Recombinant protein for preventing or treating ulcerative colitis and composition for preventing or treating ulcerative colitis containing the same

Abstract

The present invention relates to a recombinant protein for preventing or treating ulcerative colitis, which is recombined based on Milk fat globule-EGF factor 8 (MFG-E8) protein and has the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, and is expected to prevent or treat ulcerative colitis when administered to a subject for prevention or treatment. The present invention also relates to a pharmaceutical composition comprising, as an active ingredient, a recombinant protein for preventing or treating ulcerative colitis, which is recombined based on Milk fat globule-EGF factor 8 (MFG-E8) protein so as to have the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3.

Description

Technical Field

[0001] The present invention relates to recombinant proteins for preventing or treating ulcerative colitis and related embodiments for various uses including compositions for preventing or treating ulcerative colitis containing the same.

Background Art

[0002] Ulcerative colitis appears in a form in which inflammation occurs in the large intestine and inflammatory cells invade the mucosa and submucosa. Its typical symptom is bloody stool, and further symptoms include diarrhea, mucoid stool, abdominal pain, irresistible urge to defecate, constipation, etc., and systemic symptoms such as weight loss, fever, loss of appetite, general weakness, nausea, and vomiting may also appear. Normal large intestine tissue is divided into mucosa, submucosal tissue, muscular layer, and serosa. However, pathological changes occur in the mucosa and submucosal tissue due to the development of ulcerative colitis, and the exact cause is still unknown. It is a chronic relapsing disease mainly caused by environmental factors, genetic factors, and excessive immune responses of the body to bacteria normally present in the intestine. Such ulcerative colitis has conventionally had a high incidence rate mainly in meat-eating regions such as the United States and Europe. In the United States, about one million patients occur every year. Recently, however, the number of patients has been increasing in Asian regions including Korea. The number of inpatients and outpatients with ulcerative colitis in Korea with injury code K51 in 2020 has increased by about 1.7 times compared to 2010, and the medical treatment allowance cost has also increased by more than three times compared to 2010, reaching about 163.2 billion won. In addition, 5-aminosalicylic acid (5-ASA) and biological agents are used as standard drug treatment agents for ulcerative colitis, but 20% - 40% of patients fail drug treatment or undergo colectomy due to side effects. In relation to this, as a prior art of a pharmaceutical composition for treating ulcerative colitis, there is "Pharmaceutical composition for treating ulcerative colitis" (Patent Document 1) in Korean Registered Patent Publication No. 10-2319078.

Summary of the Invention

Problems to be Solved by the Invention

[0003] The present invention has been devised to solve the above problems, and an object of the present invention is to provide a recombinant protein for preventing or treating ulcerative colitis that can delay the onset of ulcerative colitis or treat the disease, and a composition containing the same as an active ingredient.

Means for Solving the Problems

[0004] To achieve the above object, the recombinant protein for preventing or treating ulcerative colitis according to the present invention is a recombinant protein based on the Milk fat globule-EGF factor 8 (MFG-E8) protein, and consists of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3. Here, the recombinant protein for preventing or treating ulcerative colitis consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 recombined based on the MFG-E8 protein can reduce the degree of crypt damage, the severity of inflammation cell invasion, and the extent of inflammation cell invasion in the mucosal layer of the large intestine tissue to be prevented or treated, as compared with the non-administration situation of the recombinant protein. In addition, the recombinant protein for preventing or treating ulcerative colitis consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 recombined based on the MFG-E8 protein can reduce the degree of erosion and the degree of epidermal hyperplasia of the mucosa in the large intestine tissue to be prevented or treated, as compared with the non-administration situation of the recombinant protein. In addition, the recombinant protein for preventing or treating ulcerative colitis, which consists of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 based on the MFG-E8 protein, can reduce the blood interleukin-6 (IL-6) concentration in the subject to be prevented or treated compared to the non-administration situation of the recombinant protein. Furthermore, the recombinant protein for preventing or treating ulcerative colitis, which consists of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 based on the MFG-E8 protein, can reduce the degree of weight loss and the degree of reduction in the length of the large intestine induced by ulcerative colitis in the subject to be prevented or treated compared to the non-administration situation. Moreover, the recombinant protein for preventing or treating ulcerative colitis, which consists of the amino acid sequence of SEQ ID NO: 1 based on the MFG-E8 protein, may be formulated for intralesional administration, intravascular administration, subcutaneous administration, intranasal administration, or intraperitoneal administration. On the other hand, in order to achieve the above object, the composition for preventing or treating ulcerative colitis according to the present invention contains, as an active ingredient, a recombinant protein for preventing or treating ulcerative colitis, which consists of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 based on milk fat globule-EGF factor 8 (MFG-E8). And, in order to achieve the above object, the present invention provides, as another aspect, a gene encoding a recombinant protein for preventing or treating ulcerative colitis, which consists of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 based on milk fat globule-EGF factor 8 (MFG-E8) protein. In addition, in order to achieve the above object, the present invention further provides, as another aspect, a recombinant vector containing a gene encoding a recombinant protein for preventing or treating ulcerative colitis, which consists of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 based on milk fat globule-EGF factor 8 (MFG-E8) protein.

Advantages of the Invention

[0005] According to the present invention, the following effects can be obtained. First, by administering the recombinant protein for preventing or treating ulcerative colitis, it is possible to expect prevention or treatment of ulcerative colitis that has developed in the subject to be prevented or treated. Second, the recombinant protein for preventing or treating ulcerative colitis can significantly reduce the degree of crypt damage of the mucosa in the large intestine tissue of the subject to be prevented or treated, the severity of inflammation cell invasion, and the extent of inflammation cell invasion. Third, the recombinant protein for preventing or treating ulcerative colitis can significantly reduce the degree of erosion and the degree of epidermal hyperplasia of the mucosa in the large intestine tissue of the subject to be prevented or treated. Fourth, the recombinant protein for preventing or treating ulcerative colitis can significantly reduce the concentration of interleukin-6 (IL-6) in the blood of the subject to be prevented or treated. Fifth, the recombinant protein for preventing or treating ulcerative colitis can significantly reduce the degree of weight loss and the degree of reduction in the length of the large intestine induced by ulcerative colitis in the subject to be prevented or treated.

Brief Description of the Drawings

[0006]

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[0007] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Preferred embodiments of the present invention will now be described in more detail with reference to the accompanying drawings, in which well-known technical details will be omitted or simplified for the sake of brevity.

[0008] 1. Explanation of the recombinant protein (NP-011) for the prevention or treatment of ulcerative colitis The process for obtaining the recombinant protein (NP-011) for preventing or treating ulcerative colitis according to the present invention and the structural characteristics of the obtained protein will be described in detail below with reference to FIGS. 1 to 5.

[0009] (1) Obtaining recombinant protein (NP-011) for the prevention or treatment of ulcerative colitis First, to explain the expression process of the recombinant protein (NP-011) for preventing or treating ulcerative colitis, we purchased Origene's MFG-E8 (NM_005928) human cDNA clone (Cat. No. RG217163) to modify the MFG-E8 protein structure. PCR was performed using this as a template to obtain DNA fragments as shown in Figure 2 or Figure 4. Subsequently, cloning was performed by inserting the DNA fragments shown in Figure 2 or 4 obtained by PCR amplification into the HindIII and SalI restriction enzyme sites (site A in Figure 5) of the pLGCF vector, which is a mammalian expression vector with a structure as shown in Figure 5. The plasmid DNA was then extracted and transfected into HEK293 cells. Two days later, the culture medium was collected and subjected to immunoprecipitation (IP) using FLAG resin. Expression of the recombinant protein (NP-011) for preventing or treating ulcerative colitis was confirmed by Western blotting. Colonies containing the intended NP-011 base sequence (the base sequence of SEQ ID NO: 2 or 4 in Figure 2 or Figure 4) were selected and used for mass production and purification. Next, the mass production process of the recombinant protein for preventing or treating ulcerative colitis (NP-011) will be explained. After confirming expression, a large amount of plasmid DNA is obtained using Maxi Prep, and then HEK293 cells are prepared and a large amount of plasmid DNA is transfected into the HEK293 cells, allowing for the mass production of the recombinant protein for preventing or treating ulcerative colitis (NP-011). Next, the selected colonies were cultured in 50 mL flasks, and NP-011 expression was verified to select the most productive clone. The culture medium was then cultured for 7 days in a 5 L fed-batch culture (pH 6.0). The culture medium was then primarily collected and used to remove impurity proteins using a Q-Sepharose column. Secondarily, pure NP-011 was purified to a purity of over 95% by centrifugation using an Sp-Sepharose column to adsorb only the NP-011 protein. In this purification process, since the FLAG gene is expressed at the C-terminus of each protein, only the target protein is bound using FLAG affinity resin, and then proteins other than the target protein can be removed by washing with washing buffer. Then, pure target protein is extracted using elution buffer, and the final protein is confirmed by SDS-PAGE. The production and purity of the target protein can be confirmed by Coomassie Blue staining and Western blotting using anti-FLAG antibody. The series of specific processes described above for cloning, mass production, and purification for the expression of the recombinant protein (NP-011) for preventing or treating ulcerative colitis, which is prepared by recombination based on the Milk fat globule-EGF factor 8 (MFG-E8) protein, are not limited thereto, and can be implemented in various ways using techniques known and obvious to those skilled in the art for constructing the specific amino acid sequence structure (SEQ ID NO: 1 (FIG. 1) or SEQ ID NO: 3 (FIG. 3)) of the recombinant protein described below. For example, various techniques can be employed for cloning the recombinant protein (NP-011) for preventing or treating ulcerative colitis, which is prepared by recombination based on the milk lipid globulin EGF-8 (MFG-E8) protein, including the structure of the expression vector, the transformation target, the production conditions and form, and the purification conditions and form. In addition, in relation to the expression of the recombinant protein (NP-011) for preventing or treating ulcerative colitis, which is prepared by recombination based on the milk globulin-like growth factor-8 (MFG-E8) protein, the structure of the DNA fragments cloned into the expression vector is also based on the structure of SEQ ID NO: 2 (Figure 2) or SEQ ID NO: 4 (Figure 4). However, depending on the implementation, a specific DNA base sequence encoding a specific signal peptide may be further linked to the DNA fragments.

[0010] (2) Structure of recombinant protein (NP-011) for preventing or treating ulcerative colitis The recombinant protein (NP-011) for preventing or treating ulcerative colitis based on milk globulin-like growth factor-8 (MFG-E8) protein, which is finally prepared through the above-mentioned processes of cloning, isolation, production, purification, etc. of the recombinant protein, has the amino acid sequence structure shown in SEQ ID NO: 1 (Figure 1) or SEQ ID NO: 3 (Figure 3) in the sequence listing below. Specifically, the recombinant protein (NP-011) for preventing or treating ulcerative colitis based on milk globulin-like growth factor factor-8 (MFG-E8) protein is recombined into a structure containing an EGF-like domain and a C1 domain, which are structural components of the MFG-E8 protein, namely, an EGF-like domain, a C1 domain, and a C2 domain. In other words, the recombinant protein (NP-011) for preventing or treating ulcerative colitis based on the milk lipid globulin EGF factor-8 (MFG-E8) protein of the present invention has a basic structure consisting of an amino acid sequence of "EGF-like domain + C1 domain," and is characterized by the structural exclusion of the C2 domain. More specifically, the recombinant protein (NP-011) for preventing or treating ulcerative colitis based on milk globulin EGF-8 (MFG-E8) protein is embodied as SEQ ID NO: 1 (Figure 1) or SEQ ID NO: 3 (Figure 3) in the sequence listing below because milk globulin EGF-8 (MFG-E8) protein has the same gene ID but exists in various versions due to mutations in single nucleotide polymorphisms (SNPs). In fact, the NCBI Human Genome Sequencing information has been updated, and the European protein database UniProtBK has updated its sequence information, which is slightly different from the Origin sequence information of milk globulin-like growth factor-8 (MFG-E8), and the 53rd amino acid in sequence number 1, M, has been modified to L, with L as the start codon. In short, the reason why the 53rd amino acid of SEQ ID NO: 3 is modified to L as underlined in Figure 3 is that, in terms of the structure of the DNA fragments as underlined in Figure 4, the triplet code corresponding to the 53rd amino acid L of SEQ ID NO: 3 in the DNA base sequence has been converted from "ATG" to "CTG" by SNP mutation. Thus, the recombinant protein (NP-011) for preventing or treating ulcerative colitis based on the milk fat globule EGF factor-8 (MFG-E8) protein of the present invention is based on all various versions due to SNP mutation, and preferably has an amino acid sequence of "EGF-like domain + C1 domain" with the "C2 domain" excluded in terms of structure. Such a recombinant protein (NP-011) for preventing or treating ulcerative colitis based on the milk fat globule EGF factor-8 (MFG-E8) protein of the present invention may be used as the main active ingredient of a pharmaceutical composition for preventing or treating ulcerative colitis. In addition, the recombinant protein (NP-011) for preventing or treating ulcerative colitis based on the milk fat globule EGF factor-8 (MFG-E8) protein of the present invention and the pharmaceutical composition having the same as the main active ingredient may be formulated for intralesional administration, intravascular (artery, vein, etc.) administration, subcutaneous administration, intranasal administration, intraperitoneal administration, or administration within a specific tissue (lung tissue). On the other hand, the recombinant protein (NP-011) for preventing or treating ulcerative colitis based on the milk fat globule EGF factor-8 (MFG-E8) protein of the present invention corresponds to the range of the structural embodiments of the DNA fragments cloned into the expression vector in relation to the expression of the aforementioned recombinant protein, and a specific signal peptide structure may be further linked by implementation. For example, the specific signal peptide structure can be implemented in a form linked to the tip of the EGF-like domain with an amino acid sequence such as "MPRPRLLAALCGALLCAPSLLVA", but is not limited thereto.

[0011] 2. Explanation of the results of the efficacy verification test of the recombinant protein (NP-011) for preventing or treating ulcerative colitis In connection with the recombinant protein (NP-011) for preventing or treating ulcerative colitis based on milk lipid-antibody EGF factor-8 (MFG-E8) protein of the present invention, the effectiveness of the recombinant protein in preventing or treating ulcerative colitis was verified through experiments. The experiments used the following experimental methods for the purpose of defining properties, etc., by means obvious to those skilled in the art.

[0012] (1) Creation of an animal model of ulcerative colitis and experimental design First, in an animal model in which ulcerative colitis is induced in mice using DSS (dextran sulfate sodium), bloody stools, weight loss, colon shrinkage, and mucosal ulcers occur. Histologically, DSS induces damage to colonic epithelial cells, causing the intestinal glands in the epithelial layer to fall off without inflammation, and the amount of various immune substances in the mucosa increases, inducing intestinal inflammation. Therefore, DSS (dextran sulfate sodium) is widely used to create experimental animal models. Specifically, 9-week-old C57BL / 6 female mice were given 2% DSS ad libitum. Since the experiment assumes that a single C57BL / 6 mouse consumes 5 ml of fluid per day in a typical DSS-induced colitis model, water containing 2% DSS was provided to each mouse in the same manner. For 7 days, the animals were given only water containing 2% DSS without any treatment, and then for 5 days from the 7th to the 11th, they were given water containing 2% DSS while receiving microinjections of various comparison substances, including NP-011 protein, at various concentrations to create an animal model of ulcerative colitis. As shown in Figures 6 and 10, the "NTC" control group, which corresponds to the control group, was supplied with only water from day 0, and the "UC CONTROL" control group, which corresponds to the positive control group, was supplied with only water containing 2% DSS from day 0 to day 7, and then with regular water from day 7 to day 14. Also, as shown in Fig. 6, the "MFG-E8" experimental group corresponding to the experimental group was supplied with only water containing 2% DSS from day 0 to day 7, then supplied with normal water from day 7 to day 14, and 160 μg / kg of MFG-E8 protein was administered 5 times once a day for 5 days from day 7. Next, as shown in Fig. 10, the "Cyclosporin A" experimental group corresponding to the experimental group was supplied with only water containing 2% DSS from day 0 to day 7, then supplied with normal water from day 7 to day 14, and 80 μg / kg of Cyclosporin A component was administered 3 times once a day at 2-day intervals from day 7. Also, as shown in Fig. 10, the "Adalimumab" experimental group corresponding to the experimental group was supplied with only water containing 2% DSS from day 0 to day 7, then supplied with normal water from day 7 to day 14, and 80 mg / kg of Adalimumab component was administered 2 times once a day on day 7 and day 11 respectively. Next, as shown in Fig. 10, the first "NP-011" experimental group corresponding to the experimental group was supplied with only water containing 2% DSS from day 0 to day 7, then supplied with normal water from day 7 to day 14, and 20 μg / kg of NP-011 recombinant protein was administered 5 times once a day for 5 days from day 7. Also, as shown in Figs. 6 and 10, the second "NP-011" experimental group corresponding to the experimental group was supplied with only water containing 2% DSS from day 0 to day 7, then supplied with normal water from day 7 to day 14, and 80 μg / kg of NP-011 recombinant protein was administered 5 times once a day for 5 days from day 7. Finally, as shown in Figs. 6 and 10, the third "NP-011" experimental group corresponding to the experimental group was supplied with only water containing 2% DSS from day 0 to day 7, then supplied with normal water from day 7 to day 14, and 320 μg / kg of NP-011 recombinant protein was administered 5 times once a day for 5 days from day 7. The following experiments using all the animal models thus prepared were conducted under the approval of the Nexell Experimental Animal Ethics Committee and complied with the animal management regulations.

[0013] (2) Verification of the alleviation of symptoms in an animal model of ulcerative colitis - related to damage to the colonic mucosa First, using animal models corresponding to the "NTC" control group, "UC CONTROL" control group, "MFG-E8" experimental group, "NP-011" second experimental group, and "NP-011" third experimental group, the cecum of the colon tissue was removed for each animal model group, and 1-2 cm of the proximal portion and 1 cm of the anus were secured and fixed in 4% paraformaldehyde. Next, tissues fixed in 4% paraformaldehyde were stained (Hematoxylin & Eosin stain) and histopathologically examined by KPNT Corporation. Pathological interpretations were determined based on INHAND, the International Standard Toxicological Pathology Dictionary. The degree of crypt damage in the large intestine mucosa, the severity of inflammation, and the extent of inflammation were measured and compared for each animal model group. The results are shown in Figure 7 and Table 1 below. (Reference: J Toxicol Pathol 2016;29(1 Suppl):1S-124S)

[0014] [Table 1]

[0015] As can be seen from the results of Figure 7 and Table 1, after induction of ulcerative colitis, the degree of mucosal crypt damage, the severity of inflammatory cell invasion, and the extent of inflammatory cell invasion were reduced in the following groups compared to the positive control group: MFG-E8 160 μg / kg, NP-011 80 μg / kg, and NP-011 320 μg / kg, confirming a reduction in the overall degree of colonic mucosal damage. Next, using animal models corresponding to the "NTC" control group, "UC CONTROL" control group, "Cyclosporin A" experimental group, "Adalimumab" experimental group, "NP-011" 1st experimental group, "NP-011" 2nd experimental group, and "NP-011" 3rd experimental group, the cecum of the colon tissue was removed for each animal model group, and 1-2 cm of the proximal side and 1 cm of the anus were secured and fixed in 4% paraformaldehyde. Next, tissues fixed in 4% paraformaldehyde were stained (Hematoxylin & Eosin stain) and histopathologically examined by KPNT Corporation. Pathological findings were determined based on INHAND, the International Standard Toxicological Pathology Dictionary. Erosion (mucsoa) and epithelial hyperplasia in the large intestine tissue were measured for each animal model group, and the lesions were compared. The results are shown in Figure 11 and Table 2 below. (Reference: J Toxicol Pathol 2016;29(1 Suppl):1S-124S)

[0016] [Table 2]

[0017] As can be seen from the results of Figure 11 and Table 2, after the induction of ulcerative colitis, compared to the positive control group, Cyclosporin A and NP-011 at 20 μg / kg reduced the degree of erosion and epidermal hyperplasia in the large intestine tissue mucosa to the same extent, followed by Adalimumab, NP-011 at 80 μg / kg, and NP-011 at 320 μg / kg, confirming a reduction in the overall degree of damage to the large intestine mucosa. (3) Verification of symptom alleviation in an animal model of ulcerative colitis - Body weight change First, using the animal models corresponding to the "NTC" control group, "UC CONTROL" control group, "MFG-E8" experimental group, "NP-011" second experimental group, and "NP-011" third experimental group respectively, the body weight was measured daily for each animal model group, and it was measured before drug administration to reduce the error caused by administration. Also, the increase or decrease in body weight was analyzed in comparison with the positive control group, and the comprehensive results for the body weight changes related thereto are as shown in FIG. 8 and Table 3 below.

[0018]

Table 3

[0019] As can be seen from the results of FIG. 8 and Table 3, after induction of ulcerative colitis, it was confirmed that a statistically significant degree of weight loss was improved from day 8 in the "NP-011" second experimental group and third experimental group compared with the positive control group (UC). (**** P<0.0001, *** P<0.001, ** P<0.01, * P<0.05) Next, using the animal models corresponding to the "NTC" control group, "UC CONTROL" control group, "Cyclosporin A" experimental group, "Adalimumab" experimental group, "NP-011" first experimental group, "NP-011" second experimental group, and "NP-011" third experimental group respectively, the body weight was measured daily for each animal model group, and it was measured before drug administration to reduce the error caused by administration. Also, the increase or decrease in body weight was analyzed based on the initial body weight, and the level of increase or decrease in body weight was also analyzed in comparison with the positive control group. The comprehensive results for the body weight changes related thereto are as shown in FIG. 12 and Tables 4 and 5 below.

[0020]

Table 4

[0021]

Table 5

[0022] As can be seen from the results in Figure 12, Tables 4 and 5, weight loss was observed in the positive control group (UC control), Adalimumab, Cyclosporine A, and NP-011 (20-80 μg / kg). Compared to the positive control group, NP-011 (80 μg / kg) and Cyclosporine A showed similar inhibitory effects on weight loss induced by colitis, followed by NP-011 (20 μg / kg), NP-011 (320 μg / kg), and Adalimumab. (**** P<0.0001, *** P<0.001, ** P<0.01, * P<0.05)

[0023] (4) Verification of symptom alleviation in an animal model of ulcerative colitis - related to changes in colon length First, using animal models corresponding to the "NTC" control group, the "UC CONTROL" control group, the "MFG-E8" experimental group, the "NP-011" second experimental group, and the "NP-011" third experimental group, the colon was excised from each animal model group and analyzed for colon length. The results are shown in Figure 9. Specifically, for colon length analysis, each animal model was euthanized by gradually increasing carbon dioxide three days after the last administration, and the colon was removed. The removed colon was photographed with a length measuring device (a 30 cm ruler), and the captured photograph was digitized using Image J. As can be seen from the results in Figure 9, after the induction of ulcerative colitis, a statistically significant increase in colon length reduction was observed in MFG-E8 and NP-011 (80, 320 μg / kg) compared to the non-administered control group, and it can be confirmed that the degree of inhibition of colon length reduction was superior in the "NP-011" second experimental group and the "NP-011" third experimental group compared to the "MFG-E8" experimental group. Here, the black cross in the center of each box in Figure 9 represents the mean, and the boxes show the distribution of values extending from 25% to 75% in each group (**** P<0.0001, *** P<0.001, ** P<0.01, * P<0.05). Next, using animal models corresponding to each of the "NTC" control group, "UC CONTROL" control group, "Cyclosporin A" experimental group, "Adalimumab" experimental group, "NP-011" first experimental group, "NP-011" second experimental group, and "NP-011" third experimental group, the large intestine was excised for each animal model group and the length of the large intestine was analyzed. The results are as shown in Fig. 13. Specifically, for the analysis of the large intestine length, each animal model was euthanized by the gradual increase of carbon dioxide 3 days after the last administration, and the large intestine was excised. The excised large intestine was photographed together with a length measuring mechanism (a 30 cm ruler), and the obtained photographs were digitized by Image J. As can be seen from the results in Fig. 13, after induction of ulcerative colitis, it can be confirmed that the length of the large intestine increased significantly statistically according to the NP-011 protein concentration (20, 80, 320 μg / kg) compared with the positive control group. Here, the central black cross in each box shown in Fig. 13 means the average, and the box shows the distribution of values from 25% to 75% in each group. (**** P<0.0001, *** P<0.001, ** P<0.01, * P<0.05)

[0024] (5) Verification of the remission of the disease condition of the ulcerative colitis animal model - related to the blood IL-6 concentration First, using animal models corresponding to each of the "NTC" control group, "UC CONTROL" control group, "Cyclosporin A" experimental group, "Adalimumab" experimental group, "NP-011" first experimental group, "NP-011" second experimental group, and "NP-011" third experimental group, the blood collected with a heparinized capillary tube for each animal model group was stored on ice for 30 minutes, centrifuged at 12,000 rpm for 15 minutes at 4°C to remove the blood clot, and the separated serum was stored at -80°C. After gradually thawing at 4°C for analysis, it was diluted with PBS (1% BSA), and the blood concentration of IL-6 was measured using an ELISA kit. The results are as shown in Fig. 14. Here, Interleukin 6 (IL-6) is a pro-inflammatory cytokine widely confirmed in the diagnosis of inflammatory diseases, including TNF-α and IL-1β. It is one of the cytokines that send signals through the JAK / STAT pathway associated with many inflammatory diseases and is necessary for differentiating naive T cells into Th17 cells, and such Th17 cells can cause inflammation. Therefore, compared with the "UC CONTROL" control group, each of the "NP-011" first experimental group, "NP-011" second experimental group, and "NP-011" third experimental group shows a low blood concentration of IL-6 and is considered to contribute to suppressing the exacerbation of inflammation. In short, it can be confirmed from Figure 14 that the blood concentration of IL-6 in each of the "NP-011" first experimental group, "NP-011" second experimental group, and "NP-011" third experimental group decreases to a statistically significant extent compared with the "UC CONTROL" control group. Here, the central black cross in each box shown in Figure 14 means the average, and the box shows the distribution of values expanded from 25% to 75% in each group. (**** P<0.0001, *** P<0.001, ** P<0.01, * P<0.05) The examples disclosed in the present invention are for the purpose of explanation rather than for limiting the technical idea of the present invention, and the scope of the technical idea of the present invention is not limited by such examples. The scope of protection should be interpreted by the appended claims, and any technical idea within the equivalent scope should be interpreted as being included in the scope of rights of the present invention.

Claims

**Claim 1** A recombinant protein based on milk fat globule - EGF factor 8 (MFG - E8) protein, characterized by comprising, as an active ingredient, a recombinant protein consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, A pharmaceutical composition for preventing or treating ulcerative colitis. **Claim 2** The recombinant protein reduces the degree of crypt damage of the mucosa in the large intestine tissue to be prevented or treated, the severity of inflammation cell invasion, and the extent of inflammation cell invasion, compared with the non - administration situation of the recombinant protein, The pharmaceutical composition for preventing or treating ulcerative colitis according to Claim 1. **Claim 3** The recombinant protein reduces the degree of erosion of the mucosa in the large intestine tissue to be prevented or treated and the degree of epidermal hyperplasia, compared with the non - administration situation of the recombinant protein, The pharmaceutical composition for preventing or treating ulcerative colitis according to Claim 1. **Claim 4** The recombinant protein reduces the blood concentration of interleukin - 6 (IL - 6) in the subject to be prevented or treated, compared with the non - administration situation of the recombinant protein, The pharmaceutical composition for preventing or treating ulcerative colitis according to Claim 1. **Claim 5** The recombinant protein reduces the degree of weight loss and the degree of reduction in the length of the large intestine induced by ulcerative colitis in the subject to be prevented or treated, compared with the non - administration situation, The pharmaceutical composition for preventing or treating ulcerative colitis according to Claim 1. **Claim 6** The recombinant protein is formulated for intralesional administration, intravascular administration, subcutaneous administration, intranasal administration or intraperitoneal administration, The pharmaceutical composition for preventing or treating ulcerative colitis according to Claim 1. **Claim 7** A gene encoding a recombinant protein contained as an active ingredient in the pharmaceutical composition according to any one of Claims 1 to 6. **Claim 8** A recombinant vector containing a gene encoding a recombinant protein contained as an active ingredient in the pharmaceutical composition according to any one of Claims 1 to 6.

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