Gene expression cassette, recombinant expression vector comprising same, and method for mass producing target protein by using same

Abstract

The present invention relates to: a gene expression cassette prepared for mass producing a target protein in plants; a recombinant expression vector comprising same; and a method for mass producing a target protein by using same. The expression cassette of the present invention significantly improves the expression of a target gene by optimally combining a newly developed terminator with a 5'UTR. As a result, efficiency of plant-based recombinant protein production is greatly increased, and various target proteins can be stably mass-produced at low cost. Therefore, the present invention is suitable for economical production of high-quality recombinant proteins, and has very high industrial applicability.

Description

Gene expression cassette, recombinant expression vector containing the same, and method for mass production of a target protein using the same

[0001] The present invention relates to a gene expression cassette for mass production of a target protein in plants, a recombinant expression vector comprising the same, and a method for mass production of a target protein using the same.

[0002] Recombinant proteins are used as core resources in various fields, including biopharmaceuticals, industrial enzymes, and diagnostic proteins; consequently, the importance of high-efficiency production technologies is rapidly increasing. The basic structure of recombinant protein production involves loading a target gene into an expression vector, introducing it into a cell, and producing the protein using the cell's transcription and translation mechanisms. These molecular biological foundational technologies are already well-established, and various cellular systems are being utilized as hosts for protein production.

[0003] Currently, the most widely used production cells are bacteria, including E. coli; animal cells, including CHO cells; and eukaryotic microorganisms such as yeast; recently, plant-based production platforms have entered the commercialization stage. Each host cell type possesses structural, economic, and technical advantages and disadvantages. Bacteria have low culture costs but have limitations in producing complex proteins. Animal cells provide post-translational modifications most similar to human proteins, but facility and operating costs are very high. Plant-based systems offer the strengths of significantly lower facility and operating costs and ease of large-scale production when using whole plants, but they suffer from the problem of low expression efficiency of recombinant proteins in plants.

[0004] Therefore, the most critical factor for the efficient production of recombinant proteins in plants is a plant expression vector capable of high expression. Currently, various plant expression vectors have been developed. The MagnICON vector is known to induce the highest expression in the field of plant transient expression, utilizing the mRNA amplification mechanism (replicon) of RNA viruses. While it demonstrated very high yields of approximately 2–4 g / kg fresh weight when targeting GFP, it is applicable only to transient expression and has structural limitations, such as the high error rate of RNA-dependent RNA polymerase (Sangita Venkataraman, Burra VLS Prasad, Ramasamy Selvarajan (2018) RNA Dependent RNA Polymerases: Insights from Structure, Function and Evolution. Viruses 10:76). Another high-expression system, the geminiviral vector, utilizes DNA replication-based amplification of DNA viruses and provides similar high yields (2-4 g / kg fresh weight) based on GFP (Tsuyoshi Yamamoto, et al (2018) Improvement of the transient expression system for production of recombinant proteins in plants. Scientific Reports 8: 4755).In addition, pEAQ-family vectors are widely used as a system that achieves very high translation efficiency by utilizing the 5' / 3' UTR of cowpea mosaic virus RNA-2 (Frank Sainsbury, Eva C Thuenemann, George P Lomonossoff (2009) pEAQ: versatile expression vectors for easy and quick transient expression of heterologous proteins in plants. Plant Biotechnol J. 7:682-693).

[0005] Despite the development of these expression vectors, there is still a need to develop high-expression plant expression vectors capable of stably producing various target proteins in plants at high yields.

[0006] The present invention aims to provide a gene expression cassette for mass production of a target protein, a recombinant expression vector comprising the same, and a method for mass production of a target protein using the same.

[0007] To achieve the above objective, the present invention provides a target gene expression cassette comprising a promoter; a target gene; and a terminator operably connected thereto, wherein the terminator is a composite terminator comprising an Hsp18.2 terminator and a tRNA-Glu terminator.

[0008] In the present invention, the Hsp18.2 terminator may be represented by the nucleotide sequence of SEQ ID NO. 2, and the tRNA-Glu terminator may be represented by the nucleotide sequence of SEQ ID NO. 7.

[0009] In the present invention, the target gene expression cassette may have a 5' UTR operably connected between the promoter and the target gene, and the 5' UTR may include an Omega sequence and three adenine sequences.

[0010] In the present invention, the 5' UTR may be represented by the nucleotide sequence of SEQ ID NO. 58.

[0011] In the present invention, the promoter may be an FM'M-UD promoter.

[0012] In the present invention, the FM'M-UD promoter may be represented by the nucleotide sequence of SEQ ID NO. 12.

[0013] The present invention also provides a recombinant expression vector comprising the above-mentioned target gene expression cassette.

[0014] In the present invention, the recombinant expression vector may be a triple recombinant expression vector further comprising a CRT1 expression cassette and a p38 expression cassette.

[0015] In the present invention, the CRT1 expression cassette may comprise a promoter; a sequence encoding CRT1; and a terminator operably connected thereto.

[0016] In the present invention, the promoter in the CRT1 expression cassette is a CSVMV promoter, and the terminator may be a composite terminator comprising an Hsp18.2 terminator and a tRNA-Glu terminator.

[0017] In the present invention, the CSVMV promoter may be represented by the nucleotide sequence of SEQ ID NO. 30, the Hsp18.2 terminator may be represented by the nucleotide sequence of SEQ ID NO. 2, and the tRNA-Glu terminator may be represented by the nucleotide sequence of SEQ ID NO. 7.

[0018] In the present invention, the CRT1 may be represented by the amino acid sequence of SEQ ID NO. 32.

[0019] In the present invention, the p38 expression cassette may be operably connected to a promoter; a sequence encoding p38; and a terminator.

[0020] In the present invention, the promoter in the p38 expression cassette may be a de35s promoter, and the terminator may be a 35s terminator.

[0021] In the present invention, the de35s promoter may be represented by the nucleotide sequence of SEQ ID NO. 35, and the 35s terminator may be represented by the nucleotide sequence of SEQ ID NO. 39.

[0022] In the present invention, the p38 may be represented by the amino acid sequence of SEQ ID NO. 38.

[0023] The present invention also provides a transgenic plant cell or a transgenic plant into which the target gene expression cassette or a recombinant expression vector containing the same has been introduced.

[0024] In the present invention, the plant cell or plant body may be selected from food crops including rice, wheat, barley, corn, soybeans, potatoes, red beans, oats, and sorghum; vegetable crops including Arabidopsis thaliana, Chinese cabbage, radish, chili pepper, strawberry, tomato, watermelon, cucumber, cabbage, Korean melon, pumpkin, green onion, onion, and carrot; special crops including ginseng, tobacco, cotton, sesame, sugarcane, sugar beet, perilla, peanuts, and rapeseed; fruit trees including apple trees, pear trees, jujube trees, peaches, grapes, citrus fruits, persimmons, plums, apricots, lemons, and bananas; and flowers including roses, carnations, chrysanthemums, lilies, sunflowers, cosmos, and tulips.

[0025] The present invention also provides a method for producing a recombinant protein, comprising the following steps:

[0026] (a) a step of culturing the above-mentioned transformed plant cell or transformed plant body; and

[0027] (b) A step of recovering recombinant protein from the cultured transformed plant cells or transformed plants.

[0028] The present invention also provides a transformed plant cell or a transformed plant body into which the triple recombinant expression vector is introduced.

[0029] In the present invention, the plant cell or plant body may be selected from food crops including rice, wheat, barley, corn, soybeans, potatoes, red beans, oats, and sorghum; vegetable crops including Arabidopsis thaliana, Chinese cabbage, radish, chili pepper, strawberry, tomato, watermelon, cucumber, cabbage, Korean melon, pumpkin, green onion, onion, and carrot; special crops including ginseng, tobacco, cotton, sesame, sugarcane, sugar beet, perilla, peanuts, and rapeseed; fruit trees including apple trees, pear trees, jujube trees, peaches, grapes, citrus fruits, persimmons, plums, apricots, lemons, and bananas; and flowers including roses, carnations, chrysanthemums, lilies, sunflowers, cosmos, and tulips.

[0030] The present invention also provides a method for producing a recombinant protein, comprising the following steps:

[0031] (a) a step of culturing the above-mentioned transformed plant cell or transformed plant body; and

[0032] (b) A step of recovering recombinant protein from the cultured transformed plant cells or transformed plants.

[0033] The expression cassette of the present invention significantly enhances the expression of a target gene by optimally combining a newly developed composite terminator with a 5' UTR. As a result, the efficiency of plant-based recombinant protein production is greatly increased, and various target proteins can be stably mass-produced at a low cost. Accordingly, the present invention is suitable for the economical production of high-quality recombinant proteins and has very high industrial utility value.

[0034] Figure 1 is a schematic diagram showing various terminator structures.

[0035] Figure 2 shows the structure (A) of an expression cassette constructed by connecting various terminators designed in Figure 1 to the FM'M-UD promoter and the target gene (hIL6), and the result (B) of analyzing the expression level of hIL6 expressed by Western blot after introducing the cassette into Nicotiana benthamiana.

[0036] Figure 3 is a schematic diagram showing the structure of two types of triple expression vectors (p38-CRT-93 and p38-CRT-99) constructed by sequentially arranging a target gene expression cassette (hIL6), a CRT1 expression cassette, and a p38 expression cassette.

[0037] Figure 4 shows the results of Western blot analysis of the amount of hIL6 expressed in Nicotiana benthamiana using two types of triple expression vectors (p38-CRT-93, p38-CRT-99) constructed in Figure 3.

[0038] Figure 5 is a schematic diagram showing the structure of four types of triple expression vectors designed by applying various 5′ modifications to the existing triple expression vector (p38-CRT-99).

[0039] Figure 6 shows the results of comparing the expression levels of EGF proteins expressed by introducing the four types of triple expression vectors constructed in Figure 5 into Nicotiana benthamiana using Western blot.

[0040] The present invention will be described in more detail below.

[0041]

[0042] In the present invention, a high-expression vector for inducing mass production of a target gene in plants was designed and its effects were verified. Specifically, in the present invention, various 3' terminators were constructed and their expressions were compared. As a result, it was confirmed that the Hsp18.2-tRNA-Glu complex terminator, formed by linking the Hsp18.2 terminator with the tRNA-Glu terminator sequence, significantly improved the expression of the target protein (e.g., IL-6). Furthermore, in a triple vector system comprising a target gene expression cassette, a CRT1 gene expression cassette that aids protein folding, and a silencing inhibitor p38 expression cassette, it was confirmed that the highest level of target protein expression was induced when the Hsp18.2-tRNA-Glu complex terminator was applied to both the target gene expression cassette and the CRT1 gene expression cassette. In addition, to secure a 5' UTR that increases the translation efficiency of the target gene, a 5' UTR (Omega + AAA) was developed by fusing the TCTAGAAA sequence to the omega sequence of the plant virus TMV, and it was demonstrated that applying this sequence significantly enhances the expression of another target protein (EGF).

[0043]

[0044] Accordingly, in one aspect, the present invention relates to a target gene expression cassette in which a promoter; a target gene; and a terminator are operably connected.

[0045] In the present invention, the terminator may be a composite terminator comprising an Hsp18.2 terminator and a tRNA-Glu terminator, and the Hsp18.2 terminator and the tRNA-Glu terminator may be sequentially connected.

[0046] In the present invention, the Hsp18.2 terminator may include the nucleotide sequence of SEQ ID NO. 2 or be composed thereof.

[0047] In the present invention, the tRNA-Glu terminator may include or be composed of the nucleotide sequence of SEQ ID NO. 7.

[0048] In one embodiment, a composite terminator comprising the Hsp18.2 terminator and the tRNA-Glu terminator may include or be composed of the nucleotide sequence of SEQ ID NO. 9.

[0049] In the present invention, the term "terminator" refers to a regulatory sequence that improves gene expression efficiency by inducing the precise termination and stabilization of transcribed mRNA. The terminator functions to prevent unnecessary transcriptional prolongation by providing a transcription termination signal, and to maintain a constant protein expression level by increasing the stability of the generated mRNA. The term "terminator" may also be referred to as a "terminator" or a "termination factor."

[0050] In the present invention, the term "composite terminator" refers to a regulatory sequence composed of two or more terminator sequences combined serially or functionally. The composite terminator developed in the present invention is designed so that the transcription termination signal and mRNA stabilization functions of each terminator element act complementarily or cumulatively, thereby serving to improve the accuracy of transcription termination, mRNA stability, and expression efficiency compared to a single terminator. In the present invention, the composite terminator may consist of two or more terminator sequences connected to operate sequentially; in this case, the two or more terminator sequences may be directly connected, or a linker sequence or restriction enzyme sequence may be included between the terminator sequences to connect the two or more terminator sequences. In this case, the linker sequence or restriction enzyme sequence may include or be composed of about 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) nucleotide sequences so that two or more terminator sequences can operate.

[0051] In the present invention, the target gene expression cassette may have a 5' UTR operably connected between the promoter and the target gene.

[0052] In one embodiment, the 5' UTR may include or be composed of the nucleotide sequence of SEQ ID NO. 13.

[0053] In another embodiment, the 5' UTR may be a modified 5' UTR comprising an Omega sequence and three adenine sequences. In the present invention, the Omega sequence may comprise or be composed of the nucleotide sequence of SEQ ID NO. 56.

[0054] For example, the modified 5'UTR may include or be composed of the nucleotide sequence of SEQ ID NO. 58.

[0055] In the present invention, "5' UTR (5' untranslated region)" refers to a non-translating nucleotide sequence located at the 5' end of the mRNA and upstream of the translation start codon. Although the 5' UTR is not translated into a protein, it can perform regulatory functions such as mRNA stability, ribosome binding and translation initiation efficiency, and selective translation regulation according to the cell environment, and may include various expression regulatory elements such as uORF and stem-loop structures as needed.

[0056] In the present invention, the promoter of the target gene expression cassette may be an FM'M-UD promoter.

[0057] In one embodiment, the FM'M-UD promoter may include or be composed of the nucleotide sequence of SEQ ID NO. 12.

[0058] In the present invention, the target gene expression cassette may include one or more selected from a gene encoding an endoplasmic reticulum targeting protein, a gene encoding an endoplasmic reticulum maintenance protein, a tag gene for purification, a linker sequence, and a gene encoding an auxiliary protein (e.g., MP or SUMO) capable of enhancing target gene expression.

[0059] In one embodiment, the target gene expression cassette may additionally include, in addition to the target gene, one or more genes selected from the group consisting of the BiP (Chaperone binding protein) gene, the MP (Mannosylated peptide region) gene, the CBM3 (Cellulose-binding module 3) gene (purification tag), the SUMO (Small ubiquitin-related modifier) ​​gene, and the gene encoding HDEL (His-Asp-Glu-Leu). For example, the target gene expression cassette may be one in which the BiP gene, the MP gene, the CBM3 gene, the SUMO gene, the target gene, and the HDEL-coding gene are sequentially linked.

[0060] In another embodiment, the target gene expression cassette may additionally include, in addition to the target gene, one or more genes selected from the group consisting of the BiP gene, the hSA (human serum albumin) gene, the 8×His gene (purification tag), and the collagen-binding motif (Col) gene. For example, the target gene expression cassette may be one in which the BiP gene, the hSA gene, the 8×His gene, the Col gene, and the target gene are sequentially linked.

[0061] In the present invention, the chaperone binding protein (BiP) is intended to target a target protein to the endoplasmic reticulum, and the BiP signal sequence is merely an exemplary configuration for inducing the target protein to the endoplasmic reticulum and can be freely replaced with other endoplasmic reticulum signal peptides, leader sequences, or signal sequence variants that perform the same or similar functions. Examples include PR1a signal sequences, calreticulin-derived signal sequences, and endoplasmic reticulum signal sequences derived from Arabidopsis species, and variants, partial sequences, or synthetic signal sequences that are functionally equivalent to these are also included within the scope of the present invention.

[0062] In the present invention, MP is a fragment consisting of 60 amino acid residues corresponding to the 231st alanine (A) to the 290th aspartic acid (D) of the PTPRC (protein tyrosine phosphatase, receptor type, C) protein, and is a functional peptide region for increasing the level of protein expression within cells. In the present invention, other peptide sequences or functional equivalents having the same or similar functions may be used in place of MP.

[0063] In the present invention, CBM3 is a cellulose binding module capable of specifically binding to microcrystalline cellulose (MCC) beads and functions as a tag for immobilizing or purifying an expressed target protein using MCC beads. This simplifies the protein purification process and enables high-efficiency purification. Functionally equivalent binding modules, affinity tags, or other purification sequences are also included within the scope of the present invention and can be replaced with other purification tags known in the art.

[0064] In the present invention, bdSUMO is a variant or functional sequence of a bacterial-derived SUMO (small ubiquitin-like modifier) ​​protein that serves to improve protein solubility and increase expression stability. In particular, it contributes to increasing the yield of the target protein by preventing protein aggregation in high-expression environments. However, the present invention is not limited to the bdSUMO sequence and also includes SUMO variants, SUMO-like sequences, or functional equivalents that perform the function of increasing protein solubility or stability.

[0065] In the present invention, HDEL is a C-terminal signal sequence for protein retention in the endoplasmic reticulum (ER) of plant cells, and has the effect of increasing the expression level of proteins that function in the ER or proteins that require ER accumulation. This contributes to increased stability and improved productivity within the ER. Furthermore, the present invention is not limited to the HDEL sequence, and functionally equivalent ER-retention signal sequences such as KDEL, SDEL, and KDEI, or their variants, are all included within the scope of the present invention.

[0066] In the present invention, hSA (human serum albumin) is a functional domain that contributes to the stability, solubility, and extension of the half-life in a blood-like environment of a target protein, and can be used to improve the overall expression level and secretion efficiency of the protein. In particular, hAS can help increase the total expression amount and recovery rate of the target protein by stabilizing the protein structure and inhibiting non-specific aggregation.

[0067] In the present invention, the Collagen-binding motif (Col) is a functional sequence for binding to a microstructural collagen or a collagen-based matrix and can be used to enhance the tissue binding or adhesion of a target protein.

[0068] In one embodiment, the target gene expression cassette may sequentially include a BiP (Chaperone binding protein) gene, an MP (Mannosylated peptide region) gene, a CBM3 (Cellulose-binding module 3) gene, a SUMO (Small ubiquitin-related modifier) ​​gene, a target gene (e.g., hIL-6), and a gene encoding HDEL (His-Asp-Glu-Leu) at the 3' end of a 5' UTR.

[0069] In one embodiment, the BiP may include or be composed of the amino acid sequence of SEQ ID NO. 15, and the sequence encoding the BiP may include or be composed of the nucleotide sequence of SEQ ID NO. 14.

[0070] In one embodiment, the MP may include or be composed of the amino acid sequence of SEQ ID NO. 17, and the sequence encoding the MP may include or be composed of the nucleotide sequence of SEQ ID NO. 16.

[0071] In one embodiment, the CBM3 may include or be composed of the amino acid sequence of SEQ ID NO. 19, and the sequence encoding the CBM3 may include or be composed of the nucleotide sequence of SEQ ID NO. 18.

[0072] In one embodiment, the SUMO may include or be composed of the amino acid sequence of SEQ ID NO. 21, and the sequence encoding the SUMO may include or be composed of the nucleotide sequence of SEQ ID NO. 20.

[0073] In one embodiment, the HDEL sequence may include or be composed of the amino acid sequence of SEQ ID NO. 25, and the sequence encoding the HDEL may include or be composed of the nucleotide sequence of SEQ ID NO. 24.

[0074] In one embodiment, the 8×His may include or be composed of the amino acid sequence of SEQ ID NO. 47, and the sequence encoding the 8×His may include or be composed of the nucleotide sequence of SEQ ID NO. 46.

[0075] In the present invention, the term "target protein" refers to a protein to be produced and may be any type of protein that can be expressed as a recombinant protein. The target gene expression cassette includes genes encoding intracellular and foreign proteins to be expressed. For example, the target protein may be one or more selected from the group consisting of thaumatin, lactoferrin, transferrin, interleukin, transcription factor, membrane protein, insulin, cytokinin, growth factor, toxin protein, hormone, hormone analog, cytokine, movement protein, lysozyme, vaccine, enzyme, enzyme inhibitor, transport protein, structural protein, receptor, receptor fragment, biological defense inducer, storage protein, exploitative protein, reporter protein, artificially designed protein, hydrophobin, antigen, antibody, and antibody fragment. The gene encoding such a target protein may include a "cloning site," which is a nucleic acid sequence into which a restriction enzyme or cleavage site has been introduced so that it can be inserted into a vector.

[0076] In one embodiment, the target protein may be hIL-6, and in another embodiment, the target protein may be EGF, but is not limited thereto.

[0077] In the present invention, the hIL-6 may include or be composed of the amino acid sequence of SEQ ID NO. 23, and the sequence encoding the hIL-6 may include or be composed of the nucleotide sequence of SEQ ID NO. 22.

[0078] In the present invention, the EGF may include or be composed of the amino acid sequence of SEQ ID NO. 55, and the sequence encoding the EGF may include or be composed of the nucleotide sequence of SEQ ID NO. 54.

[0079] In the present invention, to maximize expression efficiency in a host cell (e.g., a plant cell) encoding any protein (e.g., a target protein), the protein may be used after codon optimization in the host cell.

[0080] Between each domain of the target gene expression cassette of the present invention (e.g., BiP, MP, CBM3, SUMO, target protein, and HDEL, or BiP, hSA, 8×His, Col, and target protein), a peptide linker of an appropriate length may optionally be additionally included. The length of said peptide linker may be varied within a functionally acceptable range and may, for example, have a length of 1 to 50 amino acids, 1 to 30 amino acids, 1 to 20 amino acids, 2 to 50 amino acids, 2 to 30 amino acids, or 2 to 20 amino acids. Such peptide linker may be, for example, a glycine-serine repeat sequence, but is not limited thereto, and any other flexible linker or functionally equivalent sequence that a person skilled in the art may select according to the purpose is also included within the scope of the present invention.

[0081] In one embodiment, the linker sequence may be L1. The L1 may include or be composed of the amino acid sequence of SEQ ID NO. 45, and the sequence encoding the L1 may include or be composed of the nucleotide sequence of SEQ ID NO. 44.

[0082] In another embodiment, the linker sequence may be L2. The L2 may include or be composed of the amino acid sequence of SEQ ID NO. 49, and the sequence encoding the L2 may include or be composed of the nucleotide sequence of SEQ ID NO. 48.

[0083] In another embodiment, the linker sequence may be L3. The L3 may include or be composed of the amino acid sequence of SEQ ID NO. 51, and the sequence encoding the L3 may include or be composed of the nucleotide sequence of SEQ ID NO. 50.

[0084] In another aspect, the present invention relates to a recombinant expression vector comprising (introduced) the above-mentioned target gene expression cassette.

[0085] In the present invention, the recombinant expression vector may include (i) the target gene expression cassette; (ii) the CRT1 expression cassette; and (iii) the p38 expression cassette.

[0086] In the present invention, the CRT1 expression cassette may be operably connected to a promoter, a sequence encoding CRT1, and a terminator.

[0087] In one embodiment, the promoter in the CRT1 expression cassette may be a CSVMV promoter.

[0088] In the present invention, the CSVMV promoter may include or be composed of the nucleotide sequence of SEQ ID NO. 30.

[0089] In the present invention, "CRT1" refers to the calreticulin 1 (CRT1) chaperone protein present in the plant endoplasmic reticulum, which assists in the folding of novel synthetic proteins and performs ER quality control. 2+ It is a coupled endoplasmic reticulum resident chaperone. CRT1 is a conserved protein belonging to the CRT1 / CRT2 clade of the classical calreticulin family; in Arabidopsis thaliana, it is represented as AtCRT1 (gene ID: At1g56340), and consists of an N-domain (chaperone activity), a P-domain (proline-rich repeats), and a C-domain (Ca 2+ It consists of storage and endoplasmic reticulum maintenance signals. In the present invention, CRT1 can be utilized as a functional chaperone element to stabilize the endoplasmic reticulum environment of plant cells and to induce protein expression and quality improvement or increased stress tolerance.

[0090] In the present invention, the CRT1 may include or be composed of the amino acid sequence of SEQ ID NO. 32. In the present invention, the sequence encoding the CRT1 may include or be composed of the nucleotide sequence of SEQ ID NO. 31.

[0091] In the present invention, the terminator in the CRT1 expression cassette may be a composite terminator comprising an Hsp18.2 terminator and a tRNA-Glu terminator, and the Hsp18.2 terminator and the tRNA-Glu terminator may be sequentially operably connected. In the present invention, the Hsp18.2 terminator may comprise or be composed of the nucleotide sequence of SEQ ID NO. 2. In the present invention, the tRNA-Glu terminator may comprise or be composed of the nucleotide sequence of SEQ ID NO. 7. In one embodiment, the composite terminator comprising the Hsp18.2 terminator and the tRNA-Glu terminator may comprise or be composed of the nucleotide sequence of SEQ ID NO. 9.

[0092] In the present invention, the CRT1 expression cassette may additionally include a 5' UTR.

[0093] In the present invention, the 5' UTR in the CRT1 expression cassette may include or be composed of the nucleotide sequence of SEQ ID NO. 13.

[0094] In one embodiment of the present invention, the CRT1 expression cassette may sequentially include a BiP (Chaperone binding protein) gene, a CRT1 gene, an HA gene (purification tag), and a gene encoding HDEL (His-Asp-Glu-Leu) at the 3' end of a 5' UTR. A peptide linker may be additionally included between the binding sites of the components (BiP, CRT1, HA, HDEL) as needed.

[0095] In the present invention, HA is a tag sequence for facilitating the detection or purification of proteins, and can be usefully employed in immunological detection or affinity purification processes using anti-HA antibodies.

[0096] In one embodiment, the HA may include or be composed of the amino acid sequence of SEQ ID NO. 34, and the sequence encoding the HA may include or be composed of the nucleotide sequence of SEQ ID NO. 33.

[0097] In the present invention, the functional definition of some components (e.g., BiP, HDEL), the range of modifications and equivalents, and the length and sequence of peptide linkers are identical to those described above, so detailed descriptions that are redundant are omitted in this section.

[0098] In the present invention, the p38 expression cassette may be operably connected to a promoter, a sequence encoding p38, and a terminator.

[0099] In the present invention, the promoter in the p38 expression cassette may be a de35s promoter.

[0100] In the present invention, the de35s promoter may include or be composed of the nucleotide sequence of SEQ ID NO. 35.

[0101] In the present invention, "p38" refers to a silencing factor that inhibits gene expression or negatively regulates specific signaling pathways within plant cells; it is a regulatory protein that functions to regulate intracellular stress responses, signal regulation, or the expression levels of specific target genes. In the present invention, p38 inhibits the overexpression of specific genes or pathways, maintains intracellular balance, and can be utilized as a silencing regulatory element to enhance the efficiency of plant stress responses or expression regulation when necessary.

[0102] In the present invention, the p38 may include or be composed of the amino acid sequence of SEQ ID NO. 38, and the sequence encoding the p38 may include or be composed of the nucleotide sequence of SEQ ID NO. 37.

[0103] In the present invention, the terminator in the p38 expression cassette may be a 35s terminator.

[0104] In the present invention, the 35s terminator may include or be composed of the nucleotide sequence of SEQ ID NO. 39.

[0105] In the present invention, the p38 expression cassette may additionally include an L-UTR.

[0106] In the present invention, the L-UTR in the p38 expression cassette may include or be composed of the nucleotide sequence of SEQ ID NO. 36.

[0107] The amino acid sequences and base sequences described in this specification may be interpreted to extend to sequences having homology of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more with the provided sequences.

[0108] The above "% of sequence homology" can be verified by comparing two optimally arranged sequences with a comparison region, and a portion of the nucleotide sequence in the comparison region may include additions or deletions (i.e., gaps) compared to the reference sequence (which does not include additions or deletions) for the optimal arrangement of the two sequences.

[0109] In the present invention, the term "recombinant" refers to a cell that replicates a heterogeneous nucleic acid, expresses said nucleic acid, or expresses a protein encoded by a peptide, a heterogeneous peptide, or a heterogeneous nucleic acid. The recombinant cell may express a gene or gene fragment not found in the natural form of said cell in either a sense or antisense form. Additionally, the recombinant cell may express a gene found in a cell in its natural state, provided that said gene is modified and has been reintroduced into the cell by artificial means.

[0110] The term "recombinant expression vector" refers to a bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus, or other vector. Generally, any plasmid and vector may be used if they can replicate and stabilize within a host. An important characteristic of the recombinant expression vector is that it possesses a replication origin, a promoter, a marker gene, and a translation control element. An expression vector containing a suitable transcriptional / translational regulatory signal may be constructed by methods known to those skilled in the art. Such methods include in vitro recombinant DNA techniques, DNA synthesis techniques, and in vivo recombinant techniques. The recombinant expression vector of the present invention comprises one or more regulatory sequences (e.g., promoter, 5'UTR, intron, leader sequence, 3'UTR, species, etc.) and a target gene (for producing a recombinant protein), thereby enabling efficient and stable recombinant protein expression within a specific host cell or tissue.

[0111] The above "operably linked" may be a gene and an expression control sequence linked in such a way that gene expression is enabled when a suitable molecule is bound to the expression control sequence. The "expression control sequence" means a DNA sequence that controls the expression of the operably linked nucleotide sequence in a specific host cell. Such a control sequence includes a promoter for carrying out transcription, any operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence regulating the termination of transcription and translation. In the present invention, "operably linked" means that additional components may be included in the expression control sequence and the gene as needed. It is obvious that each component included in the expression cassette of the present invention should be understood to be operably linked to one another in a manner obvious to those skilled in the art unless otherwise noted.

[0112] In another aspect, the present invention relates to a transgenic plant cell or transgenic plant body into which the target gene expression cassette or the recombinant expression vector is introduced.

[0113] In the present invention, the plant cell or plant body may be a plant body selected from food crops including rice, wheat, barley, corn, soybeans, potatoes, red beans, oats, and sorghum; vegetable crops including Arabidopsis thaliana, Chinese cabbage, radish, chili pepper, strawberry, tomato, watermelon, cucumber, cabbage, Korean melon, pumpkin, green onion, onion, and carrot; specialty crops including ginseng, tobacco, cotton, sesame, sugarcane, sugar beet, perilla, peanuts, and rapeseed; fruit trees including apple trees, pear trees, jujube trees, peaches, grapes, citrus fruits, persimmons, plums, apricots, lemons, and bananas; and floricultural plants including roses, carnations, chrysanthemums, lilies, sunflowers, cosmos, and tulips, or a plant cell derived from said plant body.

[0114] In another aspect, the present invention relates to a method for producing a transgenic plant cell or a transgenic plant, comprising the step of introducing the target gene expression cassette or the recombinant expression vector into a plant cell or a plant body.

[0115] The introduction of the above recombinant expression vector into plant cells or plants may be performed using any one selected from the group consisting of Agrobacterium sp.-mediated methods, particle gun bombardment, sonication, electroporation, and PEG (polyethylene glycol)-mediated transformation methods.

[0116] Suitable vectors for expressing the target gene according to the present invention in plant cells may include, but are not limited to, the target gene expression cassette or the target gene expression cassette, CRT1 expression cassette, or p38 expression cassette of the present invention based on plasmids expressible in plants such as the pCambia1300 series, pRTVn, pRI 101, pGreenII series, pTM series, etc., into which the target gene expression cassette, the target gene expression cassette, the CRT1 expression cassette, or the p38 expression cassette of the present invention are introduced. Any vector capable of expressing the gene according to the present invention in plant cells may be selected and used.

[0117] A preferred example of the recombinant expression vector of the present invention is a Ti-plasmid vector capable of transferring a portion of itself, the so-called T-region, into a plant cell when present in a suitable host, such as Agrobacterium tumafaciens or Agrobacterium rhizogenes. Other types of Ti-plasmid vectors are currently used to transfer hybrid DNA sequences into plant cells or protoplasts from which new plants can be produced by appropriately inserting the hybrid DNA into the plant genome. A particularly preferred form of the Ti-plasmid vector is a so-called binary vector as claimed in EP 0120 516 B1 and U.S. Patent No. 4,940,838. Other suitable vectors that can be used to introduce DNA according to the present invention into a plant host may be selected from viral vectors, such as those derived from double-stranded plant viruses (e.g., CaMV) and single-stranded viruses, Gemini viruses, etc., for example, incomplete plant viral vectors. The use of such vectors can be advantageous, especially when it is difficult to properly transform plant hosts.

[0118] More specifically, the recombinant expression vector according to the present invention is a recombinant expression vector comprising a gene expression cassette in which a promoter, a modified 5' UTR that increases the translation efficiency of a target gene (e.g., one containing an omega sequence and three adenine sequences), and a complex terminator that significantly enhances the expression of a target gene (e.g., a complex terminator including an Hsp18.2 terminator and a tRNA-Glu terminator) are sequentially operably linked, based on a basic backbone of a conventional vector used for protein expression.

[0119] The recombinant expression vector may include a ribosome binding site and a transcription terminator as a translation initiation site.

[0120] The recombinant expression vector will preferably include one or more selector markers. The markers are nucleic acid sequences having characteristics that can typically be selected by chemical methods, and include all genes capable of distinguishing transformed cells from non-transformed cells. Examples include, but are not limited to, herbicide resistance genes such as glyphosate or phosphinothricin, antibiotic resistance genes such as kanamycin, G418, bleomycin, hygromycin, and chloramphenicol, and the aadA gene.

[0121] In the recombinant expression vector of the present invention, the promoter may be, but is not limited to, a double enhancer CaMV, CSVMV, MacT, RbcS, Glu13a, GluB1, Glub4, Prolamin, FM'M-UD, CaMV 35S, de35S, actin, ubiquitin, pEMU, amylase, or Clp promoter. The term "promoter" refers to a region of DNA upstream from a structural gene and refers to a DNA molecule to which RNA polymerase binds to initiate transcription. A "plant promoter" is a promoter capable of initiating transcription in a plant cell. A "constitutive promoter" is a promoter that is active under most environmental conditions, developmental states, or cell differentiation. Since the selection of transformants can be made by various tissues at various stages, a constitutive promoter may be preferred in the present invention. Therefore, a constitutive promoter does not limit the selectivity.

[0122] Each expression cassette according to the present invention may further include a sequence encoding a tag peptide depending on the purpose. The tag peptide may be any tag peptide known in the art known for the purpose of isolating and purifying recombinant proteins without limitation, and specifically, the protein tag may be one or more selected from the group consisting of Avi tag, Calmodulin tag, polyglutamate tag, E tag, FLAG tag, HA tag, His tag, Myc tag, CBM3 tag, S tag, SBP tag, IgG-Fc tag, CTB tag, Softag 1 tag, Softag 3 tag, Strep tag, TC tag, V5 tag, VSV tag, and Xpress tag.

[0123] Any host cell known in the art that can stably and continuously clone and express the recombinant expression vector of the present invention in a prokaryotic cell may be used, such as, for example, strains of the genus Bacillus such as E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, and Bacillus churingensis, and intestinal bacteria and strains such as Salmonella typhimurium, Serratia marcescens, and various Pseudomonas species.

[0124] In another aspect, the present invention relates to a method for producing a recombinant protein comprising the following steps:

[0125] (a) a step of culturing the above-mentioned transformed plant cell or transformed plant body; and

[0126] (b) A step of recovering recombinant protein from the cultured transformed plant cells or transformed plants.

[0127] In the present invention, the plant may be any one selected from food crops including rice, wheat, barley, corn, soybeans, potatoes, wheat, red beans, oats, and sorghum; vegetable crops including Arabidopsis thaliana, Chinese cabbage, radish, chili pepper, strawberry, tomato, watermelon, cucumber, cabbage, Korean melon, pumpkin, green onion, onion, and carrot; specialty crops including ginseng, tobacco, cotton, sesame, sugarcane, sugar beet, perilla, peanuts, and rapeseed; fruit trees including apple trees, pear trees, jujube trees, peaches, grapes, citrus fruits, persimmons, plums, apricots, lemons, and bananas; and floricultural plants including roses, carnations, chrysanthemums, lilies, sunflowers, cosmos, and tulips, and the plant cell may be a cell derived from the plant, but is not limited thereto. In a preferred embodiment, the plant may be rice, and in another preferred embodiment, the plant may be tobacco.

[0128] In the present invention, the recombinant protein is a term referring to a protein intended to be produced in a host cell, and may be any type of protein that can be expressed as a recombinant protein. For example, the recombinant protein may be one or more selected from the group consisting of GLP-1 analog, thaumatin, lactoferrin, transferrin, interleukin, transcription factor, membrane protein, insulin, cytokinin, growth factor, toxin protein, hormone, hormone analog, cytokine, movement protein, lysozyme, vaccine, enzyme, enzyme inhibitor, transport protein, structural protein, receptor, receptor fragment, biological defense inducer, storage protein, exploitative protein, reporter protein, artificially designed protein, hydrophobin, antigen, antibody, and antibody fragment. The gene encoding such a recombinant protein may include a "cloning site," which is a nucleic acid sequence into which a restriction enzyme or cleavage site is introduced so that it can be inserted into a vector.

[0129] In one embodiment, the interleukin includes, but is not limited to, human interleukin 6.

[0130] In one embodiment, the growth factor includes, but is not limited to, EGF.

[0131] Specifically, the method for producing a recombinant protein according to the present invention may comprise: (a) a step of producing the recombinant expression vector; (b) a step of introducing the recombinant expression vector into a plant to produce a transgenic plant cell or plant body; (c) a step of culturing the transgenic plant cell or plant body; and (d) a step of isolating and purifying the recombinant protein from the transgenic plant cell or plant body or the culture medium thereof.

[0132] The method for producing a recombinant protein from the above-described transformed plant cell or plant body involves transforming a plant cell or plant body with a recombinant expression vector according to the present invention, culturing the transformed cell or plant body for a suitable period of time to express the desired recombinant protein, and then obtaining the protein from the transformed cell or plant body. At this time, any method known in the art for expressing the recombinant protein may be used.

[0133] In a method for producing a recombinant protein, in order to introduce the recombinant expression vector into a plant, a culture of a transformant into which the recombinant expression vector has been introduced may be introduced into a plant cell or a plant body. For example, the culture of the transformant may be introduced by syringe infiltration into the leaves of a plant or by vacuum infiltration. The Agrobacterium introduced in this way receives a signal from the acetosyringone substance and delivers a construct containing the promoter-target protein-terminator of the vector into the plant cell.

[0134] In a method for producing recombinant proteins, the recombinant protein can be recovered through various separation and purification methods known in the art. Typically, to remove cell debris, the cell lysate may be centrifuged, followed by precipitation, for example, salting out (precipitation of ammonium sulfate and sodium phosphate), solvent precipitation (precipitation of protein fractions using acetone, ethanol, etc.), and dialysis, electrophoresis, and various types of column chromatography may be performed. The target protein of the present invention may be purified by applying techniques such as ion exchange chromatography, gel-permeation chromatography, HPLC, reverse-phase HPLC, affinity column chromatography, or ultrafiltration, either individually or in combination.

[0135]

[0136] The present invention will be described in more detail below through examples. These examples are intended solely to illustrate the present invention, and it will be obvious to those skilled in the art that the scope of the present invention is not to be interpreted as being limited by these examples.

[0137]

[0138] Example 1. Design of various 3' terminators

[0139] Various 3' terminator sequences presented in Table 1 and Figure 1 were designed.

[0140]

[0141] Name Components Sequence Number RDRD29B Terminator 13Hsp 18.2 Terminator 26RbcS3A Terminator 37Hsp18.2-N+ gcgc + Hsp18.2-N + RD29B-L Terminator 58Hsp18.2 Terminator + S28 Terminator + tRNA-Glu Terminator 89Hsp18.2 Terminator + tRNA-Glu Terminator 910RD29B-L Terminator + gaatttcgag + S28 Terminator + tRNA-Glu-m Terminator 10

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[0154]

[0155] Example 2. Confirmation of the effect of various 3' terminators on target protein expression

[0156] The various 3' terminators and the control terminator (RD29B) designed in Example 1 were chemically synthesized by Genuniversal (USA). Each synthesized terminator sequence was synthesized to include XhoI and EcoRI restriction enzyme cleavage sites at the 5' and 3' ends for subcloning into the pCambia1300 vector.

[0157] To construct a recombinant expression vector, the pCambia1300 vector (abcam, USA) was first cleaved with PstI and XbaI restriction enzymes to introduce the FM'M-UD promoter (refer to Korean Registered Patent No. 10-2849148). Subsequently, the target protein gene (BIP-MP-CBM3-bdSUMO-hIL6-HDEL) (Md Reyazul Islam 　The 3' terminator sequence was introduced downstream of the promoter by cleaving it with XbaI and XhoI restriction enzymes. The 3' terminator sequence was introduced by cleaving it with XhoI and EcoRI restriction enzymes so as to be positioned immediately after the stop codon of the target gene.

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[0174] Throughout the embodiments of the present invention, the T4 DNA ligase used for constructing the recombinant vector was a product of Takara Bio Inc. (Japan), and all ligation reactions were performed according to the manufacturer's protocol. In addition, the restriction enzymes used for cutting the vector and insert were products of New England Biolabs (NEB, USA), and all restriction enzyme reactions were also carried out in accordance with the manufacturer's guidelines.

[0175] After introducing into E. coli DH5α-transformed cells (competent cells; ThermoFisher scientific, USA), a recombinant expression vector was selected in a selection medium containing kanamycin. The obtained colonies were confirmed to have been correctly cloned by performing colony PCR and sequencing analysis.

[0176] The constructed recombinant expression vector was introduced by transforming the Agrobacterium tumefaciens strain GV3101 (GoldBio, USA). The obtained transformants were cultured overnight, and the culture medium was OD 600 A suspension for infiltration was prepared by adjusting the value to 0.7. The prepared suspension was infiltrated into 5-6 week old Nicotiana benthamiana leaves using a syringe with the needle removed, so that the target protein expression cassette was delivered to the plant cells in the form of T-DNA.

[0177] Leaves were harvested 4 days after infiltration, and the harvested leaves were ground under liquid nitrogen. Total protein from the ground tissue was extracted using an extraction solution composed of 50 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, and a 1% protease inhibitor cocktail. The extract was centrifuged at 14,000 rpm for 10 minutes at 4°C, and the supernatant was recovered.

[0178] Protein samples were prepared by adding 6× sample buffer (500 mM Tris-HCl, pH 6.8; 10% SDS; 0.5% bromophenol blue; 30% glycerol; 100 mM DTT) to the recovered protein solution to a final concentration of 1×, and after heating for 10 minutes, the samples were developed using 10% SDS-PAGE. The developed proteins were transferred to a PVDF membrane, and Western blot analysis was performed using the anti-hIL6 antibody (abcam, USA).

[0179] As a result of the analysis, the target protein was identified at approximately 65 kDa. Among the expression vectors with various 3' terminators, relatively high expression was observed in cases where 3' terminators 3 (Hsp18.2) and 9 (Hsp18.2-tRNA-Glu) were introduced, and in particular, the 9th 3' complex terminator showed the most superior expression-increasing effect (Fig. 2B).

[0180]

[0181] Example 3. Analysis of the effect of the Composite 3' terminator (hsp18.2-tRNA-Glu) on target protein expression in a triple expression vector

[0182] To produce high levels of the target protein from a single expression vector, a triple expression vector containing three expression cassettes was designed.

[0183] In the triple expression vector, the target gene expression cassette, the CRT1 expression cassette which is an endoplasmic reticulum chaperone protein, and the p38 expression cassette which is a gene silencing suppressor were each linked as independent cassettes.

[0184] First, PCR was performed using a 9th 3' complex terminator composed of Hsp18.2 and tRNA-Glu as a template. For the PCR, primers were used that add an XhoI restriction enzyme site to the 5' end (SEQ No. 26; 5'-CTCGAGatatgaagatgaagatgaaatatttg-3') and primers that add MluI, ApaI, StuI, and EcoRI restriction enzyme sites to the 3' end (SEQ No. 27; 5'-GAATTCccggttaggcctccggttgggcccccggttacgcgtctccgttgccgggactcgaaC-3'). After cleaving the amplified PCR product with XhoI and EcoRI, the existing terminator was replaced with the 9th 3' complex terminator (9th 3' complex terminator (1)) by connecting it to a pMR vector (see KR102643810B1) cleaved with the same restriction enzyme.

[0185] Next, the recombinant vector was cleaved with MluI and ApaI. A DNA fragment synthesized to have MluI and ApaI restriction enzyme sites at the 5' and 3' ends, respectively, was used as the CSVMV promoter, and after cleaving it with MluI and ApaI, it was inserted by connecting it to the corresponding cleaved site of the vector.

[0186] Next, a nucleotide sequence encoding BiP:CRT1 was chemically synthesized to have an ApaI restriction enzyme site at the 5' end and a StuI restriction enzyme site at the 3' end. The DNA fragment and vector were cleaved with ApaI and StuI, respectively, and then joined to complete the BiP:CRT1 expression cassette.

[0187] To construct a downstream terminator for the BiP:CRT1 cassette, the 9th 3' complex terminator (9th 3' complex terminator (2)) was amplified again by PCR. At this time, primers containing a StuI restriction enzyme site (5'-AGGCCTatatgaagatgaagatgaaatatttg-3' (Sequence No. 28)) and a primer containing an EcoRI restriction enzyme site (5'-GAATTCctccgttgccgggactcgaaC-3' (Sequence No. 29)) were used. The amplified product was cleaved with StuI and EcoRI restriction enzymes and linked to the recombinant vector cleaved with the same enzymes.

[0188] In the next step, the vector was cleaved with XbaI and XhoI to insert the target gene expression cassette. After cleaving the BiP-MP-CBM3-bdSUMO-hIL6-HDEL expression cassette constructed in the prior study (Md Reyazul Islam et al., 2019, Plant Biotechnol J 17:1094-1105) with XbaI and XhoI, the target gene expression cassette was introduced by connecting it upstream of the 9th 3' complex terminator (1) of the vector cleaved section.

[0189] Finally, the Hygromycin gene cassette present in the vector was removed by cleaving it with BsrGI and KpnI. For the p38 expression cassette, a synthetic DNA fragment designed to include a double enhancer CaMV 35S promoter (de35S), an L-5'UTR, and a p38 coding sequence was used. This fragment was constructed to have BsrGI restriction enzyme sites at the 5' end and KpnI restriction enzyme sites at the 3' end. After digesting the fragment with BsrGI and KpnI, it was inserted by ligating it to the same restriction enzyme cleavage sites of the vector, thereby constructing a triple expression vector containing three expression cassettes.

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[0202] In this case, the target gene expression cassette introduced terminator 9 (Hsp18.2-tRNA-Glu) as a terminator, and the CRT1 expression cassette introduced terminator 3 (Hsp18.2) or terminator 9 (Hsp18.2-tRNA-Glu) designed in Example 1 as a terminator.

[0203] Two types of triple expression vectors prepared in this manner (Fig. 3) were transformed into the Agrobacterium tumefaciens strain GV3101 in the same way as in Example 2, and after infiltrating Nicotiana benthamiana leaves, the leaves were harvested on the 3rd or 5th day and Western blot analysis was performed.

[0204] As a result, when terminator 9 (Hsp18.2-tRNA-Glu) was introduced into both the target gene expression cassette and the CRT1 expression cassette, the level of target gene expression was significantly higher (Fig. 4).

[0205] Accordingly, the triple expression vector designed in this way was named the 'p38-CRT-99 vector'.

[0206]

[0207] Example 4. Construction and Verification of the Improved Triple Expression Vector

[0208] To verify whether the vector structure exhibits the same effect on other target proteins, a new target gene was introduced into the same vector system. In addition, the 5' UTR region was redesigned to improve the expression efficiency of the existing triple vector.

[0209] A novel target gene expression cassette was hSA (human serum albumin)-L (linker)-8×His-L-Col (collagen-binding motif)-EGF (Epidermal growth factor), the entire sequence of which was chemically synthesized, and the target gene DNA fragment was chemically synthesized to include BamHI and XhoI restriction enzyme sites, and then cleaved with the said restriction enzymes and cloned into the p38-CRT-99 vector.

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[0227] Meanwhile, to replace the 17 bp 5′ control (SEQ No. 13) used in the existing triple expression vector, the following three 5′UTR modified sequences were designed.

[0228]

[0229]

[0230] Each sequence was introduced by directly incorporating it into a PCR primer. Specifically, 5′UTR-BiP DNA fragments containing each modified 5′UTR were obtained by PCR using the forward primers and common reverse primers listed below. All forward primers were designed to include the SpeI restriction enzyme site at the 5' end. The reverse primers were designed to include the BamHI restriction enzyme site at the 5' end.

[0231]

[0232]

[0233]

[0234] Three DNA fragments amplified by the above primers were cleaved with SpeI and BamHI restriction enzymes, and the 'p38-CRT-99' vector of Example 3 was cleaved with XbaI and BamHI restriction enzymes and then linked to construct a vector having a 5' UTR containing three omega sequences.

[0235] A total of four types of triple expression vectors (Fig. 5) prepared in this manner were transformed into the Agrobacterium tumefaciens strain GV3101 in the same manner as in Example 2, and after infiltrating Nicotiana benthamiana leaves, the leaves were harvested on the 4th day and Western blot analysis was performed using anti-His antibody.

[0236] As a result of independently repeating the same experiment a total of three times, it was confirmed that the triple expression vector with three adenines added to the Omega sequence exhibited the highest level of expression effect (Fig. 6).

[0237]

[0238] This study was conducted with the support of the following project.

[0239] Supported by the Ministry of Science and ICT, managed by the National Research Foundation of Korea, "Bio-Medical Technology Development (R&D)" project

[0240] - Project ID: 2710001865

[0241] - Sub-project number: 00235511

[0242] - Research Project Title: Discovery, Engineering, and Establishment of a Mass Production System for Next-Generation Therapeutic / Diagnostic Biosimilars Utilizing Hagfish Acquired Immunity

[0243] - Implementing Agency: Pohang University of Science and Technology

[0244] - Research Period: 2024.01.01.–2024.12.31.

Claims

1. Promoter; Target gene; and With a target gene expression cassette connected to a terminator for operation, A target gene expression cassette in which the terminator is a composite terminator comprising an Hsp18.2 terminator and a tRNA-Glu terminator.

2. A target gene expression cassette according to claim 1, wherein the Hsp18.2 terminator is represented by the nucleotide sequence of SEQ ID NO. 2 and the tRNA-Glu terminator is represented by the nucleotide sequence of SEQ ID NO.

7.

3. In Paragraph 1, A target gene expression cassette in which a 5' UTR is operably linked between the above promoter and the target gene, The above 5' UTR is a target gene expression cassette comprising an Omega sequence and three adenine sequences.

4. In Paragraph 3, The above 5' UTR is a target gene expression cassette represented by the nucleotide sequence of SEQ ID NO.

58.

5. In Paragraph 1, The above promoter is the FM'M-UD promoter, a target gene expression cassette.

6. In Paragraph 5, The above FM'M-UD promoter is a target gene expression cassette represented by the nucleotide sequence of SEQ ID NO.

12.

7. A recombinant expression vector comprising a target gene expression cassette of any one of claims 1 to 6.

8. In claim 7, the recombinant expression vector is, A recombinant expression vector further comprising a CRT1 expression cassette; and a p38 expression cassette.

9. In paragraph 8, the CRT1 expression cassette is, Promoter; A sequence encoding CRT1; and A recombinant expression vector with a terminator operably connected.

10. In paragraph 9, the promoter in the CRT1 expression cassette is a CSVMV promoter, and The above terminator is a recombinant expression vector that is a composite terminator comprising an Hsp18.2 terminator and a tRNA-Glu terminator.

11. In Paragraph 10, The above CSVMV promoter is represented by the nucleotide sequence of SEQ ID NO. 30, and A recombinant expression vector in which the Hsp18.2 terminator is represented by the nucleotide sequence of SEQ ID NO. 2 and the tRNA-Glu terminator is represented by the nucleotide sequence of SEQ ID NO.

7.

12. In Paragraph 9, The CRT1 above is a recombinant expression vector represented by the amino acid sequence of SEQ ID NO.

32.

13. In paragraph 8, the p38 expression cassette is Promoter; A sequence that encodes p38; and A recombinant expression vector with a terminator operably connected.

14. In Paragraph 13, The promoter in the above p38 expression cassette is the de35s promoter, and The above terminator is a recombinant expression vector that is a 35s terminator.

15. In Paragraph 14, The above de35s promoter is represented by the nucleotide sequence of SEQ ID NO. 35, and The above 35s terminator is a recombinant expression vector represented by the nucleotide sequence of SEQ ID NO.

39.

16. In Paragraph 13, The above p38 is a recombinant expression vector represented by the amino acid sequence of SEQ ID NO.

38.

17. A transgenic plant cell or transgenic plant into which the purpose gene expression cassette of any one of claims 1 to 6 or a recombinant expression vector containing the same has been introduced.

18. In paragraph 17, the above plant cell or plant body Food crops including rice, wheat, barley, corn, soybeans, potatoes, red beans, oats, and sorghum; Vegetable crops including Arabidopsis thaliana, napa cabbage, radish, chili pepper, strawberry, tomato, watermelon, cucumber, cabbage, Korean melon, pumpkin, green onion, onion, and carrot; Specialty crops including ginseng, tobacco, cotton, sesame, sugarcane, sugar beet, perilla, peanuts, and rapeseed; Fruit trees including apple trees, pear trees, jujube trees, peaches, grapes, citrus fruits, persimmons, plums, apricots, lemons, and bananas; and A transformed plant cell or transformed plant body selected from floricultural plants including roses, carnations, chrysanthemums, lilies, sunflowers, cosmos, and tulips.

19. A method for producing a recombinant protein comprising the following steps: (a) a step of culturing the transformed plant cell or transformed plant body of claim 17; and (b) A step of recovering recombinant protein from the cultured transformed plant cells or transformed plants.

20. Transformed plant cells or transformed plants into which the recombinant expression vector of paragraph 8 has been introduced.

21. In paragraph 20, the above plant cell or plant body Food crops including rice, wheat, barley, corn, soybeans, potatoes, red beans, oats, and sorghum; Vegetable crops including Arabidopsis thaliana, napa cabbage, radish, chili pepper, strawberry, tomato, watermelon, cucumber, cabbage, Korean melon, pumpkin, green onion, onion, and carrot; Specialty crops including ginseng, tobacco, cotton, sesame, sugarcane, sugar beet, perilla, peanuts, and rapeseed; Fruit trees including apple trees, pear trees, jujube trees, peaches, grapes, citrus fruits, persimmons, plums, apricots, lemons, and bananas; and A transformed plant cell or transformed plant body selected from floricultural plants including roses, carnations, chrysanthemums, lilies, sunflowers, cosmos, and tulips.

22. A method for producing a recombinant protein comprising the following steps: (a) a step of culturing the transformed plant cell or transformed plant body of claim 20; and (b) A step of recovering recombinant protein from the cultured transformed plant cells or transformed plants.

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