Construction method and application of recombinant engineering bacterium EcN with antibacterial immune peptide Scy displayed on surface

Abstract

The invention discloses a construction method and application of a recombinant engineering bacterium with an antibacterial immune peptide Scy displayed on the surface. According to the present invention, the probiotic Escherichia coli Niss1917 (EcN) is adopted as the host bacterial strain, and the pnirBmisL (pnir) is adopted as the plasmid vector, such that the efficient display of the antibacterial immune peptide Scy in the crab semen from the heterologous source on the host bacterial surface is successfully achieved; according to the method, a complex purification step of the antibacterial peptide is avoided, so that the antibacterial peptide Scy can be directly applied through the engineering bacterium EcN-Scy. The engineering bacteria still show relatively high survival activity under the condition of simulating strong acid and high salt of gastrointestinal tracts; meanwhile, the composition has a protection effect on mouse intestinal mucosal barrier injury caused by dextran sodium sulfate (DSS), can remarkably relieve DSS-induced mouse weight loss, shortened colon length and other symptoms, improves the colon form, relieves liver injury, and can be used for preventing and treating liver injury through multiple ways of regulating intestinal immune cell factors, enhancing the oxidation resistance, maintaining intestinal physical barriers and the like. The intestinal health is comprehensively protected, and a new biological preparation candidate is provided for treating the inflammatory bowel disease.

Description

Technical Field

[0001] The invention relates to a surface-displaying antimicrobial immune peptide Scy Escherichia coli genetic engineering bacterium, a construction method and application thereof, and belongs to the field of genetic engineering and biotechnology. Background Art

[0002] When dealing with infectious diseases, the medical field faces the severe challenge of antimicrobial resistance. For a long time, the widespread use of antibiotics has saved countless lives, but it has also led to the rapid spread of drug-resistant strains, which has gradually weakened the efficacy of traditional antibiotics and greatly threatened public health safety.

[0003] Faced with this global problem, countries are actively seeking solutions. On the one hand, they are strengthening supervision, limiting the abuse of antibiotics, and striving to slow down the development of drug resistance; on the other hand, they are actively exploring and developing new antimicrobial agents to meet the challenges of drug-resistant bacteria. Among them, antimicrobial peptides have become a research hotspot due to their unique antimicrobial mechanism and low toxicity.

[0004] However, the scarcity and difficulty of extracting natural antimicrobial peptides limit their large-scale application. To this end, genetic engineering and biotechnology have opened up new paths for the research, development and production of antimicrobial peptides. Through genetic engineering technology, researchers can accurately introduce genes encoding antimicrobial peptides into suitable microorganisms or cells to achieve the expression of antimicrobial peptides. At the same time, using biotechnology, the production process of antimicrobial peptides can be further optimized, and their stability and yield can be improved, thereby meeting the urgent demand for antimicrobial peptides in the medical field.

[0005] However, even with the support of genetic engineering technology, the production of antimicrobial peptides still faces many challenges. One of them is how to improve the stability and yield of antimicrobial peptides while maintaining their activity. In addition, since infectious diseases often occur in specific physiological environments (such as the intestine), how to ensure that antimicrobial peptides can accurately and efficiently reach the target site and exert their effects is also an issue that needs to be addressed. Summary of the invention

[0006] In order to overcome the shortcomings of the prior art, the purpose of the present invention is to provide a method and application of constructing a recombinant engineered bacterium EcN displaying an antimicrobial immune peptide Scy on its surface. The present invention displays antimicrobial peptides on its surface by constructing an engineered bacterium. This engineered bacterium can not only maintain the activity of antimicrobial peptides in a complex physiological environment, but also has the characteristics of acid and salt resistance, and can smoothly reach the animal intestine and play a targeted role. This innovation not only overcomes the technical difficulties in the production of traditional antimicrobial peptides, but also provides the possibility of realizing the wide application of antimicrobial peptides in the treatment of infectious diseases.

[0007] A method for constructing a recombinant engineering bacterium EcN-Scy displaying an antimicrobial immune peptide Scy on its surface comprises the following steps: a) obtaining and designing a gene sequence encoding an antimicrobial immune peptide Scy from crab semen, wherein the gene sequence is codon-optimized to adapt to the expression system of Escherichia coli, and performing full gene synthesis; b) connecting the antimicrobial immune peptide Scy gene sequence obtained in step a) with the plasmid vector pnir to construct a recombinant plasmid pnir-Scy; c) transforming the recombinant plasmid pnir-Scy constructed in step b) into Escherichia coli DH5α to obtain positive clones; d) extracting and purifying the recombinant plasmid pnir-Scy from the positive clones obtained in step c), and confirming the correctness and purity of the recombinant plasmid by electrophoresis and sequencing; e) The recombinant plasmid pnir-Scy obtained in step d) is introduced into the expression host Escherichia coli Nissle1917 EcN by electroporation to obtain the recombinant engineered bacteria EcN-Scy.

[0008] The antimicrobial immune peptide Scy gene in step a) is codon-optimized, and the sequence is shown in Seq ID No.1.

[0009] The connection operation in step b) includes connecting the PCR linearized antimicrobial immune peptide Scy gene with the plasmid pnir using homologous recombination technology.

[0010] The application of the recombinant engineered bacteria EcN-Scy is used to prepare a drug that has a protective effect on intestinal mucosal barrier damage in mice caused by dextran sulfate sodium (DSS). The drug is used to alleviate DSS-induced weight loss and shortening of colon length in mice, improve colon morphology, and alleviate liver damage.

[0011] The recombinant engineered bacteria EcN-Scy is used in the preparation of drugs or functional foods for treating or preventing intestinal inflammatory diseases. The recombinant engineered bacteria protect intestinal health by regulating intestinal immune cytokines, enhancing antioxidant capacity, and maintaining intestinal physical barriers.

[0012] The application is to prepare a pharmaceutical composition for treating or preventing intestinal inflammatory diseases, which comprises the recombinant engineered bacteria EcN-Scy as an active ingredient and a pharmaceutically acceptable carrier or excipient.

[0013] Beneficial effects of the present invention: (1) The present invention constructs an engineered bacterium EcN-Scy through genetic engineering, and the bacterium has the characteristics of acid resistance and bile salt resistance; (2) The invention uses the engineered bacteria EcN-Scy for animal experiments. For mice with DSS-induced colitis, it can significantly alleviate the weight loss and shortening of the colon caused by DSS, and improve the morphological structure of the colon. In terms of serum physiology and biochemistry, oral administration of the engineered bacteria EcN-Scy can significantly alleviate the liver function damage caused by DSS in mice. Compared with the DSS-treated group, the pro-inflammatory factors tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), interleukin 6 (IL-6), and monocyte leukemia in the colon of mice treated with the engineered bacteria EcN-Scy were significantly increased. The gene expression of cell chemoattractant protein-1 (MCP) was significantly decreased; the antioxidant indicators of colon tissue were measured, and it was found that the content of NO and T-SOD in the colon of mice treated with DSS by gavage with the engineered bacteria EcN-Scy showed an upward trend, and the gene expression of MPO in the colon was significantly reduced; in terms of maintaining the intestinal physical barrier, the gene expression of colon tight junction proteins such as ZO-1 and ZO-2 was significantly increased; the engineered bacteria EcN-Scy can protect intestinal health through multiple pathways; it provides a new candidate for biological agents for the treatment of inflammatory bowel disease and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The accompanying drawings are part of the present invention and are used to further illustrate but not to limit the scope of the present invention.

[0015] Figure 1 This is a schematic diagram of agarose gel electrophoresis of gene Scy.

[0016] Figure 2 This is a schematic diagram of agarose gel electrophoresis of plasmid pnir.

[0017] Figure 3 It is a schematic diagram of the fusion plasmid pnir-Scy.

[0018] Figure 4 This is a schematic diagram of the survival rate of engineered bacteria under different pH conditions and different salt concentration conditions.

[0019] Figure 5 This is a schematic diagram of the weight changes of each group of mice after being gavage-fed with engineered bacteria.

[0020] Figure 6 This is a schematic diagram comparing the colon lengths of each group of mice fed with engineered bacteria.

[0021] Figure 7 This is a schematic diagram of HE-stained colon sections of each group of mice fed with engineered bacteria.

[0022] Figure 8 This is a schematic diagram of the levels of serum liver function indicators in each group of mice fed with engineered bacteria.

[0023] Fig. 9This is a schematic diagram of the gene expression levels of colon immune cytokines in each group of mice fed with engineered bacteria.

[0024] Fig.10 This is a schematic diagram of the levels of colon antioxidant indicators in each group of mice fed with engineered bacteria.

[0025] Fig.11 This is a schematic diagram of the levels of colon intestinal physical barrier function indicators in each group of mice fed with engineered bacteria. DETAILED DESCRIPTION

[0026] The present invention is further described in detail below with reference to the accompanying drawings and embodiments.

[0027] The invention discloses a surface-displaying antimicrobial immune peptide Scy Escherichia coli genetic engineering bacterium and a construction method and application thereof.

[0028] The engineered bacteria efficiently integrates the linearized gene Scy obtained by PCR into the linearized plasmid pnir through homologous recombination technology. The PCR results are as follows Figure 1 and Figure 2 , forming the recombinant plasmid pnir-Scy. The schematic diagram of the recombinant plasmid is shown in Figure 3 The recombinant plasmid was then electroporated into competent EcN to obtain the engineered bacteria EcN-Scy. The antimicrobial immune peptide Scy gene was codon optimized, and the sequence is shown in Seq ID No.1.

[0029] The construction of the above-mentioned recombinant E. coli strain, analysis of its stress resistance characteristics, and the protective effect of the engineered bacteria on intestinal health mainly include the following steps: (I) Construction of recombinant expression vector; (II) Construction and induced expression of engineered bacteria; (III) Analysis of stress resistance characteristics of engineered bacteria; (IV) Protective effect of engineered bacteria on DSS-induced colitis in mice.

[0030] Furthermore, step (i) the construction of the recombinant expression vector comprises the following steps: (1) Accurately obtain the linearized Scy gene and plasmid pnir through PCR technology for amplification of antimicrobial immune peptides The specific primers for the Scy gene sequence were designed as follows: Scy F 5'-tatgtgcacacggacccgggGGCCAGGCACTGAATAAACTGA -3'; Scy R 5'- tcgccatcttcatggaattcATAGCTTGCAATCCAATCTTCCA -3'.

[0031] The amplification primers of the linearized plasmid pnir are as follows: pnir F 5'-GAATTCCATGAAGATGGCGAA-3'; pnir R 5'-CCCGGGTCCGTGTGCACA-3'.

[0032] Amplify the antimicrobial immune peptide Scy gene sequence according to the following reaction system and procedure: The PCR reaction system was as follows: ddH2O, 32 μL; 5 × SF Buffer (with 10 mM MgSO4), 10 μL; dNTP Mix (10 mM each), 1 μL; upstream primer (10 μM), 2 μL; downstream primer (10 μM), 2 μL; template DNA, 2 μL; Phanta HS Super-Fidelity DNA Polymerase, 1 μL; The reaction program was as follows: pre-denaturation at 95°C for 3 min, 35 cycles of denaturation at 95°C for 10 s, annealing at 61°C for 30 s, extension at 72°C for 10 s, and final extension at 72°C for 10 min.

[0033] Amplify the plasmid pnir sequence according to the following reaction system and procedure: The PCR reaction system was as follows: ddH2O, 32 μL; 5 × SF Buffer (with 10 mM MgSO4), 10 μL; dNTP Mix (10 mM each), 1 μL; upstream primer (10 μM), 2 μL; downstream primer (10 μM), 2 μL; template DNA, 1 μL; Phanta HS Super-Fidelity DNA Polymerase, 1 μL; The reaction conditions were as follows: pre-denaturation at 95°C for 3 min, 35 cycles of denaturation at 95°C for 10 s, annealing at 60°C for 30 s, extension at 72°C for 60 s, and final extension at 72°C for 5 min.

[0034] (2) Then prepare a 1% agarose gel and perform spotting and electrophoresis on the gel. After electrophoretic separation, accurately cut the gel to recover the target band for subsequent homologous recombination to construct a recombinant plasmid.

[0035] (3) The linearized Scy purified by gel recovery and homologous recombination technology was connected to the linearized plasmid pnir. The connection reaction system and conditions are as follows: Linearized vector pnir, 100 ng; target gene Scy, 10 ng; homologous recombinase, 5 μl; ddH2O, fill to 10 μl; single fragment recombination reaction, 50℃, 10 min; cool to 4℃ or immediately put on ice.

[0036] (4) The recombinant product was transformed into competent E. coli DH5α, and after heat shock transformation, it was spread on an ampicillin-resistant plate and cultured overnight at 37°C. Single clones were picked and shaken, and verified by colony PCR and sequencing. The PCR reaction system is as follows: Green Taq Mix, 10 μl; pnir universal primer F (10 μM), 1 μl; pnir universal primer R (10 μM), 1 μl; bacterial solution, 1 μl; ddH2O, 7 μL.

[0037] Step (ii) The specific steps of constructing genetically engineered bacteria are as follows: (1) Select the recombinant strain and inoculate it in LB liquid medium containing 0.1% AMP, and culture it overnight at 37°C and 200 rpm; take 5 ml of the overnight culture solution, centrifuge it at 4000 rpm for 15 min, and extract the recombinant plasmid using a plasmid extraction kit; (2) Add the plasmid to the thawed frozen competent EcN, mix well, and transfer to the pre-cooled electroporation cell for pulse electroporation. (3) The method for preparing the competent EcN described in step (2) is: (3.1) Take out the EcN strain stored in the -80℃ refrigerator, streak a single colony on LB medium, and incubate at 37℃ overnight; pick a fresh single colony and inoculate it into 50 ml LB medium, incubate at 37℃, 200rpm shaking, and when the OD 600 When it reaches about 0.5, the culture is stopped; (3.2) Place the cultured bacterial solution in an ice bath for 30 minutes to cool the bacteria; collect the bacteria by centrifugation at an appropriate speed of 4000 rpm at 4°C and discard the supernatant; gently resuspend the bacteria in pre-cooled 0.1M CaCl2 solution, and collect the bacteria by centrifugation again, discard the supernatant, and repeat this step 3 times; (3.3) Resuspend the cells in pre-cooled 0.1 M CaCl2 solution containing 10% glycerol to make the cell concentration reach an appropriate level, and quickly freeze the aliquoted competent cells in liquid nitrogen, and then transfer to a -80℃ refrigerator for storage.

[0038] (4) Add LB liquid culture and bacteria to the electroporation pool, mix well, transfer to a sterile centrifuge tube, and culture at 37°C, 200r / min for 60 min. Then, spread on an LB plate containing amp and culture overnight. (5) Pick the single clones on the above plate and verify by colony PCR and sequencing. The PCR reaction system is as follows: Green Taq Mix, 10 μl; pnir universal primer F (10 μM), 1 μl; pnir universal primer R (10 μM), 1 μl; bacterial solution, 1 μl; ddH2O, 7 μL; (6) The EcN-Scy bacterial solution of the positive clone verified above was inoculated into 50 ml LB medium at a 1% inoculation rate, cultured overnight at 37°C and 200 rpm, and then given an anaerobic environment for 12 hours to induce fermentation of the surface displayed peptide Scy.

[0039] Step (III) Analysis of stress resistance characteristics of engineered bacteria is as follows: (1) Prepare LB culture medium with different pH values. Adjust the pH values ​​to 1.5, 2.0, and 2.5 by adding appropriate amounts of hydrochloric acid to the LB culture medium to simulate the acidic environment of the gastrointestinal tract. Prepare LB culture medium with different concentrations of bile salts (ox bile salts). The bile salt concentration can be set to 0.5%, 1.0%, and 1.5% (w / v) to simulate the gradually increasing bile salt concentration gradient in the gastrointestinal tract. At the same time, set EcN and E. coli EcN-pnir containing an empty vector (i.e., a vector without the target gene inserted) as controls to evaluate the nonspecific effects that may be introduced during the construction of the engineered bacteria.

[0040] (2) The engineered bacteria obtained above were cultured in LB medium overnight and inoculated into the above medium at a 1% inoculum. All culture media were placed in a constant temperature shaker at 37°C and 200 rpm for a certain period of time: the acid resistance test was cultured for 2 hours and the bile resistance test was cultured for 12 hours to simulate the different times experienced in the gastrointestinal tract.

[0041] (3) Take samples of the culture medium at different pH values ​​and salt concentrations before and after culture, and use a spectrophotometer to measure the OD of each sample. 600 The values ​​were used to determine the survival rate of the engineered bacteria under different conditions.

[0042] Step (IV) The protective effect of the engineered bacteria on DSS-induced colitis in mice is as follows: (1) Experimental animals: Female Balb / c mice (8 weeks old) (20 mice, purchased from Hangzhou Haowo Biotechnology Co., Ltd.) were housed in the Experimental Animal Center of Zhejiang University, with room temperature maintained at 22±2°C and humidity at 55%±5%, and a 12-h light / dark cycle. They were allowed to eat and drink freely. All experimental procedures designed for mice were in accordance with the ethical guidelines for the care and use of animals stipulated by the Experimental Animal Center of Zhejiang University.

[0043] (2) Animal grouping: After the mice adapted to the above environmental conditions for one week, 20 mice were randomly divided into 4 groups (5 mice in each group): blank control group (control), model group (DSS), DSS+EcN group, and DSS+EcN-Scy group.

[0044] (3) Animal model construction and intervention methods: On days 0-7 of the formal experiment, 2.5% DSS was added to the drinking water of the DSS group and all experimental groups containing DSS (DSS+EcN group, DSS+EcN-Scy group) to induce the colitis model; from day 8 to the end of the experiment (day 14), all groups resumed normal drinking water without adding DSS; on days 0-14 of the formal experiment, mice in the DSS+EcN group and the DSS+EcN-Scy group received corresponding bacterial liquid gavage treatment every 2 days, with a bacterial dose of 1×10^8 CFU of bacterial liquid; the weight of the mice was recorded every 2 days, and the results are shown in the figure. Figure 5 shown.

[0045] (4) Sampling: On the 15th day of the formal experiment, the mice were killed after blood was collected from their eyeballs. The blood was centrifuged at 4°C, 4000 rpm for 15 min, serum was extracted, and stored at -80°C. The mice were dissected and their colons were taken. The length of their colons was measured with a ruler. The results are as follows: Figure 6 As shown; a colon was collected and stored at -80°C for subsequent molecular experiments; a colon was collected in 4% paraformaldehyde for subsequent preparation of HE staining sections, the results are shown Figure 7 shown.

[0046] (5) Indicator analysis (5.1) Serum liver function indexes were measured by the laboratory of the Affiliated Hospital of Hangzhou Normal University. The results are as follows: Figure 8 shown.

[0047] (5.2) Detection of colon immune cytokine gene expression levels: After the tissue samples were broken, RNA was extracted using TRIZOL and other reagents, and the extracted RNA was reverse transcribed into cDNA. The gene expression levels of immune cytokines TNF-α, IL-1β, IL-6, and MCP1 were detected by quantitative PCR technology. The results were as follows: Fig. 9 As shown, compared with the control group, the expression levels of TNF-α, IL-1β, IL-6, and MCP1 genes in the colon of mice in the DSS group were significantly increased, indicating that the DSS-induced mouse colitis model was successfully constructed. After the intervention of the engineered bacteria EcN-Scy, the levels of the above indicators in the mouse colon can be significantly reduced, making the gene expression levels of the above inflammatory factors in the mouse colon closer to the control group. It shows that the engineered bacteria involved in the present invention have a good effect in preventing and treating intestinal inflammation. In addition, compared with the DSS group, the expression of inflammation-related genes such as TNF-α, IL-1β and IL-6 in the colon of mice in the DSS+EcN group showed a downward trend, and it was particularly significant that the expression of the MCP gene was greatly reduced. This change is consistent with the immunomodulatory properties of EcN as a probiotic.

[0048] (5.3) Antioxidant index detection: Colon antioxidant indicators include gene level detection of colon antioxidant indicators catalase (CAT) and myeloperoxidase (MPO), and colon antioxidant indicators nitric oxide (NO) and total superoxide dismutase (T-SOD); the gene level detection method is consistent with (5.2), and the antioxidant markers NO and T-SOD levels are detected by colorimetric test kits. The results are as follows Fig.10 As shown in the figure, the colon tissue of mice in the DSS-treated group showed a significant downward trend in CAT gene expression, while MPO gene expression was significantly upregulated. At the same time, the contents of NO and T-SOD in the colon decreased significantly. This series of changes confirmed that the DSS-induced colitis model in mice was successfully established.

[0049] Furthermore, after the intervention of the engineered bacteria EcN-Scy, the expression of the CAT gene in the mouse colon showed a trend of recovery, while the expression of the MPO gene was significantly inhibited. It is worth noting that the NO level increased significantly and the T-SOD level also tended to recover. These changes comprehensively reflect the positive regulation of the antioxidant status of the mouse colon by oral administration of engineered bacteria, making the above antioxidant indicators closer to the health level of the control group. The above-mentioned engineered bacteria EcN-Scy showed antioxidant capacity, and its intervention effect improved the antioxidant environment in the colon of DSS-induced colitis mice.

[0050] (5.4) Intestinal physical barrier function index detection: Colon physical barrier function indicators include the gene expression levels of tight junction protein 1 (ZO-1) and tight junction protein 2 (ZO-2) in colon tissue, and the detection method is the same as (5.2).

[0051] The results are as follows Fig.11 As shown in the figure, compared with the control group mice, the ZO-1 gene expression level in the colon tissue of the DSS-treated mice tended to decrease, while the ZO-2 gene expression level was significantly reduced, suggesting the potential destructive effect of DSS treatment on the tight junction structure of the mouse colon; after the intervention of the engineered bacteria EcN-Scy, the ZO-1 and ZO-2 gene expression bacteria in the mouse colon increased significantly, indicating that the engineered bacteria effectively promoted the recovery of tight junction proteins and may have a positive effect on maintaining the colon barrier function.

[0052] The applicant hereby declares that the technical solution of the present invention is specifically described through the aforementioned embodiments, but these examples are for illustrative purposes only and are not meant to limit the scope of protection of the present invention. In other words, the implementation of the present invention does not rely on any single embodiment, but has wide applicability and flexibility. Those skilled in the art should understand that any reasonable improvement of the present invention, equivalent replacement of constituent raw materials, addition of auxiliary components, and specific selection of implementation methods, etc., are deemed to be within the technical scope of the core idea of ​​the present invention, and are therefore covered by the scope of protection of the present invention and the disclosure.

[0053] Although the above content describes the preferred embodiments of the present invention in detail, the essence of the present invention is not limited to these specific details. Within the framework of the technical concept, all kinds of simple deformation or adjustment of the technical solution of the present invention belong to the innovation category pursued and protected by the present invention.

[0054] It is worth pointing out that the specific technical features mentioned in the detailed implementation methods can be flexibly combined according to actual needs without conflicting with each other. To avoid redundancy and repetition, the present invention will not list all possible combinations one by one, but all such combinations are included in the protection scope of the present invention by default.

Claims

1. A method for constructing a recombinant engineered bacterium EcN-Scy displaying an antimicrobial immune peptide Scy on its surface, characterized in that: The following steps are involved: a) obtaining and designing a gene sequence encoding an antimicrobial immune peptide Scy from crab semen, wherein the gene sequence is codon-optimized to adapt to the expression system of Escherichia coli, and performing full gene synthesis; b) connecting the antimicrobial immune peptide Scy gene sequence obtained in step a) with the plasmid vector pnir to construct a recombinant plasmid pnir-Scy; c) transforming the recombinant plasmid pnir-Scy constructed in step b) into Escherichia coli DH5α to obtain positive clones; d) extracting and purifying the recombinant plasmid pnir-Scy from the positive clones obtained in step c), and confirming the correctness and purity of the recombinant plasmid by electrophoresis and sequencing; e) The recombinant plasmid pnir-Scy obtained in step d) is introduced into the expression host Escherichia coli Nissle1917 EcN by electroporation to obtain the recombinant engineered bacteria EcN-Scy.

2. The construction method according to claim 1, characterized in that The antimicrobial immune peptide Scy gene in step a) is codon-optimized, and the sequence is shown in Seq ID No.

1.

3. The construction method according to claim 1, characterized in that: The connection operation in step b) includes connecting the PCR linearized antimicrobial immune peptide Scy gene with the plasmid pnir using homologous recombination technology.

4. The use of the recombinant engineering bacterium EcN-Scy according to claim 1, characterized in that: The invention is used to prepare a drug which has a protective effect on intestinal mucosal barrier damage of mice caused by dextran sulfate sodium (DSS). The drug is used to alleviate DSS-induced weight loss and shortening of colon length in mice, improve colon morphology, and alleviate liver damage.

5. The use of the recombinant engineered bacteria EcN-Scy according to claim 1 in the preparation of a drug or functional food for treating or preventing intestinal inflammatory diseases, characterized in that: This recombinant engineered bacteria protects intestinal health through multiple pathways, including regulating intestinal immune cytokines, enhancing antioxidant capacity, and maintaining the intestinal physical barrier.

6. The use according to claim 5, characterized in that: A pharmaceutical composition for treating or preventing intestinal inflammatory diseases is prepared, comprising the recombinant engineered bacteria EcN-Scy as an active ingredient and a pharmaceutically acceptable carrier or excipient.

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