EXOGENOUS PROTEIN-EXPRESSING MICROORGANISM AND USE THEREOF

Abstract

Disclosed are a VIP gene expressing microorganism, a composition or a kit for preventing or treating a disorder causing gastrointestinal tract damage comprising the same, and a method for preventing or treating a disorder causing gastrointestinal tract damage using the same.

Claims

1. A recombinant microorganism, comprising a promoter, and a heterologous gene encoding vasoactive intestinal peptide (VIP) operably linked to the promoter, wherein the microorganism belongs to the genus of Lactobacillus.

2. The microorganism of claim 1, which is Lactobacillus paracasei, Lactobacillus brevis, or Lactobacillus plantarum.

3. A recombinant microorganism, comprising a constitutive promoter, and a heterologous gene encoding vasoactive intestinal peptide (VIP) operably linked to the promoter, wherein the microorganism is a lactic acid bacterium.

4. The microorganism of claim 3, wherein the constitutive promoter consists of the sequence as set forth in SEQ ID No. 10.

5. The microorganism of claim 3, wherein the lactic acid bacterium belongs to the genus of Lactobacillus, or Lactococcus.

6. The microorganism of claim 5, wherein the lactic acid bacterium is Lactobacillus paracasei, Lactobacillus brevis, Lactobacillus plantarum, or Lactococcus lactis.

7. The microorganism of claim 1, further comprising an operably linked signal sequence between the promoter and the heterologous gene.

8. The microorganism of claim 7, wherein the signal sequence encodes a signal peptide consisting of the amino acid sequence as set forth in SEQ ID No. 11.

9. A composition for preventing or treating a disorder causing gastrointestinal tract damage in human, which comprises a recombinant microorganism comprising a promoter, and a heterologous gene encoding vasoactive intestinal peptide (VIP) operably linked to the promoter, wherein the microorganism is a lactic acid bacterium.

10. The composition of claim 9, wherein the disorder causes gastrointestinal tract inflammation.

11. The composition of claim 10, wherein the disorder is one or more selected from the group consisting of inflammatory bowel disease (IBD), and colitis.

12. The composition of claim 11, wherein the inflammatory bowel disease is ulcerative colitis, or Crohn's disease.

13. The composition of claim 9, which is an oral dosage form.

14. The composition of claim 9, wherein the lactic acid bacterium belongs to the genus of Lactobacillus, or Lactococcus.

15. The composition of claim 14, wherein the lactic acid bacterium is Lactobacillus paracasei, Lactobacillus brevis, Lactobacillus plantarum, or Lactococcus lactis.

16. The composition of claim 9, wherein the promoter is a constitutive promoter.

17. The composition of claim 16, wherein the constitutive promoter consists of the nucleotide sequence of SEQ ID No. 10.

18. The composition of claim 9, wherein the recombinant microorganism further comprises an operably linked signal sequence between the promoter and the heterologous gene.

19. The composition of claim 18, wherein the signal sequence encodes a signal peptide consisting of the amino acid sequence of SEQ ID No. 11.

20. The composition of claim 9, further comprising a therapeutic agent for treating a disorder causing gastrointestinal tract damage.

21. (canceled)

22. (canceled)

Description

BRIEF DESCRIPTION OF DRAWINGS

[0058] FIG. 1 is a construction map of pMT447 vector.

[0059] FIG. 2 shows the results of western blotting of culture supernatant of L. plantarum LMT1-9, L. paracasei LMT1-21, and L. brevis LMT1-46 strains transformed with pMT447 vector.

[0060] FIG. 3 shows the therapeutic efficacy of VIP expressed from L. plantarum LMT1-9 transformed strain as DAI score in DSS induced mouse intestinal inflammation model.

[0061] FIG. 4 shows the therapeutic efficacy of VIP expressed from L. paracasei LMT1-21 transformed strain as DAI score in DSS induced mouse intestinal inflammation model.

[0062] FIG. 5 shows the therapeutic efficacy of VIP expressed from L. brevis LMT1-46 transformed strain as DAI score in DSS induced mouse intestinal inflammation model.

[0063] FIG. 6 shows the therapeutic efficacy of VIP expressed from transformed strain as DAI AUC score in DSS induced mouse intestinal inflammation model.

[0064] FIG. 7 shows the therapeutic efficacy of VIP expressed from transformed strain histopathologically in DSS induced mouse intestinal inflammation model.

[0065] FIG. 8 shows the therapeutic efficacy of combination of VIP expressed from transformed LAB strains and etanercept (Enbrel, Pfizer Korea) as DAI score in DSS induced mouse intestinal inflammation model.

[0066] FIG. 9 shows the therapeutic efficacy of combination of VIP expressed from transformed LAB strains and Enbrel as DAI AUC score in DSS induced mouse intestinal inflammation model.

[0067] FIG. 10 shows the therapeutic efficacy of combination of VIP expressed from transformed strain and Enbrel histopathologically in DSS induced mouse intestinal inflammation model.

BEST MODE

[0068] Hereunder, detailed descriptions are provided with reference to examples. However, the examples are provided only for illustrative purpose, but not intended to limit the scope of the present disclosure in any manner.

Example 1: Lactic Acid Bacteria Expressing VIP Gene, and their Therapeutic Efficacy for IBD

[0069] In the present example, VIP gene was introduced to lactic acid bacterial cells, and it was tested if the gene was expressed and extracellularly secreted. Furthermore, the lactic acid bacterial cells transformed with VIP gene were orally administered to an individual and tested for improvement in symptoms to show their efficacy for treating IBD. As VIP, a human VIP variant consisting of the amino acid sequence of SEQ ID No. 1 was used. The VIP variant has 4 amino acid substitutions in human natural VIP consisting of the amino acid sequence of SEQ ID No. 2, thereby to have an increased half-life in the human bloodstream.

[0070] 1. Construction of VIP Gene-Containing Vector

[0071] DNA synthesized by Macrogen, Inc. (South Korea) was used as VIP gene consisting of the nucleotide sequence of SEQ ID No. 14. The synthesized VIP gene fragment and Lactic Acid Bacteria (LAB)-E. coli shuttle vector pMT54-PR4-SP4 (SEQ ID No. 13) were cleaved with restrictive endonucleases SalI and XhoI, respectively. The cleaved gene fragment and vector in the reaction mixture were purified using a gel purification kit (GeneAll Biotechnology, Co., Ltd.), and then, were incubated in the presence of an alkaline phosphatase to obtain dephosphorylated cleaved VIP gene fragment and cleaved pMT54-PR4-SP4 vector.

[0072] The vector DNA of 1 μl, the VIP gene DNA of 3 μl, 0.5 μl of T4 DNA ligase (Takara), 1 μl of a buffer solution, and 5.5 μl of distilled water were mixed together in a test tube to make a total volume to 10 μl, and then, the mixture was incubated at 16° C. for 12 hours to link the VIP gene with the vector fragment to obtain the VIP gene-containing vector. E. coli competent cells (TOP10 Competent Cells) were transformed with the vector according to the method of Sambrook et al. (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edn, 1989).

[0073] The transformed E. coli was spread onto an LB (Luria-Bertani)-agar plate containing 10 μl/ml of chloramphenicol, and then, incubated. As a result, formed colonies were selected, cultivated, and the vector was isolated from the culture. The vector was designated as pMT447. The pMT447 vector comprises the VIP gene operably linked to PR4 promoter and SP4 signal sequence. Therefore, the cells transformed with pMT447 vector expresses and secretes the VIP gene extracellularly in a high efficiency.

[0074] FIG. 1 shows the construction of pMT447 vector. In FIG. 1, the promoter is the sequence (SEQ ID No. 10) of PR4 promoter, SP is the nucleotide sequence (SEQ ID No. 11) of SP4 signal peptide, and VIP is the sequence (SEQ ID No. 14) of gene encoding human VIP variant of SEQ ID No. 1. HA and 6×His is the nucleotide sequence of human influenza hemagglutinin tag and 6 histidine tag for detection, isolation, and purification of protein. E. coli ori, Rep, and CM are the replication origin of E. coli, the replication origin of lactic acid bacteria, and gene for chloramphenicol resistance marker, respectively.

[0075] 2. Introduction of Recombinant Vector to Lactic Acid Bacterial Cells

[0076] The recombinant vector pPM447 constructed in the above 1 were introduced to Lactobacillus plantarum LMT1-9 (KCTC 13421BP), Lactobacillus paracasei LMT1-21 (KCTC 13422BP), and Lactobacillus brevis LMT1-46 (KCTC 13423BP), respectively, and then, were tested for the secretion of VIP protein.

[0077] Each strain was cultivated to reach OD.sub.600 of 0.5 in a flask containing 50 mL of MRS medium (Difco Co., USA), and then, centrifuged at 4° C., 7,000 rpm for 10 minutes. Cell pellets were washed with 25 mL of cold EPS (ice-cold electroporation solution) (containing 1 mM K.sub.2HPO.sub.4, 1 mM KH.sub.2PO.sub.4, pH 7.4, 1 mM MgCl.sub.2, and 0.5 M sucrose) twice. MRS is also called as De Man, Rogosa and Sharpe agar medium.

[0078] After washing, the cells were re-suspended in 1 ml of cold EPS to prepare competent cells for electroporation and kept in a deep freezer of −80° C. The competent cells of 40 μl and the respective vector DNA (1 μg/μl) of 1 μl were added to a cuvette, and allowed to stand in ice for 5 minutes. Electric pulse was imposed on the cells under the condition of 25 pf, 8 kV/cm, 400 ohms, and then, the cells were immediately added to 1 ml of MRS liquid medium and cultivated at 37° C. for one hour. Then, the cultivated cells were spread onto MRS medium (also known as ‘MRS-CM’) containing 10 μg/ml of chloramphenicol and cultivated at 37° C. for 48 hours.

[0079] The obtained pMT447 vector-introduced strains were statically cultivated in MRS liquid medium at 37° C. for 16 hours, respectively. The culture was inoculated to 3% (v/v) in MRS liquid medium, and then, statically cultivated for 8 hours at the same temperature. The culture of 1 ml was centrifuged at 7,000 rpm for 5 minutes to take its supernatant. To the supernatant of 1 ml was added trichloroacetic acid of 100 μl to obtain a mixture and the mixture was allowed to stand at 4° C. for one hour to concentrate the culture. The culture was centrifuged at 13,000 rpm for 4° C., and the pellets were washed with 1 ml of cold acetone, dried at room temperature for 10 minutes, and eluted with Tris-HCl buffer (pH 8.8) of 100 μl.

[0080] To the elute were added 4× loading buffer (Thermo) and 10× reducing agent (Thermo), and electrophoresis was performed on SDS-PAGE gel. The gel was transferred onto nitrocellulose membrane with Trans blot semi-dry cell (bio-rad) to perform western blotting. Specifically, the membrane was blocked with TBST buffer containing 1% skim milk for one hour, reacted with anti-HA antibody (Santa cruz) at room temperature for 2 hours, and then, washed with TBST for 5 minutes three times and detected with ECL. In pMT447 vector, VIP gene was operably liked with hemagglutinin (HA) gene at its 3′ terminal, so was expressed in HA tagged form.

[0081] FIG. 2 shows the results of western blotting of culture supernatants of pMT447 vector-transformed L. plantarum LMT1-9, L. paracasei LMT1-21, and L. brevis LMT1-46 strains. In FIG. 2, lanes 2, 3, and 4 represent pMT447 vector-transformed L. plantarum LMT1-9, L. paracasei LMT1-21, and L. brevis LMT1-46, respectively, and lane 1 represents a standard protein ladder.

[0082] As shown in FIG. 2, the transformed strains efficiently secreted hVIP variants.

[0083] 3. Efficacy of VIP-Secreting Lactic Acid Bacteria for IBD in DSS Induced Model

[0084] The transformed L. plantarum LMT1-9, L. paracasei LMT1-21, and L. brevis LMT1-46 as described in 2 were orally administered to mouse colitis model induced with dextran sulfate sodium salt (DSS), and the survival rate and disease activity index (DAI) score of mice were measured to evaluate the therapeutic efficacy of the strains on intestinal inflammation.

[0085] (1) Administration of Recombinant Microorganisms Alone

[0086] Specifically, the transformed strains were statically cultivated primarily in a flask containing MRS medium of 10 ml containing 10 μg/ml of chloramphenicol at 37° C. for 24 hours. On the next day, the cultivated strains were inoculated on 300 to 600 ml of MRS CM medium to reach OD.sub.600 of 0.01, and secondarily cultivated under the same condition as the primary cultivation. After 16 to 18 hours, when OD.sub.600 of the culture reached 2 to 3, the culture was centrifuged at 7,000 rpm for 10 minutes to discard the supernatant, and cells were suspended in 1×PBS so as to be administered at 1×10.sup.9 cfu as a single dose per mouse. This cell-containing PBS solution was administered orally (1×10.sup.9 cfu as a single dose per mouse) with drinking water once a day for 16 days.

[0087] First, male Balb/c mice with the age of 8 to 10 weeks (20 to 25 g, 10 mice per group) (ORIENTBIO) were used as experimental animals. The day when strains were administered initially was set Day −7, and DSS diluted to 2% (v/v) in distilled water was supplied as drinking water on Day 0 to Day 6 to all groups but PBS group. Mice took drinking water voluntarily, but at an amount of 6 ml or less a day generally. The mice were sacrificed on Day 9.

[0088] After DSS was administered, survival rate was measured every other day and body weight, hair-raising, movement, and diarrhea were checked to obtain DAI score.

[0089] Disease activity index (DAI) scoring was performed with reference to Ameho, Gut 1997; 41:487˜493 and Wallace, Gastroenterology 1989; 96: 29˜36. The results were shown in FIG. 3 to FIG. 6.

[0090] FIG. 3 shows the therapeutic efficacy of VIP expressed from the transformed L. plantarum LMT1-9 strain in DSS-induced mouse intestinal inflammation model as DIA score. In FIG. 3, 1-9 VIP represents pMT447-transformed L. paracasei LMT1-9, and 1-9 vector represents control vector (parental vector having no VIP gene)-transformed L. plantarum LMT1-9.

[0091] FIG. 4 shows the therapeutic efficacy of VIP expressed from the transformed L. plantarum LMT1-21 strain in DSS-induced mouse intestinal inflammation model as DIA score. In FIG. 4, 1-21 VIP represents pMT447-transformed L. plantarum LMT1-21, and 1-21 vector represents control vector (parental vector having no VIP gene)-transformed L. plantarum LMT1-21.

[0092] FIG. 5 shows the therapeutic efficacy of VIP expressed from the transformed L. brevis LMT1-46 in DSS-induced mouse intestinal inflammation model as DIA score. In FIG. 5, 1-46 VIP represents pMT447-transformed L. brevis LMT1-46, and 1-46 vector represents control vector (parental vector having no VIP gene)-transformed L. brevis LMT1-46.

[0093] In FIG. 3 to FIG. 5, CyA is the abbreviation of cyclosporine A, which was used as a positive control of the experiments. PBS represents a control group treated with DSS, not with the bacteria. Normal represents a group treated neither DSS nor the bacteria. FIG. 6 shows the therapeutic efficacy of VIP expressed from the transformed strains as DAI AUC score in DSS-induced mouse intestinal inflammation model. Each strain and chemical are as described above and DAI AUC represents area under curve of DAI score until Day 8.

[0094] As shown in FIGS. 3 to 6, the strains transformed to express VIP showed the therapeutic efficacy in DSS-induced intestinal inflammation model.

[0095] FIG. 7 shows the therapeutic efficacy of VIP expressed from the transformed strains histopathologically in DSS-induced mouse intestinal inflammation model. 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 for FIGS. 1 to 6.

[0096] As shown in FIG. 7, mice administered with VIP-expressing Lactobacillus strains showed the therapeutic efficacy compared with the control group in DSS-induced intestinal inflammation mouse model, as demonstrated by histopathological examination on the colon with a microscope.

[0097] Specifically, as shown in FIG. 7, histopathological examination was performed with a microscope on the colon in DSS-induced intestinal inflammation mouse model. As a result, while monocyte infiltration, mucosal damage, etc. were the severest in DSS group and no significant effect was observed in LMT 1-9 vector, LMT 1-21 vector, and LMT 1-46 vector administration groups, histological damage and inflammatory infiltration were reduced in LMT 1-9 VIP, LMT 1-21 VIP, and LMT1-46 VIP administration groups. This suggests that the VIP-expressing Lactobacillus strains have inflammation ameliorating and protective effects in DSS-induced inflammatory bowel disease mouse model.

[0098] (2) Effects of the Combination of the Recombinant Microorganism and a TNF-Alpha Blocker

[0099] FIG. 8 shows the therapeutic efficacy of the combination of VIP expressed from transformed LAB strains and etanercept (Enbrel, Pfizer Korea) as DAI score. In FIG. 8, Normal represents non-treatment group. PBS represents only DSS-treatment group. Enbrel represent DS S and Enbrel-treatment group. 1-46 VIP represents a group administered with L. brevis LMT1-46 transformed with pMT447 in DSS-induced mice. 1-46 VIP+Enbrel represents a group administered with L. brevis LMT1-46 transformed with pMT447 and Enbrel together in DSS-induced mice.

[0100] FIG. 8 shows the results obtained by the following procedures. To evaluate the therapeutic efficacy of VIP expressed from transformed strains, the transformed stains were orally administered to DSS-induced mouse intestinal inflammatory model to measure the survival rate and DAI score. The transformed strains were primarily cultivated in 10 ml of MRS medium containing 10 μl/ml chloramphenicol for one day, and the cultivated strains were inoculated onto 300 to 600 ml of MRS CM medium to reach OD.sub.600 of 0.01. After 16 to 18 hours, when OD.sub.600 of the culture was reached 2-3, the strains were recovered considering the number of mice and administration times. The culture supernatant was discarded by centrifugation at 7,000 rpm for 10 minutes, and the recovered strains were suspended in 1×PBS to be administered at 1×10.sup.9 cfu as a single dose per mouse. The strain administration was performed once a day for 16 days in total.

[0101] The initial administration day was set Day −7 to start the therapeutic efficacy evaluation test. DSS was diluted to 2% (v/v) in distilled water, which was supplied to all groups but PBS group as drinking water over Day 0˜Day 6. On Day 9, mice were euthanized. The survival rate was checked every other day since DSS treatment, and body weight, hair raising, animal's movement, diarrhea were checked to calculate DAI score. To evaluate the efficacy of the combination with Etanercept (TNF blocker; Enbrel), Enbrel was intraperitoneally administered to the group at a dosage of 10 mg/kg on Day 0, 2, 4, and 6.

[0102] FIG. 9 shows the therapeutic efficacy of the combination of VIP expressed from transformed LAB strains and Enbrel as DAI AUC score in DSS-induced mouse intestinal inflammation model. Each strain and each chemical were as described above. DAI AUC represents the area under the curve of DAI score until Day 8.

[0103] As shown in FIG. 8 and FIG. 9, the group treated with combination of the VIP expressing strains and Enbrel showed reduced DAI score compared with the strain treatment group or Enbrel treatment group.

[0104] Enbrel used in this experiment is only an illustrative agent for treating inflammatory bowel disease, and other agents for treating inflammatory bowel disease could be used in combination with VIP expressed from transformed LAB strains.

[0105] (3) Histopathology Test

[0106] The animals were euthanized on the 9.sup.th day after starting the experiment. Two parts with the distance of 2 cm in the distal area of the colon were extracted at the length of 0.5 cm, respectively, and were fixed in 10% formalin solution and paraffin embedded. The embedded tissue was cut with a microtome to pieces having the length of 5 μm to prepare slides and stained with H&E. The stained tissue was tested for severity with referring to the evaluation method of Erben et al., Int J Clin Exp Pathol 2014; 7(8):4557-4576, which was performed three, or four or more random parts per sample.

[0107] The evaluation method may be briefly described as below. Intestinal epithelial cells damage and inflammatory cell infiltration was separately evaluated and two scores were summed to obtain scores ranging from 0 to 8.

[0108] Inflammatory cell infiltration was evaluated according to the following standard: [0109] 0=normal, [0110] 1=mild-infiltrate around crypt basis, [0111] 2=moderate-infiltrate reaching to L. muscularis mucosae, [0112] 3=marked-extensive infiltration reaching the muscularis mucosae and thickening of the mucosa, [0113] 4=severe-infiltration of the L. submucosa.

[0114] Epithelial cell damage was evaluated according to the following standard: [0115] 0=normal, [0116] 1=focal erosions-loss of goblet cells, [0117] 2=erosions-loss of goblet cells in large areas, [0118] 3=erosion/ulceration-loss of crypts (focal), [0119] 4=extended ulceration-granulation tissue, loss of crypts in large areas.

[0120] FIG. 10 shows the therapeutic efficacy of the combination of VIP expressed from transformed LAB strains and Enbrel histopathologically in DSS-induced mouse intestinal inflammation model. In FIG. 10, Normal, PBS, Enbrel, LMT1-46 VIP, and LMT1-46 VIP+Enbrel are as described for FIG. 9.

[0121] As shown in FIG. 10, the group administered with the combination of VIP-expressing strains and Enbrel showed improved therapeutic effect compared with the group administered with Enbrel only.

[0122] Specifically, as shown in FIG. 10, histopathology examination was performed with a microscope on the colon in DSS-induced intestinal inflammation model. As a result, DSS group showed the severest monocyte infiltration and mucosal damage, and Enbrel treatment group showed not so much improvement on such pathologies. In contrast, LMT1-46 VIP treatment group as well as LMT 1-46 VIP & Enbrel combination treatment group showed reduced histological damage and inflammatory infiltration lesions as in CyA treatment group used as a positive control. This supports that the VIP-expressing Lactobacillus strains have inflammation ameliorating and protective effects in DSS-induced inflammatory bowel disease mouse model.