Recombinant vector for producing platelet-derived growth factor bb recombinant protein in plants and method for producing platelet-derived growth factor bb recombinant protein in plants

Abstract

The present application provides a kind of recombinant vector, the recombinant vector is for the recombinant vector for producing platelet-derived growth factor BB (PDGF-BB) dimer protein, the recombinant vector includes bSA:PDGF-B gene sequence region, the bSA:PDGF-B gene sequence region includes the gene of the coding sequence of bovine serum albumin (bSA), His label coding sequence, the N-terminal domain coding sequence of platelet-derived growth factor (PDGF), protease recognition site coding sequence and platelet-derived growth factor B (PDGF-B) monomer protein coding sequence in sequence.

Description

Technical Field

[0001] This invention relates to a recombinant vector for producing platelet-derived growth factor-BB (PDGF-BB) recombinant protein in plant cells and a method for producing PDGF-BB recombinant protein in plant cells. Background Technology

[0002] Cultured meat refers to edible meat obtained by proliferating cells from living animals in a bioreactor using cell engineering techniques. It is meat produced at the laboratory level through cell proliferation without the processes of livestock farming and slaughter. Consuming cultured meat can reduce unethical livestock farming and slaughter practices for food, and compared to existing livestock farming methods, its production can reduce energy consumption by 55%, greenhouse gas emissions by 96%, and land use by 99%.

[0003] In pharmaceutical and industrial bioprocesses that utilize stem cell differentiation and proliferation, such as cultured meat, cell culture medium is an essential element. For example, in the cultured meat industry, cell culture medium accounts for the largest portion (over 70%) of the total production cost. Previously, the commonly used cell culture medium was based on fetal bovine serum (FBS). However, FBS is expensive and faces various difficulties during its isolation and extraction process, including pathogen infection, difficulty in achieving uniform quality management based on the extraction target, safety issues arising from unidentified unknown components, and ethical concerns related to the slaughter of pregnant cows required to obtain the serum. Furthermore, considering the fundamental purpose of cultured meat—obtaining meat through cell culture without slaughtering livestock—using FBS as the primary raw material for the culture medium is conceptually contradictory.

[0004] Therefore, there is an urgent need to develop a new generation of cell culture medium, namely a "serum-free chemically composed culture medium," in which the components constituting the cell culture medium are replaced by chemically identified components, thereby completely excluding fetal bovine serum. Therefore, the production of safe and inexpensive growth factors for cultured meat cell culture is crucial to the success of the cultured meat industry.

[0005] The culture of meat-grade cells requires various growth factors, one of which is platelet-derived growth factor (PDGF). PDGF is a crucial factor inducing mitosis in various cell types, including fibroblasts, smooth muscle cells, connective tissue cells, osteoblasts, and chondrocytes. It has been reported that PDGF can induce the proliferation of mouse myoblasts and also induce muscle cell regeneration by activating the differentiation of muscle stem cells. These characteristics, associated with cell proliferation, differentiation, and angiogenesis promotion, are considered essential components for the stem cell industry, such as the production of cultured meat, utilizing the differentiation and proliferation of muscle stem cells, as well as for the generation of functional tissues for tissue engineering applications.

[0006] Platelet-derived growth factors (PDGFs) acquire their activity by forming stable homodimers (PDGF-AA, PDGF-BB) or heterodimers (PDGF-AB) through homologous or heterologous intermolecular disulfide bonds. The dimerized PDGF binds to two homotyrosine kinase receptors, PDGF receptor α (PDGFRα) and PDGFRβ (PDGFRβ), thereby triggering intracellular signal transduction. Examples of pathways associated with PDGF-BB activation include MEK / ERK, Src, and PI3K / AKT. The four platelet-derived growth factors (A, B, C, and D) are characterized by a highly conserved growth factor domain (GF domain) consisting of 100 amino acids.

[0007] The focus of this invention is the homodimer of platelet-derived growth factor BB (PDGF-BB). Platelet-derived growth factor BB is a monomer composed of 109 amino acids with a molecular weight of 13 kilodaltons (kDa). Dimerized PDGF-BB has a molecular weight of 26 kilodaltons, but its size may be partially increased due to glycosylation.

[0008] Currently, recombinant growth factors are mainly used for research, primarily produced using human-derived genes in microorganisms such as *E. coli* or animal cell lines. However, microorganisms like *E. coli* pose a high risk of endotoxin infection and, in many cases, form inclusion bodies, making disulfide bond formation difficult, thus fundamentally limiting microbial production. Furthermore, animal cell-derived products carry a higher risk of viral infection. Therefore, there is an urgent need to develop a novel recombinant protein production system with high stability and low-cost, large-scale production capabilities. Plants have emerged as a novel recombinant protein production system, and they do not suffer from the aforementioned problems. In particular, plant-based recombinant proteins are a very suitable route for producing growth factors for meat culture media, especially from the perspective of meat culture for food. However, multiple technologies for producing recombinant proteins in plants have not yet been developed.

[0009] In this invention, in order to produce PDGF-BB, which is necessary for the culture medium used in cultured meat, a recombinant vector containing the PDGF-B gene was designed in plants, and the expression of the vector in plants was induced to produce the recombinant protein, thereby developing a technique for producing PDGF-BB dimers in mature form. Summary of the Invention

[0010] (a) Technical problems to be solved The purpose of this invention is to provide a recombinant vector that can induce high expression of platelet-derived growth factor BB (PDGF-BB) protein in plants.

[0011] Another object of the present invention is to provide a method for producing recombinant platelet-derived growth factor BB (PDGF-BB) protein in plants using the recombinant vector.

[0012] (II) Technical Solution According to one embodiment of the present invention, a recombinant vector is provided, the recombinant vector being a recombinant vector for producing platelet-derived growth factor BB (PDGF-BB) dimer protein, the recombinant vector comprising a bSA:PDGF-B gene sequence region, the bSA:PDGF-B gene sequence region comprising, in sequence, a gene encoding a bovine serum albumin protein (bSA) protein (bSA) protein, a His tag (tag) gene, a gene encoding a platelet-derived growth factor (PDGF) N-terminal domain gene, a gene encoding a protease recognition site gene, and a gene encoding a platelet-derived growth factor B (PDGF-B) monomer protein.

[0013] The bovine serum albumin (bSA) coding sequence may be a sequence that encodes a region containing wild-type bovine serum albumin (bSA-f) or a deletion mutant of bovine serum albumin, wherein the deletion mutant of bovine serum albumin contains more than 300 N-terminal amino acid residues of bovine serum albumin.

[0014] The recombinant vector may further contain a gene consisting of a 5' UTR base sequence, a base sequence encoding a BiP signal peptide, and a GB1 domain arranged sequentially at the 5' end.

[0015] According to another embodiment of the present invention, a method for producing platelet-derived growth factor BB (PDGF-BB) dimer protein in plants is provided, the method comprising the steps of: (a) introducing a recombinant vector according to the present invention into a bacterial strain to prepare a transformed strain; and (b) using the transformed strain to express the recombinant platelet-derived growth factor BB protein in a plant.

[0016] The method for producing platelet-derived growth factor BB (PDGF-BB) dimer protein in plants may further include (c) the step of pulverizing the plant and extracting the recombinant platelet-derived growth factor BB (PDGF-BB) protein.

[0017] Step (c) may include the following steps: (i) mixing pulverized plant material with a protein extraction buffer to prepare a plant material protein extract; (ii) purifying platelet-derived growth factor BB (PDGF-BB) dimer recombinant protein from the plant material protein extract; and (iii) using a protease to isolate mature platelet-derived growth factor BB (PDGF-BB) dimer protein from the recombinant protein.

[0018] (III) Beneficial Effects This invention provides a recombinant vector for producing platelet-derived growth factor BB (PDGF-BB) protein and a method for producing platelet-derived growth factor BB (PDGF-BB) protein using a novel method that is completely different from existing methods.

[0019] Compared to the use of existing animal cells or microorganisms to produce platelet-derived growth factor BB (PDGF-BB) protein, the present invention can reduce the risk of pathogen contamination and result in high production yield.

[0020] Furthermore, compared with existing methods that use plants to produce platelet-derived growth factor BB protein, the present invention can significantly improve the yield of platelet-derived growth factor BB (PDGF-BB) protein.

[0021] Therefore, according to the present invention, a method for producing platelet-derived growth factor BB (PDGF-BB) protein at low cost and on a large scale can be provided, wherein the platelet-derived growth factor BB (PDGF-BB) protein is an absolutely important growth factor for cell proliferation and differentiation. Attached Figure Description

[0022] Figure 1 This schematically illustrates the structure of a recombinant vector prepared according to one embodiment of the present invention.

[0023] Figure 2 This is an example of the effect of *Nicotiana benthamiana* (a type of tobacco) on the expression of platelet-derived growth factor BB (PDGF-BB). N.benthamiana The total extract was separated by SDS / PAGE, and the results were analyzed by Western blot using HRP-conjugated anti-human IgG antibody.

[0024] Figure 3 This illustrates the results of purifying a recombinant protein containing platelet-derived growth factor BB (PDGF-BB) protein from a total extract of *Nicotiana benthamiana* expressing PDGF-BB using microcrystalline cellulose, developing the recombinant protein by SDS / PAGE, and confirming the protein staining with Coomassie brilliant blue (CBB). Figure 3 A) and the results of Western blot analysis using HRP-conjugated anti-human IgG antibody ( Figure 3 (B).

[0025] In the Figure 3In this context, M represents a marker, WT represents a protein extract from wild-type *Nicotiana benthamiana* leaves (non-transformed wild-type extracts), Total represents the total soluble protein extract from *Nicotiana benthamiana* leaves induced with transient expression according to one embodiment of the invention, UB represents the supernatant (unbound fraction) recovered after mixing the total soluble protein extract obtained according to one embodiment of the invention with microcrystalline cellulose beads (MCC beads), W1 represents the wash-off fraction obtained by washing the microcrystalline cellulose beads again with a washing buffer, Elution 1 represents the elution fraction obtained by mixing the washed microcrystalline cellulose beads with a sample buffer and boiling the mixture for 10 minutes, and Elution 2 represents the elution fraction obtained by mixing the first elution with a sample buffer and boiling for 10 minutes. Debris refers to the precipitate obtained during the grinding of leaf tissue to obtain total soluble protein.

[0026] Figure 4 This demonstrates the treatment of recombinant protein bound to microcrystalline cellulose with enterokinase to produce platelet-derived growth factor BB in its mature form, and the use of HRP-conjugated anti-human IgG antibody. Figure 4 A) and anti-platelet-derived growth factor (anti-PDGF antibody), Figure 4 The results of Western blot analysis of B)

[0027] Figure 5 This demonstrates the treatment of a recombinant protein containing platelet-derived growth factor BB protein isolated using microcrystalline cellulose with a His-tagged enterokinase, followed by Ni 2+ -NTA affinity column chromatography was used to confirm the results of pure PDGF-BB obtained by removing the enterokinase.

[0028] Figure 6 The diagram schematically illustrates the structure of a recombinant vector prepared according to another embodiment of the invention, showing the structure of a recombinant vector further comprising the gene of wild-type bovine serum albumin (bSA-f) or the bovine serum albumin deletion mutant bSA300.

[0029] Figure 7This is a comparison of the expression levels of recombinant platelet-derived growth factor BB (PDGF-BB) protein bound to either bovine serum albumin (bSA-f) or a deletion mutant of bovine serum albumin (bSA300). More specifically, Figure 7 A shows the results of Western blot analysis using HRP-conjugated anti-human IgG antibody. Figure 7 B shows the results of confirming protein expression using Coomassie Brilliant Blue (CBB) staining. Figure 7 C represents the fractionation of total leaf extract from leaves of *Nicotiana benthamiana* expressing bSA300:bPDGF-B and bSA-f:bPDGF-B, into water-soluble proteins contained in the supernatant. Figure 7 The "S" in C) and the precipitating protein ( Figure 7 The expression levels of the protein were then confirmed by Western blot analysis after the "P" in the C was extracted and expanded using SDS-PAGE.

[0030] Figure 8 This demonstrates the results confirming whether PDGF-B prepared by expression of a recombinant vector containing either wild-type bovine serum albumin (bSA-f) or a deletion mutant of bovine serum albumin (bSA300) forms an active form of PDGF-BB dimer.

[0031] Figure 9 This demonstrates the use of Ni 2+ - The result of purifying bSA-f:bPDGF-B from total protein extract using NTA resin.

[0032] Figure 10 This demonstrates the results of confirming the activity of PDGF-B prepared and purified according to Examples 6 to 9 using NIH-3T3 cells. Best practice

[0033] Hereinafter, preferred embodiments of the present invention will be described with reference to the accompanying drawings. However, embodiments of the present invention can be modified in various other forms, and the scope of the present invention is not limited to the embodiments described below.

[0034] Figure 1 This schematically illustrates the structure of a recombinant vector prepared according to one embodiment of the present invention.

[0035] Reference Figure 1 A more detailed description will be given of a recombinant vector according to one embodiment of the present invention.

[0036] In this invention, a "vector" refers to a DNA construct containing a DNA sequence that can be operatively linked to a suitable regulatory sequence and can express DNA in a suitable host. The vector can be a plasmid, a phage particle, or simply a potential genomic insert. When transformed into a suitable host, the vector can replicate and function independently of the host genome, or in some cases, it can integrate into the genome itself.

[0037] Genes encoding proteins can be transiently expressed or stably transformed in transgenic plants or plant cells using vectors.

[0038] According to one embodiment of the present invention, a recombinant vector for producing platelet-derived growth factor BB (PDGF-BB) dimer protein is provided, comprising the bSA:PDGF-B gene sequence region.

[0039] The recombinant vector can be a binary vector, a DNA viral vector, or an RNA viral vector, but is not limited to these.

[0040] The bSA:PDGF-B gene sequence region may sequentially include: the gene encoding bovine serum albumin (bSA), the His tag encoding sequence, the N-terminal domain encoding sequence of platelet-derived growth factor (PDGF), the protease recognition site encoding sequence, and the gene encoding sequence of platelet-derived growth factor B (PDGF-B) monomer protein.

[0041] The recombinant vector may further contain a gene consisting of a 5' UTR base sequence, a base sequence encoding a BiP signal peptide, and a GB1 domain arranged sequentially at the 5' end.

[0042] The recombinant vector can be constructed using methods known to those skilled in the art. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology. The DNA sequence can be effectively linked to an appropriate promoter within the recombinant vector to guide mRNA synthesis.

[0043] The 5'UTR base sequence may contain the base sequence of SEQ ID NO:1.

[0044] The gene encoding the BiP signal peptide may be a gene encoding a leader sequence of binding immunoglobulin protein (BiP) that can target the target protein to the endoplasmic reticulum in plant cells.

[0045] The gene encoding the base sequence of the BiP signal peptide may contain the base sequence of SEQ ID NO:2.

[0046] The GB1 domain can be a domain characterized by increasing the expression level of the target protein in plants.

[0047] The gene encoding the base sequence of the GB1 domain may contain the base sequence of SEQ ID NO:3.

[0048] The gene encoding the His tag sequence may contain the base sequence of SEQ ID NO:4.

[0049] Furthermore, according to one embodiment of the present invention, the recombinant vector may further include a base sequence encoding one or more identical or different linker polypeptides at the 3' end of the His tag coding sequence.

[0050] The base sequence encoding the linker polypeptide may contain one or more of SEQ ID NO:10 to SEQ ID NO:12 and SEQ ID NO:15.

[0051] The coding sequence of the protease recognition site can be a base sequence that encodes an amino acid sequence that recognizes and cleaves the protease, and the protease is not specifically limited as long as it is a known protease.

[0052] For example, the sequence encoding the protease recognition site can be a sequence encoding the recognition site of enterokinase (EK).

[0053] The sequence encoding the enterokinase (EK) recognition site may contain the base sequence of SEQ ID NO:5.

[0054] The platelet-derived growth factor B (PDGF-B) protein may be a mammalian platelet-derived growth factor B (PDGF-B) protein, but is not limited to it.

[0055] For example, the platelet-derived growth factor B (PDGF-B) protein may be derived from animals selected from humans, apes, cattle, horses, dogs, cats, pigs, sheep, goats, mice, and rats.

[0056] More specifically, the platelet-derived growth factor B (PDGF-B) protein may be a bovine platelet-derived growth factor B (bPDGF-B) protein.

[0057] The gene encoding the bovine platelet-derived growth factor B (bPDGF-B) monomer protein may contain the base sequence of SEQ ID NO:6.

[0058] The N-terminal domain (NTD) of the platelet-derived growth factor (PDGF) may be a domain containing 57 amino acids at the N-terminus of PDGF, and may be a domain that promotes the formation of platelet-derived growth factor B (PDGF-B) as a monomer into platelet-derived growth factor BB (PDGF-BB) protein as a dimer.

[0059] The gene encoding the N-terminal domain of the platelet-derived growth factor (PDGF) may contain the base sequence of SEQ ID NO:7.

[0060] In addition, the recombinant vector may further include a promoter at the 5' end and a transcription terminator at the 3' end.

[0061] The promoter may be selected from and applied as a suitable promoter. For example, it may further include any one of the following promoters: the 35S promoter derived from cauliflower mosaic virus, the 19S RNA promoter derived from cauliflower mosaic virus, the Mac promoter, the actin promoter from plants, and the ubiquitin protein promoter. It is preferred to include the Mac promoter, and more preferably the MacT promoter, but it is not limited thereto.

[0062] The MacT promoter may contain the base sequence of SEQ ID NO:8.

[0063] The transcription terminator may include existing known transcription terminators, such as HSP-t, AtExt4-t, or RD29B-t, or may include chimeric transcription terminators that fuse two or more transcription terminators or genes.

[0064] More specifically, the chimeric transcription terminator can be a 3PR transcription terminator, which is a chimeric transcription terminator that fuses the 35S transcription terminator, the soybean PINII transcription terminator, and the RB7 sequence as a matrix attachment domain.

[0065] The 3PR transcription terminator may contain the base sequence of SEQ ID NO:9.

[0066] The whole-length bovine serum albumin precursor protein contains 607 amino acids. During secretion, a signal peptide consisting of 18 N-terminal amino acid residues is cleaved from the precursor protein, resulting in an initial protein product containing 589 amino acid residues. Further cleavage of 6 amino acids yields mature bovine serum albumin containing 583 amino acids.

[0067] In one embodiment of the present invention, the bovine serum albumin (bSA) coding sequence may be a sequence encoding a region comprising wild-type bovine serum albumin (bSA-f) or a deletion mutant of bovine serum albumin, wherein the deletion mutant of bovine serum albumin comprises more than 300 N-terminal amino acid residues of bovine serum albumin.

[0068] More specifically, the gene encoding the sequence containing the region of the deletion mutant of said bovine serum albumin may contain the base sequence of SEQ ID NO:13 encoding 300 N-terminal amino acid residues of bovine serum albumin (bSA300), and the gene encoding said wild-type bovine serum albumin (bSA-f) may be the gene encoding mature bovine serum albumin containing 583 amino acids.

[0069] The gene encoding the wild-type bovine serum albumin (bSA-f) may contain the base sequence of SEQ ID NO:14.

[0070] The gene of the present invention can be modified in various ways within the scope of the coding region of the protein expressed by the coding region of the protein without changing the amino acid sequence, and can also be modified in various ways in the parts other than the coding region without affecting gene expression. Such modified genes are also included in the scope of the present invention.

[0071] According to another embodiment of the present invention, a recombinant protein is provided for producing platelet-derived growth factor BB (PDGF-BB) dimer protein, the recombinant protein comprising a bSA:PDGF-B polypeptide region, the bSA:PDGF-B polypeptide region comprising, in sequence, bovine serum albumin (bSA), a His tag, an N-terminal domain of platelet-derived growth factor (PDGF), a protease recognition site, and platelet-derived growth factor B (PDGF-B) monomer protein.

[0072] The N-terminus of the recombinant protein may further include a BiP signal peptide and a GB1 domain.

[0073] The BiP signal peptide may contain the amino acid sequence of SEQ ID NO:17.

[0074] The GB1 domain may contain the amino acid sequence of SEQ ID NO:18.

[0075] The His tag may contain the amino acid sequence of SEQ ID NO:19.

[0076] The protease recognition site can be an amino acid sequence that recognizes and cleaves the protease, and the protease is not particularly limited as long as it is a known protease.

[0077] For example, the protease recognition site could be an enterokinase (EK) recognition site.

[0078] The enterokinase (EK) recognition site may include the amino acid sequence of SEQ ID NO:20.

[0079] The platelet-derived growth factor B (PDGF-B) protein may be a mammalian platelet-derived growth factor B (PDGF-B) protein, but is not limited to it.

[0080] For example, the platelet-derived growth factor B (PDGF-B) protein may be derived from platelet-derived growth factor B (PDGF-B) proteins selected from animals including humans, apes, cattle, horses, dogs, cats, pigs, sheep, goats, mice, and rats.

[0081] More specifically, the platelet-derived growth factor B (PDGF-B) protein may be a bovine platelet-derived growth factor B (bPDGF-B) protein.

[0082] The monomeric protein of the platelet-derived growth factor B (bPDGF-B) may contain the amino acid sequence of SEQ ID NO:21.

[0083] The N-terminal domain of the platelet-derived growth factor (PDGF) may contain 57 amino acids at the N-terminus of the platelet-derived growth factor (PDGF).

[0084] In addition, the N-terminal domain of the platelet-derived growth factor (PDGF) may contain the amino acid sequence of SEQ ID NO:22.

[0085] The bovine serum albumin (bSA) may be wild-type bovine serum albumin (bSA-f) or a deletion mutant of bovine serum albumin containing more than 300 N-terminal amino acid residues.

[0086] More specifically, the bovine serum albumin deletion mutant can be 300 N-terminal amino acid residues of bovine serum albumin (bSA300). The 300 N-terminal amino acid residues of bovine serum albumin (bSA300) can contain the amino acid sequence of SEQ ID NO:26.

[0087] The wild-type bovine serum albumin (bSA-f) may contain the amino acid sequence of SEQ ID NO:27.

[0088] In addition, it should be understood that the proteins, peptides and / or amino acid sequences included in this invention include at least the following range: functional variants or homologues having the same or similar functions as the proteins or peptides.

[0089] In this invention, the functional variant can be a protein or peptide generated by substitution, deletion or addition of one or more amino acids compared to the amino acid sequence of the protein and / or the peptide.

[0090] For example, the functional variant may comprise a protein or peptide that has amino acid variations through the substitution, deletion, and / or insertion of at least one, for example, one to ten, one to twenty, or one to thirty amino acids.

[0091] The functional variant may substantially retain the biological properties of the protein or peptide before the change (e.g., substitution, deletion, or addition). For example, the functional variant may retain 60%, 70%, 80%, 90%, or more than 100% of the biological activity of the protein or peptide before the change.

[0092] In this invention, the homologue can be a protein or peptide that has at least about 80% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence homology with the amino acid sequence of the protein and / or the peptide.

[0093] In this invention, homology generally refers to the similarity, analogousness, or association between two or more sequences. The "percent of sequence homology" can be calculated by comparing two aligned sequences within a comparison window that determines the number of positions containing the same nucleic acid bases (e.g., A, T, C, G, I) or the same amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met). To provide the number of matching positions in the comparison window (i.e., the window size), the number of matching positions is divided by the total number of positions, and the result is multiplied by 100 to provide the percentage of sequence homology. Alignment used to determine the percentage of sequence homology can be performed using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software, through a variety of methods known in the art. Those skilled in the art can determine appropriate parameters for sequence alignment, which includes any algorithm required to achieve maximum alignment in the full-length sequences being compared or in the target sequence regions.

[0094] According to another embodiment of the present invention, a method for producing platelet-derived growth factor BB (PDGF-BB) dimer protein is provided, the method comprising the steps of: (a) introducing a recombinant vector according to the present invention into a bacterial strain to prepare a transformed strain; and (b) using the transformed strain to express the recombinant platelet-derived growth factor BB protein in a plant.

[0095] The term "transformation" as used in this invention refers to the introduction of DNA into a host so that the DNA can be replicated as an extrachromosomal factor or by completing chromosomal integration.

[0096] The strain used in the preparation of the transformed strain can be *Agrobacterium tumefaciens* (Gastrobacterium tumefaciens). Agrobacterium tumefaciens The plant species may be dicotyledonous plants such as Arabidopsis thaliana, soybean, tobacco, eggplant, pepper, potato, tomato, Chinese cabbage, radish, cabbage, lettuce, peach, pear, strawberry, watermelon, cantaloupe, cucumber, carrot or celery; or monocotyledonous plants such as rice, barley, wheat, rye, corn, sugarcane, oats or onion, but are not limited to these species.

[0097] The method for preparing the transformed strain can be any method known in the art, such as the CaCl2 method, the Hanahan method, and the electroporation method, but is not limited thereto.

[0098] The step of expressing the platelet-derived growth factor B recombinant protein in the plant can be performed using any method known in the art for transferring DNA into the plant for transient expression or stable transformation in the plant or plant cells. Such methods include, but are not limited to, methods such as immersing the plant in a transformed suspension of *Agrobacterium tumefaciens*.

[0099] In addition, the method for producing the platelet-derived growth factor BB (PDGF-BB) dimer protein may further include: (c) the step of pulverizing the plant and extracting the recombinant platelet-derived growth factor BB (PDGF-BB) protein.

[0100] More specifically, step (c) may include the following steps: (i) The pulverized plant material is mixed with a protein extraction buffer solution to prepare a plant protein extract; (ii) Purifying recombinant platelet-derived growth factor BB (PDGF-BB) protein from the plant protein extract; and (iii) Using a protease, the mature platelet-derived growth factor BB (PDGF-BB) dimer protein was isolated from the recombinant protein.

[0101] The protein purification method used in the purification step may be any method known in the art, such as affinity chromatography using His tags, but is not limited thereto. Detailed Implementation

[0102] The present invention will now be described in more detail through embodiments. These embodiments are for illustrative purposes only, and the present invention is not limited thereto.

[0103] Example 1. Construction of a recombinant vector for producing platelet-derived growth factor BB (PDGF-BB) protein A recombinant vector for producing platelet-derived growth factor BB (PDGF-BB) protein was constructed using the following procedure.

[0104] First, starting with a 5'UTR base sequence known to improve translation efficiency, the following gene was fused to construct a recombinant vector: BiP, where BiP is an Arabidopsis thaliana gene encoding an endoplasmic reticulum leader sequence for endoplasmic reticulum accumulation. ArabidopsisThe gene contains: a genomic DNA fragment; the GB1 domain, which enhances protein stability and significantly increases production; the M domain, which enhances protein expression through glycosylation; the CBM3 domain, which enhances protein water solubility and binds to microcrystalline cellulose beads; the enterokinase recognition site, which is the protein that releases PDGF-BB; and the gene encoding the PDGF-B protein.

[0105] In this embodiment, platelet-derived growth factor B (bPDGF-B, base sequence: SEQ ID NO:6, amino acid sequence: SEQ ID NO:21) derived from bovine platelet-derived growth factor B (PDGF-B) is used.

[0106] At this point, a linker peptide containing one or more copies of GGS is added between each domain to maintain the independence of each domain.

[0107] More specifically, the GB1 domain contains a base sequence encoding a linker peptide L1 (base sequence: SEQ ID NO: 10, amino acid sequence: SEQ ID NO: 23) between the GB1 domain and the M domain, the M domain contains a base sequence encoding a linker peptide L2 (base sequence: SEQ ID NO: 11, amino acid sequence: SEQ ID NO: 24) between the M domain and the CBM3 domain (base sequence: SEQ ID NO: 16, amino acid sequence: SEQ ID NO: 29), and the CBM3 domain contains a base sequence encoding a linker peptide L3 (base sequence: SEQ ID NO: 12, amino acid sequence: SEQ ID NO: 25) between the CBM3 domain and the enterokinase recognition site.

[0108] To induce high-level expression, the recombinant vector contains a MacT promoter, known as a high-expression promoter, and a 3PR transcription terminator, which is a chimeric transcription terminator that fuses the 35S transcription terminator, the soybean PINII transcription terminator, and the matrix attachment domain.

[0109] The base sequence of the recombinant vector constructed through the process described above was confirmed by nucleotide sequencing.

[0110] A schematic structure of the recombinant vector confirmed by nucleic acid sequencing results is shown below. Figure 1 As shown.

[0111] Example 2. Expression and confirmation of the gene encoding recombinant platelet-derived growth factor BB protein The following process is used in plants (Nicotiana benthamiana) N. benthamiana The leaflet expresses the recombinant vector.

[0112] The recombinant vector was introduced into Agrobacterium strain GV3101 to obtain transformed Agrobacterium, which was then cultured overnight in 5 ml of medium. 1 ml of the overnight culture was added to 50 ml of LB medium containing 50 μg kanamycin and 50 μg rifampicin and cultured for 16 hours. The culture was then centrifuged at 4°C and 4500 g for 8 minutes to obtain a pellet containing Agrobacterium cells. The pellet was then suspended in buffer (10 mM MES, 10 mM MgSO4, pH 5.7) and the concentration was adjusted to 0.8 at OD600 to prepare an Agrobacterium suspension solution.

[0113] In addition, the suspension of *Agrobacterium* was mixed with 400 μM acetosyringone and incubated for 3 hours.

[0114] A suspension of *Agrobacterium* transformed with P38 as a gene silencing inhibitor was prepared using the same method, and then mixed with the *Agrobacterium* suspension containing PDGF-BB at a 1:1 ratio to prepare the final *Agrobacterium* suspension for infiltration.

[0115] Leaves of 4-5 week old Nicotiana benthamiana plants were infiltrated with the aforementioned Agrobacterium suspension to induce transient expression.

[0116] As a control group, leaves of Nicotiana benthamiana plants were infiltrated with a suspension of only Agrobacterium tumefaciens transformed with P38, i.e., a suspension of Agrobacterium tumefaciens without the introduction of the recombinant vector, thereby inducing transient expression (hereinafter referred to as "wild type").

[0117] The soaked leaves were harvested on days 3, 5, and 7 (DPI) after soaking. The harvested leaves were completely pulverized in liquid nitrogen and plant protein extracts were prepared using a protein extraction buffer solution (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Triton X-100, 1 mM DTT, and 1% protease inhibitor cocktail) equivalent to 5 times the weight of the leaves.

[0118] Total soluble proteins from plant protein extracts were developed by SDS-PAGE, followed by Western blot analysis using HRP-conjugated anti-human IgG antibody. Additionally, SDS-PAGE gels containing the same proteins were stained with Coomassie Brilliant Blue (CBB) to confirm band formation.

[0119] Figure 2 These are photographs showing the results of Western blot analysis, such as... Figure 2 As shown, the recombinant platelet-derived growth factor BB (PDGF-BB) protein was confirmed by the platelet-derived growth factor BB antibody at a position of approximately 50 kDa, which is the expected size.

[0120] Example 3. Purification of recombinant platelet-derived growth factor BB protein To isolate recombinant platelet-derived growth factor BB (PDGF-BB) protein, the following purification steps were performed.

[0121] After completely pulverizing 20g of leaf tissue induced with transient expression in Example 2 in liquid nitrogen, a protein extraction buffer solution (50mM tris(hydroxymethyl)aminomethane hydrochloride, pH 7.5, 150mM NaCl, 0.2% Triton X-100, 1% protease inhibitor mixture, 1mM DTT, 5% glycerol) equivalent to 5 times the weight of the leaf tissue was added to prepare a total protein extract of Tobacco Benzovia leaves.

[0122] The total protein extract was centrifuged at 18000g for 10 minutes to ensure the supernatant. The supernatant was then centrifuged at 18000g for 10 minutes to distinguish it from the supernatant and used as the total soluble protein extract.

[0123] The total soluble protein extract was mixed with 0.5 g of microcrystalline cellulose beads (MCC beads) and incubated for 2 hours. The supernatant (unbound component (UB)) and the microcrystalline cellulose beads were then washed 5 times with a washing buffer (40 mM tris(hydroxymethyl)aminomethane hydrochloride, pH 7.5). The resulting liquid was used as the washing fraction. The washing fractions were named W1 to W5 according to the number of washes.

[0124] 200 μl of the microcrystalline cellulose beads were mixed with 500 μl of sample buffer, and the mixture was then boiled for 10 minutes to obtain an elution fraction containing recombinant platelet-derived growth factor BB (PDGF-BB) protein. Based on the number of elutions, the elution fractions were named the first elution fraction (elution 1, E1) and the second elution fraction (elution 2, E2).

[0125] 10 μl of the first and second eluent components (E1 and E2) were respectively mixed with those from wild-type Nicotiana benthamiana (… N.benthamiana Total soluble protein extracts (WT) extracted from leaves, total soluble protein extracts (Total) extracted from leaves of *Nicotiana benthamiana* induced with transient expression according to Example 2, UB, and W1 were developed by SDS-PAGE and stained with Coomassie Brilliant Blue (CBB) to confirm the proteins. The results of the protein confirmation are shown in... Figure 3 In A.

[0126] Furthermore, 5 μl of the first elution fraction (E1) and the second elution fraction (E2), together with the total soluble protein extract (WT) extracted from wild-type *Nicotiana benthamiana* leaves, the total soluble protein extract (total) extracted from *Nicotiana benthamiana* leaves induced with transient expression according to Example 2, UB, W1, and W2, were subjected to SDS-PAGE, and Western blot analysis was performed using HRP-conjugated anti-human IgG antibody. The results of the Western blot analysis are shown in... Figure 3 In B.

[0127] Example 4. Isolation of platelet-derived growth factor BB by protease treatment Platelet-derived growth factor BB (PDGF-BB) recombinant protein, bound to microcrystalline cellulose beads, was treated with enterokinase, a protease, to isolate the PDGF-BB protein, the target protein, from other domains bound to the N-terminus of the recombinant platelet-derived growth factor BB (PDGF-BB) recombinant protein.

[0128] Therefore, 200 μl of microcrystalline cellulose bead slurry containing PDGF-BB protein was suspended in 200 μl of enterokinase buffer (20 mM tris(hydroxymethyl)aminomethane hydrochloride, pH 7.4, 2 mM CaCl2, 50 mM NaCl).

[0129] Add 0.1 μl of enterokinase to the suspension of the enterokinase buffer and perform an overnight cleavage reaction at 25°C using a shaking incubator (Enterokinase treatment example, Enterokinase (EK) treated)).

[0130] As a comparative example, microcrystalline cellulose beads conjugated with PDGF-BB protein were suspended in an enterokinase buffer solution without the addition of enterokinase, and then cultured overnight at 4°C (Comparative Example 1, C1) or 25°C (Comparative Example 2, C2), the same as the overnight cleavage reaction time in the above-mentioned enterokinase treatment examples.

[0131] The enterokinase treatment example ( Figure 4 The suspension of A (with "+EK"), Comparative Example 1 ( Figure 4 The suspension of "C1" in A and Comparative Example 2 ( Figure 4 The suspensions of C2 (from A) were boiled, then developed by SDS-PAGE, and analyzed by Western blotting using HRP-conjugated anti-human IgG antibody. The results of the Western blotting analysis are shown in... Figure 4 In A.

[0132] Reference Figure 4 In Comparative Example A, the recombinant protein in Comparative Example 1 and Comparative Example 2 was not cleaved, but in the enterokinase treatment example, a band was confirmed to be generated between 35kDa and 45kDa, thus confirming that the recombinant protein was partially cleaved by enterokinase treatment.

[0133] In addition, to ensure the isolation of PDGF-BB protein from recombinant protein treated with enterokinase, after enterokinase treatment, the protein is separated into a supernatant containing the protein isolated from the microcrystalline cellulose beads (hereinafter, "isolated protein fraction (released protein fraction)") and a component bound to the microcrystalline cellulose beads (hereinafter, "bounded protein fraction").

[0134] The combined protein components ( Figure 4The "+EK (bound)" in B, the isolated protein components ( Figure 4 In B, "+EK(Released)") and in Comparative Example 2 ( Figure 4 The suspensions of "C2" in section B were boiled, then developed by 15% SDS-PAGE, and analyzed by Western blot using anti-platelet-derived growth factor (anti-PDGF antibody). The results of the Western blot analysis are shown in... Figure 4 In B.

[0135] Reference Figure 4 In Comparative Example 2, PDGF monomers and dimers cleaved and separated by enterokinase treatment were detected in the isolated protein fraction. Conversely, in Comparative Example 2, no protein cleavage occurred, thus confirming the detection of PDGF-B in the full-length recombinant protein. Furthermore, the band formed between 35 kDa and 45 kDa in the bound protein fraction is presumably due to the peptide region containing the GB1 domain being detected during Western blotting.

[0136] The experiment confirmed the isolation of a mature form of PDGF-BB from the recombinant protein.

[0137] Example 5. Obtaining Platelet-Derived Growth Factor BB (PDGF-BB) When platelet-derived growth factor BB (PDGF-BB) recombinant protein is treated with enterokinase to isolate PDGF-BB, the supernatant contains both PDGF-BB and enterokinase. Therefore, to ensure pure PDGF-BB, enterokinase must be removed.

[0138] Therefore, in addition to treatment with enterokinase whose C-terminus is tagged with His, the same method as described in the project <Example 4. Isolation of platelet-derived growth factor BB by protease treatment> was used to ensure the binding of the protein components ( Figure 5 The “+EK (binding)” in the text), and the separated protein components ( Figure 5 The “+EK (release)” in the example and Comparative Example 2 ( Figure 5 The suspensions of "C2" in the above are boiled. 500 μl of each suspension is then passed through a solution of 50 μl of Ni. 2+ A column composed of NTA resin is used to remove His-tagged enterokinase, thereby obtaining bovine platelet-derived growth factor (BB) protein.

[0139] 500 μl of each suspension passed through the column was loaded with 10 μl of HSA containing a reference protein, followed by SDS-PAGE electrophoresis and Coomassie Brilliant Blue (CBB) staining for protein confirmation. The confirmation results of the obtained platelet-derived growth factor BB (PDGF-BB) protein are shown below. Figure 5 middle.

[0140] Example 6. Construction of a recombinant vector for platelet-derived growth factor BB (PDGF-BB) using bovine serum albumin (bSA). First, the wild-type base sequence of bovine serum albumin containing the stop codon was ensured through chemical synthesis (Gene Universal).

[0141] Based on the wild-type bovine serum albumin (bSA) base sequence, a forward primer with a 5' BamHI restriction endonuclease recognition site and a reverse primer with a 3' XmaI restriction endonuclease recognition site were used to ensure the presence of either a mutant gene (bSA300) containing the N-terminal amino acid residues of the 300 bSA protein or wild-type bovine serum albumin (bSA-f). The base sequences of the bSA300 mutant and the bSA-f gene are identical to those in SEQ ID NO:13 and SEQ ID NO:14, respectively.

[0142] In ensuring the base sequence of the bSA300 mutant or the bSA-f gene, a base sequence encoding the 8xHis tag and linker peptide L4 (base sequence: SEQ ID NO:15, amino acid sequence: SEQ ID NO:28) is added to the C-terminal base sequence region encoding the bSA300 domain or bSA-f.

[0143] In the recombinant vector (CBM3:bPDGF-B) of Example 1 prepared by fusing CBM3 and PDGF-B using BamHI and XmaI restriction endonucleases, the bSA300 mutant gene or the bSA-f gene prepared above was used for substitution insertion. The NTD:EK:bPDGF-B domain was inserted into the 3' end using XmaI and XhoI restriction endonucleases, thereby preparing bovine serum albumin:PDGF-B recombinant vectors (bSA300:bPDGF-B, bSA-f:bPDGF-B). The structures of the prepared bSA300:bPDGF-B and bSA-f:bPDGF-B recombinant vectors are as follows: Figure 6 As shown.

[0144] Example 7. Using bovine serum albumin (bSA) to increase the expression level of the recombinant vector of platelet-derived growth factor BB (PDGF-BB). The bovine serum albumin recombinant vectors (bSA300:bPDGF-B and bSA-f:bPDGF-B) of Example 6 and the recombinant vector (CBM3:bPDGF-B) of Example 1, prepared by fusing CBM3 and PDGF-B, were expressed using the same method as described in Example 2, and the expression levels of the recombinant proteins were confirmed. The results are shown in [Figure / Table / Insert Figure ... Figure 7 middle.

[0145] More specifically, Figure 7 Figure A shows the results of Western blot analysis using HRP-conjugated anti-human IgG antibody to confirm the expression levels of CBM3:bPDGF-B and bSA300:bPDGF-B. Figure 7 B shows the results of Coomassie Brilliant Blue (CBB) staining used to analyze the expression levels of these two recombinant proteins.

[0146] As described Figure 7 A and the Figure 7 As shown in Figure B, the fact that the expression of the bSA300:bPDGF-B recombinant protein is significantly higher than that of the CBM3:bPDGF-B recombinant protein can be confirmed. In particular, the difference described above is clearly shown in the Coomassie Brilliant Blue staining results.

[0147] In addition, Figure 7 In C, total leaf extract was obtained from leaves of *Nicotiana benthamiana* expressing bSA300:bPDGF-B and bSA-f:bPDGF-B, and fractionated into water-soluble proteins contained in the supernatant. Figure 7 The "S" in C) and the precipitated protein ( Figure 7 The expression levels of the protein were then confirmed by Western blot analysis after the "P" in the C was extracted and expanded using SDS-PAGE.

[0148] like Figure 7 As shown in Figure C, bSA-f:bPDGF-B was confirmed to show almost the same level of expression as bSA300:bPDGF-B. However, bSA-f:bPDGF-B showed a weaker band directly below it, which was judged to be a slightly broken form.

[0149] Example 8. Verification of dimer formation of bSA300:PDGF-B fusion protein To confirm whether platelet-derived growth factor B prepared by expression of the bSA300:bPDGF-B recombinant vector forms an active form of dimer.

[0150] Transient expression of the bSA300:bPDGF-B recombinant protein, prepared by expression of the bSA300:bPDGF-B recombinant vector, was induced in Nicotiana benthamiana, and then leaf tissues were ensured on days 3 and 5 to ensure total soluble protein extract.

[0151] 5 μg of the total soluble protein extract was mixed with a sample buffer solution containing 12% SDS and 0.6 M DTT to prepare a sample for ionizing the dimer into individual monomers (hereinafter, the ionization condition sample).

[0152] In addition, 5 μg of the total soluble protein extract was mixed with a sample buffer solution without 12% SDS and 0.6 M DTT to prepare the sample (hereinafter, non-free condition sample).

[0153] All proteins are released by boiling the free-condition sample for 10 minutes, and then loaded into a gel. Non-free-condition samples are loaded into the gel without additional processing.

[0154] After SDS-PAGE development, Western blot analysis was performed using HRP-conjugated anti-human IgG antibody. The results are shown below. Figure 8 middle.

[0155] like Figure 8 As shown, the sample under free conditions was confirmed ( Figure 8 In the “reduction” phase, most PDGF-B fusion proteins exist as monomeric PDGF-B, while in non-free samples ( Figure 8 The PDGF-B fusion protein exists in a dimer form within the "non-reducing" domain. This confirms that the bSA300 domain only increases the expression level of the PDGF-B fusion protein without affecting dimer formation.

[0156] Example 9. Purification of bSA300:PDGF-B from total soluble protein extract using Ni-NTA resin To purify bSA300:PDGF-B as the target protein, 20 g of leaf tissue induced to transiently express the vector described in Example 6 was completely pulverized in liquid nitrogen. Then, a protein extraction buffer solution (25 mM tris(hydroxymethyl)aminomethane hydrochloride, pH 7.5, 100 mM NaCl, 0.2% Tween 20, 2 mM PMSF, 2 mM CaCl2, 0.5% L-proline, 0.5% AC, 0.5% PVPP) was mixed in with the mixture and heated in a water bath at 4°C for 30 minutes.

[0157] Add 1 mM sodium phytate (Na) to the mixture again. + -phytate), then centrifuge at 18000g for 10 minutes to ensure supernatant, centrifuge the supernatant again at 18000g for 10 minutes to ensure supernatant, repeat this process twice to ensure total soluble protein extract.

[0158] The total soluble protein extract was mixed with Ni 2+ - NTA resin beads were mixed and incubated for 2 hours, then the supernatant and Ni were separately ensured. 2+ -NTA beads, the Ni 2+ The Ni2+-NTA beads were washed five times with a washing buffer (40 mM tris(hydroxymethyl)aminomethane hydrochloride, pH 7.5, 10 mM imidazole) to ensure the presence of the wash fraction (W1 to W5 based on the number of washes). The Ni2+-NTA beads were then mixed with 500 μl of elution buffer (40 mM tris(hydroxymethyl)aminomethane hydrochloride, pH 7.5, 500 mM imidazole) and incubated for 10 minutes to ensure the presence of fraction E containing the bSA-f:PDGF2 recombinant protein.

[0159] The total soluble protein extract (total extracts, T), flow-through fraction (FT), residue obtained after the first centrifugation (fragments, D), washing fraction (W), fraction eluted with elution buffer (E), and boiled Ni were compared. 2+ The fraction (B) obtained from the -NTA beads was developed by SDS-PAGE and analyzed by Western blotting using HRP-conjugated anti-human IgG antibody. The results of the Western blotting analysis are shown in... Figure 9 middle.

[0160] Example 10. Confirmation of the activity of PDGF-B produced by plants To confirm the activity of bSA300:bPDGF-B obtained according to Example 5, cell activity was measured in NIH-3T3 cells that are known to express the receptor for PDGF-B. The specific experimental procedure is as follows.

[0161] The day before the experiment, in a 12-well plate, at a depth of 2.5 × 10⁻⁶ cm⁻¹ per well... 5NIH-3T3 cells were seeded in growth medium (DMEM, 10% FBS, 1% penicillin / streptomycin). On the day of the experiment, the cells were cultured in serum-free DMEM medium for 4 hours.

[0162] To confirm the efficacy and concentration-dependent activity of the PDGF-B prepared according to an embodiment of the present invention, cells were treated with the isolated and purified PDGF-B protein at concentrations of 0 ng / ml, 20 ng / ml and 200 ng / ml, respectively, and cultured for 10 minutes.

[0163] HEK293 cells treated with the PDGF-B protein were washed with ice-cold PBS and then placed in RIPA lysis buffer (150 mM sodium chloride, 1.0% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 7.4, a mixture of protease inhibitors (Roche) and a mixture of phosphatase inhibitors (Roche)) for cell lysis. The cells were then centrifuged at 14000g for 15 minutes at 4°C to obtain the supernatant.

[0164] Protein quantification of the supernatant was performed using the BCA assay. 20 μg of the supernatant was developed by SDS-PAGE, and Western blot analysis was performed using phosphorylated Akt (pAkt, S473 site, Cell Signaling Technology), Akt antibody (Cell Signaling Technology), and HRP-conjugated anti-rabbit IgG antibody. The results of the Western blot analysis are shown below. Figure 10 middle.

Claims

1. A recombinant vector for producing platelet-derived growth factor BB (PDGF-BB) dimer protein, said recombinant vector comprising the bSA:PDGF-B gene sequence region. The bSA:PDGF-B gene sequence region sequentially contains the gene encoding bovine serum albumin (bSA), His tag, platelet-derived growth factor (PDGF) N-terminal domain, protease recognition site, and platelet-derived growth factor B (PDGF-B) monomer protein.

2. The recombinant vector according to claim 1, wherein, The bovine serum albumin (bSA) coding sequence is a sequence that encodes a region containing wild-type bovine serum albumin (bSA-f) or a deletion mutant of bovine serum albumin, wherein the deletion mutant of bovine serum albumin contains more than 300 N-terminal amino acid residues of bovine serum albumin.

3. The recombinant vector according to claim 1, wherein, The gene whose His tag encodes a sequence contains the base sequence of SEQ ID NO:

4.

4. The recombinant vector according to claim 1, wherein, The N-terminal domain of the platelet-derived growth factor (PDGF) contains 57 amino acids at the N-terminus of PDGF.

5. The recombinant vector according to claim 1, wherein, The gene encoding the N-terminal domain of platelet-derived growth factor (PDGF) contains the base sequence of SEQ ID NO:

7.

6. The recombinant vector according to claim 2, wherein, The sequence encoding the region containing the deletion mutant of said bovine serum albumin is a gene encoding 300 N-terminal amino acid residues of bovine serum albumin (bSA300), and the gene encoding said 300 N-terminal amino acid residues of bovine serum albumin (bSA300) contains the base sequence of SEQ ID NO:

13.

7. The recombinant vector according to claim 2, wherein, The gene encoding the sequence of the wild-type bovine serum albumin (bSA-f) contains the base sequence of SEQ ID NO:

14.

8. The recombinant vector according to claim 1, wherein, The platelet-derived growth factor B (PDGF-B) monomeric protein is selected from platelet-derived growth factor B (PDGF-B) monomeric proteins derived from animals such as humans, apes, cattle, horses, dogs, cats, pigs, sheep, goats, mice, and rats.

9. The recombinant vector according to claim 1, wherein, The platelet-derived growth factor B (PDGF-B) monomeric protein is a bovine platelet-derived growth factor B (bPDGF-B) monomeric protein, and the gene encoding the bovine platelet-derived growth factor B (bPDGF-B) monomeric protein contains the base sequence of SEQ ID NO:

6.

10. The recombinant vector according to claim 1, wherein, The recombinant vector further includes a gene consisting of a 5'UTR base sequence arranged sequentially at the 5' end, a base sequence encoding a BiP signal peptide, and a GB1 domain.

11. A recombinant protein for the production of platelet-derived growth factor BB (PDGF-BB) dimer protein. The recombinant protein contains the bSA:PDGF-B polypeptide region. The bSA:PDGF-B polypeptide region sequentially comprises bovine serum albumin (bSA), a His tag, the N-terminal domain of platelet-derived growth factor (PDGF), a protease recognition site, and platelet-derived growth factor B (PDGF-B) monomeric protein.

12. The recombinant protein according to claim 11, wherein, The bovine serum albumin (bSA) is wild-type bovine serum albumin (bSA-f) or a deletion mutant of bovine serum albumin containing more than 300 N-terminal amino acid residues.

13. The recombinant protein according to claim 12, wherein, The deletion mutant of bovine serum albumin is 300 N-terminal amino acid residues of bovine serum albumin (bSA300), which contains the amino acid sequence of SEQ ID NO:

26.

14. The recombinant protein according to claim 12, wherein, The wild-type bovine serum albumin (bSA-f) contains the amino acid sequence of SEQ ID NO:

27.

15. The recombinant protein according to claim 11, wherein, The platelet-derived growth factor B (PDGF-B) monomeric protein is derived from platelet-derived growth factor B (PDGF-B) monomeric proteins selected from animals including humans, apes, cattle, horses, dogs, cats, pigs, sheep, goats, mice, and rats.

16. The recombinant protein according to claim 11, wherein, The platelet-derived growth factor B (PDGF-B) monomeric protein is a bovine platelet-derived growth factor B (bPDGF-B) monomeric protein, and the platelet-derived growth factor B (bPDGF-B) monomeric protein contains the amino acid sequence of SEQ ID NO:

21.

17. A method for producing platelet-derived growth factor BB (PDGF-BB) dimer protein in plants, comprising the following steps: (a) Introducing the recombinant vector of any one of claims 1 to 10 into a strain to prepare a transformed strain; and (b) Using the transformed strain, recombinant platelet-derived growth factor B protein was expressed in the plant.

18. The method for producing platelet-derived growth factor BB (PDGF-BB) dimer protein in plants according to claim 17, wherein, The method further includes (c) pulverizing the plant and extracting recombinant platelet-derived growth factor BB (PDGF-BB) protein. Step (c) includes the following steps: (i) The pulverized plant material is mixed with a protein extraction buffer solution to prepare a plant protein extract; (ii) Purifying platelet-derived growth factor BB (PDGF-BB) dimer recombinant protein from the plant protein extract; and (iii) Using a protease, the mature platelet-derived growth factor BB (PDGF-BB) dimer protein was isolated from the recombinant protein.

19. The method for producing platelet-derived growth factor BB (PDGF-BB) dimer protein in plants according to claim 17, wherein, The plant species are selected from tobacco, Arabidopsis thaliana, soybean, eggplant, pepper, potato, tomato, Chinese cabbage, radish, cabbage, lettuce, peach, pear, strawberry, watermelon, cantaloupe, cucumber, carrot, celery, rice, barley, wheat, rye, corn, sugarcane, oats and onion.

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