Multicistronic Constructs for Various Human Disease Therapies.

The development of multicistronic vectors has opened up new opportunities to address the fundamental issues of molecular and cellular biology related to the need for the simultaneous delivery and joint expression of several genes. To date, the examples of the successful use of multicistronic vectors have been described for the development of new methods of treatment of various human diseases, including cardiovascular, oncological, metabolic, autoimmune, and neurodegenerative disorders. The safety and effectiveness of the joint delivery of therapeutic genes in multicistronic vectors based on the internal ribosome entry site (IRES) and self-cleaving 2A peptides have been shown in both in vitro and in vivo experiments as well as in clinical trials. Co-expression of several genes in one vector has also been used to create animal models of various inherited diseases which are caused by mutations in several genes. Multicistronic vectors provide expression of all mutant genes, which allows the most complete mimicking disease pathogenesis. This review comprehensively discusses multicistronic vectors based on IRES nucleotide sequence and self-cleaving 2A peptides, including its features and possible application for the treatment and modeling of various human diseases.

Effects of different 2A peptides on transgene expression mediated by tricistronic vectors in transfected CHO cells.

Multicistronic vectors can increase transgene expression and decrease the imbalance of gene expression in the Chinese hamster ovary (CHO) cell expression system. Small, self-cleaving 2A peptides have a high cleavage efficiency and are essential for constructing high-expression multicistronic vectors. In this study, we investigated the effects of two different 2A peptides on transgene expression in CHO cells via their mediating action on tricistronic vectors. The enhanced green fluorescent protein (eGFP) and red fluorescent protein (RFP) genes were linked by the porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A) peptides in a multicistronic vector. We transfected CHO cells with these vectors and screened for the presence of blasticidin-resistant colonies. Flow cytometry and real-time quantitative PCR (qPCR) were used to detect the expression levels of eGFP and RFP and the copy numbers of stably transfected cells. The results showed that P2A could enhance eGFP and RFP expression by 1.48- and 1.47-fold, respectively, compared to T2A. The expression levels of the genes were not proportional to their copy numbers. In conclusion, we found that P2A can effectively drive transgene expression in CHO cells and a potent 2A peptide can be used for recombinant protein production in the CHO cell system.

Optimized CRISPR/Cas9-mediated in vivo genome engineering applicable to monitoring dynamics of endogenous proteins in the mouse neural tissues

To analyze the expression, localization, and functional dynamics of target proteins in situ, especially in living cells, it is important to develop a convenient, versatile, and efficient method to precisely introduce exogenous genes into the genome, which is applicable for labeling and engineering of the endogenous proteins of interest. By combining the CRISPR/Cas9 genome editing technology with an electroporation technique, we succeeded in creating knock-in alleles, from which GFP (RFP)-tagged endogenous proteins are produced, in neurons and glial cells in vivo in the developing mouse retina and brain. Correct gene targeting was confirmed by single-cell genotyping and Western blot analysis. Several gene loci were successfully targeted with high efficiency. Moreover, we succeeded in engineering the mouse genome to express foreign genes from the endogenous gene loci using a self-cleaving 2A peptide. Our method could be used to monitor the physiological changes in localization of endogenous proteins and expression levels of both mRNA and protein at a single cell resolution. This work discloses a powerful and widely applicable approach for visualization and manipulation of endogenous proteins in neural tissues.

Generation of a CRISPR/Cas9-Based Vector Specific for Gene Manipulation in Leishmania major

Gene manipulation strategies including gene knockout and editing are becoming more sophisticated in terms of mechanism of action, efficacy and ease of use. In classical molecular methods of gene knockout, homologous arms are designed for induction of crossing over event in double strand DNA. Recently, CRISPR/Cas9 system has been emerged as a precise and powerful tool for gene targeting. In this effort, we aimed to generate a CRISPR/Cas9-based vector specific for targeting genes in Leishmania parasites. U6 and DHFR promoters and neomycin-resistance gene were amplified from genome of L. major (MHRO/IR/75/ER) and pEGFP-N1, respectively. U6 promoter was cloned in pX330 vector which is named as pX330-U6. DHFR promoter and neo resistance gene sequence fragments were fused using a combination of SOE (Splicing by overlap extension)-PCR and T/A cloning techniques. To generate pX-leish, fused fragments su-bcloned into the pX330-U6. Two sgRNAs were designed to target the gp63 gene and cloned in pX-leish. The pX-leish vector was designed for simultaneous expression of cas9 and G418 resistance proteins along with a self-cleaving 2A peptide for efficient separation of the two proteins. In this study pX-leish was designed with 3 features: 1) Compatible promoters with Leishmania parasites. 2) Insertion of antibiotic selection marker 3) Designing an all-in-one vector containing all components required for CRISPR/Cas9 system. This modified system would be valuable in genome manipulation studies in Leishmania for vaccine research in future.

Overexpression of the DOF-Type Transcription Factor Enhances Lipid Synthesis in Chlorella vulgaris

In the present study, DNA binding with one finger (DOF)-type transcription factors were screened from the Chlorella vulgaris genome database. The candidate DOF transcription factor was endogenously overexpressed in C. vulgaris to improve neutral lipid production. The protein expression vector contains the heat shock protein 70 and ribulose 1,5-bisphosphate carboxylase/oxygenase small subunit promoters and self-cleaving 2A peptide to reduce the transgene-silencing effect of C. vulgaris. A total of 74 phleomycin-resistant transformants were obtained. Under nitrogen-deficient conditions, the transformant CvDOF#3 showed approximately 1.5-fold higher neutral lipid content per cell compared to the original strain and also showed a His-tagged DOF candidate protein expression of 0.6%. Microscopic observations revealed that CvDOF#3 cells were larger in size. However, the observed differences in average cell diameter between CvDOF#3 and control cells were not statistically significant. These results indicated that the protein expression vector harboring the dual promoters and the 2A peptide, when used in combination with enzymatic cell wall degradation and glass bead transformation, could be useful for transgene and protein expression in C. vulgaris. Further experiment is necessary to confirm the expression efficiency of the HSP70 and RBCS dual promoter and 2A peptide strategy after construction of homologous recombination system in C. vulgaris. Our findings suggested that the overexpression of the endogenous DOF-type transcription factor can be used for improving the lipid content in C. vulgaris.

Transposon mediated reprogramming of buffalo fetal fibroblasts to induced pluripotent stem cells in feeder free culture conditions

Commonly, induced pluripotent stem (iPS) cells are generated by viral transduction of four core reprogramming genes, but recent evidences suggest that slightly different combination of transcription factors improve the efficiency and quality of generated iPS cells. However, vectors like retro- and lentiviral may cause insertional mutagenesis due to its integrating ability. Hence, alternate methods with safety concerns are needed to be investigated. Therefore, the present study was undertaken to reprogram buffalo fibroblasts using non-viral piggyBac (PB) transposon mediated transfer of six transcription factors. To generate buffalo iPS cells, fibroblasts were isolated from buffalo fetus at passage 2. The cells were co-electroporated with a PB transposon having CAGGS promoter driven cassette of Oct4, Sox2, Klf4, cMyc, Nanog, and Lin28 transcription factors separated by self-cleaving 2A peptide and a helper plasmid pCMV-PB transposase. After 12–14 days post electroporation, fibroblast cells morphology was observed to change to round structures which formed loose aggregates of cells on day 18. Putative iPS cell colonies were propagated in feeder free system and characterized through expression of pluripotency markers such as alkaline phosphatase, SSEA-1, SSEA-4, SSEA-5, TRA-1-81, Oct4, Nanog and Sox2 and endogenous genes supported the stemness property of the generated cells. These cells differentiated in vitro to form embryoid bodies and were found to express three germ layers markers. In conclusion, generation of buffalo iPS cells using transposon system provides insights into viral-free iPS technology which will facilitate genetic modification of the buffalo genome and help in the production of transgenic animals using genetically modified iPS cells.

Developmental analyses of mouse embryos and adults using a non-overlapping tracing system for all three germ layers.

Genetic lineage-tracing techniques are powerful tools for studying specific cell populations in development and pathogenesis. Previous techniques have mainly involved systems for tracing a single gene, which are limited in their ability to facilitate direct comparisons of the contributions of different cell lineages. We have developed a new combinatorial system for tracing all three germ layers using self-cleaving 2A peptides and multiple site-specific recombinases (SSRs). In the resulting TRiCK (TRiple Coloured germ layer Knock-in) mice, the three germ layers are conditionally and simultaneously labelled with distinct fluorescent proteins via embryogenesis. We show that previously reported ectopic expressions of lineage markers are the outcome of secondary gene expression. The results presented here also indicate that the commitment of caudal axial stem cells to neural or mesodermal fate proceeds without lineage fluctuations, contrary to the notion of their bi-potency. Moreover, we developed IMES, an optimized tissue clearing method that is highly compatible with a variety of fluorescent proteins and immunostaining, and the combined use of TRiCK mice and IMES can facilitate comprehensive analyses of dynamic contributions of all three germ layers.

An Effective Way of Producing Fully Assembled Antibody in Transgenic Tobacco Plants by Linking Heavy and Light Chains via a Self-Cleaving 2A Peptide.

Therapeutic monoclonal antibodies (mAbs) have evolved into an important class of effective medicine in treatment of various diseases. Since the antibody molecule consists of two identical heavy chains (HC) and two light chains (LC), each chain encoded by two different genes, their expressions at similar levels are critical for efficient assembly of functional recombinant mAbs. Although the plant-based expression system has been tested to produce fully assembled recombinant mAbs, coordinately expressing HC and LC at similar levels in a transgenic plant remains a challenge. A sequence coding for a foot-and-mouth disease virus (FMDV) 2A peptide has been successfully used to link two or more genes, which enable the translated polyprotein to be “self-cleaved” into individual protein in various genetically modified organisms. In the present study, we exploited the usage of F2A in Ebola virus monoclonal antibody (EBOV mAb) production. We found that transgenic tobacco plants carrying a transcription unit containing HC and LC linked by 2A not only produced similar levels of HC and LC but also rendered a higher yield of fully assembled EBOV mAb compared to those expressing HC and LC in two independent transcription units. Purified EBOV mAb bound to an Ebola epitope peptide with apparent Kd-values of 90.13–149.2 nM, indicating its proper assembly and high affinity binding to Ebola epitope peptide. To our knowledge, this is the first report showing mAb production by overexpressing a single transcription unit consisting of HC, LC and 2A in stable transformed tobacco plants.