KR102052108B1 - Recombinant plasmid with auxotrophy marker, microorganisms for delivering anti-cancer agent to gastrointestinal tract and anticancer pharmaceutical composition - Google Patents

Abstract

The present invention relates to a recombinant plasmid for producing an anti-cancer drug delivery strain comprising a P8 protein coding gene fragment of SEQ ID NO : 1 and an alanine racease gene of SEQ ID NO : 11, to microorganisms for drug delivery, and to a pharmaceutical composition for preventing or treating gastrointestinal diseases, comprising the same and, more specifically, to a lactic acid bacteria drug delivery system which over-expresses and secretes lactic acid bacteria-derived P8 anti-cancer protein by introducing the same into the lactic acid bacteria expression and secretion system, thereby reducing cost and enhancing safety when culturing a large amount of effective microorganisms for drug delivery through keeping cells with the recombinant plasmid without the addition of anti-biotics.

Description

Recombinant PLASMID WITH AUXOTROPHY MARKER, MICROORGANISMS FOR DELIVERING ANTI-CANCER AGENT TO GASTROINTESTINAL TRACT AND ANTICANCER PHARMACEUTICAL COMPOSITION}

The present invention relates to recombinant plasmids and strains transformed thereby, more specifically to improved recombinant plasmids for the production of anticancer drug delivery strains, and thereby to transform the gastrointestinal disease therapeutically active proteins by the oral route transformed by the gastrointestinal tract. To a microbial strain for topical delivery.

Lactic acid bacteria are microorganisms widely distributed in the natural world to produce lactic acid by using carbohydrates anaerobically. Due to the advantage that lactic acid bacteria are non-pathogenic bacteria, the range of their use has recently been extended to industrial fields as well as medical fields. These lactic acid bacteria are known to play an important role in maintaining the health of the stomach, it has been reported that can be improved by taking the lactic acid bacteria.

It is known that a therapeutic effect can be expected by administering lactic acid bacteria to colon disease patients including colorectal cancer and colitis, and techniques for using lactic acid bacteria as a therapeutic agent for colorectal diseases have been developed. For example, Korean Patent Publication No. 1313849 discloses an anticancer composition comprising Lactobacillus plantarum PEMO 08 (KFCC-11028) as an active ingredient, and Korean Patent No. 1050307 is deposited with accession number KCTC 18120P. a, it has a butanol extract of Bifidobacterium Adolfo LES sentiseu SPM0212 strain having the cancer cell proliferation inhibitory activity discloses an anticancer pharmaceutical composition that as an active ingredient. However, the conventional techniques for using lactic acid bacteria for colon or gastrointestinal diseases use lactic acid bacteria themselves, and there is little attempt to use the lactic acid bacteria strain itself as a drug delivery system by recombining genes encoding anti-cancer active proteins to lactic acid bacteria.

Applicant is a drug that can be transformed by a plasmid containing a gene encoding a therapeutically active peptide (P8 protein) and delivered safely in the intestine through oral administration to express and secrete the therapeutic peptide in the gastrointestinal tract in Korean Patent No. 1915951. A microorganism for delivery has been proposed. To maintain the microorganisms, antibiotics should be added to the culture. However, due to the characteristics of microorganisms, foreign DNA introduced from the outside tends to be lost during subculture of microorganisms, and there is a problem in that cost increases when culturing a large amount of microorganisms for drug delivery. In addition, in the course of culturing the drug delivery microorganisms to prepare an oral preparation, a problem arises in that the plasmid-dissociated cells are mixed to reduce the efficacy of the drug. Therefore, in order to solve this problem, it is required to develop a system in which only cells having a recombinant plasmid can live without adding antibiotics.

The present invention is to overcome the above-mentioned problems of the prior art, one object of the present invention is to provide a recombinant plasmid that makes it possible to mass culture microorganisms for anticancer drug delivery without the use of antibiotics.

Another object of the present invention is to provide a microorganism for delivering a therapeutic polypeptide to a specific part of the gastrointestinal tract effective for gastrointestinal diseases that can be cultured in large quantities without the addition of antibiotics.

It is still another object of the present invention to provide a pharmaceutical composition for preventing or treating gastrointestinal disease, which includes a microorganism as a drug carrier capable of expressing and secreting a therapeutic polypeptide effective at a high level at a predetermined position of the gastrointestinal tract.

Still another object of the present invention is to provide a method for culturing a large amount of microorganisms for drug delivery for treating gastrointestinal diseases without adding antibiotics.

One aspect of the present invention for achieving the above object,

It relates to a recombinant plasmid for producing an anticancer drug delivery strain comprising a P8 protein coding gene fragment of SEQ ID NO: 1 and an alanine racease gene of SEQ ID NO: 11.

The recombinant plasmid comprises a heterologous nucleic acid encoding a P8 protein of SEQ ID NO: 1 having a therapeutic effect on gastrointestinal diseases, wherein the heteronucleic acid is one derived from Pediococcus pentosaceus operably linked to a heterologous nucleic acid. It may include a gene encoding the above promoter and secretion signal peptide.

The secretory signal peptide may be a USP45 secretory signal peptide, Usp45 N4 or Lactobacillus brevis S-layer protein signal peptide.

The recombinant plasmid may have a cleavage map (SEQ ID NO: 9) of FIG. 1 or 2.

Another aspect of the present invention for achieving the above object relates to an anticancer drug delivery strain transformed by the above-described recombinant plasmid of the present invention and comprising an inactivated alanine racease (alaR) gene.

The recombinant strain may be a Pediococcus pentosaceus strain. The Pediococcus pentosaceus strain may be a Pediococcus pentosaceus PPΔalr-P8-alr strain (Accession Number: KCCM12500P).

The gastrointestinal disease may be colon cancer, colon polyp, colitis, ischemic gastrointestinal disease, dysentery, intestinal vascular dysplasia, diverticulosis, irritable bowel syndrome, or Crohn's disease.

Another aspect of the present invention for achieving the above object,

Preparing a recombinant plasmid comprising a gene fragment encoding a protein exhibiting gastrointestinal therapeutic activity and a gene encoding a protein essential for bacterial viability:

Preparing a trophogenic strain in which the essential protein coding gene of the recombinant plasmid is inactivated:

It relates to a method for producing an anticancer drug delivery strain comprising the step of transforming the trophic strain with the recombinant plasmid to produce an anticancer drug delivery recombinant strain.

According to the recombinant plasmid of the present invention, instead of the conventional selection marker using an antibiotic resistance gene, a urea-containing marker using a gene related to the biosynthesis of essential amino acids is included, and the human-derived natural substance confirmed to inhibit the proliferation and metastasis of cancer cells. The strain encoding the protein (P8 protein) can provide an advantageous effect to allow amplification on an antibiotic basis.

The microbial strain for drug delivery transformed by the plasmid of the present invention can effectively function as a drug carrier by continuously expressing and secreting therapeutic peptides at high levels while staying in the intestine.

According to the pharmaceutical composition of the present invention, not only shows excellent cancer cell proliferation inhibitory effect in colorectal cancer cells, but also is a safe substance that is not toxic to normal cells, and can be very useful for preventing and treating colorectal cancer without side effects.

Local delivery of therapeutic peptides by in situ synthesis by recombinant microorganisms via the oral route of administration in the present invention allows precise control of the dosage and timing required for treatment, thus eliminating the need for systemic administration.

The present invention can be usefully used for topical delivery of a substance that is unstable or difficult to produce in large quantities, and according to the invention there is a continuous topical use of a therapeutic peptide at a higher concentration than that obtained by systemic delivery without the problem of side effects. Can be delivered as

1 is a cleavage map of a recombinant plasmid for producing an anticancer drug delivery strain comprising a P8 protein coding gene fragment of SEQ ID NO: 1 and an alanine racema gene of SEQ ID NO: 11 of an embodiment of the present invention.
Figure 2 is a cleavage map of the recombinant plasmid (SEQ ID NO: 9) for producing an anticancer drug delivery strain of the present invention.
Figure 3 is a schematic diagram illustrating a process of cloning using the recombinant plasmid of an embodiment of the present invention after preparing a Pediococcus pentosaceus strain in which the alanine racease gene is deleted in another embodiment of the present invention.
Figure 4 is a photograph showing the experimental results confirming the nutritional composition of the anticancer drug delivery microbial strain of another embodiment of the present invention.
FIG. 5 is a diagram illustrating the complementarity test in which the recombinant plasmid of the present invention is introduced into a pediococcus pentosaceus strain in which an alanine racease gene is deleted.
FIG. 6 is a photograph illustrating a process of transplanting tumor cells by using tumor tissue to display tumors.
Figure 7a-c is a result of the experiment the tumor growth inhibitory effect of oral administration of a nutritional requirement Phedi O Lactococcus pen soil three mouse PP △ alr-P8-alr strain of the present invention to a mouse in an embodiment of the present invention It is a graph showing.
Figure 8 is required in the present invention to a mouse in an embodiment of the present invention Nutritional Composition Phedi O Lactococcus pen soil three mouse PP △ alr-P8-alr case of oral administration, the strain and the anticancer drug, showing a result of observing the tumor size changes It is a photograph.
9 is a graph showing in comparison the tumor size in the case of an orally administered auxotrophic Lactococcus Phedi o pen soil three mouse PP △ alr-P8-alr strain of the present invention to a mouse in an embodiment of the present invention.
Figure 10 is a view showing the result of the weight measurement of the tumor of the test group and the control group in the embodiment of the present invention.
11 is a graph showing the tumor growth inhibition rate of the test group and the control group in the embodiment of the present invention.
Figure 12 is a picture showing an AOM / DSS mouse after oral administration experiments auxotrophic Phedi o Isolation of Lactococcus pen soil three mouse PP △ alr-P8-alr strain of the present invention to survive in the intestine.

Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The term “nucleic acid” refers to a deoxyribonucleotide or ribonucleotide polymer in single-stranded or double-stranded form and, unless otherwise specified, refers to a natural nucleotide that hybridizes to a nucleic acid in a manner similar to a naturally occurring nucleotide. Known analogs. Unless otherwise stated, a particular nucleic acid sequence includes its complementary sequence.

As used herein, the term "promoter" refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. The promoter consists of upstream and adjacent elements.

The term "biologically active molecule" refers to a substance that is involved in gene therapy or modulates an immune response, and is capable of modulating intracellular signal transduction mechanisms or expression of other specific genes. Contains substances. Such substances may include growth factors, cancer treatment agents, tumor suppressor agents, cytokines, interferons, and the like.

As used herein, “heterologous sequence” or “heterologous nucleic acid” means originated from a foreign source (or species) or modified from its native form when derived from the same source. Thus, the heterologous nucleic acid operably linked to a promoter may be derived from a different source than the source from which the promoter is derived, or modified from its own form if it is the same source.

The term “operably linked” refers to a functional link between a nucleic acid expression control sequence (eg, a promoter, signal sequence or series of transcription factor binding sites) and a heterologous nucleic acid sequence, wherein the expression control sequence corresponds to the heterologous nucleic acid sequence. Affects transcription and / or translation of the nucleic acid.

As used herein, the term "vector" refers to a polynucleotide designed to introduce a nucleic acid into one or more host cells. Vectors can replicate automatically in a variety of host cells and include cloning vectors, expression vectors, shuttle vectors, plasmids, phage particles, cassettes and the like. Since plasmids are the most commonly used form of current vectors, "plasmid" and "vector" are used interchangeably in the present specification.

As used herein, the term "introduction" refers to "transfection", or "transfection" or "transduction", wherein the nucleic acid sequence is the genome of the cell (eg, chromosome, plasmid). , Plastid or mitochondrial DNA), converted to automatic replicon, or transiently expressed.

As used herein, the term "alanine racemase" refers to an enzyme that catalyzes the interconversion of L-alanine and D-alanine. Alanine racemase

The gene encoding may be designated as "alr" or "alrA" gene.

As used herein, the terms "protein" and "polypeptide" are used interchangeably. "Polypeptide" means a polymer of amino acids, not a molecule of a particular length. This term is meant to include post-translational modifications of the polypeptide, such as glycosylation, phosphorylation and acetylation.

As used herein, the term "prevention" means a medical or sanitary procedure aimed at preventing rather than treating or curing a disease.

As used herein, the term "treatment" refers to a medical or sanitary procedure aimed at the treatment or cure of a disease.

As used herein, the term “therapeutically effective amount” means an amount of a therapeutic substance or composition effective for treating a disease or disorder in a subject, eg, a human or animal, ie, to achieve the desired local or systemic effect and performance. Point to.

As used herein, the term "pharmaceutically acceptable" means compatible with the other ingredients of the pharmaceutical composition and not deleterious to its receptor.

One aspect of the present invention relates to a recombinant plasmid for producing an anticancer drug delivery strain comprising a P8 protein coding gene fragment of SEQ ID NO: 1 and an alanine racemic gene of SEQ ID NO: 11. The recombinant plasmid comprises a heterologous nucleic acid encoding a P8 protein of SEQ ID NO: 1 having a therapeutic effect on gastrointestinal diseases, wherein the heteronucleic acid is one derived from Pediococcus pentosaceus operably linked to a heterologous nucleic acid. Genes encoding the above promoters and secretory signal peptides are included. The recombinant plasmid of the present invention may have a cleavage map of FIG. 1 or 2 and may have a sequence of SEQ ID NO. The plasmid of the present invention can be prepared by cloning the one or more promoters of the alanine racease gene and the promoters of SEQ ID NO: 3 to SEQ ID NO: 8 to the pCBT24 plasmid.

There are 20 amino acids in higher organisms, or mammals, of which nine are essential amino acids that must be obtained from food. Among the essential amino acids, D-alanine is a component of the cell wall in microorganisms. Alanine racemase enzymes catalyze the interconversion of L-alanine and D-alanine and are encoded by the alr gene. The alr-deficient strain is auxotroph for D-alanine, which is required for cell wall biosynthesis. When the alr gene was removed from the chromosome of the Pediococcus pentosaceus CBT SL4 (-7) (Accession No .: KCTC 10297BP) strain, it was not possible to synthesize D-alanine, thereby producing a D-alanine auxotrophic strain. These strains can only live in cultures containing D-alanine. Recombinant plasmids of the present invention include genes encoding P8 protein with excellent anticancer activity and alr genes, which can be screened and amplified based on an unscented drug, and are advantageous for preparing a trophogenic strain capable of delivering P8 protein to the human body. Can be used.

A "P8 protein" refers to lactic acid bacteria of Lactobacillus protein fragment of the molecular weight of 8 kDa size extracted from Versus ramno In one embodiment, the cancer protein is derived from a Lactobacillus ramno Versus Cell lysate solutions. Anticancer drug delivery microorganisms expressing and secreting P8 protein in the gastrointestinal tract according to the present invention may provide a synergistic effect by the anticancer action of lactic acid bacteria itself in addition to the anticancer effect of P8 protein. Lactic acid bacteria strains can provide the effect of inhibiting the production of carcinogens by improving the intestinal microflora and inhibiting the proliferation of cancer cells by activating intestinal immune function. In the present invention, the anti-cancer effect of P8 protein is estimated to inhibit the growth of cancer cells by preventing the cell division by inhibiting cell cycle-related metabolism such as P8 protein is introduced into the cell and cyclin B1, cyclin D1, cdk1 and the like.

SEQ ID NO: Justice order SEQ ID NO: 1 P8 protein
Sequence gcaacagtagatcctgaaaagacattgtttctcgatgaaccaatgaacaaggtatttgactggagcaacagcgaagcacctgtacgtgatgcgctgtgggattattacatggaaaagaacagccgtgataccatcaagactgaagaagaaatgaaaccagtcctagacatgtccgacgatgaggagaaaagccctagcagaaaaggttctca SEQ ID NO: 2 P8 protein
amino acid
order ATVDPEKTLFLDEPMNKVFDWSNSEAPVRDALWDYYMEKNSRDTIKTEEEMKPVLDMSDDEVKALAEKVLKK

In the present invention, a exogenous promoter for expression in lactic acid bacteria of the drug active protein used in the recombinant plasmid is Phedi O Paracoccus the ermE promoter derived from a pen soil three mouse (erythromycin resistance gene) (SEQ ID NO: 3), PK promoters (pyruvate kinase ) (SEQ ID NO: 4), GK promoter (glucokinase) (SEQ ID NO: 5), GPFK promoter (6-phosphofructokinase) (SEQ ID NO: 6), G6Pi promoter (glucose 6-phosphate isomerase) (SEQ ID NO: 7), L-LDH promoter (L-lactate dehydrogenase) (SEQ ID NO: 8) and combinations thereof.

primer Sequence (5 '-> 3') ermE
(SEQ ID NO: 3) GGATCCTTTTTAGTATTTTTAATTAATTGTAATCAGCACAGTTCATTATCAACCAAACAAAAAATAAGTGGTTATAATGAATCGTTAATAAGCAAAATTCATATAACCAAATTAAAGAGGGTTATAATGAAAAAAAAGATTATCTCAGCTATTTTAATGTCTACAGTGATACTTTCTGCTGCCAG PK
(SEQ ID NO: 4) GGATCCCTAAAGATCGCGTTTTAGCAAGTAAGATGGGTGCTTACGCTGTTGAGCTACTCCTTGAAGGTAAGGGTGGTTTAGCAGTTGGAATCTTAGAAAATAAGGTTCAAGCTCATAACATGCTTGACTTGTTTGATGCAAAACATCAAGCAGATGATTCACTTTACCAATTAAGTGAAGATTTATCATTCTAGAGTTCTATTAATATTTGGATAAAATGACTTAAGAAGTCTTTTATAATTTAAAATCAAGGGAGAGATTCTGTAATGAAAAAAAAGATTATCTCAGCTATTTTAATGTCTACAGTGATACTTTCTGCTGCAG GK
(SEQ ID NO: 5) GGATCCATAATCTGGTAAATTAGTTGAGATGGTATTATGAAAACACTTTATGATGTGCAACAACTTTTAAAGCAATTCGGAATATTTGTTTACGTTGGAAAACGTAAATGGGATATTGAATTGATGAGTATTGAATTGAAAAATTTGTACAAAGCAGGAGTCGTCGATAAACCGACTTATGTTAAAGCTCAGTTGGTTTTACGACATGAGCATCATATTGAAGAGGTTAGAGATAACCAACAAAAATAATGGAGGGTTTCGAAGTAATGAAAAAAAAGATTATCTCAGCTATTTTAATGTCTACAGTGATACTTTCTGCTGCAG GPFK
(SEQ ID NO: 6) GGATCCCCAGTTATTTTAGTTTATGAGGATACTAATGAACATAATGAGTTGTCTGAAAAATTTTATTTAAATGACAGTTCTGAAGTAAAAGAACAATTAGCAGAATTGCTAGGAAGTCAACATATTTCGTTAATTAAAAAATAAATTTTGAATAAAGCACTTACATTCGATTAATTAAGAAAATGGTACAGACAACTGTTTTCAAAAGTGATAAAATCAACAATGAAGTTTTGAAAAAACTCAATATTTCTGTTTGAGGTGAAAAGATGAAAAAAAAGATTATCTCAGCTATTTTAATGTCTACAGTGATACTTTCTGCTGCAG G6Pi
(SEQ ID NO: 7) GGATCCATGCCGGCTAAAGTGGTGGATAAATTGAATCATCCCCAAGAACTGGAATAAGATAAAATTGTAGTGCTTTCAGGCTTTACCAGCCATCTTTTGAAAAAATTAATTTCTTTCAAAAGTGCGTGTGACAGGTGATCAACTAGATTAAATGGGGAGGGTATCCCAGTAAATATTAGGTTAAATCGGATAGGCTTAACCAAATTAAGTAATTTTATTGTATAATGGTACAGATAAAGAATTTTAAACAAAAGGGGTAGTTATTAATGAAAAAAAAGATTATCTCAGCTATTTTAATGTCTACAGTGATACTTTCTGCTGCAG L-LDH
(SEQ ID NO: 8) GGATCCTCATTTTCATGTTATTTTTCCACCCTCAACACGCAAAAACGGCTGAAAGAGCAAAAACCCCTCAGCTGTCCACGTTTATTTTCATGTAATATTACCATATTATTGACCCCAAGCGGGTCTTTTAACCTCTAACTTATCAATCACTTTACTAACTATACCCGAACTTCATAAAATTTTTACTCAACTTTCTTTTATGAAAATGCTATACTTAGTATTGTTTGATAAATTCAAATATTATATGAAAAAAGGGGATTGATCTTATGAAAAAAAAGATTATCTCAGCTATTTTAATGTCTACAGTGATACTTTCTGCTGCAG

These promoters promote the expression of heterologous nucleic acids encoding biologically active foreign proteins when introduced into microbial cells. Among these promoters, the ermE promoter, PK promoter, GK promoter, GPFK promoter, G6Pi promoter were selected from the glycolytic metabolic pathway of Pediococcus pentosaceus , and the L-LDH promoter was selected from the secondary metabolite lactate production pathway. And are potent promoters that allow high levels of heterologous nucleic acid expression in lactic acid bacteria (eg, Pediococcus pentosaceus ). Such a promoter allows the microorganism of the present invention to function as a drug carrier to produce and secrete a sufficient amount of P8 protein at specific sites in the gastrointestinal tract.

The recombinant plasmid of the present invention can not only increase the expression level of heterologous nucleic acid but also improve secretion. As used herein, the expression “secreted” of a protein means that the protein is delivered extracellularly from a microbial cell, including when the entire molecule of the protein is actually present in the medium in a completely free form, All molecules are present in the cell surface layer, and some of the protein molecules are present in the medium, and the rest of the molecules are present in the cell surface layer.

Recombinant plasmids of the present invention may also include an origin of replication capable of replicating in microbial cells. This is because the manipulation of the plasmid is more efficient in the lactic acid bacteria strain. Examples of preferred origins of replication include ColE1, Ori, oriT and the like.

Recombinant plasmids of the present invention may comprise secretory signal peptide sequences suitable for allowing the therapeutic polypeptide to be secreted or released from a microbial drug delivery system (eg, Pediococcus pentosaceus ). To enable secretion of therapeutic peptides, fragments encoding secretory signal peptides suitable for use in Pediococcus pentosaceus can be added at the 5 'end or 3' end of the heterologous nucleic acid sequence.

The secretory signal peptide may comprise a secretory signal peptide that directs strong protein secretion, such as Usp45 N4 in which the lysine at position 4 of the USP45 secretion signal, wild type Usp45 secretion signal is substituted with asparagine, or Lactobacillus brevis S-layer protein signal peptide. Can be. Such secretion signal peptides can provide the secretion of the foreign protein of interest. Such secretory signal peptide sequences allow further control of the production and secretion of the desired therapeutic polypeptide.

The recombinant plasmid of the present invention may further comprise a second nucleic acid sequence encoding a second promoter, a second secretory signal peptide and a therapeutic polypeptide, in addition to any of the six species described above downstream of said promoter.

Recombinant plasmids of the present invention must have a first promoter upstream of the heterologous nucleic acid to be expressed, and can also include a second promoter downstream of the heterologous nucleic acid to be expressed. These first and second promoters may be the same or different from each other. Non-specific promoters capable of promoting expression of foreign genes in most cells or tissues may also use specific or selective promoters such as tissue or organ specific promoters, tumor specific promoters, developmental or differentiation specific promoters. For example, a specific promoter can be used as the first promoter and a nonspecific promoter can be used as the second promoter. As such, the microorganism transformed by the recombinant plasmid containing the double promoter can express the desired heterologous polypeptide or protein in a very high yield.

The plasmid of the present invention is, for example, a pCBT24-2-PK-COSP8-PK-oriP8 plasmid by cloning the alanine racease gene and one or more promoters of the promoters of SEQ ID NO: 3 to SEQ ID NO: 8 pCBT24-2-PK-COSP8-PK Can produce -oriP8-alr.

Another aspect of the present invention is an anti-cancer drug delivery strain transformed by the recombinant plasmid described above and inactivated alanine racease (alaR) gene, which encodes a polynucleotide encoding a P8 protein and a secreted signal peptide. The present invention relates to a strain that is transformed with a recombinant plasmid containing a gene to allow for antibiotic-based amplification while expressing and secreting P8 protein in the gastrointestinal tract.

The microorganism strain may be a strain belonging to the genus Lactobacillus ( Lactobacillus ), Lactococcus ( Latococcus ), Leuconostoc , or Pediococcus ( Pediococcus ), Bifidobacterium ( Bifidobacterium ). Specific examples of the strains can include strains belonging to the genus Lactobacillus ramno Versus, Lactobacillus eksi FIG filler's, Lactobacillus para casei, Lactobacillus Planta room, Phedi O Lactococcus pen gravel three-house, or Lactobacillus brevis .

Preferably the strain may be a Pediococcus pentosaceus strain. Pediococcus pentosaceus is excellent in acid resistance and resistance to gastrointestinal tract, surviving through the gastrointestinal tract to reach the mesentery membrane in a living form and settle well, can continue to function as a drug carrier.

It is preferable that the strain used as the drug carrier is Pediococcus pentosaceus PPΔalr-P8-alr strain (KCCM12500P) belonging to the genus Pediococcus deposited with accession number KCCM12500P, but it expresses and expresses the P8 protein of the present invention. All secreting anticancer drug delivery strains are included in the scope of the present invention.

By introducing the recombinant plasmid of the present invention, the microorganism of the present invention can express a heterologous nucleic acid encoding a therapeutic peptide of interest by a microbial strain at a predetermined position of the gastrointestinal tract and produce a therapeutic peptide.

Another aspect of the present invention relates to a pharmaceutical composition for preventing or treating gastrointestinal diseases, including the above-mentioned microbial strain, which produces and secretes a P8 protein having an amino acid sequence represented by SEQ ID NO: 2, and specifically, Pediococcus pentosa It relates to a pharmaceutical composition for preventing or treating gastrointestinal diseases, including SUSUS PPΔalr-P8-alr strain (Accession Number: KCCM12500P).

The Pediococcus pentosaceus PPΔalr-P8-alr strain (Accession Number: KCCM12500P) comprises a heterologous nucleic acid encoding a therapeutic peptide having a therapeutic effect against at least one gastrointestinal disease, wherein the heteronucleic acid is heterologous And one or more promoters derived from Pediococcus pentosaceus operably linked to the nucleic acid and a gene encoding a secretory signal peptide.

The heterologous nucleic acid may preferably encode a polypeptide for therapeutic or disease related purposes. Heterologous nucleic acids may also encode antigenic polypeptides for vaccine use. The heterologous nucleic acid in the present invention may be a gene encoding hormones, cytokines, enzymes, coagulation factors, transport proteins, receptors, regulatory proteins, structural proteins, transcription factors, antigens, antibodies and the like. Specific examples include thrombopoietin, growth hormone, growth hormone releasing hormone, growth hormone releasing peptide, interferon, interferon receptor, colony stimulating factor, glucagon-like peptide, G-protein related receptor, interleukin, interleukin receptor, enzyme, Interleukin binding protein, cytokine binding protein, macrophage activator, macrophage peptide, B cell factor, T cell factor, protein A, allergic inhibitor, cell necrosis glycoprotein, immunotoxin, lymphotoxin, tumor necrosis factor, tumor suppressor, Metastatic growth factor, α-1 antitrypsin, albumin, α-lactalbumin, apolipoprotein-E, erythropoietin, high glycated erythropoietin, angiopoietin, hemoglobin, thrombin, thrombin receptor active peptide, thrombo Modulin, blood factor VII, VIIa, VII, VII and XIII, plasminogen activator, fibrin-binding peptide, urokinase, streptoky Agent, hirudin, protein C, C-reactive protein, renin inhibitor, collagenase inhibitor, superoxide dismutase, leptin, platelet derived growth factor, epidermal growth factor, epidermal cell growth factor, angiostatin, angiotensin, bone formation Growth factor, bone stimulating protein, calcitonin, insulin, atriopeptin, cartilage inducer, elkatonin, connective tissue activator, tissue factor pathway inhibitor, follicle stimulating hormone, luteinizing hormone, luteinizing hormone releasing hormone, nerve Growth factor, parathyroid hormone, relaxin, secretin, somatomedin, insulin-like growth factor, corticosteroid, glucagon, cholecystokinin, pancreatic polypeptide, gastrin releasing peptide, corticotropin releasing factor, thyroid stimulating hormone, autotaxin, lactoferrin , Myostatin, receptor, receptor antagonist, cell surface antigen, virus derived antigen, monoclonal antibody, polyclonal Genes encoding antibodies and antibody fragments, and the like, but are not limited thereto.

Gastrointestinal disorders suitable for treatment as the pharmaceutical composition of the present invention include diseases or disorders affecting the oral cavity, esophagus, stomach, small intestine, large intestine, rectum, and organs such as pancreas, liver and gallbladder. Examples include dry mouth, mouth ulcers, gingival disease, esophageal reflux disease, gastric ulcer, inflammatory bowel disease, inflammatory colitis, Crohn's disease, mycositis, gastric cancer, colorectal cancer. In particular, the strain of the present invention can be advantageously used for the treatment of colon cancer, colon polyps, colitis, ischemic gastrointestinal disease, dysentery, intestinal vascular dysplasia, diverticulosis, irritable bowel syndrome, or Crohn's disease.

Another aspect of the invention is transformed with a recombinant plasmid comprising a strain of the invention, ie a polynucleotide encoding a P8 protein operably linked to an exogenous promoter, an alanine racease gene and a gene encoding a secretory signal peptide. The present invention relates to a pharmaceutical composition for preventing or treating gastrointestinal diseases, including an anticancer drug delivery microorganism that expresses and secretes P8 protein in the gastrointestinal tract.

The secretory signal peptide may be a secretory signal peptide such as USP45 secretory signal peptide, Usp45 N4 or Lactobacillus brevis S-layer protein signal peptide.

The strain further comprises a heterologous nucleic acid sequence encoding a second promoter, a second secretory peptide and a second therapeutic peptide downstream of the promoter, wherein the second promoter may be the same as or different from each other with the first promoter. . The heterologous nucleic acid sequence encoding the first therapeutic peptide and the heterologous nucleic acid sequence encoding the second therapeutic peptide may be the same or different. In another example, the base sequence of the heterologous nucleic acid encoding the first therapeutic peptide and the base sequence of the heterologous nucleic acid encoding the second therapeutic peptide may differ from each other while encoding the same therapeutic peptide having the same amino acid sequence.

The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier or pharmaceutically acceptable salt. On the other hand, the pharmaceutical composition is 5-fluorouracil (5-FU, fluorouracil), UFT (tegafur-uracil), capecitabine (capecitabine), irinotecan (irinotecan), oxaliplatin (oxaliplatin), bevacizumab (bevacizumab, And chemotherapy treatments such as tradename Avastin) and cetuximab (erbitux).

The route of administration of the composition of the present invention is preferably to use the most effective at the time of treatment, and may be administered through oral administration or parenteral administration such as oral, intra-respiratory, rectal, subcutaneous, intramuscular and intravenous. .

Gastric tolerant oral formulations may be formulated for oral administration, which formulations can include controlled release of host cells and provide controlled release of the desired therapeutic peptide encoded therein. For example, oral formulations (including tablets, pellets, granules, and powders) are resistant to dissolution or destruction in the stomach, but not to the intestines, thereby enabling the transition through the stomach to favor disintegration, dissolution and absorption in the intestines. It may be coated with a thin layer of an excipient (typically a polymer, a cellulose derivative and / or a lipophilic substance).

Oral formulations may be designed to enable sustained release of the host cell and its recombinant protein, for example, as controlled release, sustained release, prolonged release, sustained action tablets or capsules. These formulations generally contain conventionally known excipients such as lipophilic, polymeric, cellulosic, insoluble, swellable excipients. Controlled release formulations can also be used for any other delivery site, including intestinal, colonic, bioadhesive or sublingual delivery (ie dental mucosal delivery) and bronchial delivery.

Suitable formulations for oral administration include emulsions, syrups, capsules, tablets, powders, granules and the like. Liquid preparations such as emulsions and syrups include sugars such as water, sucrose, sorbitol and fructose, glycols such as polyethylene glycol and propylene glycol, oils such as sesame oil, olive oil and soybean oil, preservatives such as p-hydroxybenzoic acid esters, Flavors, such as strawberry flavor and peppermint, can be manufactured as an additive. Capsules, tablets, powders, granules, etc. include excipients such as lactose, glucose, sucrose and mannitol, disintegrants such as starch and sodium alginate, glidants such as magnesium stearate and talc, binders such as polyvinyl alcohol, hydroxypropyl cellulose and gelatin. And plasticizers such as glycerin and surfactants such as fatty acid esters can be produced as additives.

Suitable formulations for parenteral administration include injections, site preparations, and sprays. Injectables are prepared using a salt solution, a glucose solution, or a carrier composed of a mixture of both. Alternatively, the powder injection may be prepared by lyophilizing the strain according to the conventional method and adding sodium chloride thereto. The site agent is prepared using a carrier such as cacao butter, hydrogenated fat or carboxylic acid. In addition, the nebulizer is prepared using a compound or the like or a carrier which does not irritate the oral cavity and airway mucosa of the recipient, and also disperses the compound as fine particles to facilitate absorption.

The amount of the strain producing the therapeutic peptide, which is an active ingredient of the pharmaceutical composition according to the present invention, may vary depending on the age, sex, weight, and disease of the patient, but is preferably 0.001 to 100 mg / kg, preferably 0.01 to 10 mg. / kg may be administered once to several times daily.

In addition, the dosage of the strain according to the present invention can be increased or decreased depending on the route of administration, the degree of disease, sex, weight, age and the like. Therefore, the above dosage does not limit the scope of the present invention in any aspect.

Another aspect of the present invention provides a recombinant plasmid comprising a gene fragment encoding a P8 protein of SEQ ID NO: 1 showing gastrointestinal therapeutic activity and an alanine racease gene of SEQ ID NO: 11 encoding an essential protein required for bacterial viability. Step: Preparing a ureotropic strain strain in which the alanine racemate gene of the recombinant plasmid is inactivated: Anti-cancer drug delivery comprising the step of transforming the trophic strain with the recombinant plasmid to produce an anti-cancer drug delivery recombinant strain It relates to a method for producing a drug delivery strain.

The recombinant plasmid is derived from Pediococcus pentosaceus , and is a plasmid having a cleavage map (SEQ ID NO. 9) of FIG. 1 or 2, and the anticancer drug delivery recombinant strain is Pediococcus pentosaceus PP. △ a lr-P8-alr strain (accession number: KCCM12500P) may be.

Inactivated essential protein coding genes of the strain include alanine racemase (alaR), thymidylate synthase (thyA), asparagine synthase (asnH), CTP synthase (pyrG), tryptophan synthase (trpBA), and It may be selected from the group consisting of a combination of these.

When explaining the production method of the strain of the present invention in more detail as follows. Phedi five focus three pen mouse soil SL4 (-7) (accession number: KCTC 10297BP) attached approximately 1kb DNA from a racemic alanine horseshoe gene 5 'ends, 3' ends present in the strain on both ends of the ampicillin resistance gene, and this pCBT24 Cloning to -2 plasmid to prepare pCBT24-2-alrHr1,2. This was transformed into PedioFocus pentosaceus SL4 (-7), followed by passage for about one month.As a result of homologous recombination, amp ^R was inserted into the alanine racease gene position and lacked the alanine gene. Phedi focus O, to produce a pen soil three mouse strains DDS Δalr PP. The alanine gene deficiency Pedy five focus pen Tosa three mouse strains DDS Δalr PP can live only in a medium containing D- alanine.

Transformed with a plasmid (pCBT24-2-PK-COSP8-PK -oriP8-alr plasmid) an alanine gene lacking focus Pedy o pen soil three mouse DDS Δalr PP strain of the present invention to Phedi O Lactococcus pen soil three mouse PPΔalr- A P8-arl strain (Accession No. KCCM12500P) was prepared. Three alanine gene is the focus Phedi o pen gravel-deficient mouse DDS Δalr PP strains could grow on the other hand only the culture medium by the addition of D- alanine, O Phedi focus pen soil three mouse PPΔalr-P8-alr strain is a racemic alanine horseshoe pCBT24 Because it is produced in the 2-PK-COSP8-PK-oriP8-alr plasmid, it can also be grown in medium without D-alanine. Conversely, if the pCBT24-2-PK-COSP8-PK-oriP8-alr plasmid is lost or absent, it will fail to produce D-alanine because it cannot produce alanine racemase. Using this feature, only the P. pheophos pentosaceus PPΔalr-P8-alr strain , which must include the pCBT24-2-PK-COSP8-PK-oriP8-alr plasmid, lives in culture, and the non-cells induce death. Antibiotic Free System (AFS) system can be built. Thus , Pediofocus Pentosaceus PPΔalr-P8-alr strains contain at least 99% of the pCBT24-2-PK-COSP8-PK-oriP8-alr plasmids, and more than 99% of the cells can produce P8 in culture. have.

The method of the present invention may further comprise the step of culturing the transformed recombinant strain in a medium in which the essential protein necessary for the viability of the bacteria encoded by the inactivated gene is not added.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example

Example 1 Preparation of Transgenic Strains

1.1 Preparation of Plasmid

The pCBT24-2-PK-COSP8-PK-oriP8-alr plasmid (SEQ ID NO: 9) was cloned by cloning the alanine racease gene (SEQ ID NO: 11) and the PK promoter of SEQ ID NO: 4 to pCBT24-2-PK-COSP8-PK-oriP8. Was produced.

1.2 Preparation of Alanine Racemase Gene (alr) Deficiency Strains

Ampicillin resistance gene comprising the DNA sequence (Hr1, Hr2, about 1 kb) at both ends of the alr to remove the alanine racemase gene (alr) from the Pediococcus pentosaceus CBT SL4 strain (Accession Number: KCTC 10297BP) A DNA fragment (alrHr1-amp ^R -alrHr2) comprising (amp ^R ) was prepared and cloned into a pCBT24-2 vector. pCBT24-2-alrHr1,2 was transformed into the Pediococcus pentosaceus CBT SL4 strain, and the transformants growing in the medium containing erythromycin were selected first. Subsequently, the transformants which grew only in the medium containing no D-alanine were finally selected while continuing passage (see FIGS. 5 and 6). The Pediococcus pentosaceus CBT SL4 (-7) strain, which lacked the selected alanine racemic gene (alr), was named DDS Δalr PP.

1.3. Preparation of Pediococcus pentosaceus PPΔalr-P8-alr Strain

Pediococcus pentosaceus CBT SL4 (-7) strain (DDS Δalr PP), lacking the alanine racease gene (alr), was transformed with pCBT24-2-PK-COSP8-PK-oriP8-alr plasmid 2-PK-COSP8-PK-oriP8-alr / DDSΔalr PP was produced, which was named Pediococcus pentosaceus PPΔalr-P8-alr strain. The expression and secretion of P8 protein in Pediococcus pentosaceus PPΔalr-P8-alr strains were confirmed by Western blot.

A strain transformed by the pCBT24-2-PK-COSP8-PK-oriP8-alr plasmid to express and secrete P8 protein in the gastrointestinal tract was named Pediococcus pentosaceus PPΔalr-P8-alr strain, Seoul, Korea The deposit was made on April 23, 2019 with the deposit number KCCM12500P at the Korea Microorganism Conservation Center (KCCM), Yurim Building, 45, Hongjene-2ga-gil, Seodaemun-gu, Seoul.

Example 2: Anticancer Effect Test of New Strains

2.1 Tumor Proliferation Animal Model Construction

Colorectal cancer cell line DLD-1 received from Korea Cell Line Bank (Seoul, Korea), 10% fetal bovine serum (FBS; Invitrogen, NY, USA), penicillin (0.02 UI / ml; Sigma, MO, USA), streptomycin ( RPMI 1640 medium (Sigma, MO, USA) with 0.02 μg / ml; Sigma, MO, USA), glutamine (2 mM: Sigma, MO, USA), non-essential amino acids (1%; Sigma, MO, USA) ) Was maintained by passage in 37 ° C., 5% CO ₂ incubator. Tumor cell suspensions obtained by floating cultured DLD-1 cells were 0.2 ml (2 × 10 ⁶ ) in the dorsal subcutaneous of male athymic nude mice (BALB / cAnN.Cg-Foxn1nu / CrlNarl; 5 weeks old). cell / mouse) to form a solid tumor to produce a first generation cell derived xenograft model (CDX). Tumor lumps were removed at the expense of the tumor tissue formed in the subfamily (1st generation CDX), the blood vessels and fat layers distributed on the surface were removed, and only fresh tumor tissues were selected to remove subcutaneous animals using trocars. Tissue derived xenograft (TDX) (see FIG. 8).

2.2 Experimental group separation and substance administration

After tumor mass transplantation, when individual tumor volume reached 150-200 mm 3, body weight and tumor size were measured and grouped randomly. 7 animals per group, negative control (Mock; tumor transplantation control), DDSPP (empty vector) 1x10 ¹⁰ cfu / Head, positive control (anti-cancer drug administration 90 mg / Kg; Gemcitabine Hydrochloride, Tokyo Chemical Industry), as shown in Table 2 TCI Co. LTD, JAPAN), original broth DDS PP Δalr ( Pediococcus pentosaceus PPΔalr-P8-alr strain) {(PK- ^ori P8-PK- ^cos P8) + alr (+)-Em (+) } Ver.II 1x10 ⁹ cfu / Head group, DDS PP Δalr {(PK- ^ori P8-PK- ^cos P8) + alr (+)-Em (+)} Ver.II 1x10 ¹⁰ cfu / Head group, lyophilization DDS PP Δalr {(PK- ^ori P8-PK- ^cos P8) + alr (+)-Em (+)} Ver.II 1 × ^{10 9} cfu / Head group, DDS PP Δalr {(PK- ^ori P8-PK- ^cos P8) + alr (+)-Em (+)} Ver.II 1 × ^{10 10} cfu / Head administration group was administered daily for 5 weeks.

group process Dosage Number of objects G1 CDX + PBS Treatment 7 G2 AOM / DSS + anticancer agent (5-Fu, gemcitabine) 90 mg / kg 7 G3 AOM / DSS + DDS PP (Empty vector) 1x10 ¹⁰ CFU / Head 7 G4 AOM / DSS + OB, DDS _△ alr (PK-P8-PK-P8) + Em (+)-ver.II 1x10 ⁹ CFU / Head 7 G5 AOM / DSS + OB, DDS _△ alr (PK-P8-PK-P8) + Em (+)-ver.II 1x10 ¹⁰ CFU / Head 7 G6 AOM / DSS + Powder, DDS PP _△ alr (PK-P8-PK-P8) + Em (+)-ver.II 1x10 ⁹ CFU / Head 7 G7 AOM / DSS + Powder, DDS PP _△ alr (PK-P8-PK-P8) + Em (+)-ver.II 1x10 ¹⁰ CFU / Head 7 Total target population 49

2.3 Measurement results of tumor tissue size change

The test was terminated after six weeks of administration of the test substance. It is confirmed that there is a difference in the degree of tumor proliferation of each group by the administration of the test substance, and the results of measuring the change in tumor tissue size of each group by the administration of the test substance before the end of the test using vernier calipers are shown in FIG. 9. It was.

Measurements negative control (Mock: 3946 ㎣), DDSPP (empty vector) 1x10 10 cfu / Head (3926 ㎣), positive control (an anticancer agent: gemcitabine, 712.3 ㎣), original broth DDS PP Δalr ( Phedi O Lactococcus pen soil three mouse PPΔalr-P8-alr strain) {(PK- ^ori P8-PK- ^cos P8) + alr (+)-Em (+)} Ver.II 1 × ^{10 9} cfu / Head administration group (2123 ㎣), DDS PP Δalr {(PK ^{- ori P8-PK- cos P8)} + alr (+) - Em (+)} Ver.II 1x10 10 cfu / Head group (1975 ㎣), lyophilization DDS PP Δalr {(PK- ori P8-PK- cos P8) + alr (+)-Em (+)} Ver.II 1 × ^{10 9} cfu / Head group (3035 μs), DDS PP Δalr {(PK- ^ori P8-PK- ^cos P8) + alr (+)-Em (+) } Ver.II 1 × ^{10 10} cfu / Head administration group (2596 mm 3). As shown in FIG. 5A, tumor size was significantly reduced in the benzohydroxymethoxychalcone compound (DK49) administration group than the control group (NT) from day 27. Was observed.

9, confirmed that Mock than Phedi O Lactococcus pen soil three mouse PP △ alr-P8-alr the size of the tumors was reduced upon strain administration, in particular is the tumor volume significantly reduced when the same dose of the culture medium It was.

2.4 Confirmation of Proliferation Inhibitory Effects of Tumor Tissues after Administration

After completion of the test, the tumor formed in the subject was measured for the width and length of the tumor using calipers, and the average volume of the tumor was calculated by substituting Equation 1 below (see FIGS. 7B, 7C, and 8).

[Equation 1]

Tumor volume (mm ³ ) = (tumor width ² x tumor length) / 2

Using the tumor growth inhibition rate (TGI%) defined as the average tumor volume percentage (T / Cx100) of the test and control groups based on the average tumor volume value of the control group (Mock) in the average tumor volume value of each group obtained The therapeutic efficacy was evaluated and the result is shown in FIG.

9, DDS PP (empty vector) 1x10 ¹⁰ cfu / Head (-0.4%), positive control (anticancer drug: gemcitabine, 84.2%), original broth DDS PP Δalr {(PK- ^ori P8-PK- ^cos P8 ) + alr (+)-Em (+)} Ver.II 1x10 ⁹ cfu / Head group (46.66%), DDS PP Δalr {(PK- ^ori P8-PK- ^cos P8) + alr (+)-Em (+ )} Ver.II 1x10 ¹⁰ cfu / Head group (51.2%), lyophilization DDS PP Δalr {(PK- ^ori P8-PK- ^cos P8) + alr (+)-Em (+)} Ver.II 1x10 ⁹ cfu / Head (22.95%), DDS PP Δalr {(PK- ^ori P8-PK- ^cos P8) + alr (+)-Em (+)} Ver.II 1 × ^{10 10} cfu / Head group (34.58%) .

After completion of the test, in order to confirm the effect of inhibiting tumor growth by administration of the test substance, the tumors of each individual were sacrificed and the tumors were extracted. The weight of the extracted tumor tissue was measured using an electronic balance. As a result of tumor weight measurement of each test group and control group, as shown in FIG. 10, a negative control group (Mock: 3.275 g), a DDS PP (empty vector) 1 × 10 ¹⁰ cfu / Head (3.607g), and a positive control group (an anticancer agent: gemcitabine, 0.5647 g), original broth DDS PP ΔAlr {(PK- ^ori P8-PK- ^cos P8) + alr (+)-Em (+)} Ver.II 1 × 10 ⁹ cfu / Head administration group (1.705 g) , DDS PP ΔAlr {(PK- ^ori P8-PK- ^cos P8) + alr (+)-Em (+)} Ver.II 1 × 10 ¹⁰ cfu / Head administration group (1.492 g), lyophilization DDS PP ΔAlr { (PK- ^ori P8-PK- ^cos P8) + alr (+)-Em (+)} Ver.II 1 × 10 ⁹ cfu / Head administration group (3.13 g), DDS PP ΔAlr {(PK- ^ori P8-PK ^cos P8) + alr (+)-Em (+)} Ver.II 1 × 10 ¹⁰ cfu / Head administration group (2.419 g). Tumor growth inhibition rate was calculated by substituting the weight value of the tumor obtained above into Equation 2.

[Formula 2]

Tumor growth inhibition rate, IR (%) = (1- T / C) × 100

T: Average tumor weight of test substance or positive control

C: Average tumor weight of negative controls

As a result, DDS PP (empty vector) 1x10 ¹⁰ cfu / Head (-10.1%), positive control (anticancer drug: gemcitabine, 82.7%), original broth DDS PP Δalr {(PK- ^ori P8-PK- ^cos P8) + alr (+)-Em (+)} Ver.II 1 × ^{10 9} cfu / Head group (47.9%), DDS PP Δalr {(PK- ^ori P8-PK- ^cos P8) + alr (+)-Em (+)} Ver .II 1x10 ¹⁰ cfu / Head group (54.4%), lyophilization DDS PP Δalr {(PK- ^ori P8-PK- ^cos P8) + alr (+)-Em (+)} Ver.II 1x10 ⁹ cfu / Head group ( 4.4%), DDS PP Δalr {(PK- ^ori P8-PK- ^cos P8) + alr (+)-Em (+)} Ver.II 1 × ^{10 10} cfu / Head administration group (26.1%) (see FIG. 11). ).

Culturing Phedi five focus pen soil three mouse PPΔalr-P8-alr of the present invention and live cells (original broth) and lyophilized to produce a powder concentrate (lyophilization) colon cancer cells (DLD-1) is administered to the transplant Xenograft mouse antitumor efficacy of the resulting live cells (1x10 ⁹⁾ were the 56% level compared to conventional chemotherapy (gemcitabine), anti-cancer efficacy of a powder concentrate (1x10 ⁹⁾ was 28%. When the number of cells was increased 10-fold, the anticancer efficacy of viable cells (1x10 ¹⁰ ) was 60% compared to the existing anticancer drug (gemcitabine), and the anticancer efficacy of the original (1x10 ¹⁰ ) was 41%.

Example 3. Confirmation of Mouse Intestinal Survival of Recombinant Strains

In order to determine whether a drug delivery system for secretion / expression of the P8 protein administered to mice Pedy five caucuses pen Tosa three funny PP △ alr-P8-alr strain (KCCM12500P) can reach live to the intestines of mice, the mice Pediococcus pentosaceus PPΔalr-P8-alr strain was administered daily for 1 week, and after 1 week, the small intestine material of the rat was recovered, which was obtained by MRS (erythromycin (10 μg / ml) plate or MRS (Erithro). Mycin or kanamycin, 10 μg / ml) to determine how much each strain is present.

After a week-long experiment, the small intestine material of the mice was recovered, washed twice using PBD buffer and centrifuge, and the remaining supernatant was concentrated to 20% TCA on an agar plate containing erythromycin or kanamycin. It smears and the result is shown in FIG.

In FIG. 12, (A) is a photograph showing the result of smearing the Pediococcus pentosaceus strain ( EV / PP (-7)) on the (MRS + Em + Kan) medium as a negative control, and (B) is a P8 protein coding. Pediococcus pentosaceus strain ( PP8 / PP (-7)) transfected with a gene was photographed on (MRS + Em + Kan) medium, and (C) is a pedi recovered from the small intestine of an experimental animal. A photograph of the result of smearing on Okoccus pentosaceus PPΔalr-P8-alr strain (MRS + Em + Kan) medium, and (D) shows PP8-alr-Em (-), in which the erythromycin resistance gene was deleted. It is a photograph showing the result of smearing / Δalr PP strain on MRS (Kanamycin) MRS plate.

Referring to FIG. 12, it was confirmed that the Pediococcus pentosaceus PPΔalr-P8-alr strain was well fixed in the large intestine of mice.

The use of lactic acid bacteria as a therapeutic agent for colorectal diseases, including colorectal cancer, colitis, irritable bowel syndrome, Crohn's disease, etc., is a well-known technique. There has been little development of technology for use. The present invention has introduced a lactic acid bacteria-derived protein into the lactic acid bacteria expression / secretion system to develop a lactobacillus drug delivery system that overexpresses and secretes it and applied it to an animal model to demonstrate its effectiveness. Therefore, the present invention is expected to be widely used in the medical field as a natural protein bowel disease treatment.

Although the preferred embodiment of the present invention has been described in detail above, the present invention is not limited to the above-described embodiment. Those skilled in the art will appreciate that various modifications and changes can be made based on the above-described embodiments. Therefore, the true protection scope of the present invention will be defined in the claims and the equivalent range to be described later.

Korea Microbial Conservation Center (overseas) KCCM12500P 20180423

<110> CELLBIOTECH CO., LTD. <120> MICROORGANISMS FOR DELIVERING ANTI-CANCER AGENT HAVING AUXOTROPHY          MARKER AND PHARMACEUTICAL COMPOSITION FOR PREVENTING OR TREATING          CANCER COMPRISING THE SAME <130> P19-CBT-01 <160> 11 <170> KoPatentIn 3.0 <210> 1 <211> 219 <212> DNA <213> Lactobacillus rhamnosus <400> 1 gcaacagtag atcctgaaaa gacattgttt ctcgatgaac caatgaacaa ggtatttgac 60 tggagcaaca gcgaagcacc tgtacgtgat gcgctgtggg attattacat ggaaaagaac 120 agccgtgata ccatcaagac tgaagaagaa atgaaaccag tcctagacat gtccgacgat 180 gaggtcaaag ccctagcaga aaaggttctc aagaagtaa 219 <210> 2 <211> 219 <212> PRT <213> Lactobacillus rhamnosus <400> 2 Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala   1 5 10 15 Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala              20 25 30 Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala          35 40 45 Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala      50 55 60 Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala  65 70 75 80 Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala                  85 90 95 Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala             100 105 110 Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala         115 120 125 Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala     130 135 140 Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala 145 150 155 160 Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala                 165 170 175 Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala             180 185 190 Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala         195 200 205 Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala     210 215 <210> 3 <211> 184 <212> DNA <213> Artificial Sequence <220> <223> Promotor ermE <400> 3 ggatcctttt tagtattttt aattaattgt aatcagcaca gttcattatc aaccaaacaa 60 aaaataagtg gttataatga atcgttaata agcaaaattc atataaccaa attaaagagg 120 gttataatga aaaaaaagat tatctcagct attttaatgt ctacagtgat actttctgct 180 gcag 184 <210> 4 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> Promotor PK <400> 4 ggatccctaa agatcgcgtt ttagcaagta agatgggtgc ttacgctgtt gagctactcc 60 ttgaaggtaa gggtggttta gcagttggaa tcttagaaaa taaggttcaa gctcataaca 120 tgcttgactt gtttgatgca aaacatcaag cagatgattc actttaccaa ttaagtgaag 180 atttatcatt ctagagttct attaatattt ggataaaatg acttaagaag tcttttataa 240 tttaaaatca agggagagat tctgtaatga aaaaaaagat tatctcagct attttaatgt 300 ctacagtgat actttctgct gcag 324 <210> 5 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> Promotor GK <400> 5 ggatccataa tctggtaaat tagttgagat ggtattatga aaacacttta tgatgtgcaa 60 caacttttaa agcaattcgg aatatttgtt tacgttggaa aacgtaaatg ggatattgaa 120 ttgatgagta ttgaattgaa aaatttgtac aaagcaggag tcgtcgataa accgacttat 180 gttaaagctc agttggtttt acgacatgag catcatattg aagaggttag agataaccaa 240 caaaaataat ggagggtttc gaagtaatga aaaaaaagat tatctcagct attttaatgt 300 ctacagtgat actttctgct gcag 324 <210> 6 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> Promotor GPFK <400> 6 ggatccccag ttattttagt ttatgaggat actaatgaac ataatgagtt gtctgaaaaa 60 ttttatttaa atgacagttc tgaagtaaaa gaacaattag cagaattgct aggaagtcaa 120 catatttcgt taattaaaaa ataaattttg aataaagcac ttacattcga ttaattaaga 180 aaatggtaca gacaactgtt ttcaaaagtg ataaaatcaa caatgaagtt ttgaaaaaac 240 tcaatatttc tgtttgaggt gaaaagatga aaaaaaagat tatctcagct attttaatgt 300 ctacagtgat actttctgct gcag 324 <210> 7 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> Promotor G6Pi <400> 7 ggatccatgc cggctaaagt ggtggataaa ttgaatcatc cccaagaact ggaataagat 60 aaaattgtag tgctttcagg ctttaccagc catcttttga aaaaattaat ttctttcaaa 120 agtgcgtgtg acaggtgatc aactagatta aatggggagg gtatcccagt aaatattagg 180 ttaaatcgga taggcttaac caaattaagt aattttattg tataatggta cagataaaga 240 attttaaaca aaaggggtag ttattaatga aaaaaaagat tatctcagct attttaatgt 300 ctacagtgat actttctgct gcag 324 <210> 8 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> L-LDH <400> 8 ggatcctcat tttcatgtta tttttccacc ctcaacacgc aaaaacggct gaaagagcaa 60 aaacccctca gctgtccacg tttattttca tgtaatatta ccatattatt gaccccaagc 120 gggtctttta acctctaact tatcaatcac tttactaact atacccgaac ttcataaaat 180 ttttactcaa ctttctttta tgaaaatgct atacttagta ttgtttgata aattcaaata 240 ttatatgaaa aaaggggatt gatcttatga aaaaaaagat tatctcagct attttaatgt 300 ctacagtgat actttctgct gcag 324 <210> 9 <211> 6516 <212> DNA <213> Pediococcus pentosaceus <400> 9 aagttaatgt aagccttaag gtttcaacta aagcaattac ggtcaaccat aaccatagta 60 ttggattgtc attttattgg ctataaaata gtaaatcagt gaatttcatt ggatccgcta 120 aagatcgcgt tttagcaagt aagatgggtg cttacgctgt tgagctactc cttgaaggta 180 agggtggttt agcagttgga atcttagaaa ataaggttca agctcataac atgcttgact 240 tgtttgatgc aaaacatcaa gcagatgatt cactttacca attaagtgaa gatttatcat 300 tctagagttc tattaatatt tggataaaat gacttaagaa gtcttttata atttaaaatc 360 aagggagaga ttctgtaatg aaaaaaaaga ttatctcagc tattttaatg tctacagtga 420 tactttctgc tgcagccccg ttgtcaggtg tttacgctga agtaattatt atggctaccg 480 ttgatccaga aaagactcta ttccttgatg aaccaatgaa taaagttttt gattggtcta 540 acagtgaggc tccggtgcga gatgccttat gggattacta tatggaaaag aatagccgtg 600 acacgatcaa aacagaagaa gaaatgaaac ctgtattaga tatgtcagat gacgaagtta 660 aagcattagc ggagaaagtc ttgaaaaagt aacctgacaa gaaccagtct gctattgata 720 gactattttt gtccgtgaaa tcctcgcgta tttccgtgag gagcatagta tatttagcga 780 tcttcaaatt ttaagtatat tgattcatat ggaatttgat gagtttgaag ctaagaaagg 840 tcaacgcgcg tatgaatttt tagcagggca cttttcggtt aaagaatcat atagtaaagc 900 ttacgggaca ggattaggta aaaaacttaa ttttcaagac atcgaaatta attacgatgg 960 ggttggaaaa cctaagattg ttaaacatcc gtttagtgga accgtacatg tgtcaatttc 1020 ccattctgac caccatgttg taacacaggt aattttagag ggggataatt gaaatggttg 1080 tagggataca tcgtcccagc aagctagtta ttaatgctga ggcaattaga aataatgtaa 1140 aaaatgagat tagtcgatta gatggacaca gtgaattatt tgccgtggtt aaggctaatg 1200 gctatggaca tggaattgta gaaacagccc aatttacgaa acaagctggg gcaactggtt 1260 tttgtgttgc tatcttagac gaagcgttgg ctttgcgaaa tgctgggcta acagaaacta 1320 tcctagtttt aggagttaca gatgttaaat atgctaaatt agctgctgaa aatgatatct 1380 cgttaacggt gggtgatcaa gcatggctag atgaagctac gcaaattcta gatcaaaagc 1440 cgctgaaaat tcatttaggt attgatacag gaatgggacg gattggtttt caagatggag 1500 cttcttttaa gaaagcggcc gattatcttg aacaaagtca acagttcaat tttgaaggtg 1560 tgtttacaca ctttgcaaca gcagatgaaa aggatacgac ttatttcaat ttacaggtgg 1620 aacgttttaa ccatttcatc agccaattaa ctcgacgtcc acgttatgtg catgtttcta 1680 atactgctac cagcttgtgg catgcagctt gtaatggtaa tatgattcgt tttggagtcg 1740 gcatttatgg gatgaatcct tctggaaaaa cactggaaag tccttttgat ttacagccag 1800 ctatgagtct ggagtctgaa ctatcttttt ctaaactagt gcaaaaaggc cgttcaatta 1860 gttatggagc gacttataca gctaaagaag atgaatggat tgggacgatc ccgattggtt 1920 atgcagatgg ttatgaacgt cgtttacaag gattccatgt tttgattgat ggtcaatttt 1980 gcgaaatagt aggacgaatt tgtatggatc aaatgatggt tcgcttacca aagtcatatc 2040 ctgtgggaac taaagtaatt ttaacgggaa aatcaggcga aaaatcaatt accatgacag 2100 acattgccga atatgctggg accattaatt atgaaatcac atgtggattc acacaacgtt 2160 taccacgaat ctatactgaa aatggtgtag taaattaaga gctccgccta tttttatagg 2220 ttaatgtcat gataataatg gtttcttagc gattcacaaa aaataggcac acgaaaaaca 2280 agttaaggga tgcagtttat gcatccctta acttacttat taaataattt atagctattg 2340 aaaagagata agaattgttc aaagctaata ttgtttaaat cgtcaattcc tgcatgtttt 2400 aaggaattgt taaattgatt ttttgtaaat attttcttgt attctttgtt aacccatttc 2460 ataacgaaat aattatactt ttgtttatct ttgtgtgata ttcttgattt ttttctactt 2520 aatctgataa gtgagctatt cactttaggt ttaggatgaa aatattctct tggaaccata 2580 cttaatatag aaatatcaac ttctgccatt aaaagtaatg ccaatgagcg ttttgtattt 2640 aataatcttt tagcaaaccc gtattccacg attaaataaa tctcattagc tatactatca 2700 aaaacaattt tgcgtattat atccgtactt atgttataag gtatattacc atatatttta 2760 taggattggt ttttaggaaa tttaaactgc aatatatcct tgtttaaaac ttggaaatta 2820 tcgtgatcaa caagtttatt ttctgtagtt ttgcataatt tatggtctat ttcaatggca 2880 gttacgaaat tacacctctt tactaattca agggtaaaat ggccttttcc tgagccgatt 2940 tcaaagatat tatcatgttc atttaatctt atatttgtca ttattttatc tatattatgt 3000 tttgaagtaa taaagttttg actgtgtttt atatttttct cgttcattat aaccctcttt 3060 aatttggtta tatgaatttt gcttattaac gattcattat aaccacttat tttttgtttg 3120 gttgataatg aactgtgctg attacaatta attaaaaata ctaaaaatgc ccatattttt 3180 tcctccttat aaaattagta taattatagc acgaaaagga tctaggtgaa gatccttttt 3240 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc 3300 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg 3360 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact 3420 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg 3480 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg 3540 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac 3600 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca 3660 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga 3720 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc 3780 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct 3840 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg 3900 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct 3960 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc 4020 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc 4080 gaggaaatcg atactgaatt ggcgaaagcc aaagtttcta taaaaccttg ctttcctgct 4140 taacggcgag tgaaaaagcg gttaagctgg ctcagcttgg acggggttcg gggcgttagc 4200 gtccgtatta aatgtggctt accataacca acgaacagag tgaggtgcaa ggagctgtgc 4260 gactggagtt taatgtgagc cggtttttgg ctcactcctt tgtgtttttt gtttctagat 4320 tttaatctcg tacagcggtg cctcttttat acctctttta taaacctctt ttaaacctct 4380 tttagacccc tcttgagcct tactctccca aggctcacag aaggttatca agtacctttt 4440 gtctgtttat caagtacctt ttgtctgttt atcaagtacc ttttgtctgt ttatcaagta 4500 ccttttgtct gtttatcaag tacctttata agttctgtac ttgataaaaa ggtactttta 4560 ttttaatatg tgtttgaggt gataatcatg gctaatgagt tagttaagta tgatcctgag 4620 ttgaatacta ttcccttgag aaaatttacc ccaattgaga tgaatttatt tttttcaatt 4680 atttcccgca tgcgggatca agggaataaa actgttcgtt tctcttttga ccagttaaaa 4740 gagcttagta actataaacc aaccgcaaat aaacgtttta ttgatgatat tgaaaataca 4800 taccaaaaga tcctcagcct taggtttggc cgtagaagta agagtggctt aaatcgtgaa 4860 ttttttgtta tgtttactga atttgaaatt aaaggtgaag ctgaagaacc ttatgttgat 4920 attcagattt atcccaaagc attgcacttg ctaaacgatt tagaaagttg ggttcgttat 4980 gccctaacag aatttagaaa tttaaaaagc agttacgcta aaacaatgtt tcgtctaatt 5040 aagcaatttc gaactactgg ctattcttat ttctctaaag aagatttttt tgaattgctt 5100 gatataccta aaagttattg gaatagtcct tcaaaggttg acaaaaaggt tattaagcca 5160 attagagaag aattaacccc gctttttaga gggctaacga ttagaaaaaa atatggtaaa 5220 ggcagaggaa aaccagttat cggttattct tttacttgga aacctgaaag caaggacgca 5280 aatgattttt ctcaaggcaa atttcaagat gagcgtcaaa aactctttaa cattcagcac 5340 aatgatgaat tatcagataa agaaaagtgg cgtgcaattg acaaagttaa atgcttgcct 5400 ttaggaacaa ctgaaaaaca ggtactggct gaaaaacaag ctgaacatga tcaaaaaatc 5460 agagatcaag caagacaaga atttctcgct gatctccgaa aggggtttta aaatcatgtc 5520 taaaactatt agagaacttg ctgatgaatt gaatgtctcc aaacagacta ttcaatatca 5580 ctaccaaaga ctaccagcaa agaaccaaca aaagaatagt cagggcacaa accttattag 5640 tcctacagca gaaagaatta taagaagcaa ggtagcaaag cctttgctag cgctaaagat 5700 cgcgttttag caagtaagat gggtgcttac gctgttgagc tactccttga aggtaagggt 5760 ggtttagcag ttggaatctt agaaaataag gttcaagctc ataacatgct tgacttgttt 5820 gatgcaaaac atcaagcaga tgattcactt taccaattaa gtgaagattt atcattctag 5880 agttctatta atatttggat aaaatgactt aagaagtctt ttataattta aaatcaaggg 5940 agagattctg taatgaaaaa aaagattatc tcagctattt taatgtctac agtgatactt 6000 tctgctgcag ccccgttgtc aggtgtttac gctgaagtta ttattatggc tacagttgat 6060 ccagaaaaaa cactttttct tgatgaacca atgaacaaag tttttgattg gtcaaactca 6120 gaagctccag ttcgtgatgc tctttgggat tactacatgg aaaaaaactc acgtgataca 6180 attaaaacag aagaagaaat gaaaccagtt cttgatatgt cagatgatga agttaaagct 6240 cttgctgaaa aagttcttaa aaaataacct gacaagaacc agtctgctat tgatagacta 6300 tttttgtccg tgaaatcctc gcgtatttcc gtgaggagca tagtatattt agcgatcttc 6360 aaattttaag tatattgatt gtcgaccgga aatgatcttt ttaacatcca agataaagaa 6420 agcaaaatag ctaaacagaa gattgttaaa tctggtagta ataaagatgg catacacaca 6480 aatagagcta ttaaacgttg gtggaaattc tggtaa 6516 <210> 10 <211> 389 <212> PRT <213> Pediococcus pentosaceus <400> 10 Met Ser Thr Lys Pro Phe Tyr Arg Asp Thr Trp Ala Glu Ile Asp Leu   1 5 10 15 Ser Ala Ile Lys Glu Asn Val Ser Asn Met Lys Lys His Ile Gly Glu              20 25 30 His Val His Leu Met Ala Val Val Lys Ala Asn Ala Tyr Gly His Gly          35 40 45 Asp Ala Glu Thr Ala Lys Ala Ala Leu Asp Ala Gly Ala Ser Cys Leu      50 55 60 Ala Val Ala Ile Leu Asp Glu Ala Ile Ser Leu Arg Lys Lys Gly Leu  65 70 75 80 Lys Ala Pro Ile Leu Val Leu Gly Ala Val Pro Pro Glu Tyr Val Ala                  85 90 95 Ile Ala Ala Glu Tyr Asp Val Thr Leu Thr Gly Tyr Ser Val Glu Trp             100 105 110 Leu Gln Glu Ala Ala Arg His Thr Lys Lys Gly Ser Leu His Phe His         115 120 125 Leu Lys Val Asp Thr Gly Met Asn Arg Leu Gly Val Lys Thr Glu Glu     130 135 140 Glu Val Gln Asn Val Met Ala Ile Leu Asp Arg Asn Pro Arg Leu Lys 145 150 155 160 Cys Lys Gly Val Phe Thr His Phe Ala Thr Ala Asp Glu Lys Glu Arg                 165 170 175 Gly Tyr Phe Leu Met Gln Phe Glu Arg Phe Lys Glu Leu Ile Ala Pro             180 185 190 Leu Pro Leu Lys Asn Leu Met Val His Cys Ala Asn Ser Ala Ala Gly         195 200 205 Leu Arg Leu Lys Lys Gly Phe Phe Asn Ala Val Arg Phe Gly Ile Gly     210 215 220 Met Tyr Gly Leu Arg Pro Ser Ala Asp Met Ser Asp Glu Ile Pro Phe 225 230 235 240 Gln Leu Arg Pro Ala Phe Thr Leu His Ser Thr Leu Ser His Val Lys                 245 250 255 Leu Ile Arg Lys Gly Glu Ser Val Ser Tyr Gly Ala Glu Tyr Thr Ala             260 265 270 Glu Lys Asp Thr Trp Ile Gly Thr Val Pro Val Gly Tyr Ala Asp Gly         275 280 285 Trp Leu Arg Lys Leu Lys Gly Thr Asp Ile Leu Val Lys Gly Lys Arg     290 295 300 Leu Lys Ile Ala Gly Arg Ile Cys Met Asp Gln Phe Met Val Glu Leu 305 310 315 320 Asp Gln Glu Tyr Pro Pro Gly Thr Lys Val Thr Leu Ile Gly Arg Gln                 325 330 335 Gly Asp Glu Tyr Ile Ser Met Asp Glu Ile Ala Gly Arg Leu Glu Thr             340 345 350 Ile Asn Tyr Glu Val Ala Cys Thr Ile Ser Ser Arg Val Pro Arg Met         355 360 365 Phe Leu Glu Asn Gly Ser Ile Met Glu Val Arg Asn Pro Leu Leu Gln     370 375 380 Val Asn Ile Ser Asn 385 <210> 11 <211> 1170 <212> DNA <213> Pediococcus pentosaceus <400> 11 atgagcacaa aaccttttta cagagatacg tgggcggaaa ttgacttgtc cgcgataaag 60 gaaaatgtca gcaatatgaa aaaacatatc ggtgaacatg tccacttgat ggcagttgtg 120 aaagcaaacg cctacgggca tggtgatgca gaaacagcaa aggctgctct tgacgcaggt 180 gcttcatgct tggccgtggc cattttggat gaagcgattt cactgcgcaa aaagggattg 240 aaggcgccta tattggtgct tggcgcggtt cccccggagt atgtggcaat cgctgctgag 300 tatgacgtga ccttaacagg ttattctgtt gaatggcttc aggaggcagc ccgccacacg 360 aaaaaaggtt ctcttcattt tcatctgaag gtcgatacgg ggatgaacag acttggtgta 420 aaaacagagg aagaagttca gaacgtgatg gcaattcttg accgcaaccc tcgtttaaag 480 tgcaaagggg tatttaccca ttttgcgaca gcggatgaaa aagaaagagg ctatttctta 540 atgcagtttg agcgctttaa agagctgatt gctccgctgc cgttaaagaa tctaatggtc 600 cactgcgcga acagcgccgc tggactccgg ctgaaaaaag gcttttttaa tgcagtcaga 660 ttcggcatcg gcatgtatgg ccttcgcccg tctgctgaca tgtcggacga gataccgttt 720 cagctgcgtc cggcatttac cctgcattcg acactgtcac atgtcaaact gatcagaaaa 780 ggcgagagcg tcagctacgg agccgagtac acagcggaaa aagacacatg gatcgggacg 840 gtgcctgtag gctatgcgga cggctggctc cgaaaattga aagggaccga catccttgtg 900 aagggaaaac gcctgaaaat tgccggccga atttgcatgg accaatttat ggtggagctg 960 gatcaggaat atccgccggg cacaaaagtc acattaatag gccggcaggg ggatgaatat 1020 atttccatgg atgagattgc aggaaggctc gaaaccatta actatgaggt ggcctgtaca 1080 ataagttccc gtgttccccg tatgtttttg gaaaatggga gtataatgga agtaagaaat 1140 cctttattgc aggtaaatat aagcaattaa 1170

Claims (15)

A recombinant plasmid for producing an anticancer drug delivery strain comprising a P8 protein coding gene fragment of SEQ ID NO: 1 and an alanine racease gene of SEQ ID NO: 11 and having a cleavage map of FIG. 1 or 2.
The method of claim 1, wherein the recombinant plasmid,
A heterologous nucleic acid encoding a P8 protein of SEQ ID NO: 1 having a therapeutic effect on a gastrointestinal disease, wherein said heteronucleic acid is secreted with one or more promoters derived from Pediococcus pentosaceus operably linked to the heterologous nucleic acid; Recombinant plasmid for producing an anticancer drug delivery strain comprising a gene encoding a peptide.
The recombinant plasmid of claim 1, wherein the secretory signal peptide is USP45 secretory signal peptide, Usp45 N4 or Lactobacillus brevis S-layer protein signal peptide.
delete An anticancer drug delivery strain transformed by the recombinant plasmid of any one of claims 1 to 3 and comprising an inactivated alanine racease (alaR) gene.
The anticancer drug delivery strain according to claim 5, wherein the recombinant strain is Pediococcus pentosaceus strain.
The anti-cancer drug delivery strain according to claim 6, wherein the Pediococcus pentosaceus strain is Pediococcus pentosaceus PPΔalr-P8-alr strain (Accession Number: KCCM12500P).
According to claim 5, wherein the gastrointestinal disease is colorectal cancer, colon polyps, colitis, ischemic gastrointestinal disease, dysentery, intestinal vascular dysplasia, diverticulosis, irritable bowel syndrome, or Crohn's disease anti-cancer drugs that express and secrete P8 protein Transfer strain.
Phedi O Lactococcus pen soil three mouse PP △ alr-P8-alr strain (accession number: KCCM12500P) gastrointestinal disease prevention or treatment a pharmaceutical composition comprising a.
10. The method of claim 9, wherein the gastrointestinal disease is colorectal cancer, colon polyps, colitis, ischemic gastrointestinal disease, dysentery, intestinal vascular dysplasia, diverticulosis, irritable bowel syndrome, or Crohn's disease. .
A recombinant fragment comprising a gene fragment encoding a P8 protein of SEQ ID NO: 1 showing gastrointestinal therapeutic activity and an alanine racease gene of SEQ ID NO: 11 encoding an essential protein necessary for viability of bacteria, and having a cleavage map of FIG. 1 or 2 Steps to prepare the plasmids:
A step of preparing a trophyotropic strain in which the alanine racemic gene of the recombinant plasmid is inactivated:
The method for producing an anticancer drug delivery strain comprising the step of transforming the trophic strain with the recombinant plasmid to produce an anticancer drug delivery recombinant strain.
The method for producing an anticancer drug delivery strain according to claim 11, wherein the recombinant plasmid is derived from Pediococcus pentosaceus and is a plasmid having a cleavage map of FIG. 1 or 2.
The method for producing an anticancer drug delivery strain according to claim 11, wherein the anticancer drug delivery recombinant strain is a Pediococcus pentosaceus PPΔalr-P8-alr strain (Accession Number: KCCM12500P).
The method of claim 11, wherein the inactivated essential protein coding gene of the strain is alanine racease (alaR), thymidylate synthase (thyA), asparagine synthase (asnH), CTP synthase (pyrG), tryptophan syntha A method for producing an anticancer drug delivery strain, characterized in that it is selected from the group consisting of an agent (trpBA), and combinations thereof.
12. The method of claim 11, wherein the method further comprises the step of culturing the transformed recombinant strain in a medium to which the essential protein necessary for the viability of the bacteria encoded by the inactivated gene is added to the anti-cancer drug delivery Preparation of strains.

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