Cucumber mosaic virus-based recombinant vector

Abstract

Embodiments relate to an improved recombinant vector based on cucumber mosaic virus. The recombinant vector according to the embodiments can significantly improve the expression efficiency of foreign proteins in plants, has remarkably higher foreign protein expression efficiency than existing recombinant vectors, and thus can be widely used for the production of useful proteins and research thereof in various plant species.

Description

Cucumber mosaic virus-based recombinant vector

[0001] Various embodiments of the present disclosure relate to recombinant genetic vectors based on cucumber mosaic virus (CMV).

[0002] Molecular Farming, a technology that produces useful proteins from plants, is a key topic in low-carbon green bio research and is gaining increasing attention as an alternative technology for rapidly responding to pandemic diseases such as COVID-19.

[0003] Plant virus vectors can be used as part of molecular farming technology to produce useful proteins in plants. Plant viruses possess strong infectivity and the ability to mass-produce viral proteins in plant cells. Furthermore, these plant viruses possess diverse characteristics, including a small genome size that facilitates genetic manipulation, strong reproductive capacity, systemic infection in plants, and harmlessness to humans. These characteristics, coupled with advances in genetic engineering, are highlighting the value of plant viruses as tools for plant biotechnology.

[0004] By genetically manipulating the genome of a plant virus, a gene transfer vector capable of expressing a foreign gene in plant cells can be developed, which can be used to mass-produce desired recombinant proteins in plants safely, quickly, and inexpensively without genetic modification of the plants.

[0005] Through research, the inventors of the present invention developed a viral vector capable of mass-producing recombinant proteins in tobacco plants using cucumber mosaic virus (CMV), and evaluated the recombinant protein expression efficiency of the CMV-based vector using green fluorescent protein (GFP), etc., and confirmed that it had high expression efficiency in tobacco plants.

[0006] In addition, the present inventors have further developed a CMV vector-based recombinant protein expression technology for mass production of useful proteins in tobacco plants through further research.

[0007] Embodiments provide improved recombinant vectors based on cucumber mosaic virus (CMV).

[0008] The problems to be solved through the embodiments are not limited to the problems described above, and problems not mentioned can be clearly understood by a person having ordinary skill in the technical field to which the embodiments belong from this specification and the attached drawings.

[0009] In one embodiment, a cucumber mosaic virus recombinant vector may include a first vector comprising RNA1 of cucumber mosaic virus (CMV), a second vector comprising RNA2 of CMV, and a third vector comprising modified RNA3 of CMV, wherein the modified RNA3 may have a coat protein (CP) gene deleted to introduce a target gene.

[0010] In one embodiment, the deletion of the coat protein gene may comprise the deletion of five consecutive codons of the CP open reading frame (ORF) downstream (3') from the translation initiation codon, including the translation initiation codon of the CP ORF.

[0011] In one embodiment, the modified RNA3 may be positioned in the upstream (5') region of the translation initiation codon of the target gene to be introduced, where one of the multi-cloning sites (MCS) that is cleaved by a restriction enzyme to introduce the target gene is introduced.

[0012] In one embodiment, the third vector may comprise nucleotides comprising sequence number 15.

[0013] In one embodiment, the second vector may comprise a gene silencing suppressor.

[0014] In one embodiment, the gene silencing suppressor may comprise the B2 gene of flock house virus (FHV).

[0015] In one embodiment, the second vector may comprise nucleotides comprising sequence number 2.

[0016] In one embodiment, the B2 gene can be introduced with the C-terminus of the 2b ORF of CMV RNA2 removed and a self-cleaving peptide (LLNFDLLKLAGDVESNPG / P) of foot and mouth disease virus (FMDV) and a MluI site at that location.

[0017] In another embodiment, the gene silencing suppressor may comprise the P19 gene of tobacco bush stunt virus (TBSV).

[0018] In another embodiment, the second vector may comprise nucleotides comprising sequence number 14.

[0019] In another embodiment, the P19 gene can be introduced with the C-terminus of the 2b ORF of CMV RNA2 removed and a self-cleaving peptide (LLNFDLLKLAGDVESNPG / P) of foot and mouth disease virus (FMDV) and a MluI site at that location.

[0020] The transformed microorganism according to one embodiment may be transformed by at least one of the cucumber mosaic virus recombinant vectors described above.

[0021] A method for producing a protein according to one embodiment may include the steps of constructing at least one of the cucumber mosaic virus recombinant vectors in which a target gene is introduced into the above-described modified RNA3, transforming the recombinant vector into a microorganism, and inoculating the transformed microorganism into a plant.

[0022] According to the cucumber mosaic virus-based recombinant vector according to the embodiments, the efficiency of expression of foreign proteins in plants can be significantly increased, and thus, it can be widely used for production of useful proteins and research thereof in various plant species.

[0023] The effects of the embodiments are not limited to the effects described above, and effects not mentioned can be clearly understood by a person having ordinary skill in the art to which the embodiments belong from this specification and the attached drawings.

[0024] FIG. 1 is a diagram showing the structure of a cucumber mosaic virus-based vector according to one embodiment.

[0025] Figure 1a is a diagram showing the structures of pCMV-GTN-R1, pCMV-GTN-R2, and pCMV-GTN-R3.

[0026] Figure 1b is a diagram showing the structure of pCMV-R3V.

[0027] Figure 1c is a diagram showing the structure of pCMV-R2V-B2.

[0028] Figure 1d is a drawing showing the structure of pCMV-R3V-GFP, in which a green fluorescent protein (GFP) gene is inserted into the pCMV-R3V vector to confirm the efficiency of expression of a foreign gene of pCMV-R3V.

[0029] Figure 1e is a diagram showing the structures of pCMV-Fny-R1 and pCMV-Fny-R2, infectious clones of the CMV-Fny strain for use as controls.

[0030] Figure 1f is a diagram showing the structure of PZP-GFP for use as a control.

[0031] Figure 2 is a drawing showing the results of evaluating the efficiency of expression of foreign genes of CMV-based vectors in plants, and is a drawing showing the observation of GFP expression over time at the inoculation site using a fluorescence measurement device (FOBI system) after inoculating each combination of Agrobacterium suspensions.

[0032] Figure 2a is a diagram comparing and analyzing the GFP expression efficiency between the CMV-GTN-GFP combination (pCMV-GTN-R1 + pCMV-GTN-R2 + pCMV-R3V-GFP) and PZP-GFP.

[0033] Figure 2b is a diagram comparing and analyzing the GFP expression efficiency between the CMV-GTN-GFP combination and the CMV-Fny-GFP combination (pCMV-Fny-R1 + pCMV-Fny-R2 + pCMV-R3V-GFP).

[0034] Figure 3 is a drawing showing the results of evaluating the GFP expression efficiency of a CMV-GTN-based vector in plants, and a drawing comparing and analyzing the expression efficiency between the CMV-GTN-GFP combination and the CMV-GTN-B2-GFP combination (pCMV-GTN-R1 + pCMV-R2V-B2 + pCMV-R3V-GFP).

[0035] Figure 3a is a drawing showing the expression of GFP over time at the inoculation site after inoculation with the Agrobacterium suspension of each combination using a fluorescence measurement device (FOBI system).

[0036] Figure 3b is a drawing showing the SDS PAGE analysis of total protein extracted from the inoculation site on the second day after inoculation.

[0037] Figure 3c is a diagram showing the results of quantitative comparative analysis of the GFP band of Figure 3b using ImageJ.

[0038] Figure 3d is a diagram showing the results of quantitative analysis of the GFP expression amount of a recombinant CMV GTN-based vector relative to plant fresh weight.

[0039] FIG. 4 is a diagram showing the structure of a cucumber mosaic virus-based vector according to another embodiment.

[0040] Figure 4a is a diagram showing the structure of pCMV-R3V.

[0041] Figure 4b is a diagram showing the structure of pCMV-R3V2.

[0042] Figure 4c is a drawing showing the structure of pCMV-R3V2-GFP, in which a green fluorescent protein (GFP) gene is inserted into the pCMV-R3V2 vector, to confirm the foreign gene expression efficiency of pCMV-R3V2.

[0043] Figure 4d is a diagram showing the structure of pCMV-R3V2-IF-GFP, in which a green fluorescent protein (GFP) gene was inserted into the same position as Figure 4c through In-Fusion cloning to remove the non-viral sequence of pCMV-R3V2-GFP shown in Figure 4c.

[0044] Figure 5 is a drawing showing the results of evaluating the expression efficiency of foreign genes of CMV RNA3-based vectors in plants, and is a drawing analyzing the expression efficiency of pCMV-R3V2-GFP and pCMV-R3V2-IF-GFP compared to pCMV-R3V-GFP.

[0045] Figure 5a is a drawing showing that the control group (pCMV-R3V-GFP) was inoculated on one side of the leaf and the experimental group (pCMV-R3V2-GFP or pCMV-R3V2-IF-GFP) was inoculated on the other side of the leaf.

[0046] Figure 5b is a drawing showing the expression of GFP over time at the inoculation site after inoculation of a CMV RNA3-based vector into a plant, observed using a fluorescence measurement device (FOBI system).

[0047] Figure 5c is a diagram showing the results of quantitative analysis of the GFP expression amount of a recombinant CMV RNA3-based vector relative to plant fresh weight.

[0048] Figure 5d is a drawing showing the SDS PAGE analysis of total protein extracted from the inoculation site on the second day after inoculation.

[0049] FIG. 6 is a diagram showing the structure of a cucumber mosaic virus-based vector according to another embodiment.

[0050] Figure 6a is a diagram showing the structure of pCMV-R2V.

[0051] Figure 6b is a drawing showing the structure of pCMV-R2V-B2, a B2 expression recombinant vector produced using pCMV-R2V.

[0052] Figure 6c is a drawing showing the structure of pCMV-R2V-P19, a P19 expression recombinant vector produced using pCMV-R2V.

[0053] Figure 7 is a drawing showing the results of evaluating the efficiency of foreign gene expression of CMV RNA3-based vectors in plants, and a drawing comparing and analyzing the effects of pCMV-GTN-R2, pCMV-R2V-B2, and pCMV-R2V-P19 on the efficiency of foreign gene expression.

[0054] Figure 7a is a drawing showing the expression of GFP over time at the inoculation site after inoculation of a CMV RNA3-based vector into a plant, observed using a fluorescence measurement device (FOBI system).

[0055] Figure 7b is a diagram showing the results of quantitative analysis of the GFP expression amount of a recombinant CMV RNA3-based vector relative to plant fresh weight.

[0056] The terms used in the examples are selected from widely used, current terms, taking into account the functions of the present invention. However, these terms may vary depending on the intentions of those skilled in the art, precedents, the emergence of new technologies, etc. Furthermore, in certain cases, the applicant may arbitrarily select terms, and in such cases, their meanings will be described in detail in the description of the relevant invention. Therefore, the terms used in the present invention should be defined not simply based on their names, but based on their meanings and the overall content of the present invention.

[0057] When a part of the specification is said to "include" a component, this does not exclude other components, but rather implies the inclusion of other components, unless otherwise specifically stated. Furthermore, terms such as "-unit" and "-module" used in the specification refer to a unit that processes at least one function or operation, which may be implemented in hardware, software, or a combination of hardware and software.

[0058] As used herein, when an expression such as "at least one" precedes an array of elements, it modifies the entire array of elements, not just each individual element. For example, the expression "at least one of a, b, and c" should be interpreted to include a, b, c, or a and b, a and c, b and c, or a and b and c.

[0059] In addition, when describing the embodiments disclosed in this specification, if it is determined that a detailed description of a related known technology may obscure the gist of the embodiments disclosed in this specification, the detailed description thereof will be omitted. In addition, the attached drawings are only intended to facilitate easy understanding of the embodiments disclosed in this specification, and the technical ideas disclosed in this specification are not limited by the attached drawings, and should be understood to include all modifications, equivalents, and substitutes included in the spirit and technical scope of the present disclosure.

[0060] Terms that include ordinal numbers, such as first, second, etc., may be used to describe various components, but the components are not limited by these terms. These terms are used solely to distinguish one component from another.

[0061] When a component is referred to as being "connected" or "connected" to another component, it should be understood that it may be directly connected or connected to that other component, but that there may be other components intervening. Conversely, when a component is referred to as being "directly connected" or "connected" to another component, it should be understood that there are no other components intervening.

[0062] Singular expressions include plural expressions unless the context clearly indicates otherwise.

[0063] In this specification, cucumber mosaic virus (CMV) is the virus that occurs most frequently and causes serious damage in domestic pepper cultivation. CMV has a wide host range, infecting 885 species of plants in 65 genera, and is a positive-sense single-stranded RNA virus composed of three segmented genomes. CMV isolates can be classified into subgroups IA, IB, and II according to the base sequence. Among the CMV isolates, P0 type CMV-Fny (CMV-Fny strain) belongs to subgroup IA, and P1 type CMV-GTN (CMV-GTN strain) belongs to subgroup IB. In this specification, the CMV-GTN refers to a highly pathogenic P1 type, which was purely isolated from the pepper variety 'Cheongyang' grown in a pepper farm in Goesan-gun, Chungcheongbuk-do in 2013 (Res. Plant Dis. 21(2): 99-102(2015)).

[0064] As used herein, the term "recombinant vector" refers to a vector capable of expressing a target foreign gene in a host cell, and which includes essential regulatory elements operably linked to enable expression of the gene insert. Suitable vectors include, in addition to expression regulatory sequences such as a promoter, operator, initiation codon, termination codon, polyadenylation signal, and enhancer, a signal sequence or leader sequence for membrane targeting or secretion, and can be manufactured in various ways depending on the purpose.

[0065] As used herein, the term "operatively linked" means that a gene required for expression and its regulatory sequence are linked to each other in a manner that enables gene expression.

[0066] As used herein, the term "identity" refers to the overall relatedness between polymer molecules, for example, between nucleic acids (e.g., DNA molecules and / or RNA molecules) and / or between polypeptides. For example, polypeptides are considered to be "substantially identical" to one another if their amino acid sequences are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. Calculating the percent identity of two nucleic acid or polypeptide sequences can be performed, for example, by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced into one or both of the first and second sequences for optimal alignment, and non-identical sequences can be disregarded for comparison purposes). For example, the length of the aligned sequences for comparison purposes is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of the reference sequence. The nucleic acid or polypeptide sequences at corresponding positions are then compared. Determination of the percent identity between the two sequences and the comparison of the sequences can be accomplished using mathematical algorithms. As is well known to those skilled in the art, amino acid or nucleic acid sequences can be compared using any of a variety of algorithms available in commercial computer programs, such as BLASTN for nucleotide sequences, and BLASTP, gapped BLAST, and PSIBLAST for amino acid sequences.

[0067] Below, embodiments of the present disclosure are described in detail with reference to the attached drawings so that those skilled in the art can easily implement them. Regardless of the drawing numbers, identical or similar components are assigned the same reference numerals, and redundant descriptions thereof are omitted.

[0068] The present disclosure may be implemented in various embodiments as described above or may be implemented in various different forms and is not limited to the embodiments described herein.

[0069] Hereinafter, embodiments of the present disclosure will be described in detail with reference to the drawings.

[0070]

[0071] Example

[0072] 1. Production of cucumber mosaic virus-based vectors

[0073] Hereinafter, a method for producing a recombinant vector according to an embodiment will be described with reference to FIG. 1. FIG. 1 schematically illustrates a method for producing a cucumber mosaic virus-based recombinant vector according to an embodiment. Specifically, FIG. 1 schematically illustrates a CMV infectious cDNA clone, a recombinant vector, and a recombinant clone constructed for GFP overexpression.

[0074] The pCass-Rz vector was used as a binary vector for constructing a gene overexpression vector based on cucumber mosaic virus (CMV). The pCass-Rz vector contains the left border of T-DNA, the double 35S promoter of CaMV, a multiple cloning site (MCS; StuI, KpnI, XbaI, BamHI), a cis-cleaving ribozyme sequence (Rz), a 35S terminator (T), and the right border of T-DNA in that order, so that it can be transformed into Agrobacterium and delivered to plants through agroinfiltration. In addition, the pCass-Rz vector contains a kanamycin resistance gene, so that transformants can be selectively cultured when transformed into E. coli and Agrobacterium.

[0075] To construct a CMV-based recombinant protein overexpression vector, an infectious cDNA clone of a previously isolated CMV-GTN strain (Cucumber mosaic virus isolated from pepper; Res. Plant Dis. 21(2): 99-102 (2015)) (see Virus Evolution, Volume 6, Issue 2, July 2020, veaa070) was used. The CMV genome consists of RNA segments RNA1, RNA2, and RNA3. RNA1 encodes the 1a gene involved in RNA replication, RNA2 encodes the 2a gene, a replication enzyme, and the 2b gene, a gene silencing suppressor. RNA3 encodes the viral movement protein (MP) and coat protein (CP) genes.

[0076] Genomic DNAs of RNA1, RNA2, and RNA3 of highly pathogenic CMV-GTN strains isolated from domestically packaged peppers were amplified by RT-PCR. The PCR products for each genomic RNA were individually cloned using restriction enzyme sites present in the multi-cloning site (MCS) of the pCass-RZ vector. The resulting infectious cDNA clones for CMV-GTN RNA1, RNA2, and RNA3 were designated pCMV-GTN-R1 (SEQ ID NO: 3), pCMV-GTN-R2 (SEQ ID NO: 4), and pCMV-GTN-R3 (SEQ ID NO: 5), respectively.

[0077] Using an infectious clone of the CMV-GTN strain, a CMV-based vector was developed to overexpress a foreign protein by manipulating the genomic sequence of the CP open reading frame (ORF) of RNA3, which is expected to have high expression efficiency of the foreign gene.

[0078] Specifically, the CP gene was removed from the RNA3 CP ORF, and SpeI and MluI sites were introduced as a multi-cloning site (MCS) to enable insertion of a foreign gene (gene of interest), and a 2xFLAG tag and a 6xHIS tag were introduced behind the MSC for detection and extraction of the expressed foreign protein. Therefore, when cloning using the MCS, the expressed protein will contain the 2xFLAG tag and the 6xHIS tag at the C-terminus. The CMV-GTN RNA3-based vector clone constructed in this way was named pCMV-R3V (SEQ ID NO: 1).

[0079] CMV RNA2 encodes the 2b gene, which is a gene silencing suppressor. In this example, a recombinant CMV RNA2 infectious clone was constructed by replacing the 2b gene with the B2 gene of flock house virus (FHV), which is known to be a more potent gene silencing suppressor, and was named pCMV-R2V-B2 (SEQ ID NO: 2).

[0080] Specifically, the C-terminus of the 2b ORF of CMV RNA2 was removed, and a self-cleaving peptide (LLNFDLLKLAGDVESNPG / P), an MluI site, and the FHV B2 sequence of foot and mouth disease virus (FMDV) were introduced at that location. The B2 gene is expressed through translation of the 2b ORF, and is cleaved by the FMDV self-cleaving peptide, resulting in the inclusion of an additional proline (P) amino acid at the N-terminus.

[0081]

[0082] 2. Evaluation of expression efficiency of recombinant vectors

[0083] (1) Clonal production for expression efficiency evaluation

[0084] In order to evaluate the efficiency of expression of foreign genes in plants using the CMV-GTN-based vector manufactured in 1 above, a clone was constructed by inserting the green fluorescent protein (GFP) gene using the SpeI and MluI restriction enzyme sites of pCMV-R3V. To this end, the GFP gene was amplified by PCR using primers (5'-GGACTAGTATGGTGAGCAAGGGCGAGGAG-3' and 5'-AACGACGCGTGAGGATCCCCTTGTACAGCTC-3'), and then treated with SpeI and MluI restriction enzymes to insert it into the SpeI and MluI restriction enzyme sites of the pCMV-R3V vector. The clone into which GFP was inserted in this way was named pCMV-R3V-GFP (see Fig. 1). Here, pCMV-R3V-GFP refers to the sequence of SEQ ID NO: 6.

[0085] In addition, in order to produce infectious clones for the CMV-Fny strain, which is not a highly pathogenic CMV strain, pCMV-Fny-R1 and pCMV-Fny-R2 were produced for CMV-Fny RNA1 and RNA2, respectively, using the pCass-Rz vector and the same cloning method as pCMV-GTN-R1 and pCMV-GTN-R2. Here, pCMV-Fny-R1 refers to the sequence of SEQ ID NO: 7, and pCMV-Fny-R2 refers to the sequence of SEQ ID NO: 8.

[0086] For use as a control, PZP-GFP was constructed by inserting GFP into the PZP vector. The PZP vector contains the left border of T-DNA, the double 35S promoter of CaMV, the translation enhancer element (TE), the multiple cloning site (MCS; StuI, SpeI), the 35S terminator (T), and the right border of T-DNA in that order, allowing the target gene to be delivered to plants through agroinfiltration after transformation into Agrobacterium. In addition, the PZP vector contains a spectinomycin resistance gene, allowing for selective culture of transformants when transformed into E. coli and Agrobacterium. The GFP gene was inserted using the StuI and SpeI restriction enzyme sites of the PZP vector to construct a PZP-GFP clone. Here, PZP-GFP refers to the sequence of SEQ ID NO: 9.

[0087] Plasmid DNAs of pCMV-GTN-R1, pCMV-GTN-R2, pCMV-R3V-GFP, pCMV-R2V-B2, pCMV-Fny-R1, pCMV-Fny-R2 and PZP-GFP clones having the structures of Figs. 1a to 1f were each transformed into Agrobacterium strain EHA105. The transformed Agrobacterium was cultured in 5 ml of YEP liquid medium (containing 100 μg / ml kanamycin and 50 μg / ml rifampicin) at 30°C with shaking for 16 h. Then, 1 ml of the primary culture was inoculated into 50 ml of fresh YEP medium (containing 100 μg / ml kanamycin, 50 μg / ml rifampicin, and 20 μM acetosyringone) and cultured with shaking for 6 h at 30°C. The culture was centrifuged at 4800 G for 10 min to precipitate Agrobacterium and resuspended in MMA infiltration buffer (MS salts, 10 mM MES, pH 5.6, 200 μM acetosyringone) to an OD of 0.7 at 600 nm. The suspension was then shaken and cultured at 30°C for 4 hours.

[0088] The Agrobacterium suspensions transformed with each clone were mixed in equal proportions as indicated below or were infiltrated under pressure into the abaxial surface of tobacco plant (N. benthamiana) leaves using a 1 ml syringe.

[0089] 1) CMV-GTN-GFP: Agrobacterium suspensions (OD 0.7) transformed with pCMV-GTN-R1, pCMV-GTN-R2, and pCMV-R3V-GFP were mixed in equal proportions and infiltrated.

[0090] 2) PZP-GFP: Infiltrate the Agrobacterium suspension (OD 0.7) transformed with PZP-GFP alone.

[0091] 3) CMV-Fny-GFP: Agrobacterium suspensions (OD 0.7) transformed with pCMV-Fny-R1, pCMV-Fny-R2, and pCMV-R3V-GFP were mixed in equal proportions and infiltrated.

[0092] 4) CMV-GTN-B2-GFP: Agrobacterium suspensions (OD 0.7) transformed with pCMV-GTN-R1, pCMV-R2V-B2, and pCMV-R3V-GFP were mixed in equal proportions and infiltrated.

[0093]

[0094] (2) Evaluation of GFP expression efficiency

[0095] 1) Expression efficiency of the CMV-GTN-GFP combination vector

[0096] After inoculating each combination of Agrobacterium suspensions, GFP expression over time at the inoculation site was observed using a fluorescence measurement device (FOBI system).

[0097] Figure 2a is a diagram comparing and analyzing the GFP expression efficiency between the CMV-GTN-GFP combination (pCMV-GTN-R1 + pCMV-GTN-R2 + pCMV-R3VGFP) and PZP-GFP.

[0098] Compared to PZP-GFP produced using a binary vector containing the 35S promoter widely used in plant transformation, the CMV-GTN-GFP combination (pCMV-GTN-R1 + pCMV-GTN-R2 + pCMV-R3V-GFP) was confirmed to express a very large amount of GFP (see Fig. 2a). The amount of GFP expressed by the CMV-GTN-GFP combination was highest on the second day after inoculation (2 dpi) and was confirmed to gradually decrease over time (see Fig. 2a).

[0099] Through the above results, it was confirmed that a vector containing pCMV-R3V represented by sequence number 1 can significantly promote the expression of foreign proteins in plants.

[0100] In addition, in order to confirm whether the newly produced highly pathogenic CMV-GTN strain in the example has a higher foreign protein expression efficiency as a vector than other CMV strains, the CMV-GTN-GFP combination and the CMV-Fny-GFP combination (pCMV-Fny-R1 + pCMV-Fny-R2 + pCMV-R3V-GFP) were compared.

[0101] Figure 2b shows a comparative analysis of the GFP expression efficiency between the CMV-GTN-GFP combination and the CMV-Fny-GFP combination (pCMV-Fny-R1 + pCMV-Fny-R2 + pCMVR3V-GFP). After inoculating the Agrobacterium suspension of each combination, GFP expression at the inoculation site over time was observed using a fluorescence measurement device (FOBI system).

[0102] As a result, it was confirmed that the CMV-GTN-GFP combination had a significantly superior GFP expression efficiency compared to the CMV-Fny-GFP combination (see Fig. 2b). These results show that the efficiency of foreign gene overexpression as a vector may vary depending on the CMV strain, and indicate that the CMV-GTN strain used in the examples has an excellent effect as a foreign gene overexpression vector.

[0103]

[0104] 2) Expression efficiency of the CMV-GTN-B2-GFP combination vector

[0105] Next, to determine whether the expression efficiency increases when pCMV-R2V-B2, which replaces the CMV 2b gene with the FHV B2 gene to express a more potent gene silencing suppressor, was used, the CMV-GTN-GFP combination and the CMV-GTN-B2-GFP combination (pCMV-GTN-R1 + pCMV-R2V-B2 + pCMV-R3V-GFP) were compared.

[0106] Figure 3a shows the GFP expression over time at the inoculation site after inoculation with the Agrobacterium suspension of each combination using a fluorescence measurement device (FOBI system). Figure 3b shows the SDS PAGE analysis of the total protein extracted from the inoculation site on the second day after inoculation. Figure 3c shows the results of a quantitative comparative analysis of the GFP band of B using ImageJ. Figure 3d shows the results of a quantitative analysis of the GFP expression amount of the recombinant CMV GTN-based vector relative to the fresh weight of the plant.

[0107] The expression efficiency in inoculated tobacco plants was confirmed using a fluorescence in situ hybridization (FOBI) device. The CMV-GTN-B2-GFP combination showed a slightly higher expression efficiency than the CMV-GTN-GFP combination (see Fig. 3a). To more precisely compare the GFP expression efficiency between the two combinations, total proteins were extracted from the inoculation site on the second day after inoculation and subjected to SDS-PAGE analysis. The results confirmed that the amount of GFP protein expressed using the CMV-GTN-based vector accounted for a very high proportion of the total extracted proteins, and the CMV-GTN-B2-GFP combination induced slightly more GFP expression than the CMV-GTN-GFP combination (Fig. 3b). Analysis using ImageJ, an image quantitative analysis program, confirmed that the CMV-GTN-B2-GFP combination had a GFP expression efficiency that was approximately 30% higher (see Fig. 3c).

[0108] To analyze the GFP expression level by CMV-GTN-based vectors in comparison with the fresh weight of plants, cytoplasmic proteins were extracted from the inoculation site on the second day after inoculation, and GFP quantification analysis was performed using a GFP Quantification Kit (abcam, UK, product number ab235672). The CMV-GTN-GFP combination showed a GFP expression efficiency of 2846 ng per mg of fresh weight, and the CMV-GTN-B2-GFP combination showed a GFP expression efficiency of 3444 ng per mg of fresh weight, which was approximately 20% higher (see Fig. 3d).

[0109] Therefore, the GFP gene was cloned into each vector and expressed in tobacco (Nicotiana benthamiana) plants. As a result of comparative analysis of the expression levels, it was found that the highly pathogenic CMV-GTN-based vector showed a significantly higher level of GFP expression compared to the CMV-Fny strain, which is generally widely used in research. In addition, it was confirmed that the GFP expression level increased by approximately 12% when pCMV-R2V-B2 was used compared to the wild-type CMV RNA2 infectious clone (pCMV-GTN-R2).

[0110] Through the above results, it can be seen that the vector of the CMV-GTN-B2-GFP combination (pCMV-GTN-R1 + pCMV-R2V-B2 + pCMV-R3V-GFP) according to the embodiment shows greatly improved expression efficiency in the expression of foreign proteins in plants.

[0111]

[0112] 3. Advancement of CMV-based gene transfer technology

[0113] (1) Improvement of CMV-based vectors for authentic N-terminal expression of recombinant proteins.

[0114] 1) Manufacturing of vectors capable of producing proteins according to their original amino acid sequences

[0115] pCMV-R3V, a CMV RNA3-based vector manufactured in the above 1., was constructed by manipulating the genome for CP ORF, and it was confirmed that high-level recombinant protein expression was possible using this.

[0116] However, in the process of utilizing the CP ORF in the development of pCMV-R3V, five codons including the translation initiation codon of the CP ORF and two codons resulting from the addition of restriction enzyme sites for gene cloning were included to ensure stable expression (pCMV-R3V; see Fig. 4a).

[0117] Accordingly, recombinant proteins expressed using the pCMV-R3V vector will have seven artificial amino acids added to the N-terminus. This can affect the properties and function of the protein, and may also have a detrimental effect on safety certification and other aspects of industrialization of recombinant proteins.

[0118] Therefore, in order to improve the CMV RNA3-based vector to contain the native N-terminus without artificially adding amino acids during expression of the recombinant protein and to verify the expression efficiency, a clone was constructed by removing five codons of the CP ORF and positioning the restriction enzyme site 5' upstream of the translation initiation codon (pCMV-R3V2; see Fig. 4b). Here, pCMV-R3V2 refers to the sequence of SEQ ID NO: 10.

[0119] Meanwhile, in the sequence of pCMV-R3V2, the sequence excluding the 2xFLAG tag and 6xHIS tag (base sequence 1267-1338) introduced for protein detection and extraction, and the base sequence 1641-11911, which is a 'vector base sequence' unrelated to CMV, can be called 'pCMV-R3V2_valid'. pCMV-R3V2_valid refers to the sequence of sequence number 15.

[0120]

[0121] 2) Evaluation of expression efficiency of improved vectors

[0122] To evaluate the efficiency of expression of foreign genes in the pCMV-R3V2 vector, a clone (pCMV-R3V2-GFP; see Fig. 4c) was constructed by inserting the green fluoresence protein (GFP; 729 bp) gene using the SpeI and MluI restriction enzyme sites located in the MCS. Here, pCMV-R3V2-GFP refers to the sequence of SEQ ID NO: 11.

[0123] In the case of pCMV-R3V2-GFP, there is a possibility that the expression efficiency of the downstream gene may be affected because a non-viral sequence of 6 nucleotides (SpeI restriction enzyme site) is maintained in the intercistronic region preceding the translation initiation codon during the cloning process. Therefore, to remove this non-viral sequence, an additional clone (pCMV-R3V2-IF-GFP; see Fig. 4d) was created by inserting GFP into the same position through In-Fusion cloning. Here, pCMV-R3V2-IF-GFP refers to the sequence of SEQ ID NO: 12.

[0124] The expression efficiency of pCMV-R3V2-GFP and pCMV-R3V2-IF-GFP was analyzed by comparing them with the pCMV-R3V-GFP prepared in 1 above.

[0125] The produced clone construct was transformed into Agrobacterium (strain EHA105), and the expression efficiency was compared and analyzed by agroinfiltrating the test combination below into tobacco plants (Nicotiana benthamiana) and analyzing the expression level of GFP protein over time after inoculation using a fluorescence detection device (FOBI system).

[0126] Sequential test combination 1pCMV-GTN-R1 + pCMV-R2V-B2 + pCMV-R3V-GFP2pCMV-GTN-R1 + pCMV-R2V-B2 + pCMV-R3V2-GFP3pCMV-GTN-R1 + pCMV-R2V-B2 + pCMV-R3V2-IP-GFP

[0127] Since the combination expressed GFP only at the inoculation site, the inoculated leaves were observed. The control group was inoculated on one side of the leaf and the experimental group on the other side, and the GFP expression levels were compared and analyzed (see Fig. 5a). The pCMV-GTN-R1 + pCMV-R2V-B2 + pCMV-R3V-GFP combination was used as the control group. As a result of comparing the GFP expression efficiency using a fluorescence detection device, it was confirmed that both pCMV-R3V2-GFP and pCMV-R3V2-IF-GFP, which were constructed to unify the translation initiation codon, showed GFP expression efficiency at the same level as pCMV-R3V-GFP (see Fig. 5b).

[0128] To more precisely and quantitatively compare the GFP expression efficiency of each CMV-based vector, cytoplasmic proteins were extracted from the inoculation site on the second day after inoculation and GFP quantitative analysis was performed using a GFP Quantification Kit (abcam, UK, product number ab235672).

[0129] As a result of the analysis, pCMV-R3V-GFP, pCMV-R3V2-GFP, and pCMV-R3V2-IF-GFP all showed GFP expression efficiency of approximately 3200 ng per 1 mg of fresh weight, and there was no significant difference (see Fig. 5c).

[0130] Therefore, we confirmed that the genetic manipulation introduced to unify the translation initiation codon and ensure native N-terminal expression of the recombinant protein did not affect the expression efficiency of the CMV RNA3-based vector. Accordingly, considering the ease of future gene cloning, we decided to use pCMV-R3V2-GFP, which contains a restriction enzyme site, for additional clone production.

[0131] To confirm whether only one protein of the correct size was expressed from pCMV-R3V2-GFP, which was constructed for the purpose of translation initiation codon unification, total protein was extracted from the inoculation site and analyzed by SDS-PAGE. Total protein extraction was performed using TRIzol (Invitrogen) on the second day after inoculation, and finally, total protein extracted from 20 mg of fresh leaves for each sample was loaded, separated by size on 10% SDS-PAGE, and detected through Coomassie blue staining.

[0132] GFP expressed from pCMV-R3V-GFP has 7 amino acids derived from the vector added to the N-terminus, whereas GFP expressed from pCMV-R3V2-GFP has no additional sequence at the N-terminus. As expected, SDS-PAGE analysis showed that GFP expressed from pCMV-R3V2-GFP was 7 amino acids smaller than GFP expressed from pCMV-R3V2-GFP, and no bands other than the major GFP band were detected, confirming that only the targeted protein can be expressed accurately through translation initiation codon unification (see Fig. 5d).

[0133]

[0134] (2) Evaluation of foreign gene expression efficiency of P19-expressing CMV recombinant RNA2 clone

[0135] Below, a recombinant vector constructed using pCMV-R2V (SEQ ID NO: 13) (see Figure 6a) is described.

[0136] In the above 1., it was confirmed that when the 2b gene, an RNA silencing suppressor expressed in CMV RNA2, was replaced with the FHV B2 gene (318 bp), a more potent RNA silencing suppressor (pCMV-R2V-B2; see Fig. 6b), the efficiency of foreign gene expression in the CMV-based vector could be increased.

[0137] Accordingly, we constructed a CMV recombinant RNA2 clone (pCMV-R2V-P19; see Fig. 6c) expressing the P19 gene (516 bp) of tobacco bush stunt virus (TBSV), another potent RNA silencing suppressor, as a replacement for 2b, to evaluate its effect on the efficiency of foreign gene expression of CMV-based vectors. Here, pCMV-R2V-P19 refers to the sequence of SEQ ID NO: 14.

[0138] The expression efficiency of pCMV-R2V-P19 was analyzed by comparing it with pCMV-R2V-B2, and the test combination below was agroinfiltrated into tobacco plants (Nicotiana benthamiana) and the expression level of GFP protein was analyzed over time using a fluorescence detection device (FOBI system).

[0139] Sequential test combination 1pCMV-GTN-R1 + pCMV-GTN-R2 + pCMV-R3V2-GFP2pCMV-GTN-R1 + pCMV-R2V-B2 + pCMV-R3V2-GFP3pCMV-GTN-R1 + pCMV-R2V-P19 + pCMV-R3V2-GFP

[0140] In order to confirm whether the expression efficiency is increased when using pCMV-R2V-P19, which replaces the B2 gene of CMV RNA2 with the TBSV P19 gene, the combinations of pCMV-GTN-R1 + pCMV-GTN-R2 + pCMV-R3V2-GFP, pCMV-GTN-R1 + pCMV-R2V-B2 + pCMV-R3V2-GFP, and pCMV-GTN-R1 + pCMV-R2V-P19 + pCMV-R3V2-GFP were compared through fluorescence observation. As a result, it was confirmed that pCMV-R2V-P19 had a slightly higher expression efficiency than pCMV-R2V-B2 (see Fig. 7a). In order to more precisely and quantitatively compare the increase in GFP expression efficiency by pCMV-R2V-P19, cytoplasmic proteins were extracted from the inoculation site on the second day after inoculation and analyzed using a GFP Quantification Kit. GFP quantitative analysis was performed. The analysis results showed that when pCMV-R2V-P19 was used, the expression efficiency was increased by approximately 29.8% compared to pCMV-GTN-R2 and by approximately 15.1% compared to pCMV-R2V-B2 (see Fig. 7b).

[0141]

[0142] According to the cucumber mosaic virus-based recombinant vector according to the embodiments, the efficiency of expression of foreign proteins in plants can be significantly increased, and thus, it can be widely used for production of useful proteins and research thereof in various plant species.

[0143] By producing vaccine antigens in plants through molecular farming using plant virus vectors, the expression level can be maximized, thereby increasing the utility as an edible vaccine.

[0144] Additionally, plant virus vectors can ensure safety as they are harmless to humans and animals, and can overcome the GMO controversy and increase the possibility of commercialization.

[0145] Any or all of the embodiments of the present disclosure described above are not mutually exclusive or distinct. Any or all of the embodiments of the present disclosure described above may have their respective components or functions combined or used together.

[0146] For example, it means that a configuration A described in a particular embodiment and / or drawing can be combined with a configuration B described in another embodiment and / or drawing. That is, even if a combination between configurations is not directly described, it means that a combination is possible, except in cases where a combination is described as impossible.

[0147] The above detailed description should not be construed as limiting in any respect and should be considered illustrative only. The scope of the present invention should be determined by a reasonable interpretation of the appended claims, and all modifications within the equivalent scope of the present invention are intended to be included within the scope of the present invention.

Claims

1. A first vector comprising RNA1 of cucumber mosaic virus (CMV); A second vector containing RNA2 of CMV; and A third vector comprising a modified RNA3 of CMV; The above modified RNA3 is a CMV recombinant vector in which the coat protein (CP) gene has been removed to introduce the target gene.

2. In paragraph 1, A CMV recombinant vector, wherein the deletion of the above coat protein gene includes the deletion of five consecutive codons of the CP open reading frame (ORF) in the downstream (3') direction from the translation initiation codon, including the translation initiation codon of the CP ORF.

3. In paragraph 1, A CMV recombinant vector in which the above modified RNA3 is positioned in the upstream (5') region of the translation initiation codon of the target gene to be introduced, wherein one of the multi-cloning sites (MCS) that is cleaved by a restriction enzyme to introduce the target gene is located.

4. In paragraph 1, The third vector is a CMV recombinant vector comprising nucleotides consisting of sequence number 15.

5. In paragraph 1, The second vector is a CMV recombinant vector containing a gene silencing suppressor.

6. In paragraph 5, The above gene silencing suppressor is a CMV recombinant vector containing the B2 gene of flock house virus (FHV).

7. In paragraph 6, The second vector is a CMV recombinant vector comprising nucleotides consisting of sequence number 2.

8. In paragraph 6, The above B2 gene is a CMV recombinant vector in which the C-terminus of the 2b ORF of the RNA2 of the CMV is removed and a self-cleaving peptide (LLNFDLLKLAGDVESNPG / P) of foot and mouth disease virus (FMDV) and an MluI site are introduced at the corresponding position.

9. In paragraph 5, The above gene silencing suppressor is a CMV recombinant vector containing the P19 gene of tobacco bush stunt virus (TBSV).

10. In paragraph 9, The second vector is a CMV recombinant vector comprising nucleotides consisting of sequence number 14.

11. In paragraph 9, The above P19 gene is a CMV recombinant vector in which the C-terminus of the 2b ORF of the RNA2 of the CMV is removed and a self-cleaving peptide (LLFDLLKLAGDVESNPG / P) of foot and mouth disease virus (FMDV) and an MluI site are introduced at the corresponding position.

12. A transformed microorganism transformed by the CMV recombinant vector of paragraph 1.

13. A step of producing a CMV recombinant vector in which a target gene is introduced into the modified RNA3 of paragraph 1; A step of transforming the recombinant vector into a microorganism; and A method for producing a protein, comprising the step of inoculating the transformed microorganism into a plant.

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