EcN engineering bacterium for targeted therapy of intestinal inflammation as well as construction method and application of EcN engineering bacterium

Abstract

The invention discloses an EcN engineering bacterium for targeted therapy of intestinal inflammation as well as a construction method and application thereof, and the construction method of the EcN engineering bacterium for targeted therapy of intestinal inflammation comprises the following steps: step 1, synthesizing a PD-1 agonist coding gene, inserting a pBAD carrier after seamless cloning, fusing the pBAD carrier with an OmpA outer membrane protein coding gene, and constructing a recombinant plasmid pBAD-OmpA-MP1-M; 2, the recombinant plasmid pBAD-OmpA-MP1-M obtained in the step 1 is introduced into an EcN strain through an electroporation method, and a strain EcNMP1-M with PD-MP1-M expressed on the surface is obtained; and step 3, mixing the EcNMP1-M bacterial liquid with the intestinal polymer Eudragit L100-55 solution, and adjusting the pH value to induce the formation of a nano coating, so as to prepare the engineering bacterium EcNMP1-M-L. According to the invention, the characteristics of targeting an inflammation part and treating inflammation are realized.

Description

Technical Field

[0001] The present invention relates to the technical field of engineered bacteria, and in particular to an EcN engineered bacterium for targeted treatment of intestinal inflammation, and a construction method and application thereof. Background Art

[0002] Radiation therapy is an important component of cancer treatment. Approximately 50% of cancer patients receive radiation therapy during their disease course, and it contributes to 40% of cancer cures. However, because the intestine is one of the most radiation-sensitive organs in the human body, approximately 75% of patients experience acute intestinal toxicity during radiation therapy for abdominal and pelvic malignancies, with varying degrees of damage to the small intestine, colon, and rectum, which progresses to chronic inflammation.

[0003] Radiation enteritis and its accompanying intestinal mucosal damage are often unavoidable during pelvic and abdominal radiotherapy, and are characterized by immune imbalance, disturbances in the balance of intestinal flora, and damage to the intestinal barrier function. An important feature of radiation-induced intestinal injury is the abnormal activation of the immune response. Programmed death 1 (PD-1) is expressed after activation of T cells, B cells, macrophages, and DC cells, and has been shown to play a key role in regulating peripheral tolerance and immunotherapy. It has been reported that treatments based on the PD-1 / PD-L1 pathway are also widely used in fields such as inflammatory bowel disease, but no practical treatment strategies have been found in the field of radiation-induced intestinal injury.

[0004] Generally speaking, oral administration is considered the simplest and fastest way for drugs to reach the intestinal tract and directly contact the target site. This not only has high patient compliance, but is also an important option for local treatment of digestive tract diseases. However, due to the complex gastrointestinal and intestinal mucosal barriers, and given the potent immunosuppressive effects of PD-1 agonists, systemic administration may increase the risk of infection and cancer.

[0005] Bacteria interact closely with their surroundings, sensing changes in their microenvironment and producing corresponding macro- and small-molecule products. These bacteria can respond to a range of diseases and have been extensively explored for diagnosis and treatment. The mammalian gastrointestinal tract harbors a vast community of commensal bacteria, encompassing over 1,000 different species. Growing evidence suggests that the intestinal microbiome not only participates in food degradation and energy intake but also plays a crucial role in regulating host health and immune responses. Existing treatment studies have shown that probiotics offer the advantage of rapidly regulating microbial balance, but their effectiveness in treating other symptoms of radiation enteritis is poor.

[0006] Probiotics, such as Escherichia coli Nissle 1917 (EcN), can provide significant benefits to the host by modulating the immune system, cellular metabolism, and epithelial barrier function. Live bacterial therapies using probiotics to deliver drug molecules or mediate diagnostics have attracted widespread attention from researchers in biomedical engineering and synthetic biology. By reprogramming microorganisms to meet practical medical and personalized needs, synthetically engineered probiotics offer multiple advantages over wild-type bacteria, including in vivo diagnostics, site-specific drug delivery, sustained release, and mechanically controlled therapeutics. Furthermore, bacterial outer membrane vesicles (OMVs) are non-replicating particles naturally secreted by Gram-negative bacteria. Due to their granular nature, good biocompatibility, and ability to penetrate the intestinal barrier, these vesicles can serve as carriers for therapeutic agents. Their metabolites, SCFAs, can provide energy to colonocytes, have anti-inflammatory effects, and participate in intestinal homeostasis. Therefore, given EcN's intestinal regulatory effects and editability, they could serve as a promising platform for oral drug delivery. Using oral probiotics to target and modulate the local intestinal immune microenvironment holds great potential for reducing intestinal radiation toxicity. Summary of the Invention

[0007] The technical problem to be solved by the present invention is that the existing technology has complex gastrointestinal and intestinal mucosal barriers, and given the strong immunosuppressive effect of PD-1 agonists, systemic administration may increase the risk of infection and cancer and other technical defects. The purpose of the invention is to provide an EcN engineered bacterium for the targeted treatment of intestinal inflammation and its construction method and application. For the first time, the therapeutic strategy of engineered probiotics is applied to the treatment of radiation enteritis, targeting the immunosuppressive receptor PD1 protein of immune cells such as T cells, and using the intestinal polymer Eudragit L100-55 (L100-55) to encapsulate EcN MP1-M (Forming EcN MP1-M -L), enabling it to be released into the intestine under pH-dependent conditions. MP1-M The bacterial outer membrane vesicles (OMVs) released by the bacteria express PD1 agonists on their surface. OMVs penetrate the intestinal epithelium and enter the lamina propria, inhibiting the excessive activation of immune cells such as T cells and macrophages and reducing the expression of inflammatory factors.

[0008] To solve the above technical problems, an embodiment of the present invention provides an EcN engineered bacterium for targeted treatment of intestinal inflammation, wherein the EcN engineered bacterium includes a probiotic and a recombinant expression system (construct) introduced into the probiotic, wherein the recombinant expression system includes a cell surface-displayed PD-MP1-M protein encoding gene and an OmpA outer membrane protein encoding gene, wherein the surface of the EcN engineered bacterium is also coated with a nanocoating, and the nanocoating material is the intestinal polymer Eudragit L100-55 (L100-55).

[0009] Optionally, the PD-MP1-M protein encoding gene sequence is as shown in SEQ ID NO.1, comprising mutation sites A6I, H12D, and L24E.

[0010] Optionally, the OmpA outer membrane protein encoding gene contains an L-arabinose-inducible araBAD promoter.

[0011] The present invention also provides a method for constructing an EcN engineered bacterium for targeted treatment of intestinal inflammation, comprising the following steps:

[0012] Step 1: Synthesize the PD-1 agonist encoding gene (MP1-M), seamlessly clone it into the Nco I and Hind III sites of the pBAD vector, and fuse it with the OmpA outer membrane protein encoding gene to construct the recombinant plasmid pBAD-OmpA-MP1-M;

[0013] Step 2: The recombinant plasmid pBAD-OmpA-MP1-M obtained in step 1 was introduced into the EcN strain by electroporation to obtain the strain EcN expressing PD-MP1-M on the surface. MP1-M ;

[0014] Step 3: EcN MP1-M The bacterial solution was mixed with the enteric polymer Eudragit L100-55 solution, and the pH was adjusted to induce the formation of nanocoating to prepare the engineered bacteria EcN. MP1-M -L.

[0015] In addition, to achieve the above objectives, the present invention also provides a pharmaceutical composition, which comprises any of the above-mentioned EcN engineered bacteria for targeted treatment of intestinal inflammation.

[0016] Optionally, the pharmaceutical composition further comprises a pharmaceutically or pharmacologically acceptable carrier.

[0017] In addition, to achieve the above objectives, the present invention also provides the use of the above-mentioned EcN engineered bacteria for targeted treatment of intestinal inflammation and any of the above-mentioned pharmaceutical compositions in the preparation of intestinal flora regulating drugs or drugs or health products for preventing intestinal inflammation and damage.

[0018] Optionally, the dosage form of the medicine or health product is tablets, capsules, soft capsules, granules, pills, oral liquids, emulsions or dry suspensions.

[0019] The beneficial effects of the above technical solution of the present invention are as follows:

[0020] The present invention targets the immunosuppressive receptor PD1 protein of immune cells such as T cells, designs a strain EcN as the base strain, MP1-M as the efficient agonist of PD1, and constructs the strain EcN by displaying MP1-M on the surface of OmpA protein.MP1-M The surface of the OMVs was modified with the intestinal polymer Eudragit L100-55, and the MP1-M protein was expressed under the induction of arabinose monosaccharide. The released OMVs also expressed the PD1 agonist MP1-M on the surface, inhibiting abnormally activated T cells in inflammation. After L100-55 was dissolved in situ, they colonized in the intestine and produced a large number of OMVs, which entered the propria through the intestinal epithelium, inhibited the excessive activation of immune cells such as T cells and macrophages, and reduced the expression of inflammatory factors. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a diagram showing the single-cell analysis of immune cell dynamics in radiation enteritis and the effect of irradiation on T cells in Example 1, wherein: Figure 1 Middle A is a tSNE diagram showing the analysis results of 22,680 cells, with colors indicating Seurat clusters and major labeled cell types; Figure 1 Middle B is a stacked histogram showing the dynamic changes in the proportions of major cell types in healthy intestinal samples (control) and radiation-exposed intestinal samples at different time points; Figure 1 Middle C is the differential gene map identified in T cells by KEGG pathway analysis comparing irradiated intestinal tissues with healthy tissues; Figure 1 D and Figure 1 Middle (E) shows the changes in the expression of Pdcd1 (PD1) in all cells (D) and T cells (E) in the small intestine tissue of the control group and the irradiation group at different time points; Figure 1 Middle F is a heat map of changes in the expression of inflammation-related genes in T cells in intestinal tissue on day 3 after irradiation;

[0022] Figure 2 For the engineered bacteria EcN in Example 2 MP1-M Characterization analysis diagram, in which, Figure 2 Figure A is a schematic diagram of bacterial surface expression of PD-1 receptor agonists for T cells; Figure 2 Middle B is a Western blot of the expression levels of anchor protein OMPA and fusion protein OMPA-MP1 in EcN; Figure 2 Middle C is a schematic diagram of the binding model of MP1 and PD1 generated using AlphaFold2; Figure 2 D in the middle is the free energy change caused by the mutation evaluated by DDG calculation; Figure 2 Figure E is a schematic diagram of the protein stabilizing mutation site screening process; Figure 2 Middle F is a Western blot showing the expression level of MP1 at different mutation sites of EcN;

[0023] Figure 3 The plasmid map is shown for pBAD-OmpA;

[0024] Figure 4is EcN in Example 2 MP1-M -L characterization analysis diagram, where: Figure 4 A in the middle is EcN using Eudragit L100-55 MP1-M -Schematic diagram of the L preparation process; Figure 4 B in the figure is EcN MP1-M and EcN MP1-M -L grain size distribution curve display; Figure 4 C in the middle stands for EcN MP1-M and EcN MP1-M -SEM image of L, scale bar is 5 μm; Figure 4 D is the EcN in LB medium at 37℃ and pH=2 MP1-M and EcN MP1-M -L growth curve chart; Figure 4 E in the figure is EcN in LB medium at 37℃ and pH=6.8. MP1-M and EcN MP1-M -L growth curve chart; Figure 4 F stands for EcN MP1-M - count map of L;

[0025] Figure 4 Figure G in the middle shows the induction of 108 EcN in drinking water Luc or EcN Luc-L IVIS bioluminescence images of mouse intestine after bacterial administration. Data are expressed as mean ± standard deviation (SD) and analyzed by one-way two-sided ANOVA using GraphPad Prism software. NS, not significant; *P < 0.05; **P < 0.01;

[0026] Figure 5 This is an analysis diagram of the in vitro evaluation of extracellular vesicles (OMVs) and their immunological effects in Example 3; wherein, Figure 5 A in the middle is OMV and OMVs MP1-M Average hydrodynamic size diagram of ; Figure 5 B in the middle is OMV and OMVs MP1-M TEM image, scale bar: 100 nm; Figure 5 C in the middle represents OMV and OMVs MP1-M Western blot of MP1-M expression in ; Figure 5 Middle D is a schematic diagram of the analysis of OMV epithelial penetration in vitro; Figure 5 E in the figure represents the OMVs detected by HIEC in vitro MP1-M Immunofluorescence images of endocytosis, scale bar: 50 μm; Figure 5 F in the middle stands for OMVs MP1-M Immunofluorescence images of epithelial permeability, scale bar: 50 μm; Figure 5 GL in OMVsMP1-M Schematic diagram of flow cytometric analysis of the effects on mouse spleen immune cells;

[0027] Figure 6 is EcN in Example 4 MP1-M -L on the efficacy of RIII treatment, among which, Figure 6 Middle A is a schematic diagram of the construction of the mouse RIII model; Figure 6 Middle B is the comparison of body weight changes of mice in each group; Figure 6 Middle C is the comparison of the changes in disease activity index of mice in each group; Figure 6 Middle D is a comparison of the colon length of mice in each group; Figure 6 Middle E is the comparison of colon length measured in each group of mice; Figure 6 Middle F is the comparison of IFN-γ levels in colon tissues of each group; Figure 6 Middle G is the comparison of TNF-α levels in colon tissues of each group; Figure 6 Middle H shows hematoxylin and eosin (H&E) staining and immunohistochemical analysis of TUNEL and Claudin-3 expression in mouse colon tissues. The data are expressed as mean ± SD. **P < 0.01; ***P < 0.001; ****P < 0.0001.

[0028] Figure 7 is EcN in Example 4 MP1-M -L inhibits the proliferation of intestinal CD4+T cells and reduces the progression of RIII; Figure 7 China A and Figure 7 Middle B shows the representative flow cytometry images of each group and the corresponding quantitative analysis comparison of CD3+CD4+ T cells in the colon; Figure 7 Middle C and Figure 7 Middle D shows the representative flow cytometry images of CD80+CD86+T cells in each group and the corresponding quantitative analysis comparison graphs; Figure 7 Zhong E and Figure 7 Middle F is a comparison of the quantitative analysis of CD4+ and CD8+ T cells in the mesenteric lymph nodes of each group; Figure 7 Middle G and Figure 7 Middle H is the quantitative analysis comparison of CD3+CD4+ and CD3+CD8+ T cells in the spleen of each group; Figure 7 Zhong I and Figure 7 Middle J are the immunofluorescence images of CD4 and F4 / 80 in the colon tissues of mice in each group;

[0029] Figure 8 This is an analysis of the impact of EcN engineered bacteria on intestinal flora diversity in Example 5, where: Figure 8 Middle A is the comparison of SCFA (μg / ml) concentrations in each group; Figure 8Middle B is the Ace index plot at the OTU level evaluated by the Kruskal-Wallis H test; Figure 8 Middle C is the Venn diagram of the abundance levels of intestinal flora in different groups; Figure 8 Middle D is a comparative analysis of the abundance levels of intestinal flora between the G2 group and the G4 group; Figure 8 E in the figure is the result of the principal coordinate analysis (PCoA) of β diversity between G2 group and G4 group at the OTU level, R 2 =0.3118, p=0.004900; Figure 8 Figure F shows the Student's t-test results of Shannon index analysis for Group G2 and Group G4; Figure 8 The column G in the middle shows the LDA score diagram (log10) of the G2 and G4 groups; Figure 8 H in the middle is the Wilcoxon Rank-sum test bar graph of the inter-group differences in species abundance between G2 and G4 at the genus level;

[0030] Figure 9 is EcN in Example 6 MP1-M -L in the analysis of the treatment of inflammatory bowel disease (IBD), among which, Figure 9 Middle A is a schematic diagram of the construction of the colitis mouse model; Figure 9 Middle B is a comparison of the colon length of mice in each group; Figure 9 Middle C is the comparison of colon length measured in each group of mice; Figure 9 D in the middle is the comparison of body weight changes of mice in each group; Figure 9 Middle E is the comparison of the changes in disease activity index of mice in each group; Figure 9 Middle F is the structural staining image of the colon cross section after H&E staining, scale bar: 100 μm; Figure 9 Middle G is a comparison of the expression of IL-1β mRNA levels in the colon tissues of mice in each group; Figure 9 Middle H is a comparison of the expression of IL-6 mRNA levels in the colon tissues of mice in each group; Figure 9 Figure 1 is a comparison of the expression levels of TNFα mRNA in the colon tissues of mice in each group. DETAILED DESCRIPTION

[0031] In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0032] The inventors of the present invention found in their research that an important feature of radiation-induced intestinal injury is the abnormal activation of the immune response. Even if the immune microenvironment is rebuilt after radiation, the PD1 receptor cannot effectively exert its immunosuppressive function. Therefore, local targeted activation of PD1 may be the key to treating immune homeostasis imbalance. On this basis, the inventors used the immunosuppressive receptor PD1 protein of immune cells such as T cells as the target, designed an engineered bacterium EcN with EcN as the chassis bacteria and PD-MP1-M as the efficient agonist of PD1, and constructed an engineered bacterium EcN by displaying MP1-M on the surface of OmpA protein. MP1-M MP1-M protein was expressed under the induction of arabinose monosaccharide, and the surface of the released OMVs expressed the PD1 agonist MP1-M to inhibit abnormally activated T cells in inflammation. In addition, in order to reduce the effect of gastric acid on bacterial activity, the intestinal polymer Eudragit L100-55 (L100-55) was used to inhibit EcN MP1-M After oral administration and reaching the intestine, L100-55 dissolves in situ and releases EcN MP1-M , colonizing in the intestine and producing a large number of OMVs. OMVs enter the lamina propria through the intestinal epithelium, inhibit the excessive activation of immune cells such as T cells and macrophages, reduce the expression of inflammatory factors, and further inhibit the over-activated immune response.

[0033] Based on the above findings, embodiments of the present invention provide an EcN engineered bacterium for the targeted treatment of intestinal inflammation. The EcN engineered bacterium comprises a probiotic and a recombinant expression system (construct) introduced into the probiotic. The recombinant expression system comprises a cell surface-displayed gene encoding a PD-MP1-M protein and a gene encoding an OmpA outer membrane protein. The surface of the EcN engineered bacterium is further coated with a nanocoating made of the enteric polymer Eudragit L100-55 (L100-55).

[0034] The PD-MP1-M gene sequence was integrated into the OmpA outer membrane protein encoding gene. After seamless cloning, the OmpA outer membrane protein encoding gene was inserted into the pBAD vector to generate the recombinant plasmid pBAD-OmpA-PD-MP1-M, whose expression was regulated by the L-arabinose-inducible araBAD promoter. The recombinant plasmid was introduced into the EcN strain by electroporation to generate the EcN engineered bacteria. This EcN engineered bacteria controls expression of the PD-MP1-M gene through the araBAD promoter, thereby expressing the PD1 agonist MP1-M on the surface of released OMVs, thereby suppressing abnormally activated T cells during inflammation. By coating the surface with an L100-55 nanocoating, the engineered bacteria's stability in vivo is enhanced, allowing them to release, colonize, and produce large quantities of OMVs in the intestine.

[0035] Among them, the PD-MP1-M protein encoding gene sequence is shown in SEQ ID NO.1, which contains mutation sites A6I, H12D, and L24E.

[0036] Among them, the OmpA outer membrane protein encoding gene contains the L-arabinose-inducible araBAD promoter.

[0037] The present invention also provides a method for constructing an EcN engineered bacterium for targeted treatment of intestinal inflammation, comprising the following steps:

[0038] Step 1: Synthesize the PD-1 agonist encoding gene (MP1-M), seamlessly clone it into the Nco I and Hind III sites of the pBAD vector, and fuse it with the OmpA gene to construct the recombinant plasmid pBAD-OmpA-MP1-M.

[0039] Step 2: The recombinant plasmid pBAD-OmpA-MP1-M obtained in step 1 was introduced into the EcN strain by electroporation to obtain the strain EcN expressing PD-MP1-M on the surface. MP1-M ;

[0040] Step 3: EcN MP1-M The bacterial solution was mixed with the enteric polymer Eudragit L100-55 solution, and the pH was adjusted to induce the formation of nanocoating to prepare the engineered bacteria EcN. MP1-M -L.

[0041] In addition, to achieve the above-mentioned purpose, the present invention also provides a pharmaceutical composition, which includes the above-mentioned engineered bacteria for targeted treatment of intestinal inflammation.

[0042] Optionally, the pharmaceutical composition further comprises a pharmaceutically or pharmacologically acceptable carrier.

[0043] In addition, to achieve the above objectives, the present invention also provides the use of the above-mentioned engineered probiotics for targeted treatment of intestinal inflammation and the above-mentioned pharmaceutical composition in the preparation of intestinal flora regulating drugs or drugs or health products for preventing intestinal inflammation and damage.

[0044] Optionally, the dosage form of the medicine or health product is tablets, capsules, soft capsules, granules, pills, oral liquids, emulsions or dry suspensions.

[0045] Next, the technical solution of the present invention will be further described in conjunction with specific embodiments.

[0046] Example 1: Single-cell analysis of immune cell dynamics in radiation enteritis and design of targeted regulation by engineered bacteria

[0047] Chronic low-level expression of inflammatory factors is the main hallmark of intestinal inflammation and ultimately leads to an imbalance of immune homeostasis in the intestine. In order to further study the dynamic changes of immune cells after radiation-induced intestinal damage, the RIII single-cell dataset (GSE165318) was obtained from the GEO database. This dataset covers intestinal single-cell sequencing data on days 1, 3, 7, and 14 after irradiation and the non-irradiated control group. The datasets 1, 3, 7, and 14 days after radiation were analyzed at the single-cell level. The t-SNE dimensionality reduction technology was used to reduce the high-dimensional single-cell data. According to the classic marker genes, 22,680 cells were divided into 11 different cell types, including transit amplifying cells (TA), Paneth cells, enteroendocrine cells (EEC), goblet cells, stem cells, intestinal epithelial cells, endothelial cells, stromal cells, myeloid cells, T cells, and B cells (such as Figure 1 (as shown in A in the figure).

[0048] After irradiation, the intestinal microenvironment changed significantly. The number of immune cells such as T cells and bone marrow cells decreased significantly on the first day after irradiation, increased rapidly on the third day, and returned to the baseline level comparable to the control group on the 14th day. The stacked graph shows the changes in the proportion of cell subsets between the control group and the irradiated group at different time points, focusing on the changes in the proportion of T cells, suggesting their key role in the RIII inflammatory storm (such as Figure 1 (B) The results suggest that the intestinal immune microenvironment can be reestablished within 14 days after irradiation. However, by analyzing gene expression changes in T cells at different time points after irradiation, compared with the control group, we identified genes that were upregulated between days 1 and 14.

[0049] FindMarkers was used to calculate the differential genes of T cells in the irradiated group and the control group at different time points, and then the intersection of the differential genes was taken to obtain a total of 406 common differential genes. KEGG enrichment analysis was performed using the clusterProfiler package, and it was found that the PD-1 / PD-L1 immune checkpoint pathway was significantly enriched. Further focusing on the samples on the third day after irradiation (the time point with the most significant difference), DESeq2 was used to analyze significantly upregulated genes, and it was found that JAK-STAT, Th17 differentiation and IL-17 pathways were enriched. KEGG pathway analysis showed that multiple inflammatory signaling pathways were enriched, and the immunosuppressive PD1-PDL1 signaling pathway was one of the related pathways (such as Figure 1 These findings suggest that inflammatory factors continue to be expressed while the immune microenvironment is reconstructed.

[0050] Next, the expression of PD1 receptors on all immune cells and T cells was examined. PD1 expression was positively correlated with T cell counts, reaching the highest level on day 3 and returning to a level comparable to the control group by day 14 (e.g., Figure 1 Middle D, Figure 1In addition, the transcriptional levels of inflammatory factors in T cells were detected on the third day, and it was found that compared with the control group, radiation caused a significant increase in inflammatory factors in T cells (as shown in Figure E). Figure 1 These results suggest that even after radiation, when the immune microenvironment is reconstituted, the PD1 receptor cannot effectively exert its immunosuppressive function. Therefore, localized targeted activation of PD1 may be key to treating immune homeostasis imbalances.

[0051] Example 2, engineered bacteria EcN MP1-M Build

[0052] Based on the above findings, the idea of achieving RIII treatment by constructing engineered bacteria to target and activate the PD1 signaling pathway was clarified, and it was found that the small molecule PD-MP1 can inhibit T cell activation by strongly activating the PD1 receptor in vitro and in vivo. Figure 2 As shown in Figure A, PD-MP1 is displayed on the surface of Escherichia Coli Nissle 1917 (EcN). Figure 2 As shown in B, Western blotting results showed that protein expression was significantly reduced after insertion of MP1 into OmpA.

[0053] To further enhance the stability of PD-MP1 expression, the 3D structure of PD-MP1 was computationally simulated and its interaction with PD1 was analyzed using Alphafold2, and the model with the highest score was selected as the final representation model (e.g. Figure 2 The model was processed and the model with the lowest energy score was used for Rosetta Cartesian ddG calculation. Each residue of PD-MP1 was mutated to one of the other 19 amino acids (excluding cysteine). Mutations that resulted in a ddG of less than -1.0 kcal / mol were identified as potential candidates to evaluate the change in free energy (DDG). Figure 2 As shown in D, the change in free energy caused by the mutation was evaluated by DDG calculation, and a negative DDG value indicated that the mutation could enhance protein stability or binding affinity.

[0054] Without changing the binding affinity, the binding model selected three mutation sites on PD-MP1: alanine (A) to isoleucine (I) at position 6, histidine (H) to aspartic acid (D) at position 12, and leucine (I) to glutamic acid (E) at position 24, which were named A6I, H12D, and L24E, respectively (the protein stabilization mutation site screening process is as follows Figure 2 The wild-type and mutant PD-MP1 proteins were examined by Coomassie blue staining and Western blotting (Fig. Figure 2F). The results showed that under arabinose (ARA) induction, the L24E mutant PD-MP1 showed the highest protein expression level, indicating that the L24E mutation provides the greatest stability of PD1-MP1 protein expression.

[0055] The OmpA gene was inserted into the pBAD vector, which contains the L-arabinose-inducible araBAD promoter, via Nco I and Hind III restriction endonucleases. The PD-MP1-M gene sequence was integrated into the third transmembrane domain of the OmpA gene to obtain the construct pBAD-OmpA-PD-MP1-M. The plasmid was synthesized by Azenta ( Figure 3 The recombinant plasmid pBAD-OmpA-PD-MP1-M was introduced into EcN by electroporation. The parameters of 2.5 kV, 200 Ω resistance, 5 ms time constant and three electroporation pulses were used. The strain expressing PD-MP1-M was named EcN. MP1-M .

[0056] In addition, to enhance the stability of the engineered bacteria in the body and release them in the intestine, Eudragit L100-55 (L100-55) was selected as a material to reduce the effect of gastric acid on bacterial activity. L100-55 is an anionic copolymer derived from methacrylic acid and ethyl acrylate. It remains stable under acidic conditions and dissolves when the pH exceeds 5.5, which promotes the targeted release of bacteria in the intestine and protects them from damage caused by gastric acid. MP1-M Nano coating wrapping. Figure 4 As shown in A, at 0.04 mg mL -1 L100-55 medium was incubated with calcium-anchored EcN MP1-M and then lowered to pH = 5 to prepare EcN MP1-M -L, and the EcN was detected by particle size analyzer and scanning electron microscope (SEM) images. MP1-M -L was identified to prove that the nano coating was successfully wrapped (such as Figure 4 Middle B, Figure 4 (as shown in C).

[0057] The pH sensitivity of L100-55 complexation with calcium ions is crucial for the formation and initiated dissolution of the nanocoating. MP1-M -L activity was determined by incubating the coated bacteria in LB (pH = 5.0 or 6.8) to check EcN MP1-M-L inactivation and reactivation. Uncoated EcN was used as a control. The bacterial suspension was diluted into 200 μl fresh LB to a value of 0.2 at OD600, and then cultured at 37°C with gentle shaking. At predetermined time points, the OD value of the culture medium was recorded at a rate of 600 nm every 30 minutes in a 96-well plate. To quantify bacterial growth, 20 μl of bacteria were incubated in 980 μl fresh LB (pH 5.0 or 6.8) for predetermined time intervals. Then, 50 μl of each sample was taken, washed with fresh LB medium, and spread on an LB agar plate containing 100 μg / ml kanamycin. The colonies were counted after incubation at 37°C for 24 hours. The results are shown in the figure. Figure 4 Middle D, Figure 4 As shown in F, EcN MP1-M -L is inactivated at pH = 2 and reactivated at pH = 6.8, and its activity is similar to that of EcN. MP1-M This is also consistent with the expected in vivo therapeutic process.

[0058] Next, the luciferase gene luc was inserted after the OmpA sequence for bioluminescence imaging, and the resulting strain expressing luciferase was named EcN luc .like Figure 4 As shown in G, in vitro experiments, EcN luc -L showed bioluminescent signal 5 minutes after Ara induction, and the bioluminescent signal at 30 minutes was stronger than that at 5 minutes, indicating that EcN luc -L system can successfully express the target protein and the secretion of protein gradually increases over a period of time. MP1-M -L system to successfully express the target protein.

[0059] Example 3. Characterization and functional verification of bacterial outer membrane vesicles (OMVs)

[0060] like Figure 5 China A and Figure 5 As shown in B, OMVs were extracted by ultracentrifugation. MP1-M , particle size analysis and transmission electron microscopy showed that OMVs and OMVs MP1-M The average diameter of OMVs was approximately 100 nm, indicating that MP1-M expression on OMVs did not affect the morphology or size of the vesicles. In addition, protein expression on OMVs was evaluated, and Western blotting results showed that OMVs and OMVs were induced by ARA. MP1-M The protein was expressed in all groups, indicating that MP1-M can exert immunosuppressive effects through OMV (e.g. Figure 5 (as shown in C).

[0061] To determine EcN MP1-M Secreted OMVs MP1-MTo investigate whether OMVs have the potential to cross the intestinal epithelial barrier in vitro and in vivo, we cultured HIEC cells in the upper chamber of a Transwell system for 21 days to mimic the intestinal epithelial barrier. MP1-M Introduced into the upper chamber (such as Figure 5 The OMVs were evaluated by measuring the MYC fluorescence intensity in HIEC cells in the lower chamber. MP1M The results showed that OMVs MP1-M Crossing the epithelial cell layer in the upper chamber and entering the lower chamber (such as Figure 5 (as shown in E).

[0062] In addition, in vivo mouse enema experiments were performed to evaluate the MP1-M Ability to penetrate the colon. Healthy C57BL / 6 mice aged 6 to 8 weeks were selected for the study. After fasting for 6 hours, epithelial permeation was performed. Anesthesia was induced using 0.5-1.0% isoflurane, followed by intestinal ligation. Subsequently, 500 μL of control outer membrane vesicles (OMVs) and OMVs were added. MP1-M The ligated intestinal cavity was given and incubated for 2 hours. Throughout the experiment, efforts were made to maintain the body temperature of the mice within a stable range. Colon tissue samples were then collected for immunofluorescence analysis. After 2 hours, the colon was removed and analyzed using immunofluorescence. The results are shown in Figure 2. Figure 5 As shown in F, OMVs MP1-M Efficiently crosses the intestinal epithelial barrier.

[0063] The above results showed that after oral administration, EcN MP1-M Secreted OMVs MP1-M OMVs have the potential to cross the intestinal epithelial barrier in vitro and in vivo. MP1-M It can cross the small intestinal wall and exert immunomodulatory effects.

[0064] To further evaluate the OMVs MP1-M To investigate the effects of OMVs on immune cells in vitro, mouse spleens were homogenized and the resulting cell suspensions were exposed to OMVs or OMVs. MP1-M As shown in Figures 5G-5H, flow cytometry analysis showed that OMVs MP1-M Compared with the OMV group, the CD4 + IL - 17A + This indicates that MP1-M inhibited the proliferation and function of inflammatory Th17 cells. As shown in Figure 5I and Figure 5J, compared with the PBS control group, OMVS MP1-M CD4 + IFNγ +The proportion of Th1 cells decreased, and there was no significant difference between the two, indicating that EcN-derived OMVs can inhibit the presence of Th1 cells. In addition, as shown in Figures 5K-5L, OMVs MP1-M CD11C + MHCⅡ + The proportion of cells in the OMVs was also significantly reduced, indicating that MP1-M reduced the number of innate immune cells, especially dendritic cells (DCs). MP1-M It can not only regulate specific immune responses by changing the activity and number of Th1, Th17 and other T cells, but also reduce the number of DC cells to inhibit innate immune responses.

[0065] Example 4, EcN MP1-M -L improves radiation-induced intestinal damage

[0066] This example is used to illustrate the therapeutic effect of the engineered bacterial strain provided by the present invention on radiation enteritis caused by radiation-induced intestinal damage.

[0067] Eight-week-old female C57 BL / 6 mice were exposed to a single intraperitoneal irradiation dose of 8 Gy. Subsequently, the mice were randomly assigned to different groups and administered PBS, EcN-L, EcN-L, and EcN-L by gavage on days 3, 6, 8, and 10. MP1-M -L or 5-ASA (5-aminosalicylic acid), and the mice were Figure 6 The patients were assigned to different treatment groups as shown in A: PBS (Group 1), 8Gy+PBS (Group 2), 8Gy+EcN-L (Group 3), 8Gy+EcNMP1-ML (Group 4) and 8Gy+5-ASA (Group 5). During the entire 14-day experimental period, body weight changes were monitored daily and the disease activity index (DAI) score was recorded. The results are shown in Figure 6 Middle B, Figure 6 As shown in C, at the initial stage of 8Gy, the body weight of G2, G3, G4 and G5 groups decreased significantly, while EcN induced by arabinose MP1-M The expression of -L alleviated this effect and was consistent in the DAI score. The changes in colon length were then measured. The length of the mouse colon can reflect the severity of colitis. Figure 6 D and Figure 6 As shown in F, EcN MP1-M -L mitigates the effect of 8 Gy radiation on colon length and shows better results compared with 5-ASA.

[0068] Typical inflammatory cytokines, including interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α), were assessed in colonic tissues and quantified on day 14 by ELISA. Figure 6Medium F and Figure 6 As shown in G, the levels of IFN-γ and TNF-α in the colon tissue of mice in group G2 were significantly increased, and EcN MP1-M The levels of inflammatory cytokines were significantly decreased in mice treated with -L.

[0069] On the last day, the entire colon was resected and 0.5 cm distal sections were obtained for histological examination using H&E staining and immunofluorescence and immunohistochemical analysis. Intestinal cell death and intestinal barrier integrity were further examined by H&E, TUNEL and Claudin-3 immunohistochemical staining. Figure 6 As shown in Figure H, H&E staining showed that compared with the G2 group, the infiltration of immune cells in the G4 group was significantly reduced, apoptosis was increased in G2, and the expression of intestinal barrier markers Claudin-3, Occludin, and villin was downregulated. In contrast, these symptoms were significantly alleviated in G4.

[0070] Lamina propria lymphocytes (LPL) were isolated from the intestine, and the changes in T cell subsets and antigen-presenting cell (APC) subsets were analyzed by flow cytometry. Figure 7 Middle A- Figure 7 As shown in D, compared with the G2 group or other treated mice, EcN MP1-M -L treated mice showed a significant decrease in the proportion of CD3+CD4+ T cells and antigen presenting cells. MP1 -M -L can effectively inhibit the infiltration of CD3+CD4+T cells and antigen-presenting cells (CD80+CD86+).

[0071] In addition to affecting immune cells in the intestinal lamina propria, EcN MP1-M Whether the OMVs secreted by EcNs affect other systemic immune organs. On day 14, mesenteric lymph node cells and spleen cells were collected for further flow cytometric analysis. MP1-M -L did not significantly alter the number of CD4+ or CD8+ cells in the mesenteric lymph nodes or spleen (eg, Figure 7 Middle E- Figure 7 In addition, tissue immunofluorescence staining confirmed that EcN MP1-M Colonic tissues of RIII mice treated with -L showed decreased CD3+CD4+ T cells and F4 / 80 macrophages ( Figure 7 Medium I- Figure 7 These results indicate that EcN MP1-M-L can locally regulate immune cells in the intestinal layer, thereby inhibiting the infiltration and activity of inflammatory T cells and antigen-presenting cells. The above results show that EcN MP1-M -L exerts its effects in specific areas of the intestine and does not affect the activity or number of immune cells in the lymph nodes and spleen, with a good safety profile.

[0072] Example 5, EcN MP1-M -L Reduce intestinal flora imbalance

[0073] This example is used to illustrate whether the engineered bacterial strain provided by the present invention can change the richness of intestinal flora.

[0074] SCFA is a key metabolite that maintains the homeostasis of the intestinal environment and has important anti-inflammatory effects. First, the SCFA levels in the feces of mice from different groups were measured. The results are as follows Figure 8 As shown in A, EcN MP1-M -L significantly upregulated SCFA concentrations, indicating that EcN MP1-M -L has the effect of reducing inflammatory response, and EcN MP1-M -L may alter the abundance of intestinal microbiota.

[0075] Furthermore, in the diversity analysis, the alpha diversity of species in the sample is usually evaluated using Chao, ACE and other indices. Among them, Chao1 index can reflect the richness of the community, and ACE index can reflect the diversity of the community. 16S rDNA sequencing was performed on the intestinal flora in mouse feces. Ace and Chao diversity indices showed that EcN in G2 group MP1-M -L treatment significantly increased the diversity and abundance of intestinal microbiota (e.g. Figure 8 The number of unique and common OTUs in different groups was determined using a Venn diagram to visually present the similarities and differences in the OTU composition of each experimental group. Figure 8 The OTU Venn diagram shown in Figure C shows 260 shared OTUs across all five groups, with groups G1, G2, G3, G4, and G5 having 161, 18, 37, 75, and 71 unique OTUs, respectively. Group G4 showed a significantly greater number of unique OTUs compared to group G2. Further analysis showed that group G4 had 387 unique OTUs compared to group G2 (e.g., Figure 8 The PCoA of fecal bacterial communities showed significant differences between G2 and G4 at the OTU level (as shown in Figure 2D). Figure 8 Student's t-test was performed to analyze the Shannon index difference between G2 and G4, and consistent results were observed in the diversity analysis (as shown in Figure 5E). Figure 8The significant differences in microbial taxa between G2 and G4 were identified using Lefse, and the abundance of genera with LDA scores > 4 was significantly increased in G4 compared to G2. Only the Prevotellaceae was more abundant in G2, while other bacteria showed higher abundance in G4 (e.g. Figure 8 (shown as G in the figure).

[0076] Finally, the Wilcoxon rank sum test was used to analyze the differences in microbiome at the genus level. Figure 8 As shown in Figure H, compared with the G2 group, the proportion of Staphylococcus coli in the feces of mice with radiation-induced colitis in the G4 group was significantly reduced, while the abundance of Prevotellaceae was significantly increased. MP1-M -L can increase the richness of intestinal flora in RIII mice and promote damage repair.

[0077] Example 6, EcN MP1-M -L therapeutic effect in inflammatory bowel disease (IBD)

[0078] This example is used to illustrate the therapeutic effect of the engineered bacterial strain provided by the present invention on inflammatory bowel disease, wherein inflammatory bowel disease (IBD) is a chronic gastrointestinal disease, including Crohn's disease and ulcerative colitis (UC), which affect the gastrointestinal tract and colon, respectively. The number of lymphocytes expressing PD-1+ and PD-L1+ in the lamina propria of IBD patients (especially UC patients) is significantly increased, indicating that the PD-1 / PD-L1 pathway is involved in the pathogenesis of UC. Studies have shown that high expression of PD-L1 protein can reduce the severity of colitis symptoms induced by dextran sodium sulfate (DSS) in UC mice.

[0079] Therefore, in order to evaluate Ec NMP1-M To investigate the therapeutic effect of L-L, a 3% DSS-induced colitis mouse model was established and the mice were treated as Figure 9 The patients were divided into different treatment groups as shown in A: PBS (Group 1), DSS+PBS (Group 2), DSS+EcN MP1-M -L(Group 3),DSS+EcN MP1-M -L (Group 4) and DSS+5-ASA (Group 5). In G4, EcN MP1-M -L and 2 g / L arabinose were orally administered. During the 10-day experimental period, daily changes in body weight were monitored and DAI (Disease Activity Index) scores were recorded.

[0080] The results are as follows Figure 9 D and Figure 9As shown in E, similar to the radiation colitis model, in the IBD mouse model, DSS significantly reduced the body weight of mice in the G2 group, while arabinose-induced EcN MP1-M -L expression reduced weight changes. DAI scores further indicated that the treated mice were in better health. Changes in colon length were also measured. Figure 9 Middle B and Figure 9 As shown in C, EcN MP1-M -L reduced the effect of DSS on colon length, and the results were better than those of 5-ASA treatment. H&E staining showed that the number of infiltrating monocytes in the G4 group was significantly reduced compared with the G2 group, and the tissue structure was better preserved (such as Figure 9 (as shown in F in the figure).

[0081] The mRNA expression levels of inflammatory cytokines in intestinal tissues were detected by RT-qPCR. Figure 9 Medium G- Figure 9 As shown in Figure 1, the levels of inflammatory cytokines IL-1β, TNF-α, and IL-10 were significantly upregulated in the G2 group, but downregulated to normal levels in the G4 group. MP1-M -L strongly inhibited the progression of inflammation.

[0082] In summary, the present invention has constructed a broad-spectrum probiotic EcN MP1-M -L, which not only treats radiation colitis, but also has a significant therapeutic effect on inflammatory bowel disease (IBD), providing great potential for clinical promotion of treatment.

[0083] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. An EcN engineered bacterium for targeted treatment of intestinal inflammation, characterized in that: The EcN engineered bacteria include probiotics and a recombinant expression system introduced into the probiotics, wherein the recombinant expression system includes a cell surface-displayed PD-MP1-M protein encoding gene and an OmpA outer membrane protein encoding gene, wherein the surface of the EcN engineered bacteria is coated with a nanocoating, and the nanocoating material is the intestinal polymer Eudragit L100-55.

2. The EcN engineered bacteria for targeted treatment of intestinal inflammation according to claim 1, characterized in that The PD-MP1-M protein encoding gene sequence is shown in SEQ ID NO.1, which contains mutation sites A6I, H12D, and L24E.

3. The EcN engineered bacteria for targeted treatment of intestinal inflammation according to claim 1, characterized in that The OmpA outer membrane protein encoding gene contains the L-arabinose-inducible araBAD promoter.

4. A method for constructing an EcN engineered bacterium for targeted treatment of intestinal inflammation, characterized in that: The following steps are involved: Step 1: Synthesize the PD-1 agonist MP1-M encoding gene, seamlessly clone it into the Nco I and Hind III sites of the pBAD vector, and fuse it with the OmpA outer membrane protein encoding gene to construct the recombinant plasmid pBAD-OmpA-MP1-M; Step 2: The recombinant plasmid pBAD-OmpA-MP1-M obtained in step 1 was introduced into the EcN strain by electroporation to obtain the strain EcN expressing PD-MP1-M on the surface. MP1-M ; Step 3: EcN MP1-M The bacterial solution was mixed with the enteric polymer Eudragit L100-55 solution, and the pH was adjusted to induce the formation of nanocoating to prepare the engineered bacteria EcN. MP1-M -L.

5. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the EcN engineered bacteria for targeted treatment of intestinal inflammation according to any one of claims 1 to 3 or the engineered bacteria EcN prepared by the construction method according to claim 4. MP1-M -L.

6. The pharmaceutical composition according to claim 5, characterized in that The pharmaceutical composition further includes a pharmaceutically or pharmacologically acceptable carrier.

7. Use of the engineered probiotics for targeted treatment of intestinal inflammation according to claim 1 and the pharmaceutical composition according to any one of claims 5 to 6 in the preparation of drugs for regulating intestinal flora or drugs or health products for preventing intestinal inflammation and damage.

8. The use according to claim 7, characterized in that The dosage form of the medicine or health product is tablets, capsules, soft capsules, granules, pills, oral liquids, emulsions or dry suspensions.

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