Method for producing antimicrobial peptide

Abstract

According to the present invention, a technique which enables the production of an antimicrobial peptide Persulcatusin at reduced cost has been developed. A plant cell into which a polynucleotide sequence that encodes a gene for a protease to which a signal peptide localized in an intercellular organelle is added and a polynucleotide sequence that encodes a Persulcatusin fusion protein to which a signal peptide localized in an intracellular organelle different from the aforementioned intracellular organelle is added are introduced is produced, wherein, in the Persulcatusin fusion protein, a sequence that is cleavable with the protease is disposed between Persulcatusin and a protein that fuses to the Persulcatusin. Thus, Persulcatusin, which can serve as a therapeutic agent for bovine mastitis, can be produced at low cost.

Description

Method for producing antimicrobial peptides

[0001] The present invention relates to a method for producing an antimicrobial peptide, and more particularly to a method for producing persulcatusin, an antimicrobial peptide secreted by Ixodes persulcatus, which has antimicrobial activity mainly against Gram-positive bacteria.

[0002] One of the problems with current livestock production is the excessive use of antibiotics. It is said that the amount of antibiotics used in livestock production is greater than that used for human medical purposes, and excessive use of antibiotics in livestock has led to the emergence of drug-resistant bacteria. As a result, this not only causes great harm to the livestock industry but also poses a major threat to human public health. Therefore, new treatments that can replace antibiotics are desired.

[0003] The use of antimicrobial peptides as therapeutic agents to replace antibiotics is gaining attention. Antimicrobial peptides are a general term for peptides with antimicrobial properties, and most are peptides consisting of around 20 to 60 amino acids. While antibiotics inhibit bacterial DNA synthesis and protein production, antimicrobial peptides lyse and kill bacteria by damaging their cell membranes. Compared to antibiotics that target specific proteins, antimicrobial peptides target the bacterial cell membrane, making it less likely for resistant bacteria to develop. In addition, because they are proteins, they are less likely to persist in the environment, which also makes it less likely for resistant bacteria to develop.

[0004] In dairy farming, bovine mastitis is a disease that reduces milk production and quality, causing economic losses that are said to account for approximately 70% of total losses. Mastitis is a disease caused by microorganisms (mostly bacteria) infecting the udder, and Staphylococcus aureus, in particular, is a bacterium that causes significant economic losses due to its intractability and repeated recurrence. In recent years, the extensive use of antibiotics has exacerbated the problem of the emergence of resistant bacteria to methicillin and similar antibiotics. Therefore, as mentioned above, antimicrobial peptides, which have a significantly lower incidence of resistant bacteria compared to existing antibiotics, have attracted attention.

[0005] Persulcatusin is an antimicrobial peptide with cationic and amphipathic properties discovered in the midgut of the Ixodes persulcatus tick (Ixodes persulcatus). It has three disulfide bonds and an α-helix and β-sheet structure (Non-Patent Document 1). Persulcatusin is a peptide that exhibits antimicrobial activity primarily against Gram-positive bacteria, and has been shown to have stronger antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Staphylococcus aureus (VRSA) than other antimicrobial peptides, such as BMAP28 and nisin. Furthermore, since it has no effect on the growth, morphology, or DNA of animal cells, it is considered to be highly safe for animals. Therefore, the practical application of persulcatusin as a therapeutic agent for bovine mastitis, which is refractory and highly recurrent and caused by Staphylococcus aureus, is eagerly awaited (Non-Patent Documents 1 and 2).

[0006] Persulcatusin is a sequence consisting of 38 amino acids, and chemical synthesis or expression and purification using animal cells or bacteria is costly. Therefore, we investigated its production using plant molecular farming. In recent years, plant molecular farming, which uses plants as bioreactors, has attracted attention for the production of useful proteins such as antimicrobial peptides and antigenic proteins that act as vaccines. Plant molecular farming offers several advantages, including reduced capital investment and running costs for production, easy adjustment of production scale according to demand, and a low risk of contamination with infectious pathogens or toxins that can pose a threat to humans or livestock during the production process.

[0007] While plant molecular agriculture offers the aforementioned advantages in substance production, the high cost of extracting and purifying substances from cultivated plants poses a problem. For example, in the production of β-glucuronidase, it has been estimated that extraction and purification costs account for more than 80% of the total production cost (Non-Patent Document 3). Therefore, to commercialize substances produced by plant molecular agriculture, it is necessary to reduce extraction and purification costs by establishing a lower-cost method for extracting target proteins or by using plants as they are. Attempts to simplify extraction and reduce extraction costs include expressing target substances as oleosin fusion proteins and accumulating them in seed oil bodies (Non-Patent Document 4), secreting target substances from shake-cultured cells into liquid media (Non-Patent Document 5), and accumulating vaccines or antibodies in edible parts of food crops or plants and using the entire plant as a pharmaceutical (Non-Patent Documents 6-8). Furthermore, a method has been proposed in which protease inhibitor genes are introduced to suppress degradation within plants in order to recover as much target protein molecules as possible (Patent Document 1).

[0008] Special Publication No. 2024-513542

[0009] Miyoshi N., etal., Parasites and Vectors, 2016, Vol.9, 85.Miyoshi N, etal., Journal of Antibiotics, 2017, Vol.70, pp.142-146.Evangelista RL,et al., Biotechnology Progress, 1998, Vol.14, pp.607-614.Boothe J., et al., Plant BiotechnologyJournal, 2010, Vol.8, pp.588-606.Shaaltiel Y., et al., Plant BiotechnologyJournal, 2007, Vol.5, pp.579-590.Twyman RM, et al., Trends in Biotechnology,2003, Vol.21, pp.570-578.Makhzoum A., et al., BioDrugs, 2014, Vol.28, pp.145-159.Nochi, T., et al., Proc. Natl. Acad. Sci.USA, 2007, Vol.104, pp.10986-10991.Lehrer RI, et al., JImmunol Method, 1991 Vol.137:167-173.

[0010] An objective of the present invention is to provide a method for producing persulcatusin, a therapeutic agent for bovine mastitis, at low cost, and a plant body for use in the method.

[0011] The present invention relates to the following plant cells, vectors, and methods for producing antimicrobial peptides using the same: (1) A plant cell into which a construct has been introduced, the construct including a polynucleotide sequence encoding a protease gene to which a signal peptide that localizes in an organelle has been added, and a polynucleotide sequence encoding a Persulcatusin fusion protein to which a signal peptide that localizes in an organelle different from the organelle has been added, the Persulcatusin fusion protein having a sequence cleaved by the protease between Persulcatusin and a protein fused thereto. By expressing Persulcatusin as a fusion protein and localizing it in an organelle separate from the protease, the protease can act only when the plant is harvested and extracted, allowing active Persulcatusin to be obtained.

[0012] (2) The plant cell according to (1), wherein the protease is a tobacco etch virus (TEV) protease, and the sequence cleaved by the protease is a TEV protease recognition sequence. TEV protease is a highly specific protease that recognizes a seven-amino acid sequence. Therefore, there is no risk of cleaving portions of the fusion protein other than the recognition sequence.

[0013] (3) The plant cell according to (2), wherein the persulcatusin fusion protein is a fusion protein of persulcatusin and calmodulin, the TEV protease recognition sequence is located between persulcatusin and calmodulin, and the two are separated by cleavage with TEV protease. Calmodulin has a relatively small molecular weight, so it is thought that when persulcatusin is used as a fusion protein, its expression is not hindered. Furthermore, by using the persulcatusin fusion protein, persulcatusin has no physiological activity until cleaved by TEV protease, and the function of the plant cell is not impaired.

[0014] (4) The plant cell according to (1), wherein the intracellular organelle is a chloroplast or an apoplast, and when the fusion protein is localized in the chloroplast, a signal sequence is added so that the protease is localized in the apoplast, and when the fusion protein is localized in the apoplast, a signal sequence is added so that the protease is localized in the chloroplast. It is generally known that accumulation in chloroplasts increases the production of foreign proteins. Although chloroplasts are prokaryotic organelles, persulcatusin is a fusion protein with calmodulin, so it does not have any adverse effects on the host.

[0015] (5) The plant cell according to any one of (1) to (4), characterized in that the plant cell is derived from rice. The genome information of rice has been analyzed in detail, and a culture method using callus or the like has been established. Therefore, the cells can be cultured efficiently, leading to the production of persulcatusin.

[0016] (6) A method for purifying persulcatusin, comprising: obtaining seeds by redifferentiating the plant cells described in (5), germinating the seeds to form sprouts, and crushing the sprouts to bring the persulcatusin fusion protein into contact with a protease, cleaving the persulcatusin fusion protein, and extracting persulcatusin with antibacterial activity. Redifferentiating plant cells is no different from cultivating plants, from growing the plants to harvesting the seeds. It takes only about 10 to 12 days to germinate the harvested seeds and collect them as sprouts, and the cultivation costs are very low.

[0017] (7) A vector set for producing persulcatusin, comprising a vector containing a construct comprising a polynucleotide sequence encoding a protease to which a signal peptide that localizes in an intracellular organelle has been added, and a vector containing a construct comprising a polynucleotide sequence encoding a persulcatusin fusion protein to which a signal peptide that localizes in an intracellular organelle different from the intracellular organelle has been added and which has a sequence cleaved by the protease between persulcatusin and a protein fused thereto. In the following examples, rice is used as the host, but by using this vector set, persulcatusin can be produced using other plants as hosts.

[0018] (8) A vector for producing Persulcatusin, which comprises, in a single vector, a construct containing a polynucleotide sequence encoding a protease to which a signal peptide that localizes in an organelle has been added, and a polynucleotide sequence encoding a Persulcatusin fusion protein to which a signal peptide that localizes in an organelle different from the organelle has been added and which has a sequence cleaved by the protease between Persulcatusin and a protein fused thereto. The vector for producing Persulcatusin may be introduced into a plant body by making the fusion protein and the protease into separate vectors as described in (7), or may be introduced into a plant body as a single vector.

[0019] Schematic diagram of a construct for expressing a Persulcatusin fusion protein. (A) Western blotting analysis of the amount of Persulcatusin fusion protein produced in shake-cultured rice cells. (B) Western blotting analysis of the amount of Persulcatusin fusion protein produced in the culture supernatant and cells of shake-cultured rice cells. Analysis of the amount of fusion protein produced in shake-cultured cells. Diagram showing the results of purification of a fusion protein using a histidine tag. Diagram showing the antibacterial activity of a fusion protein against Staphylococcus aureus. An example of a construct for expressing proteins so that the fusion protein and TEV protease are localized differently in a single plant. Diagram showing the results of RT-PCR analysis of gene expression after introducing a construct that expresses TEV protease in chloroplasts into rice. Diagram showing the results of RT-PCR analysis of gene expression after introducing a construct that expresses secreted TEV protease into rice. Fig. 1 shows the results of Western blot analysis of protein expression after introducing a construct that expresses secreted TEV protease into rice. Fig. 2 shows another example of a construct that expresses proteins so that the fusion protein and TEV protease are localized differently in a single plant.

[0020] As will be explained in detail below, this technology involves preparing a fusion protein (hereinafter sometimes referred to as a Persulcatusin fusion protein, or simply as a fusion protein) that contains a sequence that is cleaved by a protease and functions as Persulcatusin upon cleavage by the protease, and a construct to which a signal sequence has been added so that the protease is localized in a separate organelle of the plant cell, and introducing this into a plant cell. Therefore, only after the plant body is subjected to a crushing and extraction process can the protease be able to act on the fusion protein, resulting in cleavage of the fusion protein and the function of Persulcatusin.

[0021] Since it is generally known that accumulation in chloroplasts increases the production of foreign proteins, one protein can be localized in the chloroplast, and the other protein can be localized in another organelle, such as the apoplast. For example, when accumulating a Persulcatusin fusion protein in chloroplasts, a construct can be prepared to which a sequence that localizes the TEV protease in the apoplast is added, and the construct can be introduced into plant cells. However, this is not limited to this, and it is sufficient that the two proteins can be localized in different organs of the plant body. Therefore, a construct can be used in which the Persulcatusin fusion protein is expressed in the apoplast and the protease is expressed in the chloroplast.

[0022] Although tobacco etch virus (TEV) protease is used here as the protease, any protease may be used, provided that a sequence recognized and cleaved by the selected protease is placed between Persulcatusin and another protein used as the fusion protein.

[0023] The persulcatusin fusion protein exhibits no or only very weak antibacterial activity while it exists as a fusion protein. Here, calmodulin is used as the protein to be fused to persulcatusin, but this is not limited to calmodulin. Any protein may be used as long as it does not exhibit antibacterial activity while it exists as a fusion protein.

[0024] As shown in the following examples, the produced protein can be purified by culturing the transfected cells in a callus state with shaking and then recovering the cultured cells, or by recovering and purifying the fusion protein secreted into the culture supernatant, since a signal sequence for secretion into the culture supernatant has been added. When purifying from the culture supernatant, the protein is very easy to purify, since the culture supernatant does not contain a large amount of protein.

[0025] Alternatively, persulcatusin may be purified from sprouts obtained from the introduced cells. Specifically, the introduced cells may be redifferentiated to obtain seeds, which may then be germinated to produce sprouts, which may then be crushed and the fusion protein extracted. It takes only 10 to 12 days to germinate the seeds and harvest the sprouts, and the cultivation costs are very low.

[0026] Furthermore, the use of rice as the plant material is extremely advantageous when used to treat bovine mastitis. Because rice is used as bedding or feed, it is thought that there is no problem with using it in the treatment of mastitis in a crudely purified form that may contain some rice protein.

[0027] [Persulcatusin Fusion Protein Construct] The present invention will now be described with reference to data. First, using a plasmid into which a gene sequence synthesized encoding a fusion protein of persulcatusin and calmodulin had been cloned as a template, the region encoding the persulcatusin fusion protein (the region indicated by the arrow in Figure 1) was amplified by PCR using the following primers. The amplified DNA fragment was cloned into the binary vector pBUH201 (a plasmid in which the kanamycin resistance gene nos-NPTII of pRI201-ON (TaKaRa) was replaced with the hygromycin resistance gene 35S-Hyg and the 35S promoter was replaced with the UBI promoter). RAmy3D-F9: CATGAAGAACACCTCGTCAC (SEQ ID NO: 1) PI-R1: TCATCTGGAATAGCACGTGC (SEQ ID NO: 2)

[0028] The abbreviations in Figure 1 represent the following genes or sequences: sp: rice amylase (RAmy3D) signal sequence (MKNTSSLCLLLLLVVLCSLTCNSGQA, SEQ ID NO: 3), H: 6x histidine tag, CaM: mouse calmodulin (GenBank accession number: AAA66182.1), TEV: tobacco etch virus protease recognition sequence (SENLYFQ, SEQ ID NO: 4), IP: persulcatusin (GenBank accession number: BAH09304.1), R B: Right border, LB: Left border, Ubi: Ubiquitin promoter (SEQ ID NO: 5), 5'UTR: Rice alcohol dehydrogenase 5' untranslated region (SEQ ID NO: 6), HSP: Heat shock protein terminator (SEQ ID NO: 7), 35S-Hyg: Hygromycin resistance gene transcriptionally controlled by the cauliflower mosaic virus 35S promoter (35S: SEQ ID NO: 8, Hyg: gene=aph(4)-Ia of CP055258). The same abbreviations are used in other figures.

[0029] The protein expressed by the constructed vector is a fusion protein of calmodulin and persulcatusin. Because a sequence recognized and cleaved by TEV protease exists between the two proteins, in the presence of TEV protease, the protein is cleaved into persulcatusin and calmodulin, thereby expressing the antibacterial activity of persulcatusin. Furthermore, the signal sequence sp (SEQ ID NO: 3) allows the expressed protein to be secreted into the apoplast. Furthermore, the construct pBsp-H-CaM-IP contains a histidine tag, allowing the expressed protein to be purified using a Ni-NTA affinity column. While a histidine tag is used here for purification, it goes without saying that tags commonly used in this field, such as FLAG and Myc tags, can also be used.

[0030] [Gene introduction] The prepared binary vector was introduced into Agrobacterium (EHA101) by electroporation. An empty vector (pBUH201) was also introduced in the same manner. L-broth solid medium supplemented with 50 μg / ml kanamycin and 50 μg / ml hygromycin was used as the medium, and the cells were cultured at 28°C in the dark for 2 days to obtain colonies.

[0031] Calli were induced from rice seeds by standard methods, and gene transfer was performed using the Agrobacterium method. A portion of each resulting Agrobacterium colony was scraped with a sterilized tip and attached to the inner wall of a 50-ml tube in 40 ml of N6CO liquid medium containing 40 mg / L acetosyringone. The bacteria were then suspended in the medium by rotating and stirring for 1 hour in a light-shielded aluminum foil, and this was used for infection.

[0032] The rice callus was added to the fungal suspension and mixed gently by inversion. After discarding the fungal suspension, three sheets of sterile filter paper soaked in 5.5 ml of N6CO liquid medium containing 40 mg / l acetosyringone were placed in a deep Petri dish (2 cm thick), and the callus was placed on top of the filter paper. The Petri dish was sealed with parafilm to prevent drying, and the callus was co-cultured at 28°C for 3 days in a light-shielded aluminum foil.

[0033] After 3 days, the calli were washed repeatedly with DDW containing 40 mg / L meropenem (Dainippon Sumitomo Pharma) to remove any Agrobacterium attached to the calli. The calli were then transferred to N6SE solid medium. The petri dishes were sealed with surgical tape and cultured at 28°C under a 24-hour light period. The calli were subcultured 3 weeks after transplantation and then cultured on selective medium for 6 weeks.

[0034] After 6 weeks, the calli surviving on the N6SE medium were transferred to MSRE solid medium and cultured at 28°C with a 24-hour light period until regenerated individuals appeared. During the regeneration induction period, the calli were subcultured on new MSRE solid medium every 3 weeks.

[0035] Shoots regenerated on MSRE solid medium were transplanted onto MSHF solid medium for root induction. After rooting, when the regenerated plants reached a sufficient size, they were transplanted into culture soil and cultivated in a greenhouse at a room temperature of 28°C under a 24-hour cycle of 16 hours light and 8 hours dark.

[0036] [Confirmation of Expression of Transgene] Genomic DNA was extracted from the leaf blades and calli of the regenerated rice plants to confirm whether the gene had been introduced, and then protein expression was confirmed. As shown in Figure 1, the persulcatusin fusion protein has a rice amylase signal sequence attached to its N-terminus. Therefore, it is expected that the persulcatusin fusion protein will be secreted into the culture supernatant of shake-cultured cells. Therefore, shake-culture was performed, and the persulcatusin fusion protein secreted into the cultured cells and the supernatant was analyzed.

[0037] pBsp-CaM-IP and pBsp-H-CaM-IP were introduced, and callus induction was performed from the resulting rice seeds. Three weeks after induction, approximately 3.0 to 4.0 g of callus was planted in N6CI liquid medium, with gellan gum removed from the composition. 30 ml of N6CI medium was placed in a 100 ml Erlenmeyer flask, and cultured with shaking at 28°C and 100 rpm. Subculture was performed every two weeks.

[0038] The day the shake-cultured cells were subcultured was designated day 0, and the cultured cells and culture supernatants were collected over three days: days 5, 10, and 14. Proteins were extracted from the cultured cells using standard methods and analyzed by Western blotting (Figure 2A). Detection was performed using an anti-calmodulin antibody as the primary antibody. Expression of the persulcatusin fusion protein was confirmed in all shake-cultured cells. The sp-H-CaM-IP fusion, which contains a histidine tag in its sequence, has the advantage of being easily purified. On the other hand, the sp-CaM-IP fusion has the advantage of expressing a high amount of fusion protein.

[0039] The expression of the sp-CaM-IP fusion protein, which showed high expression levels (hereafter, the fusion protein is abbreviated as follows: N-terminus, type of signal peptide (sp), and fusion of calmodulin and persulcatusin; those containing a histidine tag are indicated by H), in the culture supernatant and in cells was analyzed (Fig. 2B). As expected from the addition of a signal sequence, the fusion protein was also detected in the culture supernatant.

[0040] The amount of fusion protein produced in shaking culture was measured. Two strains of sp-CaM-IP fusion and sp-H-CaM-IP fusion were each cultured on shaking, and cells were collected on day 5 of culture and analyzed by Western blotting. Detection was performed using an anti-calmodulin antibody as described above (Figure 3). WT represents wild-type rice cultured cells that had not been transfected with the gene, and CaM represents purified mouse calmodulin protein. The amount of fusion protein produced per unit is shown in Table 1. Although some differences were observed between strains, the fusion without the histidine tag showed higher expression levels.

[0041]

[0042] Shaking culture cells were collected on the 5th day after subculture, and the histidine-tagged sp-H-CaM-IP fusion protein was affinity purified using Ni-NTA Agarose (Fujifilm Wako Pure Chemical Industries, Ltd.). Ni-NTA Agarose was then equilibrated with equilibration buffer (50 mM NaH ２ P.O. ４ The column was equilibrated with a washing buffer (50 mM NaHCO3, 300 mM NaCl, 10 mM imidazole (pH 8.0)) and centrifuged at 500 rpm for 5 minutes. The supernatant was removed and the protein extract was added. The column was then mixed by inversion at room temperature for 60 minutes, centrifuged again, and then washed with a washing buffer (50 mM NaHCO3, 300 mM NaCl, 10 mM imidazole (pH 8.0)). ２ P.O. ４ , 300 mM NaCl, 20 mM imidazole (pH 8.0)), and then elution buffer (50 mM NaH ２ P.O. ４The fusion protein was eluted by adding 100 mM NaCl, 300 mM NaCl, 250 mM imidazole (pH 8.0). SDS-PAGE was performed, followed by CBB staining and Western blotting (Figure 4). As is clear from the results of CBB staining, affinity purification with Ni-NTA Agarose enabled the purification of the fusion protein as an almost single band.

[0043] The fusion protein was examined for antibacterial activity against Staphylococcus aureus (Figure 5). Antibacterial activity was analyzed by spotting antibacterial compounds onto bottom agar based on the radial diffusion assay (Non-Patent Document 9). Both the sp-CaM-IP fusion protein and the sp-H-CaM-IP fusion protein showed significant antibacterial activity against Staphylococcus aureus when treated with TEV protease (with TEV). No antibacterial activity was observed with the control protein extracted from wild-type (WT) rice. IP stands for chemically synthesized persulcatusin.

[0044] As shown in Figure 5, to elicit the antibacterial activity of the Persulcatusin fusion protein, the fusion protein must be cleaved with TEV protease to separate calmodulin and Persulcatusin. If the Persulcatusin fusion protein and TEV are localized in different organs in a single plant and the fusion protein is cleaved by TEV during the extraction process, active Persulcatusin can be obtained during the extraction and purification process.

[0045] Figure 6 shows an example of a construct designed to localize the Persulcatusin fusion protein and TEV in different organs. (I) shows a construct in which a signal sequence (sp) directing targeting to the apoplast (SEQ ID NO: 9) is added to the fusion protein and a signal sequence (cp) directing targeting to the chloroplast (MAPSVMASSATTVAPFQGLKSTAGMPVARRSGNSSSFGNVSNGGRIRC, SEQ ID NO: 9) is added to the TEV protease (GenBank accession number ABF71454.1, the first methionine has been removed). (II) shows a schematic diagram of a construct in which a signal sequence (cp) directing targeting to the chloroplast (SEQ ID NO: 9) is added to the fusion protein and a signal sequence (sp) directing targeting to the apoplast (TEV protease). While the construct shown here is a fusion protein without a histidine tag, a similar construct with a histidine tag may also be used. Furthermore, if the histidine tag is located immediately after the TEV recognition sequence or at the C-terminus of Persulcatusin, the cleaved Persulcatusin can be purified using a Ni-NTA affinity column.

[0046] A construct containing a signal sequence (cp) for chloroplast transport was added to TEV protease constructs and analyzed by RT-PCR (Figure 7). When the cells transfected with the TEV protease gene were regenerated, nearly half of the plants were albino. Therefore, RNA was extracted from the calli of the albino plants and from the leaf blades of the plants that had regenerated into normal plants, and RT-PCR was performed. RT-PCR was performed using primers specifically amplifying TEV protease (SEQ ID NOs: 10 and 11) and primers specifically amplifying actin (SEQ ID NOs: 12 and 13) according to standard methods. While actin was amplified in all isolated lines, TEV protease expression was observed only in cells transfected with the vector. The primers used are as follows: TEV protease primers F-TEVp: GGTGAGTCGCTTTTCAAGGG (SEQ ID NO: 10) TEV-R1: TCAGCGCCTTCGTCTACGATTC (SEQ ID NO: 11) Actin primers RAc-1: AACTGGGATGATATGGAGAA (SEQ ID NO: 12) RAc-2: CCTCCAATCCAGACACTGTA (SEQ ID NO: 13)

[0047] Next, a secreted TEV protease construct containing a signal sequence (sp) that directs transport to the apoplast was introduced into rice cells to produce transformants, and expression analysis was performed in the same manner. RNA was extracted from rice leaf blades and analyzed by RT-PCR (Figure 8A). Numbers 1 to 19 represent independent transformants. WT: wild type; PC: PCR positive control (TEV protease: plasmid used for introduction; actin: genomic DNA); RT+: reverse transcriptase added; RT-: reverse transcriptase not added. All transformants demonstrated expression of the TEV protease gene.

[0048] The expression of secreted TEV protease protein in these transformants was analyzed by Western blot (Figure 8B). Total soluble protein was extracted from the leaf blades of the transformant rice plants used above and subjected to Western blot analysis using an anti-TEV protease antibody. The triangle in the figure indicates the TEV protease band. Although RNA expression was observed in all transformants, protein expression was observed in only a limited number of transformants. However, in those transformants in which protein expression was observed, sufficient amounts of TEV protease were produced to cleave the fusion protein and produce persulcatusin. WT: wild type; PC: 30 ng of TEV protease.

[0049] By crossbreeding plants in which the organelles localizing the TEV protease and the fusion protein are different, rice plants expressing both the fusion protein and the TEV protease can be obtained. Alternatively, genes encoding each protein can be cloned into a single vector from the beginning and then introduced. For example, by using a construct such as that shown in Figure 9, the fusion protein can be localized in the chloroplast and the TEV protease in the apoplast.

[0050] As shown above, by expressing the fusion protein and protease locally in different organelles of a plant, persulcatusin can be easily isolated and purified by extraction. Since purification is also relatively easy, persulcatusin can be used as an alternative to antibiotics for the treatment of bovine mastitis.

Claims

1. A plant cell into which a construct has been introduced, the construct comprising: a polynucleotide sequence encoding a protease gene to which a signal peptide that localizes in an organelle has been added; and a polynucleotide sequence encoding a Persulcatusin fusion protein to which a signal peptide that localizes in an organelle different from the organelle has been added, and which has a sequence between Persulcatusin and the protein fused to it that is cleaved by the protease.

2. The plant cell according to claim 1, wherein the protease is tobacco etch virus (TEV) protease, and the sequence cleaved by the protease is a TEV protease recognition sequence.

3. The plant cell according to claim 2, wherein the persulcatusin fusion protein is a fusion protein of persulcatusin and calmodulin, the TEV protease recognition sequence is located between persulcatusin and calmodulin, and the two are separated by cleavage with TEV protease.

4. The plant cell according to claim 1, wherein the intracellular organelle is a chloroplast or an apoplast, and when the fusion protein is localized in the chloroplast, a signal sequence is added so that the protease is localized in the apoplast, and when the fusion protein is localized in the apoplast, a signal sequence is added so that the protease is localized in the chloroplast.

5. The plant cell according to any one of claims 1 to 4, characterized in that the plant cell is derived from rice.

6. A method for purifying persulcatusin, comprising obtaining seeds by redifferentiating the plant cells of claim 5, germinating the seeds to form sprouts, and crushing the sprouts, whereby the persulcatusin fusion protein comes into contact with a protease, the persulcatusin fusion protein is cleaved, and the persulcatusin fusion protein is extracted as persulcatusin with antibacterial activity.

7. A vector set for producing Persulcatusin, comprising a vector containing a construct comprising a polynucleotide sequence encoding a protease gene to which a signal peptide that localizes in an intracellular organelle has been added, and a vector containing a construct comprising a polynucleotide sequence encoding a Persulcatusin fusion protein to which a signal peptide that localizes in an intracellular organelle different from the intracellular organelle has been added and which has a sequence between Persulcatusin and the protein fused to it that is cleaved by the protease.

8. A vector for producing Persulcatusin, comprising, in a single vector, a construct comprising a polynucleotide sequence encoding a protease gene to which a signal peptide that localizes in an intracellular organelle has been added, and a polynucleotide sequence encoding a Persulcatusin fusion protein to which a signal peptide that localizes in an intracellular organelle different from the intracellular organelle has been added, and which has a sequence between Persulcatusin and the protein fused to it that is cleaved by the protease.

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