KR102155449B1 - Method or mass production of LysP11 in plants, and pharmaceutical composition for prevention and treatment of swine erysipelas comprising the same - Google Patents

Abstract

The present invention relates to a method for producing LysP11 in plants and a pharmaceutical composition for the prevention and treatment of swine erysipelas comprising the same.

Description

[Method or mass production of LysP11 in plants, and pharmaceutical composition for prevention and treatment of swine erysipelas comprising the same}

The present invention relates to a method for the production of LysP11 in plants and a pharmaceutical composition for preventing and treating pig alone disease comprising the same.

Methods that can produce various recombinant proteins at low cost using plants have been developed. Plants have a lower cost of producing recombinant protein than that using animal cells, and can be used as a system for mass production of proteins that may be used industrially as well as expensive medical proteins. Accordingly, many studies are being conducted on a method for producing various recombinant proteins in plants. Plants have many advantages in producing medical proteins in that they hardly contain toxins such as endotoxins present in microorganisms such as E. coli, and there are no pathogens that can infect the human body. In addition, it is known that there is no harmful protein such as prion, so it is possible to produce a recombinant protein that is safe compared to animal cells or microorganisms. However, even with such advantages, relatively low protein expression and difficulty in isolation and purification are the biggest disadvantages compared to other hosts including animal cells in producing proteins in plant cells. Accordingly, many studies are being conducted, and there are attempts to increase the protein expression level in plant cells and efficiently separate and purify proteins by various methods.

The possibility of expression of various recombinant proteins in plants was confirmed. One of these is antibacterial protein. Various antibiotics made of chemical substances have been developed and are now widely used. However, with the emergence of antibiotic-resistant bacteria gradually, the production of new kinds of antibiotics is required. Among them, endolysin is drawing attention as one of the new antibiotics. Endolysin is a gene of bacteriophages, which can be called bacterial viruses, and proteins made from them can specifically degrade peptidoglycans in the cell wall of bacteria. Therefore, it has enzymatic properties that can dissolve bacteria, so it can become a new antibiotic. The endolysin is encoded in the genome of bacteriophages, and the bacteriophages are specific for bacteria, and the endolysin of each bacteriophage has specificity for host bacteria. This specificity is thought to be mainly manifested by the domain that binds to the C-terminal cell wall. Therefore, it is predicted that endolicin can be used to make antibiotics specific to each type of bacteria. Recently, genome sequences of various types of bacteriophage have been revealed.

The present invention is a result of intensive efforts to facilitate separation and purification of endolysin from plants, and as a result of fusion of the endolysin LysP11 (Propionibacterium acnes bacteriophages P1.1 endolysin) derived from the bacteriophage P11 of Propionibacterium into a recombinant vector, the production of LysP11 is easy. The present invention was completed by confirming that LysP11 decomposes peptidoglycan of Erysipelothrix rhusiopathiae, the causative strain of swine alone.

An object of the present invention is to provide a recombinant vector for producing LysP11 in plants.

Another object of the present invention is to provide a transformant transformed with the recombinant vector.

Another object of the present invention is to provide a method for producing LysP11 in a plant comprising the following steps:

(a) preparing the above-described recombinant vector;

(b) introducing the recombinant vector into cells to prepare a transformant;

(c) culturing the transformant;

(d) infiltrating the culture into the plant; And

(e) pulverizing the plant and binding it to cellulose beads.

Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of swine alone disease comprising LysP11.

In order to solve the above problems, the present invention (i) M domain; (ii) CBM3 (Cellulose-binding module 3); (iii) a small ubiquitin-related modifier (SUMO); And (iv) can provide a recombinant vector for producing LysP11 in a plant containing LysP11.

According to a preferred embodiment of the present invention, the gene of LysP11 may include the nucleotide sequence of SEQ ID NO: 1.

According to another preferred embodiment of the present invention, the gene of the M domain may include the nucleotide sequence of SEQ ID NO: 4.

According to another preferred embodiment of the present invention, the gene of CBM3 may include the nucleotide sequence of SEQ ID NO: 7.

According to another preferred embodiment of the present invention, the SUMO gene may include the nucleotide sequence of SEQ ID NO: 5.

According to another preferred embodiment of the present invention, BiP or HDEL may be further included in the recombinant vector.

According to another preferred embodiment of the present invention, the BiP gene may include the nucleotide sequence of SEQ ID NO: 3, and the HDEL gene may include the nucleotide sequence of SEQ ID NO: 6.

In the present invention, the recombinant vector is a 35S promoter derived from cauliflower mosaic virus, a 19S RNA promoter derived from cauliflower mosaic virus, a plant actin protein promoter, and a ubiquitin protein promoter. It may further include any one promoter selected from the group consisting of.

The present invention can also provide a transformant transformed with the recombinant vector.

According to a preferred embodiment of the present invention, the transformant may be Agrobacterium.

The present invention can also provide a method for producing LysP11 in a plant comprising the following steps:

(a) preparing the above-described recombinant vector;

(b) introducing the recombinant vector into cells to prepare a transformant;

(c) culturing the transformant;

(d) infiltrating the culture into the plant; And

(e) pulverizing the plant and binding it to cellulose beads.

According to a preferred embodiment of the present invention, the cellulose beads are carboxyl methyl cellulose (CMC), methyl cellulose (MC), amorphous cellulose (amphous cellulose, AMC), microcrystalline cellulose (microcrystalline Cellulose, MCC), hydroxy ethyl cellulose (HEC), and hydroxy propyl methyl cellulose (hydroxy propyl methyl cellulose, HPMC) can be used any one of the cellulose beads selected from the group consisting of.

The present invention can also provide a pharmaceutical composition for the prevention or treatment of swine dyslexia comprising LysP11.

The recombinant vector according to the present invention has the effect of producing LysP11 in plants with low cost and high efficiency. In detail, the recombinant vector contains BiP, which induces high accumulation of ER, M domain that increases protein expression, CBM3 containing cellulose binding domain, and HDEL, which induces high accumulation rate in ER. In addition, it is a vector prepared by sequentially connecting the 5'CaMV 35S promoter and HSP terminator, which are translation amplification sequences, to the recombinant vector. The recombinant vector can be transformed into Agrobacterium and then cultured to produce LysP11 at low cost and high efficiency in plants by a syringe infiltration method, and the produced LysP11 is isolated through cellulose beads and then purified using His:bdSENP1 There is an effect that can be done. When LysP11 was treated with Gram-positive bacteria, E. rhusiopathiae ATCC 19414, there was an effect of reducing its growth. As a result of treatment of LysP11 with E. rhusiopathiaek, which is a porcine dog bacterium, it has an effect of killing by damaging cell walls or cell membranes.

1 is a result of confirming the structure of the vector of MCS-LysP11 and the expression level in N. benthamiana using the same.
2 is a result of pure separation and purification of MCS-LysP11 from the total protein extract of N. benthamiana using MCC beads.
3 is a result of measuring the anti-viability of LysP11.
4 is a result of measuring the antimicrobial activity of LysP11 according to pH, temperature, and NaCl concentration.
5 is a result of confirming the change of E. rhusiopathiae on an electron microscope after treatment with LysP11.

As described above, production of protein using animal cells or microorganisms causes problems such as various contaminants such as viruses, cancer genes, and enterotoxins, and has limitations in that a large amount of time and cost are consumed when produced. . In order to overcome this, a method for producing protein in plants has been developed, but this also has low protein expression and difficulty in separation and purification.

Accordingly, the present inventors produced a recombinant vector by developing a method for increasing protein expression in plants and efficiently separating and purifying proteins. The recombinant vector of the present invention includes LysP11, after culturing the recombinant vector containing LysP11, infiltrating the culture medium into a plant through a syringe infiltration method, mass-producing it, and separating it through cellulose beads. , The carbonic anhydrase was purified using His:bdSENP1 produced in E. coli, resulting in a high purification rate. In addition, as a result of treatment of LysP11 prepared as a recombinant vector with E. rhusiopathiae ATCC 19414, its growth was reduced, and further, as a result of treatment with pig alone bacteria, there was an effect of damaging cell walls or cell membranes and thus killing cells.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings so that those of ordinary skill in the art can easily implement the present invention. The present invention may be implemented in various different forms, and is not limited to the embodiments described herein.

The present invention (i) M domain; (ii) CBM3 (Cellulose-binding module 3); (iii) a small ubiquitin-related modifier (SUMO); And (iv) can provide a recombinant vector for producing LysP11 in a plant containing LysP11.

In the present invention, the term "" refers to the bacteriophage P11 of Propionibacterium. One of the bacteria's antibiotics is endolysin. Endolysin is the genes of bacteriophages, which can be called bacterial viruses, and proteins made from these proteins specifically degrade peptidoglycans in the bacterial cell wall to kill bacteria. Endolysin can be used to make antibiotics specific to each type of bacteria. Among these, the sequence for the bacteriophage P11 of Propionibacterium is also disclosed, and LysP11 is an endolicin gene.

The gene of LysP11 may include the nucleotide sequence of SEQ ID NO: 1, and the nucleotide sequence variant is included within the scope of the present invention. Specifically, the gene includes a nucleotide sequence having a sequence homology of 70% or more, more preferably 80% or more, even more preferably 90% or more, most preferably 95% or more, respectively, with the nucleotide sequence of SEQ ID NO: 1 can do. The “% of sequence homology” for a polynucleotide is identified by comparing two optimally aligned sequences with a comparison region, and a portion of the polynucleotide sequence in the comparison region is a reference sequence (addition or deletion) for the optimal alignment of the two sequences. It may include additions or deletions (ie, gaps) compared to (not including).

In the present invention, a recombinant vector was constructed to produce the recombinant protein containing LysP11 in plants. The vector included a BiP leader sequence for ER targeting, an M domain for inducing high expression, and a SUMO domain for removing an N-terminal fusion domain. In addition, HDEL, an ER retention signal, was included at the C-terminal end. A modified 35S promoter with a double enhancer was used as a promoter, and an HSP terminator was used as a terminator (FIG. 1A). In order to induce expression in the leaf tissues of N. benthamiana plant, a binary vector containing the MCS-LysP11 recombinant gene was infiltrated into the Agrobacterium strain EHA. In order to induce high expression, a binary vector containing the gene silencing inhibitor P38 was introduced into the Agrobacterium strain EHA and cultured overnight and the binary vector containing the MCS-LysP11 recombinant gene was used as the Agrobacterium strain EHA. The culture was infiltrated and cultured overnight was simultaneously infiltrated. The tissue was harvested 3-5 days after infiltration and protein was extracted. Expression of the protein was confirmed through Western blot analysis of the protein extract using an anti-CBM3 antibody. MCS-LysP11 was identified at the 65-70 kD position (Fig. 1b). The MCS-LysP11 was purely separated using MCC (microcrystalline cellulose) beads as an affinity matrix. Total protein was extracted from 40 g of leaf tissue, and the protein extract was mixed with MCC beads and incubated at 4° C. for 1 hour. After that, wash 5 times with 40 mM Tris-HCl buffer (pH 7.5) to remove loosely bound non-specifically protein, boil and elute the protein bound to MCC beads, and then extract the total protein, washing- As a result of confirming the purity of the protein through Western blot analysis using the off solution with an anti-CBM3 antibody, it was confirmed that MCS-LysP11 can be purified purely (Fig. 2a). In order to secure the tag-free LysP11, MCS-LysP11-bound MCC beads were cultivated together with His:bdSENP1 cut by recognizing the SUMO domain produced in E. coli, and then removed from the culture medium using a Ni+-NTA affinity column. As a result of confirming the purity of LysP11 using SDS-PAGE, 31 kD of LysP11 was confirmed (Fig. 2b). In order to confirm whether His:bdSENP1 was contaminated with the LysP11 portion, it was confirmed through Western blot analysis using an anti-His antibody. His:bdSENP1 was not confirmed in the LysP11 part, and it was confirmed that LysP11 was purely separated and purified. Through this, 8 mg of pure LysP11 could be obtained from 1 kg of fresh weight. To confirm the antibiotic activity of LysP11, P. acnes ATCC 6919, Streptococcus pyogenes (S. pyogenes) ATCC 19615, and E. rhusiopathiae ATCC 19414 and E. coli JM109, which are Gram-negative bacteria, were treated at a concentration of 1 μg/ml. I did. Through this, it was confirmed that LysP11 showed specific antibiotic activity against E. rhusiopathiae (Fig. 3c). As a result of comparing the anti-bioactivity according to pH and temperature of LysP11, it showed high activity between pH 8 and 9, and showed high activity between 35 and 37°C. In addition, as the NaCl concentration increased, the activity was increased, and the activity was maximized at 400 mM, and the activity was decreased above that (FIG. 4).

CBM3 (cellulose-binding module 3) is a cellulose-binding domain secured from C. thermocellum and shows very high binding power to cellulose, so it has several characteristics for use as an affinity tag for pure protein separation. The CBM3 is specific to amorphous cellulose and has a high degree of binding. When the target protein is fused to CBM3, it is less susceptible to interference by the fused target protein, and also helps the folding of the fused target protein. In microorganisms, CBM3 has been reported to increase protein production yield. Cellulose to which CBM3 binds has several advantages, such as being eco-friendly as an affinity resin, and being very inexpensive. Since cellulose has little chemical reactivity, buffer solutions of various compositions can be used, and it is known that it will not react with proteins. In addition, there is also an advantage that various types of cellulose are commercially readily available. Therefore, it has various conditions that can be the optimal resin for pure protein separation. In fact, it is already used in a variety of medical and human-applied products due to its human affinity.

The gene of M doamin may include the nucleotide sequence of SEQ ID NO: 4, and nucleotide sequence variants are included within the scope of the present invention. Specifically, the gene includes a nucleotide sequence having a sequence homology of 70% or more, more preferably 80% or more, even more preferably 90% or more, most preferably 95% or more, respectively, with the nucleotide sequence of SEQ ID NO: 4 can do. The “% of sequence homology” for a polynucleotide is identified by comparing two optimally aligned sequences with a comparison region, and a portion of the polynucleotide sequence in the comparison region is a reference sequence (addition or deletion) for the optimal alignment of the two sequences. It may include additions or deletions (ie, gaps) compared to (not including).

The gene of CBM3 (cellulose-binding module 3) may include the nucleotide sequence of SEQ ID NO: 7, and nucleotide sequence variants are included within the scope of the present invention. Specifically, the gene includes a nucleotide sequence having sequence homology of at least 70%, more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95% of the nucleotide sequence of SEQ ID NO: 7 can do. The “% of sequence homology” for a polynucleotide is identified by comparing two optimally aligned sequences with a comparison region, and a portion of the polynucleotide sequence in the comparison region is a reference sequence (addition or deletion) for the optimal alignment of the two sequences. It may include additions or deletions (ie, gaps) compared to (not including).

Small ubiquitin-related modifier (SUMO) is a small protein that modifies the most studied protein after ubiquitin among the group of ubiquitin-like molecules (Ubls) in eukaryotic cells. SUMO is covalently linked by forming isopeptide with ε-amino residues of lysine present in various target proteins through the C-terminus. In order to fuse this SUMO to a target protein, a SUMO-specific protease must remove the extra site present at the C-terminal region from the SUMO precursor. At this time, the SUMO-specific protease recognizes a glycine-glycine motif specifically present at the C-terminus of the SUMO domain, and removes the extra site present at the C-terminus of the GG sequence. When the SUMO protease makes SUMO from the SUMO precursor, it recognizes the SUMO domain and cuts the C-terminal region of two glycines, so that the SUMO protease can show very high specificity. In addition, since there is almost no specificity for the type of amino acid following two glycine residues, various proteins can be attached after the di-glycine residue, from which the mature SUMO domain having di-glycine at the C-terminus is removed accurately. It has the property that certain proteins in the C-terminal region do not have extra amino acids. Therefore, by fusion of the target protein to SUMO, it is made into a fusion protein of SMO-target, and the SUMO domain is removed therefrom to make a target protein that does not have other extra amino acid residues at the desired N-terminus.

The recombination refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a peptide, a heterologous peptide, or a protein encoded by a heterologous nucleic acid. Recombinant cells may express genes or gene segments that are not found in the natural form of the cell in either a sense or antisense form. In addition, the recombinant cell can express a gene found in a cell in a natural state, but the gene is modified and introduced into the cell by artificial means.

The term “recombinant expression vector” refers to a bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus or other vector. In general, any plasmid and vector can be used as long as they can replicate and stabilize in the host. An important characteristic of the expression vector is that it has an origin of replication, a promoter, a marker gene and a translation control element.

Expression vectors containing the above recombinant expression vectors and appropriate transcription/translational control signals can be constructed by methods well known to those skilled in the art. The method includes in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombinant technology. The DNA sequence can be effectively linked to an appropriate promoter in an expression vector to guide mRNA synthesis. A suitable vector for expressing the DNA fragment and the gene encoding the target protein according to the present invention in plant cells is a pUC19-based plasmid or There is a Ti plasmid vector.

A preferred example of the recombinant vector of the present invention is a Ti-plasmid vector capable of transferring a part of itself, a so-called T-region, to plant cells when present in a suitable host. Other types of Ti-plasmid vectors are currently being used to transfer hybrid DNA sequences into plant cells, or protoplasts from which new plants can be produced that properly insert hybrid DNA into the genome of the plant. Particularly preferred of Ti-plasmid vectors. The form is a so-called binary vector as claimed in EP 0120 516 B1 and in U.S. Patent 4,940,838. Other suitable vectors that can be used to introduce the DNA according to the invention into a plant host are viral vectors such as those that can be derived from double-stranded plant viruses (e.g., CaMV) and single-stranded viruses, gemini viruses, etc. For example, it can be selected from incomplete plant viral vectors. Examples of the suitable vector are not limited thereto, but it is preferable to use a binary vector such as 326 GFP or pCAMBIA series, and those skilled in the art can express the DNA fragment according to the present invention and the gene encoding LysP11 in plant cells. Any vector that exists can be selected and used.

More specifically, the recombinant vector uses an existing vector used for protein expression as a basic skeleton, and the DNA fragment according to the present invention that enhances the expression level of a promoter and a protein of interest and a gene encoding a target protein can be sequentially operated. It is a recombinant expression vector that is closely linked. In addition, the expression vector may include a ribosome binding site and a transcription terminator as a translation initiation site. The expression vector will preferably contain one or more selectable markers. The marker is typically a nucleic acid sequence having a property that can be selected by a chemical method, and all genes capable of distinguishing a transformed cell from a non-transgenic cell are applicable. Examples include herbicide resistance genes such as glyphosate or phosphinothricin, kanamycin, G418, bleomycin, hygromycin, and chloramphenicol. Such as antibiotic resistance gene, aadA gene, and the like, but are not limited thereto.

The term “operably linked” can be a gene and an expression control factor linked in a manner that allows gene expression when an appropriate molecule is bound to an expression control factor.

The present invention may also provide a recombinant vector further comprising a chaperone binding protein (BiP) or a His-Asp-Glu-Leu (HDEL) as the above-described recombinant vector.

The BiP gene may include the nucleotide sequence of SEQ ID NO: 3, and the HDEL gene may include the nucleotide sequence of SEQ ID NO: 6, and the nucleotide sequence is 70% or more, more preferably 80% or more, respectively, of the nucleotide sequence. % Or more, even more preferably 90% or more, and most preferably 95% or more of sequence homology. The “% of sequence homology” for a polynucleotide is identified by comparing two optimally aligned sequences with a comparison region, and a portion of the polynucleotide sequence in the comparison region is a reference sequence (addition or deletion) for the optimal alignment of the two sequences. It may include additions or deletions (ie, gaps) compared to (not including).

In the recombinant vector of the invention, the promoter is composed of a 35S promoter derived from cauliflower mosaic virus, a 19S RNA promoter derived from cauliflower mosaic virus, a plant actin protein promoter, and a ubiquitin protein promoter. It may be any one promoter selected from the group, but is not limited thereto. The term “promoter” refers to a region upstream of DNA from a structural gene and refers to a DNA molecule to which RNA polymerase binds to initiate transcription. "Constitutive promoter" is a promoter that is active under most environmental conditions and developmental states or cell differentiation. Since the selection of transformants can be made by various tissues at various stages, a constitutive promoter is preferred in the present invention. Therefore, constitutive promoters do not limit the possibility of selection.

In the recombinant vector of the present invention, a conventional terminator can be used, examples of which include nopaline synthase (NOS), rice amylase RAmy1 A terminator, paseolin terminator, Agrobacterium tumafaciens octopine gene The terminator of E. coli and the rrnB1/B2 terminator of Escherichia coli, but are not limited thereto. Regarding the need for terminators, it is generally known that such regions increase the certainty and efficiency of transcription in plant cells. Therefore, the use of a terminator is very desirable in the context of the present invention.

The present invention can provide a transformant transformed with the recombinant vector.

In addition, when the vector of the present invention is transformed into eukaryotic cells, as host cells, yeast (Saccharomyce cerevisiae), insect cells, human cells (eg, CHO cell line (Chinese hamster ovary), W138, BHK, COS-7, 293 , HepG2, 3T3, RIN and MDCK cell lines) and plant cells, and the like can be used. Preferably, it may be grobacterium tuma faciens, and more specifically, it may be Agrobacterium LBA4404, GV3101, AGL1, EHA101 or EHA105.

The method of transporting the vector of the present invention into a host cell is, when the host cell is a prokaryotic cell, the CaCl ₂ method, one method (Hanahan, D., J. Mol. Biol., 166:557-580 (1983)). And an electroporation method. In addition, when the host cell is a eukaryotic cell, the vector can be injected into the host cell by microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, and gene bombardment. I can.

The manufacturing sum vector of the present invention may be represented by the cleavage map of FIG. 1A.

The real name also includes the steps of (a) constructing the above-described recombinant vector; (b) introducing the recombinant vector into cells to prepare a transformant; (c) culturing the transformant; (d) infiltrating the culture into the plant; And (e) can provide a method for producing LysP11 in a plant comprising the step of pulverizing the plant and binding to cellulose beads.

The method for producing LysP11 from the transformed plant strain can be obtained from the transformed cells after transforming the recombinant vector according to the present invention into cells and then incubating for an appropriate time so that LysP11 is expressed. At this time, any method known in the art may be used for expressing the LysP11.

The cellulose beads are carboxyl methyl cellulose (CMC), methyl cellulose (MC), amorphous cellulose (amphous cellulose, AMC), microcrystalline cellulose (MCC), hydroxy ethyl cellulose ( Any one cellulose bead selected from the group consisting of hydroxy ethyl cellulose (HEC) and hydroxy propyl methyl cellulose (HPMC) may be used, and most preferably, microcrystalline cellulose (MCC). I can.

In the present invention, (d) in the step of infiltrating the culture into plants, the culture of the recombinant vector containing P38 in addition to the recombinant vector culture for producing the carbonic anhydrase of the present invention can be simultaneously infiltrated. p38 is a gene silencing suppressor. In addition, it may be gene silencing suppressor P19, HC-Pro, TVA 2b, etc.

The method of infiltrating the plant may include a chemical cell method, a vacuum method, or a syringe infiltration method, and most preferably a syringe infiltration method, but is not limited thereto.

The plants include rice, wheat, barley, corn, beans, potatoes, wheat, red beans, oats, food crops including sorghum; Vegetable crops including Arabidopsis, Chinese cabbage, radish, pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, pumpkin, green onion, onion, and carrot; Specialty crops including ginseng, tobacco, cotton, sesame, sugar cane, sugar beet, perilla, peanut, and rapeseed; Fruit trees including apple trees, pear trees, jujubes, peaches, grapes, tangerines, persimmons, plums, apricots, and bananas; It may be selected from flowers including roses, carnations, chrysanthemums, lilies, and tulips, and preferably Nicotiana benthamiana.

It may provide a method for purifying LysP11, further comprising: (e) treating the enzyme after the step.

The enzyme may be bdSENP1, and may be produced in E. coli.

The present invention can also provide a pharmaceutical composition for the prevention or treatment of swine dyslexia comprising LysP11.

In order to confirm the stellar activity of LysP11 against E. rhusiopathiae, E. rhusiopathiae treated with LysP11 was observed with an electron microscope. As a result of observing E. rhusiopathiae treated with ysP11 for 20 minutes, the cell wall was deformed and the cytoplasmic membrane was thinned. It was confirmed that the morphology of the cells was greatly deformed, and when LysP11 was treated for 60 minutes, the cell walls or membranes were damaged, some of the cytoplasmic contents disappeared, and it was confirmed that the cells did not have a normal shape. A hole was formed in the cell wall, and it was observed that the cytoplasmic contents escaped through this hole. Through this, it was confirmed that LysP11 degrades the peptidoglycan of E. rhusiopathiae (FIG. 5).

Porcine dogmatosis is a major infectious disease in pigs with symptoms such as fever, skin lesions, arthritis and endocarditis, and the causative bacteria is Erysipelothrix rhusiopathiae. Even if the specimen has elapsed for a long time, the pig alone bacteria can be easily cultured, and the pig alone bacteria can survive for several months in pork or animal tissues. Even when exposed to pig manure or nature, it can survive for 6 months at an environmental temperature below 12℃. This disease is a difficult disease to eradicate and is the first type of legal infectious disease. It is characterized by sudden death, fever, loss of appetite, diamond-shaped skin spots, arthritis, endocarditis and miscarriage of pregnant sows. The causative bacteria are generally found in the tonsils of normal pigs and are caused by environmental factors such as high temperature, viral infection, fatigue, and vaccines.

Although an embodiment of the present invention has been described above, the spirit of the present invention is not limited to the embodiment presented in the present specification, and those skilled in the art who understand the spirit of the present invention can add components within the scope of the same idea. It will be possible to easily propose other embodiments by changing, deleting, adding, etc., but it will be said that this is also within the scope of the present invention.

Production of MSC-Lysp11

In order to mass-produce LysP11 recombinant protein in plants, a binary vector, MCS, was used. The vector includes a BiP leader sequence for ER targeting, an M domain that induces high expression (from amino acids Ala 231 to Asp 290 of human tyrosine phosphatase receptor type C (CD45)), and a SUMO domain for removing the N-terminal fusion domain. Included. In addition, HDEL, an ER retention signal, was included at the C-terminal end. The sequence number of the gene is shown in Table 1 below. LysP11 was optimized for the codon for expression in Nicotiana benthamiana. LysP11 optimized for the codon was positioned between the SUMO domain and HDEL. The recombinant protein gene for LysP11 expression thus divided was named MCS-LysP11 (FIG. 1A). The modified 35S promoter with double enhancer was used as the promoter, and the HSP terminator was used as the terminator (1300-MCS-LysP11).

Introduction of recombinant vector into Agrobacterium strain EHA105 and expression in Nicotiana benthamiana

In order to induce expression in the leaf tissues of N. benthamiana plant, a binary vector containing the MCS-LysP11 recombinant gene was introduced into the Agrobacterium strain EHA. This was cultured overnight, and the culture was incubated at OD600 to 1.0. And in order to induce high expression of MCS-LysP11, a binary vector containing P38, a gene silencing inhibitor, was introduced into the Agrobacterium strain EHA and cultured overnight, and the culture was again cultured at OD600 to 1.0. The two MCS-LysP11 and p38 cultures were mixed at a ratio of 1:1 to infiltrate the leaves of N. benthamiana to induce transient expression. The tissue was harvested 3-5 days after infiltration and protein was extracted. Expression of the protein was confirmed through Western blot analysis of the protein extract using an anti-CBM3 antibody. MCS-LysP11 was identified at the 65-70 kD position (Fig. 1b).

Pure separation of the recombinant protein LysP11 using cellulose beads

In order to investigate the activity of LysP11, pure separation and purification were performed. First, MCS-LysP11 was purely separated using MCC (microcrystalline cellulose) beads as an affinity matrix. Total protein was extracted from 40 g of leaf tissue, and the protein extract was mixed with MCC beads and incubated at 4° C. for 1 hour. Thereafter, the loosely bound non-specifically protein was removed by washing 5 times with 40 mM Tris-HCl buffer (pH 7.5). Subsequently, the protein bound in close contact with the MCC beads was boiled and eluted, and the purity of the protein was confirmed through Western blot analysis using an anti-CBM3 antibody with a total protein extract and a washing-off solution. As a result, it was confirmed that pure separation and purification of MCS-LysP11 can be performed using MCC (Fig. 2a). To secure LysP11 without tag, MCS-LysP11-conjugated MCC beads were incubated with His:bdSENP1 produced in E. coli for 6-8 hours at 4°C. His:bdSENP1 recognizes and cuts the SUMO domain, so LysP11 could be purely isolated from MCS-LysP11. After treatment, His:bdSENP1 was removed from the culture medium using a Ni+-NTA affinity column. As a result of confirming the purity of LysP11 using SDS-PAGE, 31 kD of LysP11 was confirmed (Fig. 2b). In order to confirm whether His:bdSENP1 was contaminated with the LysP11 portion, it was confirmed through Western blot analysis using an anti-His antibody. His:bdSENP1 was not confirmed in the LysP11 part, and it was confirmed that LysP11 was purely separated and purified. Through this, 8 mg of pure LysP11 could be obtained from 1 kg of fresh weight.

Confirmation of the antibiotic activity of LysP11

To confirm the antibiotic activity of LysP11, the culture media of various bacteria were treated at a concentration of 1 μg/ml, and the growth of the bacteria was confirmed at OD600. The gram-positive bacteria P. acnes ATCC 6919, Streptococcus pyogenes (S. pyogenes) ATCC 19615, and E. rhusiopathiae ATCC 19414 and the gram negative bacteria E. coli JM109 were used. Among these, only E. rhusiopathiae ATCC 19414 showed a 52% reduction in OD 600 in 60 minutes treatment time, and a reduction in OD 600 by 68% when 90 minutes treatment (FIG. 3 ). Subsequently, the bacterial activity treated with LysP11 was confirmed. The activity was confirmed by a method of confirming the colony forming unit by securing the bacteria at each time and culturing them in MS plates. It was confirmed that the bacteria treated for 90 minutes decreased the colony-forming ability of 5 or more in the log value. Through this, it was confirmed that LysP11 showed specific antibiotic activity against E. rhusiopathiae (Fig. 3c). As a result of comparing the anti-bioactivity according to pH and temperature of LysP11, it showed high activity between pH 8 and 9, and showed high activity between 35 and 37°C. In addition, as the NaCl concentration increased, the activity was increased, and the activity was maximized at 400 mM, and the activity was decreased above that (FIG. 4).

LysP11 confirmed stellar activity against E. rhusiopathiae

In order to confirm the stellar activity of LysP11 against E. rhusiopathiae, E. rhusiopathiae treated with LysP11 was observed with an electron microscope. In E. rhusiopathiae treated with LysP11 for 20 minutes, it was confirmed that the cell wall was deformed, the cytoplasmic membrane was thinned, and the shape of the cell was greatly deformed. When LysP11 was treated for 60 minutes, the cell wall or cell membrane was damaged, some of the cytoplasmic contents disappeared, and it was confirmed that the cells did not have a normal shape. In addition, a hole was formed in the cell wall, and cytoplasmic contents were observed to escape through this hole. Through this, it was confirmed that LysP11 degrades the peptidoglycan of E. rhusiopathiae (FIG. 5).

<110> POSTECH ACADEMY-INDUSTRY FOUNDATION <120> Method or mass production of LysP11 in plants, and pharmaceutical composition for prevention and treatment of swine erysipelas comprising the same <130> 1065300 <160> 7 <170> KoPatentIn 3.0 <210> 1 <211> 463 <212> DNA <213> Artificial Sequence <220> <223> sequence of LysP11 <400> 1 agatttattc cggcagcaca tcattcagca ggatcaaaca gcccagtgaa tagagttgtt 60 atccatgcaa catgtccgga cgttggattt ccttctgcat ccagaaaggg tcgggcagtg 120 tctacagcaa actatttcgc ttccccctct tctggtggta gtgcgcatta tgtctgtgat 180 atcagtgaaa cagtccagtg cttgagcgag tctacaattg ggtggcatgc gcctcccaat 240 ccacactcgc taggtataga gatttgcgct gatggaggtt cacacgcctc tttccgtgta 300 ccaggacatg cttacacgag ggaacaatgg ctcgatccta gagtttggcc tgctgtggag 360 aaggctgcca tactctgtag aagattgtgc gacaaataca atgttcctaa aaggaaactg 420 tctgctgccg acttgaaggc tggcaggcgg ggtgtatgtg gcc 463 <210> 2 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> sequence of 5 UTR <400> 2 tctagaatta ttacatcaaa acaaaaa 27 <210> 3 <211> 272 <212> DNA <213> Artificial Sequence <220> <223> sequence of BiP <400> 3 atggctcgct cgtttggagc taacagtacc gttgtgttgg cgatcatctt cttcggtgag 60 tgattttccg atcttcttct ccgatttaga tctcctctac attgttgctt aatctcagaa 120 ccttttttcg ttgttcctgg atctgaatgt gtttgtttgc aatttcacga tcttaaaagg 180 ttagatctcg attggtattg acgattggaa tctttacgat ttcaggatgt ttatttgcgt 240 tgtcctctgc aatagaagag gctacgaagt ta 272 <210> 4 <211> 224 <212> DNA <213> Artificial Sequence <220> <223> sequence of M domain <400> 4 ggatcccgat ggcaaacatc actgtggatt acttatataa caaggaaact aaattattta 60 cagcaaagct aaatgttaat gagaatgtgg aatgtggaaa caatacttgc acaaacaatg 120 aggtgcataa ccttacagaa tgtaaaaatg cgtctgtttc catatctcat aattcatgta 180 ctgctcctga tggtggagga ggttctggtg gtggatcaac tagt 224 <210> 5 <211> 240 <212> DNA <213> Artificial Sequence <220> <223> sequence of SUMO <400> 5 atgcatatta atttgaaagt gaagggacag gacggaaacg aggttttctt ccgtatcaaa 60 agatcaacac aactcaagaa actcatgaat gcttactgcg atagacaaag cgtggacatg 120 acagctatag ctttcctatt tgatgggaga aggttgagag cggaacagac tccggatgag 180 cttgaaatgg aagatggaga tgagattgac gcaatgttac atcagactgg tgccggcggt 240 240 <210> 6 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> sequence of HDEL <400> 6 cacgatgagc tc 12 <210> 7 <211> 480 <212> DNA <213> Artificial Sequence <220> <223> sequence of CBM3 <400> 7 gtatcaggta accttaaggt ggagttttac aactcgaacc cttctgatac aactaactca 60 ataaacccac agttcaaagt tacaaacaca ggcagctctg cgatcgattt gtctaaatta 120 accctcagat actattatac ggttgatgga cagaaggacc agactttctg gtgtgatcat 180 gcagctatca ttggttctaa cggtagctac aacggaatta catcaaacgt gaagggcact 240 ttcgttaaga tgtcctctag cactaacaac gcagacacat atttggagat cagttttacg 300 gggggaaccc ttgaaccagg tgctcacgtc cagattcaag gaagattcgc taaaaacgac 360 tggtcgaact atacccaaag taatgattac agttttaaat ccgcctcaca atttgttgag 420 tgggatcagg tcactgctta cctgaacggg gttctagtgt ggggaaagga acctggtccc 480 480

Claims (15)

(i) M domain;
(ii) CBM3 (Cellulose-binding module 3);
(iii) a small ubiquitin-related modifier (SUMO); And
(iv) LysP11 (Propionibacterium acnes bacteriophages P1.1 endolysin);
Including,
The LysP11 gene contains the nucleotide sequence of SEQ ID NO: 1, a recombinant vector for producing LysP11 in plants. delete The recombinant vector according to claim 1, wherein the gene of the M domain comprises the nucleotide sequence of SEQ ID NO: 4. The recombinant vector according to claim 1, wherein the gene of CBM 3 comprises the nucleotide sequence of SEQ ID NO: 7. The recombinant vector according to claim 1, wherein the SUMO gene comprises the nucleotide sequence of SEQ ID NO: 5. The recombinant vector of claim 1, further comprising BiP or HDEL to the recombinant vector. The recombinant vector according to claim 6, wherein the BiP gene includes the nucleotide sequence of SEQ ID NO: 3, and the HDEL gene includes the nucleotide sequence of SEQ ID NO: 6. The method of claim 1, wherein the recombinant vector is a 35S promoter derived from cauliflower mosaic virus, a 19S RNA promoter derived from cauliflower mosaic virus, a plant actin protein promoter, and a ubiquitin protein promoter. A recombinant vector further comprising any one promoter selected from the group consisting of. A transformant transformed with the recombinant vector of claim 1. The transformant of claim 9, wherein the transformant is Agrobacterium. (a) constructing the recombinant vector according to claim 1;
(b) introducing the recombinant vector into cells to prepare a transformant;
(c) culturing the transformant;
(d) infiltrating the culture into the plant; And
(e) crushing the plant and binding it to cellulose beads;
A method for producing LysP11 in a plant comprising a. The method of claim 11, wherein the cellulose beads are carboxyl methyl cellulose (CMC), methyl cellulose (MC), amorphous cellulose (AMC), microcrystalline cellulose (MCC). , Hydroxy ethyl cellulose (hydroxy ethyl cellulose, HEC) and hydroxy propyl methyl cellulose (hydroxy propyl methyl cellulose, HPMC) using any one cellulose bead selected from the group consisting of, a method for producing LysP11 in plants. The method of claim 12, further comprising the step of treating the enzyme after the step (e). The method of claim 13, wherein the enzyme is bdSENP1. delete

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