Inflammation-responsive genetically engineered probiotic bacteria, and preparation method and application thereof

Abstract

The application discloses an inflammation response type genetically engineered probiotic, a preparation method and application thereof, and belongs to the technical field of probiotic application. The probiotic provided by the application can perceive inflammation signals in the intestinal tract, continuously produce butyric acid, exosome CAT, SOD and anti-inflammatory proteins under the induction of the inflammation signals, and adopt a layer-by-layer self-assembly coating technology to realize efficient targeted delivery. Experimental results show that the probiotic provided by the application can improve intestinal integrity, regulate the level of immune inflammatory factors, and effectively treat DSS-induced inflammatory bowel disease in mice. The application provides a genetically engineered probiotic with multiple functions of producing butyric acid, removing ROS, repairing barriers and regulating immunity, thereby providing a new biological treatment strategy for inflammatory bowel disease, which is significantly superior to the prior art in terms of efficacy, safety, precision, durability and controllability.

Description

Technical Field

[0001] This invention relates to the field of probiotic application technology, and in particular to an inflammatory-responsive genetically engineered probiotic, its preparation method, and its application. Background Technology

[0002] Inflammatory bowel disease (IBD), such as Crohn's disease and ulcerative colitis, is a complex chronic inflammatory bowel disease. Its pathogenesis is closely related to impaired intestinal mucosal barrier function, abnormal immune regulation, and gut microbiota dysbiosis. Among current treatment strategies, the use of probiotics to regulate the gut microbiota has received widespread attention as a therapy with few side effects and high patient compliance.

[0003] Existing probiotic treatment technologies for IBD mainly revolve around the following core mechanisms:

[0004] 1. Anti-inflammatory and immunomodulatory effects: Some probiotic strains can indirectly alleviate inflammation by competing for ecological niches with pathogenic bacteria, secreting antimicrobial peptides, or modulating host immune cells. Their effects depend on the interaction between the bacteria and the host gut.

[0005] 2. Provides beneficial metabolites: Butyrate is a short-chain fatty acid produced by the fermentation of dietary fiber by specific gut bacteria and is a major energy source for colonic epithelial cells. Existing research has explored the use of butyrate-producing probiotics to treat IBD. Butyrate can enhance the intestinal epithelial barrier function, inhibit histone deacetylase to exert anti-inflammatory effects, and regulate immune responses.

[0006] 3. Scavenging Reactive Oxygen Species (ROS): The presence of abundant neutrophil infiltration in IBD lesions leads to a sharp increase in ROS levels, causing oxidative stress and exacerbating tissue damage and inflammation. In existing technologies, researchers have developed engineered probiotics capable of expressing antioxidant enzymes. These enzymes are expressed intracellularly and mitigate oxidative damage by breaking down superoxide anions and hydrogen peroxide.

[0007] Despite the progress made by the aforementioned existing technologies, they still have the following limitations:

[0008] 1. Limited Mechanism of Action and Limited Efficacy: Most existing engineered probiotics can only target a single link in the complex pathogenesis network of IBD. For example, butyrate-producing bacteria alone cannot effectively cope with the acute oxidative stress storm; while strains that only express antioxidant enzymes lack butyrate support, which is crucial for intestinal barrier repair. IBD is the result of multiple pathological mechanisms working together, and probiotics with single functions are unlikely to achieve synergistic and potent therapeutic effects.

[0009] 2. Limited Scope of Action of Intracellularly Expressed Antioxidant Enzymes: In existing technologies, antioxidant enzymes such as SOD and CAT are typically expressed intracellularly in engineered probiotics. This prevents them from effectively clearing extracellular ROS that directly damage host intestinal epithelial cells. These antioxidant enzymes are confined to the bacterial cell interior, and their antioxidant capacity cannot reach the surface of the host cells and surrounding environment, which are most in need of protection, resulting in low efficiency.

[0010] 3. Lack of lesion specificity, potentially disrupting the normal gut microenvironment: Many engineered bacteria exhibit constitutive gene expression, meaning they function continuously and indiscriminately across different sites. This "indiscriminate" expression pattern may waste resources in uninflamed intestinal regions and could even disrupt normal physiological signals and the balance of the gut microbiota. Ideally, treatment should involve engineered bacteria being specifically activated only at the site of inflammation, achieving precise targeted therapy.

[0011] 4. Shortcomings of existing secretion system applications: Although some studies have utilized bacterial secretion systems to deliver proteins, they are usually used to deliver single anti-inflammatory factors or antimicrobial peptides, failing to be combined with a comprehensive, multifunctional antioxidant + repair therapy strategy, and lacking precise regulation of secretion behavior.

[0012] To achieve efficient delivery of probiotics, existing technologies have explored various bacterial coating or microencapsulation strategies, such as using biomaterials like sodium alginate and chitosan. However, these methods have the following drawbacks:

[0013] 1. Difficulty in controlling coating thickness and uniformity: Traditional coating methods are often based on bulk mixing and gelation, resulting in coating layers that are too thick and uneven. Excessively thick coating layers severely hinder the exchange of nutrients and signaling molecules, significantly inhibiting bacterial metabolic activity and proliferation, leading to decreased colonization efficiency.

[0014] 2. Limited functionality and lack of synergy: Existing coating materials primarily focus on physical isolation and protection (such as resistance to gastric acid), but lack synergistic therapeutic functions that promote bacterial colonization in the gut. The coating and therapeutic strains are merely a simple physical combination, failing to form a functionally integrated therapeutic whole.

[0015] 3. Lack of mucosal targeting and intelligent responsiveness: Most coating materials are inert in the intestine and cannot actively target the inflamed mucosa, nor can they intelligently release bacteria or drugs in response to the inflammatory microenvironment, thus limiting the precision of treatment.

[0016] In summary, there is an urgent need for an engineered probiotic that can intelligently respond to the inflammatory environment, possess multiple synergistic therapeutic mechanisms—such as barrier repair, efficient removal of extracellular ROS, continuous production of butyrate to nourish intestinal mucosal cells, and precise delivery of therapeutic effects to the lesion site. Summary of the Invention

[0017] The purpose of this invention is to provide an inflammatory-responsive genetically engineered probiotic, its preparation method, and its application, in order to solve the problems existing in the prior art. This invention provides a genetically engineered probiotic that has multiple functions, including producing butyric acid, clearing ROS, repairing the barrier, and regulating immunity, thereby providing a new biological treatment strategy for inflammatory bowel disease that is significantly superior to the prior art in terms of efficacy, safety, precision, durability, and controllability.

[0018] To achieve the above objectives, the present invention provides the following solution:

[0019] This invention provides an inflammation-responsive genetically engineered probiotic, EcN-BCST, which is obtained by transforming plasmid pRE112-B and plasmid p15A-CST into Escherichia coli EcN.

[0020] The nucleotide sequence of the plasmid pRE112-B is shown in SEQ ID NO.1;

[0021] The nucleotide sequence of the plasmid p15A-CST is shown in SEQ ID NO.2.

[0022] This invention also provides a method for preparing the above-mentioned inflammatory-responsive genetically engineered probiotic EcN-BCST, comprising the following steps:

[0023] The plasmid pRE112-B was transformed into DAP auxotrophic Escherichia coli to obtain the donor bacteria;

[0024] The donor bacteria were mixed with the E. coli EcN and screened to obtain the engineered bacteria EcN-B;

[0025] The plasmid p15A-CST was transferred into the engineered bacteria EcN-B to obtain the inflammatory-responsive genetically engineered probiotic EcN-BCST.

[0026] Furthermore, the DAP auxotrophic Escherichia coli is Escherichia coli WM3064.

[0027] This invention also provides the application of the above-mentioned inflammatory-responsive genetically engineered probiotic EcN-BCST in the preparation of intestinal-targeted probiotic products.

[0028] The present invention also provides a colon-targeted probiotic product, with the above-mentioned inflammatory-responsive genetically engineered probiotic EcN-BCST as the active ingredient.

[0029] Furthermore, it also includes coating agents;

[0030] The coating agent includes polylysine solution and carboxymethyl inulin solution.

[0031] The present invention also provides a method for preparing the above-mentioned colon-targeted probiotic product, comprising the following steps:

[0032] The inflammatory-responsive genetically engineered probiotic EcN-BCST was resuspended in polylysine solution to obtain EcN-BCST@P;

[0033] EcN-BCST@P was resuspended in carboxymethyl inulin solution to obtain EcN-BCST@PC;

[0034] The EcN-BCST@PC was resuspended in polylysine solution to obtain EcN-BCST@PCP;

[0035] The EcN-BCST@PCP was resuspended in a carboxymethyl inulin solution to obtain EcN-BCST@PCPC, which is the colon-targeted probiotic product.

[0036] Furthermore, the concentration of the polylysine solution is 2 mg / mL;

[0037] The concentration of the carboxymethyl inulin solution was 2 mg / mL.

[0038] The present invention also provides the use of the above-mentioned inflammatory-responsive genetically engineered probiotic EcN-BCST or the above-mentioned colon-targeting probiotic product in the preparation of drugs that improve intestinal integrity, regulate immunity, have anti-inflammatory and / or antioxidant effects.

[0039] The present invention also provides the application of the above-mentioned inflammatory-responsive genetically engineered probiotic EcN-BCST or the above-mentioned colon-targeting probiotic product in the preparation of a drug for treating inflammatory bowel disease.

[0040] The present invention discloses the following technical effects:

[0041] 1. It achieves multiple synergistic treatments, significantly improving efficacy.

[0042] Advantages: This invention integrates three major therapeutic mechanisms—nutritional support (butyric acid), antioxidant stress (SOD / CAT), and anti-inflammatory repair (TFF3)—into a single engineered strain, overcoming the limitations of existing single-function probiotics in terms of limited efficacy.

[0043] Effects: Butyrate provides energy to epithelial cells and strengthens the physical barrier, while creating favorable conditions for TFF3-driven cell migration and repair; while the oxidative stress environment of SOD / CAT scavenging ROS provides a stable microenvironment for the function of butyrate and TFF3. The three promote each other, forming a synergistic effect against the complex pathological mechanisms of IBD, which can significantly improve treatment efficacy and remission rate.

[0044] 2. It achieved highly efficient clearance of extracellular ROS, overcoming the bottleneck of limited scope of action.

[0045] Advantages: This invention transforms traditional intracellular antioxidants into highly efficient extracellular secretory antioxidants by constructing a fusion protein of SOD / CAT and HlyA signal peptide and introducing a complete HlyB / D secretion system.

[0046] Effects: SOD and CAT secreted outside the bacteria can directly act on extracellular ROS that damage the intestinal epithelium, greatly improving clearance efficiency and fundamentally solving the problem that antioxidant enzymes are difficult to secrete outside the cell and cannot exert their anti-ROS function in existing technologies.

[0047] 3. It achieves lesion-specific treatment, improving safety and accuracy.

[0048] Advantages: This invention utilizes the ThsS / R system derived from Shewanella halifaxensis to strictly control the expression of all therapeutic proteins under the influence of IBD inflammatory markers—thiosulfate.

[0049] Results: This design achieves a precise treatment model of "working when sick, resting when healthy." On the one hand, it concentrates treatment resources on the lesion, avoiding unnecessary resource consumption and potential interference in healthy intestinal areas; on the other hand, it greatly improves the safety of treatment and reduces the long-term risks that may arise from the continuous expression of exogenous proteins.

[0050] 4. It realizes the multifunctional and intelligent application of the endocrine system.

[0051] Advantages: This invention upgrades the Hly secretion system from a simple protein delivery tool into a comprehensive platform capable of synergistically secreting multiple different functional proteins (antioxidant enzymes and repair factors), and incorporates smart linker peptide technology to ensure the complete activation of repair factors.

[0052] Results: A single secretory system delivers three classes of therapeutic molecules simultaneously, with high efficiency and a compact structure. In particular, the design of the linker peptide with dual cleavage sites for TFF3 ensures its efficient release and natural activity in the complex intestinal environment, demonstrating "intelligent" functional delivery.

[0053] 5. It achieves a balance between efficient delivery and long-term colonization, overcoming the traditional contradictions of coating technology.

[0054] Advantages: This invention employs a single-bacterial layer-by-layer self-assembly technology based on polylysine (PLL) and carboxymethyl inulin (CMI) to construct a thin, uniform, multifunctional coating.

[0055] Effects: (1) Highly effective protection: The coating effectively protects engineered bacteria from gastric acid and bile salts, ensuring that a sufficient amount of live bacteria reach the colon. (2) Colon-targeted and sustained release: Utilizing the enzymatic hydrolysis properties of CMI in the colon, the coating is specifically degraded in the colon and the bacteria are released in a programmed manner, avoiding premature inactivation in the upper digestive tract. (3) Enhanced colonization: The positive charge provided by PLL enhances the adhesion ability of bacteria to the negatively charged intestinal mucosa, and CMI, as a prebiotic, can improve the colonization microenvironment. The two work synergistically to significantly prolong the residence time and functional duration of engineered bacteria in the intestine.

[0056] 6. Genetic stability and economic efficiency of production

[0057] Advantages: This invention ensures the genetic stability of butyrate production traits by integrating the butyrate synthesis pathway into the chromosome and knocking out the competing metabolic pathway (lactate dehydrogenase); at the same time, it simplifies the complex sensing and secretion system to a single plasmid, simplifying the construction process of engineered bacteria.

[0058] Results: This strategy not only ensures the functional stability of the strain during passage and use and reduces the risk of plasmid loss, but also makes it more suitable for large-scale industrial fermentation production and has significant commercial application potential.

[0059] In summary, this invention provides a novel IBD biological treatment strategy that significantly outperforms existing technologies in terms of efficacy, safety, durability, and controllability through a three-in-one design of "intelligent sensing, collaborative treatment, and precise delivery". Attached Figure Description

[0060] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0061] Figure 1 A schematic diagram illustrating the preparation method of EcN-BCST@PCPC, a genetically engineered probiotic for the treatment of inflammatory bowel disease;

[0062] Figure 2 The graph shows the results of the EcN-BCST butyric acid production efficiency verification; where a is the in vitro butyric acid production; b is the fecal butyric acid content on day 7; c is the fecal butyric acid content on day 10; and d is the curve of fecal butyric acid content change.

[0063] Figure 3The figure shows the results of verifying the eGFP-HlyA secretion efficacy induced by thiosulfate; where a represents the fluorescence intensity induced by different concentrations of thiosulfate; b represents the fluorescence intensity induced by different compounds; c represents the fluorescence intensity of different components; and d represents the fluorescence microscopy observation results.

[0064] Figure 4 Figure 1 shows the results of in vitro efficacy validation of SOD-HlyA and CAT-HlyA induced by thiosulfate; where a is the oxygen concentration of the uninduced bacterial suspension; b is the oxygen concentration of the 1 mM induced bacterial suspension; c is the oxygen concentration of the 1 mM induced bacterial pellet; d is the oxygen concentration of the 1 mM induced supernatant; e is the superoxide anion inhibition rate of the uninduced bacterial suspension; f is the superoxide anion inhibition rate of the 1 mM induced bacterial suspension; g is the superoxide anion inhibition rate of the 1 mM induced bacterial pellet; and h is the superoxide anion inhibition rate of the 1 mM induced supernatant.

[0065] Figure 5 The graph shows the therapeutic effect of genetically engineered probiotics on inflammatory bowel disease induced by dextran sulfate sodium (DSS) in mice; where a is the body weight change curve; b is the colon length; c is the intestinal integrity; d is the interleukin-1β content; e is the tumor necrosis factor-α content; and f is the interleukin-10 content. Detailed Implementation

[0066] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0067] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0068] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0069] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0070] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0071] This invention aims to provide a multifunctional engineered probiotic and its efficient delivery system for treating inflammatory bowel disease (IBD), in order to comprehensively address the following key technical issues:

[0072] 1. Addressing the issue of insufficient synergistic effect of single therapies: Overcoming the limitations of existing engineered probiotics that have single functions and cannot simultaneously address the multiple pathological mechanisms of IBD, such as oxidative stress, barrier damage, and immune dysregulation, a multifunctional strain capable of producing synergistic therapeutic effects is provided. This integrates synergistic nutritional support, anti-oxidative stress, anti-inflammatory, and mucosal repair therapies into a single treatment.

[0073] 2. Addressing the limited range of action of intracellular antioxidant enzymes: This addresses the limitation of existing technologies where superoxide dismutase (SOD) and catalase (CAT) are expressed only within probiotic cells and cannot effectively remove extracellular reactive oxygen species (ROS), achieving highly efficient ROS removal from key sites of the intestinal mucosa. This is achieved through the creation of a signal peptide fusion protein and the addition of an exosecticidal system.

[0074] 3. Addressing the lack of lesion specificity in treatment: Overcoming the resource waste and potential interference with the normal microenvironment caused by constitutive expression, enabling the specific activation and precise delivery of therapeutic proteins at the site of inflammatory lesions. Achieving precise regulation through the sensing of inflammatory signals.

[0075] 4. The problem of limited application strategies for existing secretion systems: Optimize the application of bacterial secretion systems, upgrading them from simple protein delivery tools into comprehensive platforms capable of synergistically delivering multiple therapeutic factors (antioxidant enzymes and anti-inflammatory / repair factors). This can be achieved by creating signal peptide fusion proteins and adding exocrine systems.

[0076] 5. Solve the problem of contradictory efficacy and activity in existing bacterial delivery strategies: Provide a novel bacterial coating technology that effectively protects probiotics from adverse gastrointestinal environments without sacrificing their colonization activity and metabolic function, and additionally endows them with targeting capabilities.

[0077] In summary, the core objective of this invention is to provide a more efficient, precise, and safer IBD biological treatment strategy by constructing an engineered probiotic system that is intelligently triggered by inflammatory signals, possesses multiple functions such as "ROS clearance, barrier repair, and immune regulation," and employs advanced coating technology to achieve highly efficient targeted delivery.

[0078] A schematic diagram of the preparation method of the IBD treatment probiotic EcN-BCST@PCPC of the present invention is shown below. Figure 1 As shown.

[0079] Example 1

[0080] 1. Construction of engineered plasmid pRE112-B

[0081] The vector backbone of the recombinant plasmid pRE112-B in this invention is pRE112, which contains the chloramphenicol resistance gene.

[0082] After cleaving pRE112 at base 3089 using the SmaI restriction enzyme, the artificially synthesized AtoB-Hbd-Crt-FabV-TesB sequence was inserted using the Gibbson Assembly method to form the pRE112-B recombinant plasmid. The full sequence of pRE112-B is shown in SEQ ID NO.1.

[0083] In the AtoB-Hbd-Crt-FabV-TesB sequence, AtoB is an acetyl-CoA acetyltransferase from Escherichia coli. Hbd is a 3-hydroxybutyryl-CoA dehydrogenase from Clostridium acetobutylicum. Crt is a short-chain enoyl-CoA hydratase from Clostridium acetobutylicum. FabV is a trans-2-enoyl-CoA reductase from Treponemadenticola. TesB is an acyl-CoA thioesterase 2 from Escherichia coli. The promoter P_fnr is the promoter of the FNR protein from Escherichia coli. The ribosome binding site for all five enzymes in the pathway is B0034. AtoB-Hbd is transcription unit 1, Crt-FabV is transcription unit 2, and TesB is transcription unit 3. The terminator for all three transcription units is B0015.

[0084] The suicide plasmid pRE112, carrying a butyrate synthase set AtoB-Hbd-Crt-FabV-TesB, was integrated into the chromosomal DNA of the substrate bacterium *Escherichia coli* Nissle 1917 (EcN) via homologous recombination. Simultaneously, the lactate dehydrogenase (LDH) of EcN was knocked out, reducing EcN's carbon source consumption and increasing butyrate production. The homologous arms are 1000 bp sequences upstream and downstream of the LDH gene. The promoter of the butyrate synthesis pathway enzymes is the anaerobic inducible promoter P_fnr, which induces butyrate production in the hypoxic environment of the terminal ileum and colon. This plasmid was named pRE112-B.

[0085] 2. Construction of engineered plasmid p15A-CST

[0086] The recombinant plasmid p15A-CST in this invention has a vector backbone of p15A containing a kanamycin resistance gene. After cleaving the 52nd base with Xmn I / Pdm I restriction enzyme, a synthetically produced ThsS-ThsR-SOD-HlyA-CAT-HlyA-TFF3-HlyA-HlyB-HlyD sequence was inserted using the Gibbson Assembly method to form the p15A-CST recombinant plasmid. The full sequence of p15A-CST is shown in SEQ ID NO.2.

[0087] In the sequence ThsS-ThsR-SOD-HlyA-CAT-HlyA-TFF3-HlyA-HlyB-HlyD, ThsS and ThsR are regulatory elements with thiosulfate sensing capabilities. These ThsS / R elements are derived from the expression product of the Shal_3128 / 9 gene of *Shewanella halifaxensis* (strain HAW-EB4). EcNs carrying this plasmid can sense an important target in colonic inflammation—excess thiosulfate—and trigger the promoter expression of downstream genes, achieving thiosulfate-responsive induction. HlyA, derived from the hemolysin A protein of *Escherichia coli*, is a major source of pathogenicity in *Escherichia coli*, secreted toxic HlyA through the synergistic secretion of HlyA via HlyB and HlyD. Designing SOD-HlyA, CAT-HlyA, and TFF3-HlyA fusion proteins with HlyA signal peptide sequences can achieve efficient bacterial exudation. The HlyB / D used is derived from *Escherichia coli*, and the HlyA signal peptide is the 60 amino acid residues at the C-terminus of the HlyA protein. SOD-HlyA uses SOD derived from human extracellular superoxide dismutase, linked to the HlyA signal peptide via a flexible sequence. CAT-HlyA uses CAT derived from human catalase, also linked to the HlyA signal peptide via a flexible sequence. TFF3-HlyA uses TFF3 derived from human trefoil factor 3. TFF3 is secreted by intestinal goblet cells and has anti-inflammatory effects. TFF3 and TLR2 are functionally related, and both block the overexpression of IL1β-induced pro-inflammatory cytokines through the PI3K / AKT pathway. TFF3 may promote intestinal mucosal remodeling through crosstalk between the ERK and JAK / STAT3 pathways.

[0088] All of the above-mentioned ThsS / R, HlyB, HlyD, SOD-HlyA, CAT-HlyA, and TFF3-HlyA were placed into a single p15A plasmid. ThsS and ThsR constitute transcription unit 1, with promoters J23100 and J23101, respectively. SOD-HlyA, CAT-HlyA, and TFF3-HlyA constitute transcription unit 2, with the promoter PphsA. HlyB and HlyD constitute transcription unit 3, with the promoter J23104. All ribosome binding sites are B0034, and all terminators are B0015. This plasmid was named p15A-CST.

[0089] 3. Recombinant plasmid pRE112-B homologously integrated the butyrate pathway into the EcN chromosomal DNA to construct EcN-B.

[0090] 3.1 Bacterial strains and culture media

[0091] Donor bacteria: Escherichia coli WM3064 (pRE112-B). WM3064 was purchased from BioBio and is a diaminopimelic acid (DAP) auxotrophic (ΔdapA) strain that can only grow in DAP-containing media. WM3064 transformed with plasmid pRE112-B was used as the donor bacteria.

[0092] Recipient bacteria: Escherichia coli Nissle 1917 (EcN) was purchased from Beina Biotechnology. EcN is prototrophic and can synthesize DAP on its own.

[0093] LB liquid medium (with 0.3 mM DAP added), LB solid plates (with 0.3 mM DAP and 25 μg / mL chloramphenicol (Cm) added).

[0094] Donor bacteria culture plate: Add DAP to LB nutrient agar to a final concentration of 0.3 mM.

[0095] Conjugation plate: LB nutrient agar was mixed with chloramphenicol (Cm) to a final concentration of 25 μg / mL, without the addition of DAP.

[0096] Sucrose reverse selection plates: Prepare salt-free LB medium (10 g / L tryptone, 5 g / L yeast extract, 15 g / L agar powder) and add sucrose to a final concentration of 10% (w / v), without adding any antibiotics or DAP.

[0097] 3.2 Plasmid pRE112-B was transformed into Escherichia coli WM3064

[0098] 3.2.1 Bacterial Culture

[0099] WM3064 bacterial culture stored at -80℃ was streaked onto an LB agar plate containing 0.3 mM DAP and incubated overnight (16 h) at 37℃. A fresh, plump single colony was picked and inoculated into 10 mL of LB liquid medium containing 0.3 mM DAP and incubated overnight (16 h) at 37℃. The overnight bacterial culture was then transferred at a 1:100 ratio to an Erlenmeyer flask containing 50 mL of LB liquid medium containing 0.3 mM DAP. When the OD of the bacterial culture... 600 Stop culturing immediately when the concentration reaches 0.5.

[0100] 3.2.2 Cell Cooling and Harvesting

[0101] Incubate the bacterial culture in an ice-water bath for 30 min to stop bacterial growth. All subsequent operations should be performed on ice or in a 4°C centrifuge, using pre-chilled solutions and supplies accordingly. Aliquot the cooled bacterial culture into pre-chilled 50 mL centrifuge tubes and centrifuge at 4°C, 4000 rpm for 10 min to collect the bacterial cells.

[0102] 3.2.3 CaCl2 treatment and preparation of competent cells

[0103] Carefully discard the supernatant, add 25 mL of pre-chilled 0.1 M CaCl2 solution, and gently resuspend the cells by pipetting or vortexing. Incubate on ice for 30 min. Centrifuge again at 4000 rpm for 10 min at 4°C. Carefully discard the supernatant, add 3 mL of pre-chilled 0.1 M CaCl2 solution, and gently resuspend the cells to obtain the chemocompetent cell suspension. Aliquot the competent cell suspension into pre-chilled sterile microcentrifuge tubes at 100 μL per tube.

[0104] 3.2.4 Adding plasmid DNA

[0105] Add 3 μL of pRE112-B plasmid DNA (300 ng) to competent cells and gently mix with a pipette tip. Incubate the mixture on ice for 30 min.

[0106] 3.2.5 Heat shock treatment

[0107] Float the centrifuge tubes in a 42°C water bath and heat shock for 90 seconds with precise time control. After heat shock, immediately transfer the centrifuge tubes back to ice and let them stand for 5 minutes.

[0108] 3.2.6 Resuscitation and Cultivation

[0109] Add 900 μL of LB liquid medium containing 0.3 mM DAP but no antibiotics to each tube. After mixing, incubate at 37°C with gentle shaking at 200 rpm for 1.5 h to allow the bacteria to recover and express the chloramphenicol resistance gene on the plasmid.

[0110] 3.2.7 Coating and Cultivation

[0111] Centrifuge the revived bacterial culture at 4000 rpm for 5 min at room temperature to concentrate the cells. Discard 900 μL of supernatant and gently resuspend the cells in the remaining culture medium. Take 100 μL of the bacterial culture and spread it evenly on an LB select plate containing 0.3 mM DAP and 25 μg / mL chloramphenicol using a sterile spreader. Place the plate upright in a 37°C incubator and incubate upside down for 24–48 h after the liquid has been completely absorbed. Single colonies that grow on the plate are potential positive clones, namely WM3064 (pRE112-B).

[0112] 3.3 Bonding Transfer

[0113] From a freshly streaked agar plate, pick a single colony of donor bacteria WM3064 (pRE112-B) and inoculate it into 5 mL of LB broth containing 0.3 mM DAP. Simultaneously, pick a single colony of recipient bacteria (EcN) and inoculate it into 5 mL of ordinary LB broth. Incubate both the donor and recipient bacteria overnight (16 h) at 37°C and 220 rpm with shaking. Take 1 mL of the overnight culture of both the donor and recipient bacteria, centrifuge at 12000 rpm for 1 min at room temperature, and discard the supernatant to remove DAP from the medium. Resuspend the bacterial cells once in 1 mL of fresh, DAP-free LB broth, centrifuge again, and discard the supernatant to remove residual DAP. Resuspend the washed donor and recipient bacterial pellets thoroughly in 100 μL of DAP-free LB broth. Add the entire mixture dropwise to the center of a DAP-free and antibiotic-free LB agar plate and spread it evenly using a sterile spreader. Place the plate upright and incubate it statically in a 37°C incubator for 8 hours.

[0114] 3.4 First homologous recombination (single crossover) screening

[0115] Add 1 mL of sterile LB medium to the plate after conjugation transfer culture. Gently scrape all bacterial growth from the plate surface with a sterile spreader and collect the bacterial suspension. Serially dilute the collected bacterial suspension. Take 100 μL of each dilution and spread it evenly onto an LB plate containing 25 μg / mL chloramphenicol and free of DAP. Invert the plate and incubate at 37°C for 24 h. Pick at least 5 appropriately sized single colonies and streak them onto LB plates containing chloramphenicol for purification. These colonies are named EcN::pRE112-B (single exchange integron).

[0116] 3.5 Induction and Screening of Second Homologous Recombination (Double Crossover)

[0117] From the single-exchange integrin, a single clone was picked and inoculated into 5 mL of antibiotic-free LB broth. The culture was incubated overnight (16 h) with shaking at 30°C and 220 rpm. The overnight culture was serially diluted. 100 μL of each dilution was spread evenly onto salt-free LB sucrose agar plates (containing 10% sucrose, DAP-free) using a spreader. The plates were inverted and incubated at 30°C for 24 h. Using a sterile toothpick, a single sucrose-resistant colony was spotted onto an LB agar plate containing 25 μg / mL chloramphenicol and another LB agar plate without antibiotics. The plates were incubated overnight at 37°C. The correct engineered bacteria should grow on the antibiotic-free plate but not on the chloramphenicol plate, confirming the plasmid backbone (containing Cm) + The sacB gene has been removed. EcN-B was obtained.

[0118] 4. Recombinant plasmid p15A-CST was transformed into EcN-B to construct EcN-BCST.

[0119] 4.1 Bacterial Culture

[0120] Single colonies were picked from fresh EcN-B plates and inoculated into 10 mL of LB liquid medium. The culture was incubated overnight (12 h) at 37°C with shaking at 200 rpm. The overnight culture was then transferred to fresh LB medium at a 1:100 ratio and incubated under the same conditions until the bacterial OD reached its maximum. 600 It reached 0.5.

[0121] 4.2 Cell Cooling and Harvesting

[0122] Incubate the bacterial culture on ice for 30 minutes, then aliquot it into pre-cooled centrifuge tubes and collect the bacterial cells by centrifuging at 4°C and 2000 g for 10 minutes using a horizontal centrifuge.

[0123] 4.3 CaCl2 treatment and preparation of competent cells

[0124] Discard the supernatant, add 1 mL of pre-cooled 100 mM CaCl2 solution, and gently resuspend the cells. Then incubate on ice again for 30 min.

[0125] 4.4 Dispensing and Storage

[0126] After centrifugation at 4°C, discard the supernatant, resuspend the bacterial cells in pre-cooled CaCl2 solution, and dispense 100 μL into each pre-cooled sterile centrifuge tube for transformation.

[0127] 4.5 Add plasmid DNA

[0128] Take a tube of 100 μL EcN-B competent cells, add 3 μL (300 ng) of p15A-CST recombinant plasmid, mix gently, and incubate on ice for 30 min.

[0129] 4.6 Heat shock treatment

[0130] Place the centrifuge tubes in a 42°C water bath for precise time-controlled heat shock for 90 seconds, then quickly return them to ice and let them stand for 2 minutes.

[0131] 4.7 Resuscitation and Cultivation

[0132] Add 1 mL of antibiotic-free LB medium to the tube, mix well, and incubate at 37°C and 200 rpm for 60 min with shaking to allow the cells to recover and express the resistance gene.

[0133] 4.8 Coating and Cultivation

[0134] Take 100 μL of the revived EcN-BCST bacterial culture and spread it on a kanamycin-containing LB agar plate. After the bacterial culture is absorbed by the plate, invert the plate and incubate it at 37℃ for 12 h, then observe the transformation results. Pick the successfully transformed colonies and transfer them to 10 mL of LB medium for later use to obtain EcN-BCST.

[0135] 4.9 Preparation of Control Group

[0136] Using the same method as described in "4.1-4.8" above, the recombinant plasmid p15A-CST was directly transferred into EcN to obtain EcN-CST.

[0137] 5. Layer-by-layer self-assembly coating of genetically engineered probiotic EcN-BCST yields EcN-BCST@PCPC

[0138] Poly-L-lysine (PLL) was used as coating material 1, and carboxymethyl inulin (CMI) was used as coating material 2. First, positively charged PLL was deposited on the surface of negatively charged EcN-BCST using electrostatic interaction to form EcN-BCST@P, thereby reversing the surface charge from negative to positive. Then, negatively charged CMI was deposited on its surface to form EcN-BCST@PC. The above process was repeated to form EcN-BCST@PCPC.

[0139] 5.1 Preparation of Coating Solution

[0140] Coating solution 1: Dissolve 80 mg of polylysine in 40 mL of a solution containing 0.5 M sodium chloride and 20 mM 2-morpholinoethanesulfonic acid to make a 2 mg / mL polylysine solution, and precisely adjust the pH to 6.

[0141] Coating solution 2: Take 80 mg of carboxymethyl inulin and dissolve it in 40 mL of a solution containing 0.5 M sodium chloride and 20 mM 2-morpholine ethanesulfonic acid to make a 2 mg / mL carboxymethyl inulin solution. Precisely adjust the pH to 6.

[0142] Washing solution: An aqueous solution containing 0.5 M sodium chloride and 20 mM 2-morpholinoethanesulfonic acid, with a pH of 6.

[0143] 5.2 Layer-by-layer self-assembly of engineered probiotics

[0144] Take 10 mL of LB medium containing EcN-BCST, and transfer the overnight culture to fresh LB medium at a ratio of 1:100. Incubate at 37°C with shaking at 200 rpm until the bacterial culture reaches OD. 600Once the concentration reaches 0.5, centrifuge at 2000 g for 10 min in a horizontal centrifuge to collect the bacterial cells, and wash twice with washing solution.

[0145] Resuspend the bacterial cells in the above coating solution 1, incubate at 37℃ and 200 rpm with shaking for 60 min, centrifuge at 2000 g for 10 min to collect the bacterial cells and wash them. Resuspend the bacterial cells in the above coating solution 2, incubate at 37℃ and 200 rpm with shaking for 60 min, centrifuge at 2000 g for 10 min to collect the bacterial cells and wash them. Repeat the above operation once more to form a multilayer self-assembled coating of polylysine-carboxymethyl inulin-polylysine-carboxymethyl inulin on the surface of the probiotics.

[0146] The bacterial culture was resuspended in washing buffer to obtain 10 mL of bacterial culture. 0.5 mL of 1.0% calcium chloride solution was added, and the culture was incubated at 37°C and 200 rpm for 10 min with shaking. The cells were collected by centrifugation at 2000 g for 10 min using a horizontal centrifuge. The cells were washed twice with washing buffer and resuspended again to obtain EcN-BCST@PCPC.

[0147] 6. Validation of EcN-BCST butyric acid yield efficiency

[0148] 6.1 In vitro butyrate production

[0149] On fresh plates, single colonies of all five strains (wild-type EcN, EcN-B, EcN-CST, EcN-BCST, and EcN-BCST@PCPC) were picked and inoculated into 5 mL of LB medium (Kanamycin was added to the medium of EcN-CST, EcN-BCST, and EcN-BCST@PCPC).

[0150] Take 1 mL of overnight culture, centrifuge at 2000 × g for 10 min at room temperature, discard the supernatant, gently resuspend the cells once with 1 mL of sterile PBS, centrifuge again and discard the supernatant. Resuspend the cells in 1 mL of modified M9 medium (1× M9 salt solution, 1 mM MgSO4, 0.1 mM CaCl2, 0.4% (w / v) glucose, 0.2% (w / v) yeast extract), and adjust the OD of all suspensions. 600The value was 0.1. Take 15 mL of sterile modified M9 culture medium and place it in a 15 mL sealed centrifuge tube. Inoculate with the standardized bacterial suspension at a ratio of 1:100. Set up a bottle of uninoculated modified M9 culture medium as a blank control. Incubate statically at 37℃ for 72 h. Repeat in triplicate. Centrifuge the bacterial suspension at 12000×g for 15 min at 4℃. Carefully aspirate the supernatant and filter it through a 0.22 μm aqueous filter membrane to obtain sterile supernatant. Take 100 μL of the filtered supernatant and place it in a 2 mL centrifuge tube. Add 10 μL of internal standard working solution (2-ethylbutyric acid final concentration 1 mM). Add 10 μL of concentrated hydrochloric acid and vortex to mix.

[0151] 6.2 Derivatization reaction

[0152] Add the following to the centrifuge tube sequentially: 500 μL acetonitrile, 100 μL pyridine, and 50 μL phenyl isocyanate. Vortex for 5 min to ensure thorough mixing. Place the centrifuge tube in a 60°C water bath and react in the dark for 30 min. Remove the centrifuge tube and cool to room temperature. Add 500 μL n-hexane and vortex for 5 min to extract the derivative. Centrifuge at 12000×g for 10 min at room temperature to separate the two phases. Carefully aspirate the upper n-hexane phase (organic phase) and transfer it to a new centrifuge tube. Transfer the clear organic phase to an HPLC vial for analysis. Use C... 18 The reversed-phase column (250 mm × 4.6 mm, 5 μm) was used. Mobile phase A was 10 mM KH2PO4 (pH 2.5), mobile phase B was acetonitrile, flow rate was 1.0 mL / min, gradient elution was used, column temperature was 30℃, detection wavelength was 254 nm, and injection volume was 20 μL.

[0153] 6.3 Production of butyric acid in the body

[0154] Healthy male C57BL / 6J mice aged 6-8 weeks were randomly divided into four groups: blank control group (PBS), positive control group (sodium butyrate), EcN group, EcN-B group, EcN-CST group, EcN-BCST group, and EcN-BCST@PCPC group, with n=5. All EcN strains and their engineered strains were cultured in LB medium (containing corresponding antibiotics) at 37℃ with shaking until mid-logarithmic growth (OD). 600 ≈0.6). Mice were administered gavage at a fixed time each day for 7 consecutive days, with a daily gavage volume of 200 μL (2 × 10⁻⁶). 8 CFU / mouse / day, 18.75 mg / mL sodium butyrate solution). Butyrate content was determined from feces of mice in each group on days 7 and 10 after gavage.

[0155] 6.4 Experimental Results

[0156] The results are as follows Figure 2 As shown, by Figure 2 As shown in Figure a, EcN-B can effectively produce butyric acid in vitro, and the enhanced function of EcN-BCST has no significant effect on the butyric acid yield of EcN-B. Furthermore, the layer-by-layer self-assembly coating also has no significant effect on the butyric acid yield.

[0157] In vivo experimental results as follows Figure 2 As shown in Figure bd, it can be seen that with the support of CST, EcN-BCST is better able to compete for more ecological niches in the colon and produce more butyric acid. The coating, to some extent, limits the ability of EcN-BCST to produce butyric acid quickly, but it effectively improves the colonization ability of EcN-BCST. Even after stopping the gavage of engineered probiotics, it can still continue to produce a lot of butyric acid.

[0158] 7. In vitro efficacy verification of thiosulfate-induced eGFP-HlyA secretion

[0159] 7.1 Construction and culture of EcN-BeGFP strain

[0160] The CDS of SOD-HlyA and CAT-HlyA in p15A-CST were replaced with eGFP-HlyA, and the resulting culture was transferred into EcN-B to obtain EcN-BeGFP, which served as the experimental group, with EcN-B as the negative control. EcN-BeGFP and EcN-B strains were streaked onto LB agar plates containing the corresponding antibiotics and incubated overnight at 37°C. A single colony from each strain was picked and inoculated into 5 mL of LB medium (containing the corresponding antibiotics) and incubated overnight at 37°C and 220 rpm. The overnight bacterial culture was then transferred at a 1:100 ratio to 50 mL of LB medium (containing the corresponding antibiotics) and incubated at 37°C and 220 rpm until OD500 was reached. 600 It is 0.5.

[0161] 7.2 Induction

[0162] To OD 600 Different amounts of sodium thiosulfate were added to bacterial cultures with a pH of 0.5 to achieve final thiosulfate concentrations of 0.001 mM, 0.01 mM, 0.1 mM, 0.5 mM, 1 mM, 2 mM, 3 mM, 5 mM, and 10 mM, respectively; and the concentrations were adjusted towards OD. 600Sulfate, sulfite, thiosulfate, tetrathionine, glutathione, nitrate, nitrite, hydrogen peroxide, and butyric acid were added to a bacterial culture with a concentration of 0.5 mM, respectively, to a final concentration of 1 mM. The specificity of thiosulfate induction was tested. The culture was then incubated at 37℃ and 220 rpm in the dark for 8 h. 200 μL of the induced culture was transferred to a 96-well plate, and the fluorescence intensity was immediately measured using a microplate reader. The parameters were set as follows: excitation light: 488 nm, emission light: 510-530 nm. OD was also measured simultaneously. 600 Normalized fluorescence value.

[0163] 7.3 Fluorescence detection of bacterial suspension, bacterial precipitate, and supernatant

[0164] Take 10 mL of LB medium containing EcN-B and EcN-BeGFP, respectively, and add sodium thiosulfate to a final concentration of 250 μM. For the non-induction group, add an equal volume of PBS. Incubate at 37℃ and 220 rpm in the dark for 8 h. Divide the culture medium into 5 mL aliquots. Centrifuge one 5 mL aliquot at 4000×g for 10 min, separate the supernatant into another centrifuge tube, and resuspend the precipitate in 5 mL PBS to obtain the bacterial pellet (resuspended solution). Take 200 μL of each EcN-B and EcN-BeGFP bacterial suspension, 200 μL of the supernatant, and 200 μL of the bacterial pellet, respectively, and plate them in a 96-well plate. Immediately measure the fluorescence intensity using a microplate reader. Simultaneously measure the OD... 600 Use normalized fluorescence values. Operate in the dark throughout the entire process.

[0165] Take 20 μL of bacterial suspension, 20 μL of supernatant, and 20 μL of bacterial precipitate for EcN-B and EcN-BeGFP, respectively, and prepare slides on glass slides. Observe bacterial fluorescence using a confocal microscope. Parameter settings: excitation light: 488 nm, emission light: 510-530 nm. Operate in the dark throughout the process.

[0166] 7.4 Experimental Results

[0167] Experimental results are as follows Figure 3 As shown, the results indicate that the ThsS / R system can sense thiosulfate and effectively induce the expression of downstream proteins, exhibiting a quantity-response relationship. Figure 3 (a). Meanwhile, ThsS / R exhibits strong specificity, showing no response to other sulfur-containing compounds but cross-reacting with tetrathionate, possibly related to its ability to be converted to thiosulfate. Figure 3 (b) The Hly secretion system can effectively secrete proteins carrying the HlyA signal peptide outside the bacteria, thus better realizing the function of engineered strains. Figure 3 (c and d).

[0168] 8. In vitro efficacy verification of thiosulfate-induced SOD-HlyA and CAT-HlyA

[0169] 8.1 Preparation of different components of engineered probiotics

[0170] EcN, EcN-B, EcN-CST, EcN-BCST, and EcN-BCST@PCPC were cultured under anaerobic conditions until OD. 600 =0.5, the bacterial culture was divided into two equal parts. One part was cultured anaerobically for 8 h, centrifuged, and then incubated at 37°C for 30 min with a solution containing 1 mg / mL lysozyme and 0.1% Triton X-100 to lyse it, and this was used as an induction-free suspension. The other part was treated with sodium thiosulfate to a final concentration of 1 mM and cultured anaerobically for 8 h.

[0171] Each group of bacterial suspensions induced with sodium thiosulfate was divided into two equal portions. One portion was centrifuged and incubated at 37°C for 30 min with a solution containing 1 mg / mL lysozyme and 0.1% Triton X-100 to induce lysis, and was used as a 1 mM induction suspension. The other portion was centrifuged at 12000×g for 15 min at 4°C, the supernatant was carefully aspirated, and the bacterial precipitate was collected. The supernatant was filtered through a 0.22 μm filter membrane and placed on ice, and was used as a 1 mM induction supernatant.

[0172] The bacterial pellet was resuspended and washed once with pre-cooled potassium phosphate buffer, centrifuged again, and resuspended in a solution of 1 mg / mL lysozyme and 0.1% Triton X-100, vortexing several times during resuscitation. Then, it was centrifuged at 12000×g for 15 min at 4°C, and the supernatant was collected as the 1 mM induced bacterial pellet.

[0173] 8.2 Oxygen Concentration Detection

[0174] The oxygen concentration was measured after 3 min by directly measuring the oxygen generation rate in 0.6 mM H2O2 solution added to the solution. The activity of catalase (CAT) in different components of the engineered probiotics induced by thiosulfate was quantitatively analyzed.

[0175] 8.3 Detection of Superoxide Anion Inhibition Rate

[0176] Add 2.95 mL of Tris-HCl buffer (50 mM, pH 8.2) and 0.05 mL of pyrogallol working solution (60 mM) to a quartz cuvette. Measure the absorbance at 325 nm using UV spectrophotometry, recording the absorbance every 30 seconds for 5 minutes. Calculate the superoxide anion generation inhibition rate and analyze the superoxide dismutase (SOD) activity.

[0177] 8.4 Experimental Results

[0178] The results are as follows Figure 4 As shown, CAT produced by the engineered probiotic EcN-BCST induced by 1 mM sodium thiosulfate effectively decomposed hydrogen peroxide, while the layer-by-layer self-assembled coating of EcN-BCST@PCPC had no significant effect on CAT activity. Furthermore, the activity of the supernatant was roughly equal to the activity of the enzyme within the bacteria. Figure 4 (ad).

[0179] The situation with SOD is roughly the same as that with CAT. Figure 4 (eh). The above content demonstrates that CAT and SOD function normally, and that the exosecretory system of engineered probiotics can effectively secrete CAT and SOD from the bacteria to catalyze reactions of substrates in the environment.

[0180] 9. Engineered probiotics for the treatment of dextran sulfate-induced inflammatory bowel disease in mice.

[0181] 9.1 Mouse grouping

[0182] Six- to eight-week-old male C57BL / 6J mice were randomly divided into seven groups of five mice each. Inflammatory bowel disease (IBD) was induced in the mice using dextran sulfate (DSS). The specific groupings are as follows:

[0183] Group 1: Normal drinking water, PBS administered by gavage, serving as the healthy control group; Group 2: DSS, PBS administered by gavage, serving as the DSS model group; Group 3: DSS+EcN; Group 4: DSS+EcN-B; Group 5: DSS+EcN-CST; Group 6: DSS+EcN-BCST; Group 7: DSS+EcN-BCST@PCPC; Groups 3-7 served as the treatment group.

[0184] 9.2 Experimental Methods

[0185] Mice were acclimatized for one week. From day 7 before the experiment (Day-7) to day 0 of the experiment, all treatment groups were administered bacterial solution by gavage daily (gavage dose was 1×10⁻⁶). 9 CFU / 200 μL / animal), healthy control group and DSS model group were given the same volume of PBS by gavage.

[0186] From the start of the experiment (Day 0) to the 7th day after the start of the experiment (Day 7), except for the healthy control group, all other groups freely drank 3% (w / v) DSS solution (molecular weight 36000-50000) for 7 days. The healthy control group drank normal water throughout the experiment, and all groups were continuously gavaged.

[0187] From Day 7 to Day 14 of the experiment, all mice were allowed to drink normal water, and all groups continued to be administered gavage until the end of the experiment. On Day 14, mice were administered FITC-labeled dextran (FITC-DSS) via gavage. Four hours after gavage, the mice were sacrificed, and blood was collected from their hearts to assess intestinal integrity and function. Colons were harvested to measure colon length, and the levels of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interleukin-10 (IL-10) in the colon tissue were measured using ELISA.

[0188] 9.3 Experimental Results

[0189] The results are as follows Figure 5 As shown, engineered probiotics can exert a synergistic effect of butyrate production, ROS resistance, and anti-inflammation. Furthermore, through layer-by-layer self-assembly coating, they can effectively alleviate weight loss induced by DSS-induced IBD in mice. Figure 5 (a) Changes in colon length can also reflect its therapeutic effect. Figure 5 (b) Intestinal integrity was assessed using FITC-labeled dextran, demonstrating that the engineered probiotics with layered self-assembly coatings effectively mitigated DSS-induced damage to the intestinal structure and improved intestinal integrity. Figure 5 (c). ELISA testing of colonic tissue revealed significantly decreased levels of pro-inflammatory factors IL-1β and TNF-α, while significantly increased levels of anti-inflammatory factor IL-10. Furthermore, EcN-BCST@PCPC showed the best therapeutic effect. Figure 5 (df).

[0190] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An inflammatory-responsive genetically engineered probiotic, EcN-BCST, characterized in that, The inflammatory-responsive genetically engineered probiotic ECN-BCST is obtained by transferring plasmid pRE112-B into DAP auxotrophic Escherichia coli to obtain donor bacteria, mixing the donor bacteria with the E. coli EcN, screening to obtain engineered bacteria EcN-B, and then transferring plasmid p15A-CST into engineered bacteria EcN-B. The nucleotide sequence of the plasmid pRE112-B is shown in SEQ ID NO.1; The nucleotide sequence of the plasmid p15A-CST is shown in SEQ ID NO.

2.

2. A method for preparing the inflammatory-responsive genetically engineered probiotic EcN-BCST as described in claim 1, characterized in that, Includes the following steps: The plasmid pRE112-B was transformed into DAP auxotrophic Escherichia coli to obtain the donor bacteria; The donor bacteria were mixed with the Escherichia coli EcN and screened to obtain the engineered bacteria EcN-B. The plasmid p15A-CST was transferred into the engineered bacteria EcN-B to obtain the inflammatory-responsive genetically engineered probiotic EcN-BCST.

3. The preparation method according to claim 2, characterized in that, The DAP auxotrophic Escherichia coli is Escherichia coli WM3064.

4. The application of the inflammatory-responsive genetically engineered probiotic EcN-BCST as described in claim 1 in the preparation of intestinal-targeted probiotic products, characterized in that, The preparation method of the gut-targeting probiotic product includes the following steps: The inflammatory-responsive genetically engineered probiotic EcN-BCST was resuspended in polylysine solution to obtain EcN-BCST@P; EcN-BCST@P was resuspended in carboxymethyl inulin solution to obtain EcN-BCST@PC; The EcN-BCST@PC was resuspended in polylysine solution to obtain EcN-BCST@PCP; The EcN-BCST@PCP was resuspended in a carboxymethyl inulin solution to obtain EcN-BCST@PCPC, which is the intestinal-targeted probiotic product.

5. A colon-targeted probiotic product, characterized in that, The inflammatory-responsive genetically engineered probiotic EcN-BCST described in claim 1 is the active ingredient; The preparation method of the colon-targeted probiotic product includes the following steps: The inflammatory-responsive genetically engineered probiotic EcN-BCST was resuspended in polylysine solution to obtain EcN-BCST@P; EcN-BCST@P was resuspended in carboxymethyl inulin solution to obtain EcN-BCST@PC; The EcN-BCST@PC was resuspended in polylysine solution to obtain EcN-BCST@PCP; The EcN-BCST@PCP was resuspended in a carboxymethyl inulin solution to obtain EcN-BCST@PCPC, which is the colon-targeted probiotic product.

6. A method for preparing a colon-targeted probiotic product as described in claim 5, characterized in that, Includes the following steps: The inflammatory-responsive genetically engineered probiotic EcN-BCST was resuspended in polylysine solution to obtain EcN-BCST@P; EcN-BCST@P was resuspended in carboxymethyl inulin solution to obtain EcN-BCST@PC; The EcN-BCST@PC was resuspended in polylysine solution to obtain EcN-BCST@PCP; The EcN-BCST@PCP was resuspended in a carboxymethyl inulin solution to obtain EcN-BCST@PCPC, which is the colon-targeted probiotic product.

7. The preparation method according to claim 6, characterized in that, The concentration of the polylysine solutions was 2 mg / mL. The concentration of the carboxymethyl inulin solution was 2 mg / mL.

8. The use of an inflammatory-responsive genetically engineered probiotic EcN-BCST as described in claim 1 or a colon-targeting probiotic product as described in claim 5 in the preparation of a medicament for treating inflammatory bowel disease.

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