Application of streptococcus pneumoniae peptide chain endonuclease O (PepO) in preparation of medicine for treating ulcerative colitis

Abstract

The invention belongs to the technical field of medical biology, and particularly discloses application of streptococcus pneumoniae peptide chain endonuclease O (PepO) in preparation of a medicine for treating ulcerative colitis. The invention finds that the streptococcus pneumoniae endonuclease O (PepO) regulates the composition of intestinal flora, inhibits the recruitment of macrophages, down-regulates the expression of intestinal proinflammatory factors and enhances the barrier function of the intestinal tract through a TLR2 receptor in vivo, thereby relieving ulcerative colitis.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical biotechnology, and in particular to application of Streptococcus pneumoniae endopeptidase O in the preparation of a medicine for treating ulcerative colitis. Background Art

[0002] Ulcerative colitis (UC) is an autoimmune disease characterized by chronic intestinal inflammation and one of the most common inflammatory bowel diseases (IBD). Currently, UC is an incurable chronic disease. The most common sites of lesions are the sigmoid colon and rectum. In severe cases, UC can spread throughout the colon and even to the terminal ileum, severely impacting patients' lives. Due to recurrent attacks and the need for long-term treatment, direct medical costs are three times higher than those for patients without IBD, placing a heavy burden on families and society as a whole. Early-stage UC is more common in developed countries, but recent changes in lifestyle and diet have led to an increase in UC incidence in some Asian countries, including China. The prevalence in Asia is projected to increase fourfold by 2035, indicating that UC has become a global burden. Because the exact cause of UC remains unclear, current treatments focus on symptom relief. However, a large proportion of UC patients do not respond to current drug therapies or develop secondary non-response after treatment, resulting in a remission rate of only 30% to 60%. Therefore, the development of more UC treatments is urgently needed.

[0003] Immunomodulators are a class of drugs that can enhance or suppress immune system activity and are widely used to treat various immune-related diseases. Currently, immunomodulators are also widely used in the treatment of UC, aiming to suppress an overactive immune system, thereby alleviating intestinal inflammation and symptoms. These immunomodulators primarily function as immunosuppressants and often carry a higher risk of infection: For example, long-term use of anti-tumor necrosis factor drugs can increase the risk of tuberculosis reactivation, while JAK inhibitors may also increase the risk of respiratory, urinary, and herpes virus infections. S1PR modulators also carry similar risks. Traditional immunosuppressants (such as 6-mercaptopurine) have been reported to be associated with an increased risk of certain tumors. Therefore, an immunomodulator with a non-suppressive effect could be a safer and more effective treatment for UC.

[0004] Studies have shown that UC patients generally have intestinal flora disorders, which are manifested by a decrease in beneficial bacteria (such as Bifidobacterium and Lactobacillus) and an increase in potential pathogenic bacteria (such as Escherichia coli and Clostridium). As an important "microbial organ" of the human body, the intestinal flora plays a key role in maintaining intestinal homeostasis by participating in physiological processes such as nutrient metabolism, immune regulation and barrier maintenance. In addition, there is a close interaction between the intestinal flora and intestinal macrophages. The intestinal flora can affect the polarization, function and inflammatory response of macrophages through metabolites, pathogen-associated molecular patterns (PAMPs) and immunomodulatory molecules. The imbalance of this regulatory network plays an important role in the occurrence and development of UC.

[0005] Numerous studies have demonstrated that macrophages are activated in ulcerative colitis (UC) patients and release multiple inflammatory mediators, participating in the regulation of inflammatory responses and playing a crucial role in the development and progression of the disease. The roles of these cells in intestinal tissue include not only pathogen clearance and immune balance, but also repair of the intestinal mucosal barrier and immune regulation. In the context of chronic inflammation, macrophage activity can also damage host tissues. These studies suggest that macrophage regulation may be a key factor in promoting or alleviating UC.

[0006] Toll-like receptors (TLRs) are important pattern recognition receptors expressed on a variety of cells, including those of the innate immune system, such as macrophages, neutrophils, dendritic cells (DCs), natural killer (NK) cells, and mast cells. TLRs recognize pathogen-associated molecular patterns and participate in a range of immune responses, including phagocytic killing by phagocytes and antigen presentation by antigen-presenting cells. Therefore, substances targeting TLRs are considered immunomodulators and may have potential therapeutic value. However, aberrant activation of TLR signaling pathways can also alter the composition and structure of the intestinal microbiota, increasing the abundance of harmful bacteria and decreasing the abundance of beneficial bacteria, further exacerbating intestinal inflammation. However, studies have shown that the presence of TLR2 can protect against colonic inflammation and reduce intestinal damage. Many probiotics, such as Lactobacillus and Bifidobacterium, rely on the presence of TLR2 to strengthen the intestinal epithelial barrier and prevent intestinal inflammation. However, no single substance has been found to exert therapeutic effects through TLR2. Summary of the Invention

[0007] In view of the above-mentioned lack of existing immunomodulators, the purpose of the present invention is to provide the use of Streptococcus pneumoniae endopeptidase O in the treatment of ulcerative colitis, so as to solve the problems of infection risk and adverse side effects of immunomodulators for the treatment of ulcerative colitis in existing treatment strategies.

[0008] To achieve the above-mentioned and other related objectives, the present invention provides, in a first aspect, the use of Streptococcus pneumoniae endopeptidase O in the preparation of a medicament for treating ulcerative colitis. In the present invention, the gene sequence of Streptococcus pneumoniae endopeptidase O (PepO) is the nucleotide sequence shown in SEQ ID NO. 1, and the protein sequence is the polypeptide fragment shown in SEQ ID NO. 2.

[0009] A second aspect of the present invention provides an isolated or purified protein as described above, wherein the Streptococcus pneumoniae endopeptidase O has at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity with the protein encoded by the nucleotide sequence shown in SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO. 2, and is a variant having endopeptidase activity and activity for treating ulcerative colitis.

[0010] The third aspect of the present invention provides a pharmaceutical composition for treating ulcerative colitis, comprising a therapeutically effective amount of Streptococcus pneumoniae endopeptidase O and a pharmaceutically acceptable carrier, wherein the active ingredient is the above-mentioned protein.

[0011] The fourth aspect of the present invention provides a pharmaceutical composition as described above, wherein the active ingredient is a nucleic acid molecule encoding pneumococcal endopeptidase O, characterized in that its sequence includes at least one of the following sequences:

[0012] (1) the nucleotide sequence shown in SEQ ID NO. 1;

[0013] (2) a nucleotide sequence that has at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity with the sequence shown in SEQ ID NO: 1 and encodes a protein having endopeptidase activity;

[0014] (3) a sequence complementary to any sequence described in (1) or (2);

[0015] (4) RNA equivalent of the sequence shown in SEQ ID NO: 1.

[0016] The fifth aspect of the present invention provides a recombinant expression vector containing the above-mentioned nucleotide sequence.

[0017] The sixth aspect of the present invention provides an expression system, which contains the above-mentioned recombinant expression vector or the above-mentioned exogenous nucleotide sequence integrated into the genome.

[0018] A seventh aspect of the present invention provides a method for preparing a drug for treating ulcerative colitis, comprising culturing the expression system as described above under conditions suitable for expressing the protein.

[0019] In an eighth aspect, the present invention provides the pharmaceutical composition as an oral preparation, an enema preparation, a suppository, a sustained-release preparation or an enteric-coated preparation.

[0020] As described above, the use of Streptococcus pneumoniae endopeptidase O of the present invention in the preparation of a drug for treating ulcerative colitis has the following beneficial effects:

[0021] The present invention discovered that pneumococcal endopeptidase O (PepO) regulates the composition of the intestinal flora through the TLR2 receptor in vivo, inhibits macrophage recruitment, downregulates the expression of intestinal proinflammatory factors, and enhances intestinal barrier function, thereby significantly alleviating ulcerative colitis. This invention provides a new immunomodulator for the treatment of ulcerative colitis and has great development and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The figure shows the results of PCR identification of the recombinant plasmid pET28a(+)-PepO and the nucleotide sequence identification of the protein PepO (1893 bp) in Example 1 of the present invention.

[0023] Figure 2 The figure shows the detection results of the purified expression of PepO (molecular weight of 72KDa) in Example 2 of the present invention.

[0024] Figure 3 This figure shows the results of PepO alleviating ulcerative colitis in vivo in Example 3 of the present invention.

[0025] Figure 4 This figure shows the results of Example 4 of the present invention, showing that PepO relies on TLR2 receptor to alleviate ulcerative colitis in vivo.

[0026] Figure 5 This figure shows the results of Example 5 of the present invention, in vivo, in which PepO inhibits the recruitment of macrophages to the intestine.

[0027] Figure 6 This figure shows the results of Example 6 of the present invention, showing that PepO relies on intestinal flora to alleviate ulcerative colitis in vivo.

[0028] Figure 7 This figure shows the results of PepO regulating the composition of intestinal flora in vivo in Example 7 of the present invention, which depends on the TLR2 receptor. DETAILED DESCRIPTION

[0029] The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different viewpoints and applications without departing from the spirit of the present invention.

[0030] The invention discloses application of pneumococcal endopeptidase O in preparing medicine for treating ulcerative colitis. The gene sequence is nucleotide sequence SEQ ID NO.1, and the protein sequence is amino acid sequence SEQ ID NO.2.

[0031] As one of the most important components of the innate immune response in the intestine, macrophages play a crucial role in intestinal homeostasis. They may play a role in ulcerative colitis in two ways: indirectly, by secreting cytokines and chemokines, they promote the recruitment of immune cells to the intestine and participate in immune regulation. Directly, macrophages maintain intestinal stability by phagocytosing and clearing apoptotic cells, thereby preventing immune responses triggered by the accumulation of apoptotic cells and playing a crucial role in immune regulation.

[0032] The present invention found that pneumococcal endopeptidase O regulates intestinal flora through the TLR2 receptor in the body, reduces the recruitment of intestinal macrophages, and further acts on the intestine to inhibit the expression of pro-inflammatory factors such as IL-6 and TNF-α, thereby strengthening the intestinal barrier and alleviating ulcerative colitis.

[0033] Example 1

[0034] Construction of recombinant expression vector pET28a(+)-PepO.

[0035] (1) Materials:

[0036] The prokaryotic expression plasmid pET28a(+) was purchased from Novagen, and the Prime Star high-fidelity enzyme, dNTPs, buffer, and MgCl2 used in PCR were purchased from Takara Biotechnology (Dalian) Co., Ltd. The PTC-200 PCR instrument was a Perkin Elmer product.

[0037] (II) Design and synthesis of primers:

[0038] Using the genomic DNA of Streptococcus pneumoniae D39 as a template and referring to its complete sequence (GeneBank No. CP000410.2), primers were designed using Premier 5.0 and synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0039] PepO: Upstream primer: 5'-GCCATGGCACGTTATCAAGATGATTTTTAT-3 ’ , containing NcoI site;

[0040] Downstream primer: 5'-CCCTCGAGCCAAATAATCACGCGCTCCTCT-3', containing XhoI site.

[0041] (III) PCR amplification of target gene:

[0042] Amplification of the PepO gene. The nucleotide sequence of the amplified PepO gene is shown in SEQ ID NO.1.

[0043] Amplification system:

[0044]

[0045] Wherein, “P1 (5pM) 2μl” means: take the PepO upstream primer with a concentration of 5pM and add 2μl;

[0046] "P2 (5 pM) 2 μl" means: take the PepO downstream primer with a concentration of 5 pM and add 2 μl.

[0047] Conditions: 98°C for 2 min; 55°C for 45 s, 72°C for 90 s, 33 cycles; 72°C for 10 min, once. Using the above conditions, the corresponding target gene was amplified.

[0048] (IV) Construction of prokaryotic expression vector:

[0049] PCR product recovery was performed according to the Roche kit instructions. Plasmid pET28a(+) was extracted using the Omega Mini-Plasmid DNA Extraction Kit instructions. The vector DNA and PepO gene were double-digested with NcoI and BamH1, then recovered and purified using the Roche kit. The enzyme digestion reaction system was as follows:

[0050] 2 μL DNA fragments

[0051] NcoI 1 μL

[0052] BamHI 1 μL

[0053] 2 μL enzyme digestion buffer

[0054] ddH2O 14μL.

[0055] The reaction system was incubated in a 37°C water bath for 3 hours. The product was identified by agarose gel electrophoresis and recovered using a Roche kit. The digested PepO gene fragment was ligated to the pET28a(+) plasmid using T4 ligase. The ligation reaction system was as follows:

[0056] PepO fragment 8 μL

[0057] pET28a(+) fragment 2 μL

[0058] T4 ligase 0.2 μL

[0059] 2 μL ligation buffer

[0060] ddH2O 7.8μL.

[0061] The reaction system was placed in a 22°C water bath for 1 h.

[0062] (V) Transformation and identification of ligation products:

[0063] 200 μL of DH5α stored at -80°C was slowly thawed in an ice bath, 10 μL of the ligation reaction product was added, and the mixture was gently mixed and placed in an ice bath for 30 minutes; heat shock was performed at 42°C for 30 seconds, and then the mixture was placed in an ice bath for 2 minutes; 800 μL of LB medium was added to the bacterial solution, and the culture was cultured at 37°C and 180 rpm for 1 hour; the bacterial solution was centrifuged at 5000 rpm for 5 minutes, 800 μL was discarded, and the remaining 200 μL of bacterial solution was mixed and spread on an LK plate, which was then incubated in a 37°C incubator overnight.

[0064] Pick 20 single colonies and perform PCR identification using PepO upstream and downstream primers (reaction system and conditions are as described above). Select 5 PCR-positive colonies for bacterial culture and send them to Qingke Company for sequencing identification. The transformants with correct sequencing are saved for future use. The results of PCR identification of recombinant plasmid pET28a(+)-PepO and the nucleotide sequence identification of the protein PepO are shown in Figure 2. Figure 1 shown.

[0065] Example 2

[0066] Expression, Identification and Purification of Prokaryotic Expression Plasmid pET28a(+)-PepO in Escherichia coli

[0067] (1) The recombinant plasmid pET28a(+)-PepO was transformed into E. coli competent BL21(DE3). The method and conditions are as in Example

[0068] 1. Positive transformants were identified by sequencing and stored for future use.

[0069] (2) IPTG induced the massive expression of PepO recombinant protein.

[0070] Add the stored pET28a(+)-PepO-BL21(DE3) E. coli to 30 mL of LB medium and incubate at 37°C, 180 rpm, for 8 hours. Transfer the entire cell suspension to 500 mL of LB medium and enrich the suspension at 37°C, 180 rpm, for 4 hours. Subsequently, add IPTG to a final concentration of 40 mM and incubate overnight at 22°C, 180 rpm.

[0071] (3) Purification of recombinant protein.

[0072] After collecting the bacteria by centrifugation at 12000g for 10 min at 4°C, resuspend them in 30 mL of binding buffer and disrupt them by ultrasonication. The bacterial disruption solution was centrifuged at 12000g for 10 min at 4°C to separate the supernatant, which was filtered through a 0.45 μm filter membrane and the filtrate was collected for later use.

[0073] Affinity chromatography purification: Pipette 2 ml of 50% Ni2+-NTA resin suspension into the chromatography column and equilibrate the resin by flowing 20 mL of binding buffer through the resin twice; thoroughly mix the equilibrated Ni2+-NTA resin suspension with the above filtrate and incubate slowly with shaking at 4°C for 1 hour; transfer the suspension to the chromatography column and allow the liquid to flow out naturally to equilibrate the column bed; perform gradient elution using different imidazole concentrations and collect the eluates separately.

[0074] (4) The eluates with different imidazole concentrations were identified by SDS-PAGE and Coomassie Brilliant Blue staining. The eluate with the highest PepO purity and abundance was selected and pressure ultrafiltration was performed using a 20 kD ultrafiltration membrane and PBS buffer (pH = 8.0) to remove the imidazole from the protein.

[0075] The ultrafiltered protein was detected by SDS-PAGE and Coomassie Brilliant Blue, and the concentration was over 95%.

[0076] (5) Removal of residual endotoxins in the recombinant protein: Use the endotoxin removal kit provided by GenScript to remove residual endotoxins in the protein. The operation method and steps are carried out according to the manufacturer's instructions.

[0077] (6) Quantification of recombinant protein (BCA method)

[0078] The concentration of the recombinant protein was determined using a Beyotime kit, and the operation method and steps were carried out according to the manufacturer's instructions.

[0079] Example 3

[0080] In vivo, PepO alleviates ulcerative colitis.

[0081] (I) Grouping of mice

[0082] Male C57BL / 6J mice weighing 18-20 g were randomly divided into three groups with similar body weights, namely, a blank control group (Water group), an ulcerative colitis modeling group (DSS group), and a protein treatment group (DSS+PepO group, treated with PepO recombinant protein), with 6 mice in each group.

[0083] (II) Modeling of ulcerative colitis

[0084] Mice were orally administered a 3% dextran sulfate sodium (DSS) solution to establish a model. Based on a daily intake of 8 ml per mouse, DSS powder was dissolved in Wahaha mineral water. Stirring the solution in a beaker with a glass rod until completely dissolved, the solution was filtered through a 22 μm filter and then administered to the mice. The 3% DSS solution was administered orally for six consecutive days. On the sixth day, the DSS solution was replaced with Wahaha mineral water, and the mice rested for one day. Modeling was completed on the seventh day. A blank control group continued to receive Wahaha mineral water. In addition to DSS administration, the protein-treated group received an intraperitoneal injection of 10 mg / kg PepO protein per mouse on the first day of modeling.

[0085] (3) Disease Activity Index Score

[0086] During the modeling period, mice's weight changes and disease manifestations were recorded daily. The Disease Activity Index (DAI) score was calculated based on weight loss percentage, stool form, and fecal bleeding: 1%-5% weight loss was assigned a score of 1, 6%-10% was assigned a score of 2, 11%-18% was assigned a score of 3, and >18% was assigned a score of 4. Soft but formed stool was assigned a score of 1, soft stool was assigned a score of 2, very soft and wet stool was assigned a score of 3, and diarrhea was assigned a score of 4. Negative fecal occult blood was assigned a score of 1, positive fecal occult blood was assigned a score of 2, visible blood in the stool was assigned a score of 3, and obvious rectal bleeding was assigned a score of 4. The sum of these three scores was the total DAI score.

[0087] (IV) Mouse colon sampling

[0088] The mice were killed by cervical dislocation. The skin was cut open along the midline of the abdomen from the pubic symphysis to the xiphoid process with surgical scissors to open the abdominal cavity and expose the organs. The colon was gently lifted with forceps and the mesentery and blood vessels around the colon were carefully separated with surgical scissors, starting from the starting end of the colon (cecum end) and continuing to the rectum end. After the separation was completed, the colon was cut at both ends with surgical scissors, and the entire colon was removed intact and placed in a culture dish filled with sterile PBS. Blood and impurities on the surface of the colon were gently rinsed for subsequent observation or further processing.

[0089] (V) Determination of colonic inflammatory factors

[0090] After flushing blood and impurities from the colon surface and feces and mucus from the colon with sterile PBS, the intestinal tissue was quickly frozen in liquid nitrogen and ground into powder using a mortar. 1 ml of RIPA was added to lyse the intestinal tissue, and the tissue was lysed on ice for 50 min. The supernatant was centrifuged at 13,000 rpm in a 4°C low-temperature centrifuge for 10 min, and the expression level of colon inflammatory factors was measured using an ELSIA test kit.

[0091] (6) Colon tissue staining

[0092] After removing the colon and flushing it clean, use surgical scissors to take a section of intestinal tissue about 0.5 cm long from the middle of the colon. Do not cut it longitudinally, keep the original shape, flush the inside of the colon with paraformaldehyde, and soak the colon tissue in paraformaldehyde, store it at 4°C, and send it to the company for staining.

[0093] (VII) Experimental results showed that compared with the modeling group, PepO treatment significantly reduced the percentage of weight loss, colon tissue length shortening, disease activity index scores, inflammatory cytokines in colon tissue, and inflammatory cell infiltration in the colon. Furthermore, colon tissue structural integrity was improved, and goblet cell loss was reduced. These results indicate that PepO can alleviate colonic inflammation, strengthen the intestinal barrier, and alleviate ulcerative colitis.

[0094] Example 4

[0095] In vivo, PepO alleviates ulcerative colitis in a TLR2-dependent manner.

[0096] (1) Establishment of ulcerative colitis model in TLR2- and TLR4-deficient mice

[0097] Colitis was established in C57 male mice deficient in the TLR2 gene and deficient in the TLR4 gene, respectively. Experimental grouping, modeling methods, colon sampling, colon inflammatory factor determination, and colon tissue staining were the same as in Example 3. TLR2- and TLR4-deficient mice were purchased from Shanghai South Model Organisms Science Co., Ltd. These two gene-deficient mice were bred and propagated at the Experimental Animal Center of Chongqing Medical University. Genotyping was performed using the manufacturer's provided method prior to the experiments to ensure experimental reliability.

[0098] (II) Experimental results showed that in mice with TLR4 gene deficiency, PepO treatment still attenuated weight loss, increased colon length, reduced disease activity scores, decreased inflammatory cytokines in colonic tissue, reduced colonic inflammatory cell infiltration, and improved colonic tissue structural integrity. However, in mice with TLR2 gene deficiency, PepO treatment did not have the above-mentioned protective effects, with no difference between the modeling group and the protein treatment group. These results indicate that although PepO is a TLR2 / 4 dual ligand protein, its effect in alleviating ulcerative colitis is dependent on the TLR2 receptor.

[0099] Example 5

[0100] In vivo, PepO inhibits macrophage recruitment to the intestine.

[0101] (A) Determination of CCL2 at the transcriptome level.

[0102] Male C57BL / 6J mice weighing 18-20 g were used, and the experimental grouping, modeling method, colon sampling, and colon tissue staining were the same as in Example 3. After flushing the blood and impurities on the surface of the colon, the feces and mucus inside the colon with sterile PBS, the intestinal tissue was quickly frozen with liquid nitrogen, the colon tissue was crushed to powder with a mortar, 1 ml of RNAiso Plus was added to collect RNA, and the supernatant was collected by centrifugation at 13000 rpm at 4 ° C for 10 min. RNA was extracted using a matching RNA kit and reverse transcribed into cDNA, and the expression level of CCL2 in the transcriptome was detected using Q-PCR. The method for detecting CCL2 protein levels is the same as the method for determining inflammatory factors in Example 3.

[0103] (II) Experimental results showed that PepO treatment reversed DSS-induced upregulation of colonic CCL2 expression and reduced the recruitment of macrophages in intestinal tissue. These results suggest that PepO can alleviate ulcerative colitis by inhibiting the recruitment of intestinal macrophages, reducing intestinal inflammatory damage.

[0104] Example 6

[0105] In vivo, PepO alleviates ulcerative colitis in a gut-microbiota-dependent manner

[0106] (1) Antibiotic sweep experiment

[0107] Using a combination antibiotic sweep, an antibiotic solution was prepared according to a dosing regimen of 200 mg / kg ampicillin, 200 mg / kg metronidazole, 200 mg / kg neomycin, and 100 mg / kg vancomycin. The antibiotic powder was dissolved in sterile PBS and administered orally (200 μl) to each mouse for five consecutive days. After the antibiotic sweep, an ulcerative colitis model was established using the same method as in Example 3.

[0108] (2) Fecal microbiota transplantation (FMT) experiment

[0109] The feces of mice in the DSS group and DSS+PepO group of normal modeling were collected. The feces in the colon of the two groups of mice were collected in a clean bench. After collection, they were dissolved in sterile PBS to a concentration of 100 mg / ml, centrifuged at 800g for 5 minutes, and the supernatant was collected. Male C57BL / 6J mice weighing 18-20g were used. The mice were first subjected to antibiotic cleaning to remove intestinal flora, using the same method as described in (1). After the antibiotic cleaning, the mice were divided into two groups to establish a model of ulcerative colitis. At the same time as the modeling, the mice were gavaged with 200ul of fecal supernatant every day. One group was gavaged with the fecal supernatant of the DSS group, and the other group was gavaged with the fecal supernatant of the DSS+PepO group.

[0110] (III) Experimental results showed that antibiotic combination eradication of the microbiota of normal mice eliminated the protective effect of PepO against DSS-induced ulcerative colitis. In a fecal microbiota transplant (FMT) experiment, recipient mice transplanted with fecal microbiota from PepO-treated colitis mice showed significantly reduced colitis symptoms compared to mice transplanted with fecal microbiota from the control group. In addition, after antibiotic eradication, PepO could not inhibit the upregulation of intestinal CCL2, but after transplantation of fecal microbiota from PepO-treated colitis mice, the upregulation of intestinal CCL2 was reversed. The above experimental results indicate that PepO treatment of ulcerative colitis depends on the presence of intestinal microbiota.

[0111] Example 7

[0112] In vivo, PepO regulates the composition of the intestinal microbiota through the TLR2 receptor

[0113] (A) 16sRNA sequencing of fecal microbiota.

[0114] Normal 18-20 g male C57BL / 6J mice and TLR4 gene-deficient C57 male mice were used to establish colitis, and the experimental grouping and modeling methods were the same as in Example 3. After the modeling was completed, the colon feces of each group of mice were collected in an ultra-clean bench and placed in enzyme-free cryopreservation tubes, quickly frozen in liquid nitrogen, and transported on dry ice to a third-party company for fecal microbiome 16s RNA sequencing.

[0115] (II) Experimental results showed that PepO treatment significantly altered the intestinal flora of DSS-fed mice, with a significant increase in probiotic bacteria such as Lactobacillus and Akkermansia, and a significant decrease in harmful bacteria such as Clostridium and Pseudomonas. However, the TLR2 receptor deficiency in mice abolished PepO's regulatory ability. These results suggest that PepO treatment alters the composition of the mouse intestinal flora through the TLR2 receptor, increasing the abundance of beneficial bacteria and reducing the abundance of harmful bacteria.

[0116] Based on the above results, the present invention successfully prepared a novel natural immunomodulator, PepO, and described its mode of action. Its efficacy in treating ulcerative colitis was also evaluated. The results showed that PepO can modulate the composition of the intestinal flora through the TLR2 receptor in vivo, reduce macrophage recruitment, improve intestinal inflammatory damage, enhance intestinal barrier function, and effectively treat ulcerative colitis.

[0117] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Anyone skilled in the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by one of ordinary skill in the art without departing from the spirit and technical principles disclosed herein are intended to be covered by the claims of the present invention.

Claims

1. Use of Streptococcus pneumoniae endopeptidase O (PepO) in the preparation of a medicament for treating ulcerative colitis, characterized in that: The amino acid sequence of the Streptococcus pneumoniae endopeptidase O is shown in SEQ ID NO.2, and the encoding nucleotide sequence is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: The Streptococcus pneumoniae endopeptidase O has at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity with the protein encoded by the nucleotide sequence shown in SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO. 2, and is a variant having endopeptidase activity and activity for treating ulcerative colitis.

3. A pharmaceutical composition for treating ulcerative colitis, characterized in that: The invention comprises a therapeutically effective amount of Streptococcus pneumoniae endopeptidase O and a pharmaceutically acceptable carrier, wherein the active ingredient is Streptococcus pneumoniae endopeptidase O, as defined in claim 1 or 2.

4. The pharmaceutical composition according to claim 3, wherein the active ingredient is a nucleic acid molecule encoding Streptococcus pneumoniae endopeptidase O, characterized in that: Its sequence includes at least one of the following sequences (1) the nucleotide sequence shown in SEQ ID NO. 1; (2) a nucleotide sequence that has at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity with the sequence shown in SEQ ID NO: 1 and encodes a protein having endopeptidase activity; (3) a sequence complementary to any sequence described in (1) or (2); (4) RNA equivalent of the sequence shown in SEQ ID NO:

1.

5. A recombinant expression vector, characterized in that Containing the nucleotide sequence of claim 4.

6. An expression system characterized in that Contains the recombinant expression vector according to claim 5 or the exogenous nucleotide sequence according to claim 4 integrated into the genome.

7. A method for preparing a drug for treating ulcerative colitis, comprising the protein according to claim 1 or 2, characterized in that The expression system according to claim 6 is cultured under conditions suitable for expressing the protein.

8. The use according to claim 1, or the pharmaceutical composition according to any one of claims 3 or 4, characterized in that: The pharmaceutical composition is an oral preparation, an enema preparation, a suppository, a sustained-release preparation or an enteric-coated preparation.

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