KR102527954B1 - Microorganism expressing heterologous protein, and use thereof - Google Patents

Abstract

Provided are microorganisms expressing a VIP gene, compositions and kits for use in preventing or treating diseases causing damage to the gastrointestinal tract including the microorganisms, and methods for preventing or treating diseases causing damage to the gastrointestinal tract.

Description

Microorganism expressing heterologous protein, and use thereof {Microorganism expressing heterologous protein, and use thereof}

It relates to a microorganism expressing a VIP gene, a composition for use in preventing or treating a disease causing damage to the gastrointestinal tract including the microorganism, a kit, and a method for preventing or treating a disease causing damage to the gastrointestinal tract.

Vasoactive intestinal peptide (VIP) is a peptide hormone that is vasoactive in the intestine. VIP is a peptide with 28 amino acid residues belonging to the glucagon/secretin superfamily, ligands of class II G protein-coupled receptors.

U.S. Patent No. 9,561,262 discloses a method for use in treating hypertension in a patient comprising administering VIP and an anti-hypertensive agent to the patient.

Even according to the prior art, there is a need for microorganisms expressing VIP genes and their use in treating inflammatory bowel disease.

One aspect provides a recombinant microorganism comprising a genetic modification that increases the expression of a gene encoding VIP.

Another aspect provides a composition for use in preventing or treating a disease that causes damage to the gastrointestinal tract containing the microorganism.

Another aspect provides a kit for use in preventing or treating a disease that causes damage to the gastrointestinal tract comprising the microorganism and a TNF-alpha blocker.

Another aspect provides methods for preventing or treating diseases that cause damage to the gastrointestinal tract.

One aspect provides a recombinant microorganism comprising a genetic modification that increases the expression of a gene encoding VIP.

As used herein, the term "VIP" or "VIP protein" or "VIP polypeptide" refers to a biologically active polypeptide having one or more of the biological activities as described herein. In humans, the q25 region of chromosome 6 on the human genome encodes a member of the secretin family that is 170 amino acids in length. This member of 170 amino acids in length is cleaved after translation to form the vasoactive intestinal peptide (VIP). The active form of the VIP polypeptide is to lower blood pressure, increase vasodilation of blood vessel walls, relax smooth muscle in tissues of the respiratory system and gastrointestinal tract, reduce the Th1 response as well as promote the immune response through stimulating Th2. It functions by reducing blood pressure, regulating innate and acquired immune responses, or stimulating the secretion of electrolytes in the gut. In addition, VIP was found to be active in the central nervous system as a neurotransmitter and in communication with lymphocytes. The bioactivity of VIP is conveyed through three known receptors: VIP ₁ R, VIP ₂ R, and PAC ₁ R. These receptors are known to cause cAMP concentration as well as production of intracellular calcium. Like VIP, their affinity for secretins depends on the subtype and amino acid sequence of the ligand. Human native VIP has a half-life of about 2 minutes in the bloodstream, so it has a short half-life. Thus, VIPs referred to herein include variants that mimic the function of native VIPs. Such variants include those having an increased half-life than native VIP and equivalent or greater biological activity compared to native VIP.

VIPs of the present invention include, but are not limited to, human VIPs, recombinant human VIPs, murine VIPs and/or recombinant mouse VIPs. The VIP has at least 50%, at least 60%, at least 70%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 1 may have The VIP may have an amino acid sequence of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, and 9, respectively. The VIP of SEQ ID NO: 2 is a natural VIP of human origin. VIPs having amino acid sequences of SEQ ID NOs: 1, 3, 4, 5, 6, 7, 8, and 9 are human-derived natural VIP variants VIP1, VIP2, VIP3, VIP4, VIP5, VIP6, VIP7, and VIP8.

The VIP polypeptides used herein can be isolated from a variety of sources, such as human tissue or other sources, or produced by recombinant or synthetic methods. The term "VIP polypeptide" also includes variants of the VIP polypeptide. A VIP of the invention can be modified in such a way as to form a chimeric molecule comprising the VIP fused to another heterologous polypeptide or amino acid sequence.

The microorganism may contain a genetic modification that increases the expression of VIP. The genetic modification may be to increase the copy number of a gene encoding VIP. Increasing the copy number may be by introduction of a foreign gene into a microorganism. The introduction may be by transformation, transduction, transfection or electroporation. The introduced foreign gene may be integrated into the genome of the host cell or not integrated into the host cell genome.

The microorganism may contain a foreign gene encoding VIP. The gene may be operably linked with a regulatory sequence that regulates its expression. The regulatory sequence may be a promoter, operator, terminator, or nucleotide sequence encoding a signal polypeptide. The promoter may be a constitutive promoter. The promoter may have transcription initiation efficiency equal to or greater than that of a conventional promoter, for example, the P11 promoter in lactic acid bacteria. The promoter may also be an inducible promoter. The inducible promoter may be, for example, a nisin inducible promoter. The promoter may be PR4 (SEQ ID NO: 10) derived from Lactobacillus paracasei . When the signal polypeptide is linked in frame with an exogenous protein gene, the ability to secrete it outside the cell may be greater than that of a conventional signal polypeptide, for example, the USP45 signal polypeptide. The signal polypeptide may be of SP4 (SEQ ID NO: 11) from Lactobacillus paracasei . The microorganism may secrete VIP extracellularly.

The microorganism may be a bacterium. The bacteria may be gram positive bacteria or gram negative bacteria. The gram-positive bacteria may be lactic acid bacteria. The lactic acid bacteria are Lactobacillus , Lactococcus , Bifidobacteria , Streptococcus , Leuconostoc , Weissella , Pediococcus , or Entero It may belong to the genus Enterococcus . The lactic acid bacteria may be Lactobacillus paracasei , Lactobacillus brevis , Lactobacillus plantarum or Lactococcus lactis . The lactic acid bacteria may be Lactobacillus plantarum LMT1-9 (KCTC 13421BP), Lactobacillus paracasei LMT1-21 (KCTC 13422BP), or Lactobacillus brevis LMT1-46 (KCTC 13423BP).

Another aspect provides a composition for use in preventing or treating a disease that causes damage to the gastrointestinal tract comprising the microorganisms described above.

The disease may be one that causes inflammation of the gastrointestinal tract. The disease may be one or more selected from the group consisting of inflammatory bowel disease (IBD) and colitis. The inflammatory bowel disease may be ulcerative colitis or Crohn's disease.

The composition may include a pharmaceutically or food-acceptable carrier, excipient or diluent. The pharmaceutically or foodtically acceptable carrier can be any standard, such as phosphate buffered saline solution, 5% aqueous solution of dextrose, and emulsion (e.g., oil/water or water/oil emulsion). Means a pharmaceutical or food carrier. Non-limiting examples of excipients include adjuvants, binders, fillers, diluents, disintegrants, emulsifiers, wetting agents, lubricants, glidants, sweeteners, flavors and colorants. Preferred pharmaceutical or dietary carriers depend on the intended mode of administration of the active agent. Typical modes of administration include enteral (eg, oral) administration. The composition may be in unit dosage form. The composition may have an oral dosage form. The composition may be a food or pharmaceutical composition. The composition may include dry matter of the microorganisms. The composition may include a culture medium of the microorganism.

In this specification, "pharmaceutically acceptable or acceptable for food" indicates that it does not substantially cause adverse reactions when administered to a subject. The side effect may be toxicity, allergy, or immune response.

Another aspect provides a kit for use in preventing or treating diseases that cause damage to the gastrointestinal tract, comprising the recombinant microorganism described above and a drug for treating other diseases of the gastrointestinal tract. The recombinant microorganisms are as described above. The recombinant microorganism may be in the form of the composition described above. The kit may include instructions directing the use of the recombinant microorganism and other drugs for treating diseases of the gastrointestinal tract to prevent or treat diseases that cause damage to the gastrointestinal tract.

The drug for treating other gastrointestinal damaging diseases includes any drug that treats gastrointestinal damaging diseases, for example, inflammatory bowel disease. Drugs to treat damaging disorders of the gastrointestinal tract may include:

(i) Steroidal anti-inflammatory agents

dexamethasone, hexestrol, methimazole, betamethasone, triamcinolone, triamcinolone acetonide, fluocinonide, fluocinolone acetonide, predonisolone, methylpredonisolone, cortisone acetate, hydrocortisone, fluorometholone, beclomethasone dipropionate, estriol, paramethasone acetate, fludrocortisone acetate, clobetasol propionate, di florasone acetate, dexamethasone propionate, difluprednate , betamethasone dipropionate, budesonide, diflucortolone valerate, amcinonide, halcinonide, mometasone furoate, hydrocortisone butyrate propionate, flumetasone pivalate, clobetasone butyrate, dexametasone acetate, etc.

(ii) 5-aminosalicylic acid

sulfasalazine, mesalazine, olsalazine, balsalazide, etc.

(iii) immunomodulators or immunosuppressants

methotrexate, cyclophosphamide, MX-68, atiprimod dihydrochloride, BMS-188667, CKD-461, rimexolone, cyclosporine, tacrolimus, gusperimus, azathiopurine, antilymphocyte serum, freeze-dried sulfonated normal immunoglobulin, erythropoietin, colony stimulating factor, interleukin, interferon, etc.

(iv) JAK inhibitors

tofacitinib, ruxolitinib, etc.

(v) TNF inhibitors

Recombinant TNF-alpha-receptor IgG-Fc fusion protein (etanercept), infliximab, adalimumab, certolizumab pegol, golimumab, PASSTNF-α, soluble TNF-α receptor, TNF-α binding protein, anti-TNF-α antibody, CDP571, etc.

(vi) integrin inhibitors

natalizumab, vedolizumab, AJM300, TRK-170, E-6007, etc.

(vii) Interleukin-12/23 inhibitors

ustekinumab, briakinumab (anti-interleukin-12/23 antibody), etc.

(viii) non-steroidal anti-inflammatory drugs (NSAIDs)

(a) classic NSAIDs

alcofenac, aceclofenac, sulindac, tolmetin, etodolac, fenoprofen, thiaprofenic acid, meclofenamic acid, meloxicam, tenoxicam, lornoxicam, nabumeton, acetaminophen, phenacetin, ethenzamide, sulpyrine, antipyrine, migrenin, aspirin, mefenamic acid, flufenamic acid, diclofenac sodium, ketophenylbutazone , loxoprofen sodium, phenylbutazone, indomethacin, ibuprofen, ketoprofen, naproxen, oxaprozin, flurbiprofen, fenbufen, pranoprofen, floctafenine, piroxicam, tenoxicam, epirizole, tiaramide hydrochloride, zaltoprofen, gabexate mesylate, camostat mesylate, ulinastatin, colchicine, probe necid, sulfinpyrazone, bucolome , benzbromarone, allopurinol, sodium aurothiomalate, sodium hyaluronate, sodium salicylate, salicylic acid, atropine, scopolamine, levorphanol, oxymorphone or its salts, etc.

(b) cyclooxygenase inhibitors (COX-1 selective inhibitors, COX-2 selective inhibitors, etc.)

Salicylic acid derivatives (eg celecoxib, aspirin), etoricoxib, valdecoxib, diclofenac sodium, indomethacin, loxoprofen, etc.

(c) nitric oxide-releasing NSAIDs.

Another aspect provides a method for preventing or treating a disease causing damage to the gastrointestinal tract, comprising administering to a subject a preventive or therapeutically effective amount of the microorganism.

The method may further include administering to the subject an amount of a TNF-alpha blocker effective to prevent or treat a disease causing damage to the gastrointestinal tract. The step of administering the TNF-alpha blocker to the subject may be performed simultaneously with, before, or after the step of administering the microorganism to the subject. The TNF-alpha blocker may be administered by the same or different route as the route through which the microorganism is administered. For example, the TNF-alpha blocker may be administered orally or parenterally. The parenteral route may be an injection such as intravascular injection, intraperitoneal injection, or subcutaneous injection. For example, the TNF-alpha blocker may be administered intraperitoneally, and the microorganism may be administered orally. The TNF-alpha blocker may be administered intraperitoneally after the microorganism is orally administered. The TNF-alpha blocker may be administered in an amount of 5mg to 100mg, 5mg to 50mg, 5mg to 40mg, 10mg to 50mg, or 20mg to 50mg per administration.

In the method, the disease may cause gastrointestinal inflammation. These diseases include inflammatory intestinal disease, autoimmune disease, radiation-induced damage to the gastrointestinal tract or graft versus host disease (GVHD), inflammatory bowel disease : IBD), and at least one selected from the group consisting of colitis including chronic colitis. The inflammatory bowel disease may be ulcerative colitis or Crohn's disease.

As used herein, the “administering” or “administration” refers to the microorganism, a composition comprising the same, or a therapeutically Indicates the act of giving a therapeutic treatment. Thus, in the method, the microorganism may be in the form of the composition described above. An acceptable route of administration to the human body may be oral, or mucosal (eg, intestinal mucosal, oral mucosal or bucal). The administration may be administered in combination with additional therapeutic agents. The combined administration includes either simultaneous or sequential administration.

As used herein, the term "treatment" refers to prophylactic treatment or therapeutic treatment. In certain embodiments, “treatment” refers to administering a microorganism or composition to an individual for therapeutic or prophylactic purposes.

A “therapeutic” treatment is treatment administered to a subject exhibiting pathological signs or symptoms to reduce or eliminate the signs or symptoms. Signs or symptoms may be biochemical, cellular, histological, functional or physical, subjective or objective.

A "prophylactic" treatment is treatment administered to a subject who does not show signs of a disease or who exhibits only early signs of a disease to reduce the risk of developing a pathology. The microorganisms or compositions described herein can be provided as a prophylactic treatment to reduce the likelihood of developing a pathology or, if it does occur, to minimize the severity of the pathology.

As used herein, "therapeutically effective amount" refers to an amount sufficient to accomplish the stated purpose. Effective amounts can be determined empirically. An effective amount may be determined, for example, depending on the degree of extracellular secretion of VIP by the microorganism. For example, it may be the number of microorganisms capable of secreting 0.01 to 300 mg, or 0.5 to 100 mg of VIP per day for a person with a body weight of 60 kg. For example, 1x10 ⁷ to 1x10 ¹¹ cfu, 1x10 ⁷ to 1x10 ¹⁰ cfu, 1x10 ⁸ to 1x10 ¹¹ cfu, 1x10 ⁸ to 1x10 ¹⁰ cfu, 2.5x10 ⁸ to 7.5x10 ⁹ cfu, per administration per subject per subject; 5.0x10 ⁸ to 5.0x10 ⁹ cfu, 7.5x10 ⁸ to 2.5x10 ⁹ cfu, or about 1x10 ⁹ cfu.

In the above method, the subject may be a mammal. The mammal may be a human, horse, pig, cow, dog, cat, monkey, chimpanzee, sheep, or goat. The subject may be a non-human mammal.

A recombinant microorganism according to one aspect can produce and secrete VIP.

A composition or kit according to another aspect may be used for use in preventing or treating diseases that cause damage to the gastrointestinal tract.

According to the method for preventing or treating diseases causing damage to the gastrointestinal tract according to another aspect, diseases causing damage to the gastrointestinal tract can be efficiently prevented or treated.

1 is a diagram showing the configuration of the pMT447 vector.
FIG. 2 is a diagram showing Western blot results of culture supernatants of L. plantarum LMT1-9, L. paracasei LMT1-21, and L. brevis LMT1-46 strains transformed with the pMT447 vector.
Figure 3 is a diagram showing the therapeutic efficacy of VIP derived from L. plantarum LMT1-9 transformed strain in DSS-induced mouse intestinal inflammation model by DAI score.
Figure 4 is a diagram showing the therapeutic efficacy of VIP derived from L. paracasei LMT1-21 transformed strain in DSS-induced mouse intestinal inflammation model by DAI score.
Figure 5 is a diagram showing the therapeutic efficacy of VIP derived from L. brevis LMT1-46 transformed strain in DSS-induced mouse intestinal inflammation model by DAI score.
6 is a diagram showing the therapeutic efficacy of VIP derived from a transgenic strain in terms of DAI AUC score in a mouse intestinal inflammation model induced by DSS.
7 is a histopathological view of the therapeutic efficacy of VIP derived from a transgenic strain in a mouse intestinal inflammation model induced by DSS.
8 is a diagram showing the combined treatment efficacy of LAB-transformed strain-derived VIP and etanercept (Enbrel, Korea Pfizer Pharmaceuticals) in a DSS-induced mouse intestinal inflammation model by DAI score.
9 is a diagram showing the efficacy of combination treatment of VIP and Enbrel derived from a LAB transformed strain in DSS-induced intestinal inflammation model by DAI AUC score.
10 is a diagram showing the histopathological results of the combination treatment efficacy of VIP and Enbrel derived from a LAB transformed strain in a mouse intestinal inflammation model induced by DSS.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are intended to illustrate the present invention by way of example, and the scope of the present invention is not limited to these examples.

Example 1: Lactic acid bacteria cells expressing the VIP gene and their IBD therapeutic effect

In this example, it was confirmed that the VIP gene was introduced into lactic acid bacteria cells, and the gene was expressed to secrete the product outside the cells. In addition, whether the lactic acid bacteria cells transformed with the VIP gene were effective in treating IBD was orally administered to the subject, and the symptom improvement was confirmed. As VIP, a human VIP variant having the amino acid sequence of SEQ ID NO: 1 was used. The VIP variant is one in which 4 amino acid residues are substituted in the natural human VIP having the amino acid sequence of SEQ ID NO: 2, and has an increased half-life in human bloodstream.

1. Construction of a vector containing the VIP gene

The VIP gene composed of the nucleotide sequence of SEQ ID NO: 14 was used by synthesizing DNA from Macrogen (Korea). The synthesized VIP gene segment and lactic acid bacteria (LAB)-E. coli shuttle vector pMT54-PR4-SP4 (SEQ ID NO: 13) were contacted and cut in the presence of SalI and XhoI restriction enzymes, respectively. After purifying the cleaved gene fragment and vector in the reaction solution using a gel purification kit (GeneAll), the cleaved gene fragment and vector are incubated in the presence of alkaline phosphatase to dephosphorylate, and the dephosphorylated cleaved VIP gene fragment and a truncated pMT54-PR4-SP4 vector.

1 ul of vector DNA and 3 ul of VIP gene DNA thus prepared, 0.5 ul of T4 DNA ligase (Takara), 1 ul of buffer solution, and 5.5 ul of distilled water were mixed in a test tube to make a total volume of 10 ul, and then incubated at 16°C for 12 hours to generate the VIP gene. DNA and vector fragments were ligated to obtain a vector containing the VIP gene. This vector was transformed into E. coli Top10 Competent Cells using the method of Sambrook et al. (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edn, 1989).

The transformed E. coli was spread on a plate containing LB (Luria-Bertani)-agar containing 10 μg/ml of chloramphenicol and incubated. As a result, colonies formed were selected and cultured, and the vector was isolated from the culture. This vector was named pMT447. The pMT447 vector has the VIP gene operably linked to the PR4 promoter and the signal peptide sequence of SP4. Therefore, cells transformed with the pMT447 vector express the VIP gene with high efficiency and secrete it extracellularly.

1 is a diagram showing the configuration of the pMT447 vector. In Figure 1, the promoter is the PR4 promoter sequence of SEQ ID NO: 10, SP represents the nucleotide sequence (SEQ ID NO: 12) encoding the SP4 secretion signal sequence of SEQ ID NO: 11, VIP is the human VIP variant of SEQ ID NO: 1 Gene (SEQ ID NO: 14). HA and 6xHis represent the nucleotide sequences encoding the human influenza hemagglutinin tag and six histidines for detection, separation, and purification of proteins. E.coli ori, Rep, and CM represent an E. coli replication origin, a lactic acid bacteria replication origin, and a chloramphenicol resistance marker gene, respectively.

2. Introduction of recombinant vectors into lactic acid bacteria cells

The recombinant vector pMT447 vector constructed in Section 1 was introduced into Lactobacillus plantarum LMT1-9 (KCTC 13421BP), Lactobacillus paracasei LMT1-21 (KCTC 13422BP), and Lactobacillus brevis LMT1-46 (KCTC 13423BP), respectively, and VIP protein secretion was examined. confirmed.

Each strain was cultured in a flask containing 50 mL of MRS medium (Difco Co., USA) until the OD ₆₀₀ reached 0.5, and then centrifuged at 4°C and 7,000 rpm for 10 minutes. -cold electroporation solution) (containing 1 mM K ₂ HPO ₄ , 1 mM KH ₂ PO ₄ , pH 7.4, 1 mM MgCl ₂ , and 0.5 M sucrose) twice. MRS is also known as De Man, Rogosa and Sharpe agar medium.

After washing, the cells were resuspended in 1 ml of cold EPS, and competent cells to be used for electroporation were prepared and stored in a -80°C deep freezer. 40 μl of competent cells and 1 μl of each vector DNA (1 μg/μl) were placed in a cuvette and left on ice for 5 minutes. After giving an electric pulse under conditions of 25μF, 8kV/cm, 400 ohms, it was immediately added to 1ml MRS liquid medium and incubated at 37°C for 1 hour. Then, the cultured cells were plated on MRS medium (also referred to as 'MRS-CM') containing 10 μg/ml chloramphenicol and cultured at 37°C for 48 hours.

Each strain into which the obtained pMT447 vector was introduced was statically cultured in MRS liquid medium at 37° C. for 16 hours. The culture was inoculated to 3 (v / v)% in MRS liquid medium, and then cultured at the same temperature for 8 hours. 1 ml of the culture was centrifuged at 7,000 rpm for 5 minutes, and the supernatant was taken. 100 μl of trichloroacetic acid was added to 1 ml of the supernatant and allowed to stand at 4° C. for 1 hour to concentrate the culture components. After the reaction was centrifuged at 4° C. and 13,000 rpm for 10 minutes, the pellet was washed once with 1 ml of cold acetone, dried at room temperature for 10 minutes, and then eluted with 100 μl of Tris-HCl buffer (pH 8.8).

After adding 4x loading buffer (Thermo) and 10x reducing agent (Thermo) to the eluate, electrophoresis was performed on an SDS-PAGE gel. The gel was transferred to a nitrocellulose membrane using a Trans blot semi-dry cell (biorad), and Western blotting was performed. Specifically, the membrane was blocked with TBST buffer containing 1% skim milk for 1 hour, reacted with an anti-HA antibody (Santa cruz) at room temperature for 2 hours, washed three times with TBST for 5 minutes each, and then detected by ECL did In the pMT447 vector, the VIP gene is operably linked to the hemagglutinin (HA) gene at its 3' end, so that it is expressed in an HA-tagged state.

FIG. 2 is a diagram showing Western blot results of culture supernatants of L. plantarum LMT1-9, L. paracasei LMT1-21, and L. brevis LMT1-46 strains transformed with the pMT447 vector. In Figure 2, lanes 2, 3, and 4 represent L. plantarum LMT1-9, L. paracasei LMT1-21, and L. brevis LMT1-46 transformed with the pMT447 vector, respectively, and lane 1 represents the standard protein ladder. indicate

As shown in Figure 2, the transformed strain effectively secreted the hVIP variant.

3. Efficacy of VIP-secreting lactic acid bacteria against IBD in a DSS-induced model

Mice enteritis induced by dextran sulfate sodium salt (DSS) in the transformed L. plantarum LMT1-9, L. paracasei LMT1-21, and L. brevis LMT1-46 described in Section 2 After oral administration to the (colitis) model, the survival rate and disease activity index (DAI) score of the mice were confirmed, and the intestinal inflammation treatment effect by the strain was evaluated.

(1) Administration of recombinant microorganism alone

Specifically, the transformed strain was firstly cultured at 37° C. for 24 hours in a flask containing 10 ml MRS medium containing 10 μg/ml chloramphenicol. The next day, the cultured strain was inoculated into 300 to 600 ml of MRS CM medium to an OD ₆₀₀ of 0.01, and secondary culture was performed under the same conditions as the primary culture. After about 16 to 18 hours, when the OD ₆₀₀ of the culture reached 2 to 3, the culture was centrifuged at 7,000 rpm for 10 minutes to remove the supernatant, and the cells were suspended in 1xPBS for administration at 1x10 ⁹ cfu once per animal. . This cell-containing PBS solution was administered to the subject by oral administration (1×10 ⁹ cfu once per animal) and drinking water in parallel, once a day for a total of 16 days.

First, 8 to 10 week old male Balb/c mice (20 to 25 g, 10 animals in each group) (ORIENTBIO) were used as experimental animals. The first strain administration date was set to Day-7, and DSS was diluted to 2 (v/v)% in sterile water and supplied as negative water to all groups except the PBS group from Day 0 to Day 6. Mice voluntarily consume drinking water, but generally consume less than 6 ml per day. Mice were sacrificed on day 9.

After DSS administration, the survival rate of mice was checked at 2-day intervals, and the DAI score was calculated by examining body weight, hair bristles, animal movement, and diarrhea.

Disease activity index (DAI) scoring was performed by Ameho, Gut 1997; 41:487-493 and Wallace, Gastroenterology 1989; 96: 29-36. The results are shown in Figures 3 to 6.

Figure 3 is a diagram showing the therapeutic efficacy of VIP derived from L. plantarum LMT1-9 transformed strain in DSS-induced mouse intestinal inflammation model by DAI score. 3, VIPs 1-9 represent strains in which pMT447 was transformed into L. plantarum LMT1-9. Vectors 1-9 represent strains in which the control vector (parental vector without the VIP gene) was transformed into L. plantarum LMT1-9.

Figure 4 is a diagram showing the therapeutic efficacy of VIP derived from L. paracasei LMT1-21 transformed strain in DSS-induced mouse intestinal inflammation model by DAI score. 4, VIPs 1-21 represent strains in which pMT447 was transformed into L. paracasei LMT1-21. 1-21 vector represents a strain transformed with a control vector (parental vector without VIP gene) into L. paracasei LMT1-21.

Figure 5 is a diagram showing the therapeutic efficacy of VIP derived from L. brevis LMT1-46 transformed strain in DSS-induced mouse intestinal inflammation model by DAI score. In FIG. 5, 1-46 VIPs represent strains in which pMT447 was transformed into L. brevis LMT1-46. 1-46 vector represents a strain transformed with a control vector (parental vector without VIP gene) in L. brevis LMT1-46.

3 to 5, CyA is an abbreviation of cyclosporinA and was used as a positive control in the experiment. PBS is a control group treated with only DSS without any bacteria. Normal represents the group treated with neither bacteria nor DSS.

6 is a diagram showing the therapeutic efficacy of VIP derived from a transgenic strain in terms of DAI AUC score in a mouse intestinal inflammation model induced by DSS. A description of each strain and chemical is mentioned above and DAI AUC represents the area under the curve of DAI SCORE by day 8.

As shown in Figures 3 to 6, the Lactobacillus strain transformed to express VIP had a therapeutic effect in the DSS-induced intestinal inflammation model.

7 is a histopathological view of the therapeutic efficacy of VIP derived from a transgenic strain in a mouse intestinal inflammation model induced by DSS. In FIG. 7, Normal, PBS, LMT1-9 vector, LMT1-9 VIP, LMT1-46 vector, LMT1-46 VIP, LMT1-21 vector, and LMT1-21 VIP are as described in FIGS. 1 to 6.

As shown in FIG. 7, a histopathological examination of the large intestine was performed through a microscope in the DSS-induced intestinal inflammation mouse model, and it was confirmed that the mice administered with the Lactobacillus strain expressing VIP had a therapeutic effect compared to the control group. It became.

Specifically, as shown in FIG. 7, a histopathological examination of the large intestine was performed under a microscope in a mouse model of intestinal inflammation induced by DSS, and mononuclear cell infiltration and mucosal damage were most severe in the DSS group. In addition, the groups administered with LMT 1-9 vector, LMT 1-21 vector, and LMT 1-46 vector did not show significant improvement in symptoms. On the other hand, in the LMT 1-9 VIP, 1-21 VIP, and LMT1-46 VIP treatment groups, histological damage and inflammatory infiltrate lesions tended to decrease. This can be seen that the Lactobacillus strain expressing VIP has anti-inflammatory and protective effects in the DSS-induced inflammatory bowel disease mouse model.

(2) Effect of combined administration of recombinant microorganisms and TNF-alpha blockers

8 is a diagram showing the combined treatment efficacy of LAB-transformed strain-derived VIP and etanercept (Enbrel, Korea Pfizer Pharmaceuticals) in a DSS-induced mouse intestinal inflammation model by DAI score. In Fig. 8, Normal is a group that does not process anything. PBS is a group treated only with DSS. Enbrel is the group that processed DSS and Enbrel. 1-46 VIP was a group in which DSS-induced mice were fed a strain transformed with pMT447 in L. brevis LMT1-46. 1-46 VIP + enbrel is a group in which DSS-induced mice were treated with L. brevis LMT1-46 pMT447 and Enbrel.

8 is a result obtained by the following process. In order to evaluate the therapeutic efficacy of the VIP protein derived from the transformed strain, the transformed strain was orally administered to a mouse intestinal inflammation model induced by DSS, and then the survival rate or DAI score was confirmed. The transformed strain was first cultured for one day in 10ml of MRS medium containing 10ug/ml chloramphenicol, and then the cultured strain was inoculated in 300 to 600ml of MRS CM medium to have an OD600 of 0.01. After 16 to 18 hours, when the OD600 of the culture medium reached 2 to 3, the strain was recovered according to the total number of administered mice and the number of feedings. The culture supernatant was removed by centrifugation at 7,000 rpm for 10 minutes, and the recovered strain was suspended in 1x PBS so that it could be administered at 1x10 ⁹ cfu once per animal. Strain administration was performed once a day for a total of 16 days.

The treatment efficacy experiment was started by setting the first strain administration day to Day-7, and DSS was diluted to 2 (v / v)% in sterile water and supplied as negative water from day 0 to day 6 to all groups except the PBS group. . Mice were euthanized on day 9. After DSS treatment, the survival rate was checked every 2 days, and the DAI score was calculated by checking the body weight, hair bristles, animal movement, and diarrhea. In addition, to a group to evaluate the efficacy of co-administration with etanercept (TNF blocker; Enbrel), Enbrel was intraperitoneally (IP) administered at 10 mg/kg on days 0, 2, 4, and 6.

9 is a diagram showing the efficacy of combination treatment of VIP and Enbrel derived from a LAB transformed strain in DSS-induced intestinal inflammation model by DAI AUC score. A description of each strain and chemical is described above and DAI AUC represents the area under the curve of DAI SCORE by day 8.

As shown in Figures 8 and 9, when the strain expressing VIP and Enbrel were co-administered, the DAI score was reduced compared to the group treated only with the strain or Enbrel.

Enbrel used in this experiment is an exemplary drug for treating inflammatory bowel disease, and other drugs for treating inflammatory bowel disease may be used in combination with VIP derived from a LAB transformed strain.

(3) Histopathology test method

After the experimental animals were euthanized on the ninth day of the experiment, two parts of the distal colon at a distance of 2 cm were extracted 0.5 cm each, fixed in 10% formalin solution, and embedded in paraffin. The embedded tissue was cut with a microtome at intervals of 5 μm to prepare slides, and then H&E staining was performed. For the stained tissue, the severity of the tissue was evaluated by referring to the evaluation method of Erben et al., Int J Clin Exp Pathol 2014;7(8):4557-4576, and 3 or 4 or more sites were evaluated for each sample. .

Briefly, the evaluation method was evaluated separately for the degree of damage to intestinal epithelial cells and the degree of infiltration of inflammatory cells, and the result was expressed as 0 to 8 points by adding the scores of each part.

First, the degree of infiltration of inflammatory cells was evaluated according to the following criteria:

0=normal;

1=mild-infiltrate around crypt basis,

2 = moderate-infiltrate reaching to L. muscularis mucosae,

3 = marked -extensive infiltration reaching the muscularis mucosae and thickening of the mucosa,

4=severe-infiltration of the L. submucosa.

In addition, the degree of epithelial cell damage was evaluated according to the following criteria:

0=normal, 1=focal erosions-loss of goblet cells,

2 = Exfoliation - Loss of goblet cells in large areas,

3 = erosion/ulceration-loss of crypts (focal);

4 = extended ulceration-granulation tissue, loss of crypts in large areas.

10 is a diagram showing the histopathological results of the combination treatment efficacy of VIP and Enbrel derived from a LAB transformed strain in a mouse intestinal inflammation model induced by DSS. In FIG. 10, Normal, PBS, Enbrel, LMT1-46 VIP, and LMT1-46 VIP+Enbrel are as described in FIG. 9.

As shown in Figure 10, when the strain expressing VIP and Enbrel were co-administered, the treatment efficacy was improved compared to the group treated with only the strain or Enbrel.

Specifically, as shown in FIG. 10, a histopathological examination of the large intestine was performed through a microscope in the DSS-induced intestinal inflammation mouse model, and mononuclear cell infiltration and mucosal damage were most severe in the DSS group. , the group administered with Enbrel alone did not show significant improvement in symptoms. On the other hand, LMT1-46 VIP alone and LMT 1-46 VIP & Enbrel combined treatment groups showed a tendency to reduce histological damage and inflammatory infiltrate lesions, like the CyA-administered group used as a positive control group. This can be seen that the Lactobacillus strain expressing VIP has anti-inflammatory and protective effects in the DSS-induced inflammatory bowel disease mouse model.

Korea Research Institute of Bioscience and Biotechnology KCTC13421BP 20171212 Korea Research Institute of Bioscience and Biotechnology KCTC13422BP 20171212 Korea Research Institute of Bioscience and Biotechnology KCTC13423BP 20171212

SEQUENCE LISTING <110> LIVEOME INC. <120> Microorganism expressing heterologous protein, and use thereof <130> PN144077KR <160> 14 <170> PatentIn version 3.5 <210> 1 <211> 28 <212> PRT <213> Homo sapiens <400> 1 His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Arg Gln 1 5 10 15 Leu Ala Val Arg Arg Tyr Leu Asn Ser Ile Leu Asn 20 25 <210> 2 <211> 28 <212> PRT <213> artificial <220> <223> VIP1 <400> 2 His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Val Lys Lys Tyr Leu Asn Ser Ile Leu Asn 20 25 <210> 3 <211> 30 <212> PRT <213> artificial <220> <223> VIP Variant 2 <400> 3 His Ser Asp Ala Val Phe Thr Ala Asn Tyr Thr Arg Leu Arg Arg Gln 1 5 10 15 Leu Ala Val Arg Arg Tyr Leu Ala Ala Ile Gly Leu Arg Arg 20 25 30 <210> 4 <211> 25 <212> PRT <213> artificial <220> <223> VIP variant 3 <400> 4 Phe Thr Ala Asn Tyr Thr Arg Leu Arg Arg Gln Leu Ala Val Arg Arg 1 5 10 15 Tyr Leu Ala Ala Ile Leu Gly Arg Arg 20 25 <210> 5 <211> 28 <212> PRT <213> artificial <220> <223> VIP variant 4 <400> 5 His Ser Asp Gly Ile Phe Thr Asp Ser Tyr Ser Arg Tyr Arg Lys Gln 1 5 10 15 Met Ala Val Lys Lys Tyr Leu Ala Ala Val Leu Asn 20 25 <210> 6 <211> 28 <212> PRT <213> artificial <220> <223> VIP variant 5 <400> 6 His Ser Asp Ala Val Phe Thr Asp Asn Tyr Ala Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Val Lys Lys Ala Leu Asn Ser Ile Leu Ala 20 25 <210> 7 <211> 31 <212> PRT <213> artificial <220> <223> VIP variant 6 <400> 7 His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Val Ala Ala Lys Lys Tyr Leu Gln Ser Ile Lys Asn Lys Arg Tyr 20 25 30 <210> 8 <211> 31 <212> PRT <213> artificial <220> <223> VIP variant 7 <400> 8 His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Arg Gln 1 5 10 15 Leu Ala Val Arg Arg Tyr Leu Asn Ser Ile Leu Asn Gly Arg Arg 20 25 30 <210> 9 <211> 23 <212> PRT <213> artificial <220> <223> VIP variant 8 <400> 9 His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Arg Gln 1 5 10 15 Leu Ala Val Arg Arg Tyr Leu 20 <210> 10 <211> 500 <212> DNA <213> Lactobacillus paracasei LMT1-21 <400> 10 tccacaatca aggtgcttgg ctttttcgat cgcgaggtca ccatgtacat cagtcgtgag 60 agcattgtgt tgacagtgat cggcatcgtg ttcggctatc tgctcggcaa tttgctgaca 120 gcctacattt tgtatcaagc cgaaactgag gccgtggttt ttccactcac gatcagcatt 180 gtcggctacc tcacggccac gttactcatg ttggccttca ccggcgtcgt cacctggctc 240 acgcatcgtc gactccaacg ggtggacatg gtcgaagccc tgaaatcaaa cgaataacct 300 acaattttgt caggcagcgt cgtcacggcg ctgctttttt catacaaaat tcatcaaaaa 360 ttgggattaa aaacgttcat gatcgcaatt ttgaagcgca aatgaagatt gagaccaact 420 cctaacagtc ctgtaacgct gacgtaacat tgacacagta aagtagcctt tagttaatca 480 aattaagggt gaggtcaaaa 500 <210> 11 <211> 31 <212> PRT <213> Lactobacillus paracasei LMT1-21 <400> 11 Met Lys Phe Asn Lys Val Met Ile Thr Leu Val Ala Ala Val Thr Leu 1 5 10 15 Ala Gly Ser Ala Ser Ala Val Thr Pro Val Phe Ala Asp Thr Ser 20 25 30 <210> 12 <211> 93 <212> DNA <213> Lactobacillus paracasei LMT1-21 <400> 12 atgaaattca ataaagtcat gatcacgttg gttgctgcag ttaccttagc aggttctgct 60 agcgccgtaa caccagtttt cgctgataca agc 93 <210> 13 <211> 4259 <212> DNA <213> artificial <220> <223> pMT54-PR4-SP4 vector <400> 13 ggtacctccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 60 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 120 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 180 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 240 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 300 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 360 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 420 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 480 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 540 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 600 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca 660 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 720 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 780 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 840 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt 900 tttaaatcaa tctaaagtat atatgagtaa acttggtgaa ttcgcaaggt atagcccaat 960 atactttaact tcgtaaagtt tgagctctgc gaggcaaaaa cgtcagatat tgaatctaaa 1020 caagagtata ataaacacag ttacacaaat aaaaacaaaa ggagtagatc ttaaaaatgg 1080 aagcaaatcc aaagtggcaa gaaacaggaa gacaacaaaa agtaaaacaa cttgactatt 1140 cagaaacatt agcagtaaga atagacaatg tggaattata caactcagaa aagaaattat 1200 attatctagt aattcaaacc cttcttggga atccccaaaa aaatgaatgg cgtatccagg 1260 gacaaagtca aatattaaaa gaatcaaaac aaaaaataat tgtgtcataa aattattgca 1320 cattaaaagt cctaaaaatt attttgcatt aaagatttca aaactcattt aatgtaaaat 1380 aattttttct ttgataatcc catttagcaa caatctcgcg taccacttgg tcaacctttt 1440 gatcatcact atccgtatta atcaaatcac cattctcaac gtcttctaat tgcaactgct 1500 tgcgaatttc tttcagcaaa ccgccatagc taatttgccg ggaaccagcc aaagctcgtt 1560 ccaaatcatc aattacttgt aggtcttgtt cttgattatt agtcaaaata tctttagatt 1620 ttacctgata tttagccgtt tcttgagcac tagccaataa cgaattttta tggcgattca 1680 tattcggttt aactgcctca acattcacca ttggtacata agtcaatttc attgcccgtt 1740 gccaaaaacc agtccattct acttgtgaaa tatagttatc agtcccctta aaataatggt 1800 tcttcacaaa aagcaaaaca tgcatatgat ggtggtacat tggctgaccc gcttcatgat 1860 taacagtcac ttcagtcgaa cgtacatagc ccaataaatt tttggcaact tttgtatact 1920 gaaaaatttt tgcaacagct cgtcccattt gacgtaactc actcttcaat tgatcaccag 1980 ttgtattctc aaccgtcaac gttaaaaata agaaccggcc cgtctttcgt tgtttaacag 2040 cttccgtcaa aatctgtgtt aactgattgg attgcttcat tgcccgccgc caattacata 2100 acggacacaa acgagaatgg caaaaccaag tctgcgccaa tttcttgtga ccatttttat 2160 cttccacaaa acgcaaaact tcgccacatt ctttaactcg atgagctttc ttataatgca 2220 atatttgtaa atagtcacca tactgcaagt tctccaactt gcgctcccgc cacggccgaa 2280 ctttccctga ctttgaccga tcaaccaaga ctttttcatt agccaaaata aaaactcccc 2340 tcaccaacca cgtgagaaga gttaatctgt tatgtacttg cttcacttaa atcagtcaga 2400 aggcttgacg gcaaagggtt caagctttaa actatgtcta gtaaatccaa atacgatttt 2460 tacaagatta actcacgcgt tcgaggtcgg caaactttcg aagctcacgt gggttttttt 2520 tatatttatt ttataccaca ataatacgcc taaacccagt tgtgtcaagg gtttacctca 2580 ctttttgaaa atgacgttgt ttctaatagt atcaagataa gaagaaaccg tcgaaaaaac 2640 gacggtttca aaccccaaaa agcagagaat tcggtacctg gagctgtaat ataaaaacct 2700 tcttcaacta acggggcagg ttagtgacat tagaaaaccg actgtaaaaa gtacagtcgg 2760 cattatctca tattataaaa gccagtcatt aggcctatct gacaattcct gaatagagtt 2820 cataaacaat cctgcatgat aaccatcaca aacagaatga tgtacctgta aagatagcgg 2880 taaatatatt gaattacctt tattaatgaa ttttcctgct gtaataatgg gtagaaggta 2940 attactatta ttattgatat ttaagttaaa cccagtaaat gaagtccatg gaataataga 3000 aagagaaaaa gcattttcag gtataggtgt tttgggaaac aatttccccg aaccattata 3060 tttctctaca tcagaaaggt ataaatcata aaactctttg aagtcattct ttacaggagt 3120 ccaaatacca gagaatgttt tagatacacc atcaaaaatt gtataaagtg gctctaactt 3180 atcccaataa cctaactctc cgtcgctatt gtaaccagtt ctaaaagctg tatttgagtt 3240 tatcaccctt gtcactaaga aaataaatgc agggtaaaat ttatatcctt cttgttttat 3300 gtttcggtat aaaacactaa tatcaatttc tgtggttata ctaaaagtcg tttgttggtt 3360 caaataatga ttaaatatct cttttctctt ccaattgtct aaatcaattt tattaaagtt 3420 catttgatat gcctcctaaa tttttatcta aagtgaattt aggaggctta cttgtctgct 3480 ttcttcatta gaatcaatcc ttttttaaaa gtcaatatta ctgtaacata aatatatatt 3540 ttaaaaatat cccactttat ccaattttcg tttgttgaac taatgggtgc tttagttgaa 3600 gaataaagac cacattaaaa aatgtggtct aagcttctgc aggatatccg atcgtccaca 3660 atcaaggtgc ttggcttttt cgatcgcgag gtcaccatgt acatcagtcg tgagagcatt 3720 gtgttgacag tgatcggcat cgtgttcggc tatctgctcg gcaatttgct gacagcctac 3780 attttgtatc aagccgaaac tgaggccgtg gtttttccac tcacgatcag cattgtcggc 3840 tacctcacgg ccacgttact catgttggcc ttcaccggcg tcgtcacctg gctcacgcat 3900 cgtcgactcc aacgggtgga catggtcgaa gccctgaaat caaacgaata acctacaatt 3960 ttgtcaggca gcgtcgtcac ggcgctgctt ttttcataca aaattcatca aaaattggga 4020 ttaaaaacgt tcatgatcgc aattttgaag cgcaaatgaa gattgagacc aactcctaac 4080 agtcctgtaa cgctgacgta acattgacac agtaaagtag cctttagtta atcaaattaa 4140 gggtgaggtc aaaaatgaaa ttcaataaag tcatgatcac gttggttgct gcagttacct 4200 tagcaggttc tgctagcgcc gtaacaccag ttttcgctga tacaagcgtc gacctcgag 4259 <210> 14 <211> 84 <212> DNA <213> artificial <220> <223> VIP variant 1 <400> 14 cattcagatg ctgttttcac cgataactat acccgtttgc gtcgtcaatt ggctgttcgt 60 cgttatttga actcaatctt gaac 84

Claims (1)

Lactobacillus paracasei LMT1-21 (KCTC 13422BP).

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