Engineering modified probiotic-polymer conjugate and application thereof

Abstract

The invention provides an engineered probiotic-polymer conjugate and application thereof, SpyCatcher protein is displayed on the surface of probiotics, a polymer comprises a SpyTag residue and a dopamine structure part, and the probiotics and the polymer are coupled through an isopeptide bond formed between the SpyCatcher and the SpyTag. The colonization ability of the conjugate in the gastrointestinal tract is remarkably improved, the tolerance of the probiotics in the gastrointestinal tract is effectively improved, the construction method of the conjugate does not affect the inherent probiotic activity of the probiotics, and the conjugate has important significance in treatment of gastrointestinal diseases and is expected to promote the development of engineered intestinal microorganisms.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to an engineered probiotic and its application, particularly in improving the colonization ability of probiotics in the intestine. Background Technology

[0002] Within the vast gut microbiota, probiotics occupy a crucial position as the dominant flora. They participate in various vital physiological activities through intermediate metabolites or surface antigen structures, and are therefore increasingly used to regulate growth and prevent and treat diseases. However, the complex environment of the gastrointestinal tract makes precise regulation of probiotics impossible, often resulting in poor retention and low survival rates, thus hindering their full functional expression. Therefore, improving the colonization of probiotics in the gastrointestinal tract is of great research significance.

[0003] The academic community has proposed various strategies for "arming" probiotics, namely forming coatings on the cell surface, such as polydopamine, adhesion proteins, and tannins, to endow them with enhanced functionality. However, to date, almost all research has been limited to in-situ synthesis of coatings, and point-to-point specific modification has not yet been achieved. This method undoubtedly limits the range of coating materials to a great extent, and in-situ synthesis can also cause unknown damage to cells. Therefore, it is necessary to develop safer and more efficient arming methods to expand the available material spectrum and thus broaden the application scope of armed probiotics.

[0004] Lactococcus lactis is a recognized food-grade safe microorganism. Due to its strong ability to break down proteins and produce acid, it can inhibit microorganisms such as Staphylococcus and Bacillus, and is therefore widely used in the food fermentation industry. Furthermore, Lactococcus lactis possesses rich probiotic functions, playing an important role as a probiotic in maintaining intestinal flora balance, stimulating the secretion of immune molecules, and enhancing the body's immunity. In addition, it is also an important model organism, characterized by rapid growth, simple metabolism, weak antigenicity, and high expression efficiency, making it the best choice for food-grade expression hosts. However, compared with other lactic acid bacteria, Lactococcus lactis has poor adhesion in the intestine. Therefore, providing an engineered probiotic (such as Lactococcus lactis) to improve its colonization ability in the intestine is of great significance for the application and development of probiotics. Summary of the Invention

[0005] To address the shortcomings of existing technologies in the colonization ability of probiotics in the intestine, this invention proposes an engineered probiotic-polymer conjugate in which the probiotic surface displays SpyCatcher protein. The probiotic binds to a polymer containing SpyTag, significantly improving the colonization ability of the probiotic in the intestine without affecting cell activity, which is of great significance for the treatment and / or prevention of gastrointestinal diseases.

[0006] In a first aspect, the present invention provides an engineered probiotic, wherein the surface of the probiotic displays SpyCatcher protein.

[0007] Specifically, the engineered probiotics are obtained by converting probiotics into a recombinant expression vector (surface display vector) containing nucleotides encoding anchoring proteins (such as AcmA), SpyCatcher, and signal peptides.

[0008] The AcmA gene comprises, or is composed of, a nucleotide sequence having 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 99.9% or more homology with SEQ ID NO: 14.

[0009] The coding gene of SpyCatcher comprises, or is composed of, a nucleotide sequence having 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 99.9% or more homology with SEQ ID NO: 15.

[0010] The signal peptide is selected from Usp45, ATPγ or Rf1b, preferably Usp45.

[0011] In one specific embodiment of the present invention, the signal peptide is Usp45.

[0012] The Usp45 gene comprises, or is composed of, a nucleotide sequence having 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 99.9% or more homology with SEQ ID NO: 16.

[0013] The expression vector is selected from one or more of pNZ8148, pET28a, pNZ8048 or pNZ8150, preferably pNZ8148.

[0014] In one specific embodiment of the present invention, the expression vector is pNZ8148.

[0015] The recombinant expression vector includes, from 5' to 3', the gene encoding the signal peptide, the gene encoding SpyCatcher, and the AcmA gene.

[0016] In one specific embodiment of the present invention, the recombinant expression vector comprises the sequence shown in SEQ ID NO: 18.

[0017] The probiotics are selected from one or more of lactic acid bacteria, bifidobacteria, or Escherichia coli; preferably, the lactic acid bacteria are selected from Streptococcus, Enterococcus, Lactococcus, Pediococcus, or Lactobacillus; more preferably, the Lactococcus is selected from Lactococcus lactis, Lacococcus garvieae, or Lactococcus cremoris.

[0018] In one specific embodiment of the present invention, the probiotic is Lactococcus lactis, such as Lactococcus lactis NZ9000.

[0019] A second aspect of the present invention provides a method for constructing the probiotics described in the first aspect, the method comprising:

[0020] Step 1: Insert the encoding genes of AcmA, Spycathcer, and signal peptide into the expression vector to construct a surface display vector;

[0021] Step 2: Transfer the surface display carrier into the probiotics.

[0022] Step one further includes amplifying the encoding genes of the AcmA gene, Spycathcer gene, and signal peptide, preferably, the signal peptide is Usp45.

[0023] The primers for amplifying the AcmA gene comprise SEQ ID NO: 6 and SEQ ID NO: 7, or, constitute a nucleotide sequence having 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 99.9% or more homology with SEQ ID NO: 6 and SEQ ID NO: 7.

[0024] The primers for amplifying the Usp45-Spycathcer gene comprise SEQ ID NO: 8 and SEQ ID NO: 9, or, constitute a nucleotide sequence having 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 99.9% or more homology with SEQ ID NO: 8 and SEQ ID NO: 9.

[0025] Step one also includes linearizing the circular expression vector to obtain a linearized expression vector, and then amplifying the linearized vector.

[0026] In one specific embodiment of the present invention, the linearization vector is the pNZ8148 linearization plasmid.

[0027] The primers for amplifying the linearized pNZ8148 plasmid comprise SEQ ID NO: 10 and SEQ ID NO: 11, or, constitute a nucleotide sequence having 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 99.9% or more homology with SEQ ID NO: 10 and SEQ ID NO: 11.

[0028] Step two also includes electrotransferring probiotics into the surface display carrier constructed in step one.

[0029] In one specific embodiment of the present invention, the probiotic is Lactococcus lactis NZ9000.

[0030] A third aspect of the present invention provides the use of the probiotics described in the first aspect or the probiotics obtained by the construction method described in the second aspect in the preparation of products for the treatment and / or prevention of gastrointestinal diseases.

[0031] The products mentioned include one or more of the following: pharmaceuticals, health products, or food.

[0032] The product also includes excipients; the excipients are selected from one or more of the following: fillers, binders, wetting agents, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, colorants, pH regulators, antibacterial agents, or buffers.

[0033] The gastrointestinal diseases mentioned are selected from one or more of the following: diarrhea, gastritis, enteritis, colitis, Crohn's disease, appendicitis, peptic ulcer, gastric cancer, or intestinal cancer.

[0034] A fourth aspect of the invention provides a polymer comprising SpyTag residues, further comprising a dopamine structural moiety (such as...). ), and optionally other hydrophilic structural portions, such as oligomeric methacrylate (e.g., OEGMA), acrylamide, acrylic acid, acrylate, methacrylic acid, methacrylate, styrene structural portions.

[0035] Specifically, the polymer comprises the following structural parts:

[0036]

[0037] Wherein, ST represents a SpyTag residue, and the amino acid sequence of SpyTag is shown in SEQ ID NO: 17, wherein the terminal lysine is linked to the amino group shown in the above formula via a carbonyl group.

[0038] Specifically, the polymer comprises the following structural parts:

[0039]

[0040] Where m is an integer from 1 to 50 (such as 2, 4, 6, 8, 10, 12, 14, 15, 16, 18, 20, 25, 30, 40, 50), for example 1-20, 2-15, 4-12, 6-10, 8.

[0041] Specifically, the polymer may also include one or more of the following structural portions:

[0042]

[0043] Where n is an integer from 1 to 50 (such as 2, 4, 6, 8, 10, 12, 14, 15, 16, 18, 20, 25, 30, 40, 50), for example 2-50, 10-40, 20-40, 30-40, 35;

[0044] p is an integer from 1 to 20 (such as 2, 4, 6, 8, 10, 12, 14, 15, 16, 18, 20), for example 2-10, 2-8, 4 or 5;

[0045] R1 is selected from: H, C1-C10 alkyl (such as C1-C6 alkyl, C1-C3 alkyl, methyl, ethyl), especially H or methyl.

[0046] Specifically, the polymer is a random copolymer, and in some embodiments of the present invention, it includes the following structural units: and one or more of the following:

[0047] In some embodiments of the present invention, the polymer has the following structure:

[0048]

[0049] Where m is an integer from 1 to 50;

[0050] n is an integer from 1 to 50;

[0051] p is an integer from 1 to 20;

[0052] ST stands for SpyTag residues;

[0053] X2 is a halogen, such as Br or Cl.

[0054] In some embodiments of the present invention, the molecular weight of the polymer is 1-25 kDa (e.g., 1, 2, 4, 5, 6, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 25 kDa), for example 5-25 kDa, 10-20 kDa; in one embodiment of the present invention, the molecular weight of the polymer is about 17.6 kDa.

[0055] Specifically, in the polymer The molar ratio of the portion to other hydrophilic structural portions (e.g., oligomeric methacrylate (such as OEGMA), acrylamide, acrylic acid, acrylate, methacrylic acid, methacrylate, styrene structural portions) is 1:1-10 (e.g., 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:8); in one embodiment of the invention, the ratio is 1:4.

[0056] A fifth aspect of the present invention provides a method for preparing the above-described polymer, comprising the following steps:

[0057]

[0058] Step (1): Dopamine reacts with M-1 to obtain intermediate M-2;

[0059] Step (2): M-2 reacts with a hydroxyl protecting agent to obtain intermediate M-3 with the hydroxyl group protected;

[0060] Step (3): M-3 and monomer M-5 undergo polymerization reaction in the presence of initiator M-4 to obtain intermediate M-6;

[0061] Step (4): Deprotect M-6 by removing its hydroxyl groups;

[0062] Wherein, X1 and X2 are independently selected from leaving groups;

[0063] R PIt is a hydroxyl protecting group.

[0064] Specifically, monomer M-5 is a hydrophilic polymeric monomer compound, especially those suitable for ATRP polymerization, such as oligomeric methacrylate (e.g., OEGMA), acrylamide, acrylic acid, acrylate, methacrylic acid, methacrylate, and styrene.

[0065] In some embodiments of the present invention, Y is selected from one or more of the following:

[0066] In some embodiments of the present invention, the preparation method includes the following steps:

[0067]

[0068] In some embodiments of the present invention, X1 is a halogen, such as Cl.

[0069] In some embodiments of the present invention, X2 is a halogen, such as Br.

[0070] In some embodiments of the present invention, R P It is silane, such as In this case, the hydroxyl protecting group can be tert-butyldimethylchlorosilane (TBDMS-Cl).

[0071] Step (3) further includes using atom transfer radical polymerization (ATRP) to achieve the polymerization reaction, wherein the ATRP includes:

[0072] Step (3.1): After adding M-3, M-5, M-4 and catalyst to the Schlenk tube, seal it and purge with nitrogen for 10-60 minutes;

[0073] Step (3.2): Add the complexing agent Me6TREN to the Schlenk tube using a deoxygenated microsyringe, and continue to purge with nitrogen for 10-50 minutes;

[0074] Step (3.3): Remove the nitrogen gas device and place the Schlenk tube in an oil bath at 40℃-80℃ for 15h-30h.

[0075] Preferably, the nitrogen purging time in step (3.1) is 15-30 minutes, such as 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, or 60 minutes. More preferably, the nitrogen purging time is 20 minutes.

[0076] Preferably, the nitrogen purging time in step (3.2) is 20-40 minutes, for example, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, or 50 minutes. More preferably, the nitrogen purging time is 30 minutes.

[0077] Preferably, the temperature of the oil bath in step (3.3) is 50℃-70℃, for example 40℃, 50℃, 60℃, 70℃, or 80℃. More preferably, the temperature of the oil bath is 60℃.

[0078] Preferably, the oil bath reaction time in step (3.3) is 20h-25h, for example 15h, 18h, 20h, 22h, 24h, 26h, 28h, or 30h. More preferably, the oil bath reaction time is 24h.

[0079] In a sixth aspect of the invention, the use of the above-described polymer in the preparation of a reagent that enhances the colonization ability of probiotics in the gut is provided.

[0080] In a seventh aspect of the invention, an engineered probiotic-polymer conjugate is provided, wherein the probiotic surface displays SpyCatcher protein (as described in the first aspect of the invention);

[0081] The polymer contains SpyTag residues and a dopamine structural moiety (such as...). The SpyTag and SpyCatcher are coupled via isopeptide bonds.

[0082] In some embodiments of the present invention, the polymer further comprises other hydrophilic structural portions, such as oligomeric methacrylate (e.g., OEGMA), acrylamide, acrylic acid, acrylate, methacrylic acid, methacrylate, and styrene.

[0083] In some embodiments of the present invention, the polymer is the polymer described in the fourth aspect of the present invention.

[0084] An eighth aspect of the present invention provides a method for constructing the coupling described in the seventh aspect, the method comprising:

[0085] Step 1: Insert the encoding genes of AcmA, Spycathcer, and signal peptide into the expression vector to construct a surface display vector;

[0086] Step 2: Transfer the surface display carrier into the probiotics;

[0087] Step 3: Prepare a polymer containing SpyTag residues;

[0088] Step 4: Co-incubate the probiotics and polymer to obtain the conjugate.

[0089] Step four involves mixing the probiotic suspension with the polymer solution and co-incubating them. The solvents for the bacterial suspension and polymer solution can be any buffer suitable for culturing cells, such as PBS, Tris buffer, glycine buffer, and especially PBS.

[0090] The final concentration of the polymer is 1-10 mg / mL (e.g., 2, 4, 5, 6, 8, 10 mg / mL) based on the mixture of bacterial suspension and polymer solution.

[0091] The co-incubation time in step four is 0.5h-3h, preferably 1h-2.5h, for example 0.5h, 1h, 1.5h, 2h, 2.5h, 3h.

[0092] The co-incubation involves placing a mixture of probiotics and polymer in a shaker at a speed of 50-300 r / min, preferably 100-200 r / min, such as 50 r / min, 75 r / min, 100 r / min, 125 r / min, 150 r / min, 175 r / min, 200 r / min, 225 r / min, 250 r / min, 275 r / min, or 300 r / min.

[0093] The temperature of the shaker is 20℃-50℃, preferably 25℃-40℃, for example 20℃, 25℃, 30℃, 35℃, 40℃, 45℃, 50℃.

[0094] In one specific embodiment of the present invention, step four involves placing the probiotics and the polymer in a shaker at 30°C and incubating them together at 150 r / min for 2 hours.

[0095] A ninth aspect of the present invention provides a recombinant expression vector (Spycathcer surface display vector) comprising an AcmA gene, a Spycathcer gene, and a gene encoding a signal peptide.

[0096] The signal peptide encoding gene, the SpyCatcher encoding gene, and the AcmA gene are sequentially linked and inserted into the expression vector pNZ8148 to form the Spycathcer surface display vector.

[0097] In some embodiments of the present invention, the recombinant expression vector comprises the encoding nucleotide of the signal peptide-SpyCatcher-AcmA, such as Usp45-Spycatcher-AcmA (SEQ ID NO: 18).

[0098] Specifically, the expression vector is selected from one or more of pNZ8148, pET28a, pNZ8048 or pNZ8150, preferably pNZ8148.

[0099] In a tenth aspect of the present invention, there is an application of the conjugate described in the seventh aspect or the conjugate obtained by the construction method described in the eighth aspect in the preparation of a reagent to enhance the colonization ability of probiotics in the gut or in the preparation of a product for the treatment and / or prevention of gastrointestinal diseases.

[0100] The products mentioned include one or more of the following: pharmaceuticals, health products, or food.

[0101] The product also includes excipients; the excipients are selected from one or more of the following: fillers, binders, wetting agents, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, colorants, pH regulators, antibacterial agents, or buffers.

[0102] The gastrointestinal diseases mentioned are selected from one or more of the following: diarrhea, gastritis, enteritis, colitis, Crohn's disease, appendicitis, peptic ulcer, gastric cancer, or intestinal cancer, especially colitis.

[0103] In an eleventh aspect of the present invention, a method for improving the intestinal colonization ability of probiotics is provided, the method comprising co-incubating probiotics with Spycathcer surface-displaying residues (as described in the first aspect of the present invention) with a polymer containing SpyTag residues to obtain probiotics modified with a surface-coupled polymer, said polymer containing SpyTag residues and a dopamine structural moiety (as described in the first aspect of the present invention) to obtain probiotics with surface-coupled polymer modification. (For example, the polymer described in the fourth aspect of the present invention).

[0104] According to a twelfth aspect of the present invention, a composition is provided, the composition comprising the probiotics described in the first aspect, the probiotics obtained by the construction method described in the second aspect, the probiotics described in the seventh aspect, or the probiotics obtained by the construction method described in the eighth aspect.

[0105] The compositions include pharmaceutical compositions, vaccine compositions, food or feed compositions.

[0106] The dosage form of the composition is selected from tablets, pills, powders, suspensions, gels, emulsions, creams, granules, nanoparticles, capsules, suppositories, injections, or sprays.

[0107] In some embodiments of the present invention, the composition is an oral formulation comprising live cells of the probiotic.

[0108] In a thirteenth aspect of the invention, a kit is provided comprising the engineered probiotics described in the first aspect and the polymer described in the fourth aspect.

[0109] Specifically, the kit also includes reagents for incubation, such as buffer solutions (e.g., PBS).

[0110] In a fourteenth aspect of the invention, a kit is provided comprising the recombinant expression vector described in the ninth aspect and the polymer described in the fourth aspect.

[0111] Specifically, the kit also includes reagents for incubation, such as buffer solutions (e.g., PBS).

[0112] Specifically, the kit also includes reagents for electroconversion, such as electroconversion wash buffer and recovery culture medium.

[0113] Beneficial effects:

[0114] This invention provides an engineered probiotic, wherein the probiotic surface displays Spycatcher, and the probiotic further includes the use of ATRP and genetic engineering technology to achieve chemical linkage between the probiotic surface displaying Spycatcher protein and a polymer containing SpyTag residues, successfully constructing a LAB-Spy-PDA with surface-linked polydopamine, and verifying its enhanced colonization and therapeutic effects in the gastrointestinal tract.

[0115] (1) A strain of Lactococcus lactis SC-LAB with Spycatcher protein domain on its surface was constructed using genetic engineering technology, and it was demonstrated that it can effectively link with proteins containing Spytag.

[0116] (2) Polydopamine ST-PDA with Spytag end groups was obtained by bulk polymerization of ATRP at 60°C. The reaction had a high conversion rate and did not affect the function of Spytag. ST-PDA was also shown to have good structural stability in the gastrointestinal environment.

[0117] (3) Lactococcus lactis LAB-Spy-PDA with polydopamine surface-linked was prepared by Spy chemistry. A series of characterizations demonstrated that this Spy chemistry-based coupling did not delay cell growth, and the polymer could effectively enhance the tolerance of Lactococcus lactis to the gastrointestinal environment.

[0118] (4) The mouse gavage experiment and DSS-colitis treatment experiment showed that the conjugation of polydopamine can effectively enhance the adhesion and colonization of Lactococcus lactis in the intestine, and the genetic engineering modification did not affect the inherent beneficial life and biosafety of Lactococcus lactis.

[0119] In summary, the engineered probiotic-polymer conjugates provided by this invention significantly improve the colonization ability of probiotics in the gut. The method for constructing probiotic-polymer conjugates in this invention not only expands the application of protein-polymer conjugate systems, but also can be used to expand the functionality and diversity of armed probiotics, and is expected to build a powerful conjugate platform based on Spy chemistry for gut microbiota. Attached Figure Description

[0120] Figure 1 A schematic diagram illustrating the construction of Lactococcus lactis-polymer conjugates using Spy chemistry to enhance its colonization in the gut.

[0121] Figure 2 Schematic diagram of the target gene Usp45-Spycatcher-AcmA.

[0122] Figure 3 Construction process of plasmid pNZ8148-AcmASC.

[0123] Figure 4 SDS-PAGE results of engineered bacteria SC-LAB before (left) and after (right) induction.

[0124] Figure 5 Fluorescence emission spectra of SC-LAB and sfGFP-ST after co-incubation for different times.

[0125] Figure 6 SDS-PAGE of supernatants from different amounts of SC-LAB culture medium co-incubated with sfGFP-ST for 1 h.

[0126] Figure 7 Synthesis of dopamine monomers.

[0127] Figure 8 DOPM 1 H NMR spectrum.

[0128] Figure 9 DOPMS 1 H NMR spectrum.

[0129] Figure 10 ST-PDA in CDCl3 1 H NMR spectrum.

[0130] Figure 11 GPC curves of ST-PDA in a) SGF, b) SIF, c) bile salt solution and d) pH 2 / 8 solution for 20 h.

[0131] Figure 12ST-PDA incubated for 20 hours in pH 2 / 8 solution, SGF, SIF, and bile salt solution 1 H NMR spectrum.

[0132] Figure 13 SDS-PAGE results of ST-PDA, SC-sfGFP, and their conjugates.

[0133] Figure 14 Inhibitory effect of different concentrations of ST-PDA on Micrococcus luteus.

[0134] Figure 15 Synthesis of the fluorescent copolymer ST-FLPDA.

[0135] Figure 16 Fluorescence emission spectrum of TAMRA-LAB-Spy-FLPDA.

[0136] Figure 17 Laser scanning confocal microscopy image of TAMRA-LAB-Spy-FLPDA.

[0137] Figure 18 : Hydration kinetics and particle size variations of SC-LAB and LAB-Spy-PDA connected at different times.

[0138] Figure 19 ST-PDA Zeta potential changes of SC-LAB and LAB-Spy-PDA connected at different times.

[0139] Figure 20 ST-PDA TEM images of SC-LAB and LAB-Spy-PDA.

[0140] Figure 21 : Static water contact angles of SC-LAB (top) and LAB-Spy-PDA (bottom).

[0141] Figure 22 : a) Cell viability assay and b) Growth curves of SC-LAB and LAB-Spy-PDA linked at different times.

[0142] Figure 23 : Glucose consumption rate of SC-LAB and LAB-Spy-PDA coupled for different durations.

[0143] Figure 24 : Cell viability of SC-LAB and LAB-Spy-PDA after culturing with a)SGF, b)SIF and c) bile salt solutions for different time periods.

[0144] Figure 25 : TEM images of SC-LAB and LAB-Spy-PDA after culturing with SGF and SIF for 0.5 h.

[0145] Figure 26 : Free cell counts were performed after co-incubating SC-LAB and LAB-Spy-PDA with isolated small intestinal tissue.

[0146] Figure 27 : Different time points after gavage Distribution of SC-LAB and LAB-Spy-PDA in the gastrointestinal tract.

[0147] Figure 28 : Different time points after gavage Statistical analysis of fluorescence of SC-LAB and LAB-Spy-PDA at different locations in the gastrointestinal tract.

[0148] Figure 29 : Different time points after gavage Statistical analysis of fluorescence of SC-LAB and LAB-Spy-PDA in the gastrointestinal tract.

[0149] Figure 30 LSCM imaging of the contents of the small intestine, cecum and colon 24 hours after gavage.

[0150] Figure 31 4 hours and 24 hours after gavage Plate counts of SC-LAB and LAB-Spy-PDA at different locations in the gastrointestinal tract.

[0151] Figure 32 : Different time points after gavage Plate counts of SC-LAB and LAB-Spy-PDA at different locations in the gastrointestinal tract.

[0152] Figure 33 The process of establishing and treating DSS-induced colitis in mice.

[0153] Figure 34 a) Changes in body weight of mice during induction and treatment (n=7); b) Colon length of mice after treatment (n=5); c) Colon photographs of the group after treatment.

[0154] Figure 35 The expression levels of a) IL-6 and b) TNF-α in the serum of mice after treatment.

[0155] Figure 36 H&E and PAS staining results of colon tissue from mice after treatment.

[0156] Figure 37 Goblet cell count chart.

[0157] Figure 38 Immunofluorescence staining of Claudin-1 and ZO-1 in the colon tissue of mice after treatment.

[0158] Figure 39 Statistical analysis of fluorescence of Claudin-1 and ZO-1.

[0159] Figure 40 H&E staining images of the heart, liver, spleen, lungs, and kidneys of mice after treatment. Detailed Implementation

[0160] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0161] This invention provides an engineered probiotic-polymer conjugate and its application. Specifically, taking *Lactococcus lactis* as an example, it achieves point-to-point chemical linkage between Spy-mediated polydopamine and the surface of *Lactococcus lactis*. The *Lactococcus lactis*-polydopamine conjugate enhances the colonization ability of *Lactococcus lactis* in the gastrointestinal tract. The construction process is as follows: Figure 1 As shown.

[0162] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0163] Unless otherwise specified, all reagents, materials, and instruments used in the following examples are commercially available.

[0164] The starting strain of Lactococcus lactis used in the examples was Lactococcus lactis NZ9000.

[0165] 1. Reagents used in the examples and their manufacturers:

[0166] (1) Dopamine, tert-butyldimethylchlorosilane, acrylamide, N-isopropylacrylamide, hydroxyethyl acrylate, N-(2-hydroxypropyl)acrylamide, butyric anhydride and dextran sulfate (Mw=40000) were all obtained from Shanghai Titan Technology Co., Ltd.

[0167] (2) Fluorescein-O-methacrylate, 5-carboxyfluorescein succinimide ester and 5-carboxytetramethylrhodamine succinimide ester were all obtained from Shanghai Yuanye Biotechnology Co., Ltd.

[0168] (3) Trypsin (Shanghai Sigma-Aldrich Trading Co., Ltd.), pepsin (Shanghai Aladdin Reagent Co., Ltd.), ox bile salts (Shanghai Aladdin Reagent Co., Ltd.), protein molecular weight marker (White Shark Biotechnology Co., Ltd.), M17 broth (Qingdao Haibo Biotechnology Co., Ltd.);

[0169] (4) Phanta Max Master Mix (2×) and ClonExpress Ultra One Step Cloning Kit were obtained from Novizan Biosciences Co., Ltd.

[0170] (5) Small-volume agarose gel DNA recovery kit (Beijing Zhuangmeng International Biotechnology Co., Ltd.), high-purity plasmid mini-preparation kit (Beijing Zhuangmeng International Biotechnology Co., Ltd.), mouse TNF-α detection kit (Beijing Solarbio Technology Co., Ltd.), mouse interleukin-6 detection kit (Beijing Solarbio Technology Co., Ltd.).

[0171] 2. Model and company of the instruments used in the embodiments

[0172] Laser confocal microscope (ME204E, Leica), small animal live imaging system ( Lumina III (PerkinElmer), glucose concentration meter (SBA-40D, Shandong Academy of Sciences Institute of Biology), electroporator (Micropulser 165-2100, Bio-Rad), polymerase chain reaction (T100, Bio-Rad), particle size potentiometer (Zetasizer Nano, Malvern Instruments), contact angle meter (SDC-80S, China Shengding Precision Instruments Co., Ltd.), digital slide scanner (Pannoramic MIDI, 3DHISTECH).

[0173] 3. Characterization methods

[0174] 1H NMR analysis was performed using deuterated chloroform (CDCl3), deuterated dimethyl sulfoxide (DMSO-d6), or D2O as solvents.

[0175] GPC: The relative molecular weight of ST-PDA was determined using a Waters gel permeation chromatograph equipped with 7.8×300 mm and 4.6×300 mm aqueous columns (Agilent, AdvanceBio), with 0.2 M NaNO3 aqueous solution as the mobile phase, a flow rate of 0.5 mL / min, and a column temperature of 35 °C.

[0176] TEM: For ST-PDA, dissolve a small amount of sample in ddH2O, dilute to a suitable concentration, and drop 10 μL onto a copper grid, then allow to air dry. For LAB-Spy-PDA, after centrifuging to collect the bacterial cells, add an appropriate amount of 4% paraformaldehyde, fix at room temperature for 20 min, then wash three times with ddH2O, resuspend in ddH2O again, drop 10 μL onto a copper grid, dry, and then add 10 μL of 3% tungsten phosphate negative staining solution for 2 min. Finally, remove excess negative staining solution with filter paper.

[0177] All fluorescence imaging images were processed using ImageJ.

[0178] 4. Primers

[0179] Table 4 Primers used in this invention

[0180]

[0181]

[0182] 5. Culture medium, buffer solution and gel formulation

[0183] GM17 medium: Dissolve M17 broth powder in ddH2O according to the instructions, sterilize at 121℃ for 20 min, and add 0.5% sterile glucose solution before use.

[0184] GSGM: 0.5M sucrose, 25g / L glycine, M17 broth, 5g / L glucose.

[0185] Electroporation washing solution I: 0.5M sucrose, 100mL / L glycerol.

[0186] Electroporation washing solution II: 0.5M sucrose, 100mL / L glycerol, 0.05M EDTA.

[0187] Recovery medium: M17 broth, 0.5% glucose, 20mM MgCl2, 2mM CaCl2.

[0188] Example 1: Construction and characterization of SC-LAB genetically engineered bacteria

[0189] N-acetylmuraminidase (AcmA) is a major peptidoglycan hydrolase in *Lactococcus lactis*, participating in cell division and autolysis. Due to its C-terminus's cell wall anchoring function, it is often used as a carrier protein to construct *Lactococcus lactis* surface display systems. This invention fuses Spycather with AcmA to construct a *Lactococcus lactis* (SC-LAB) displaying the Spycather domain on its surface. The surface display system contains a target gene consisting of three parts: Usp45 (SEQ ID NO: 16), Spycather (SEQ ID NO: 15), and AcmA (SEQ ID NO: 14). The sequence of the constructed Usp45-Spycather-AcmA is shown in SEQ ID NO: 18, and the structure of the target gene is as follows: Figure 2 As shown in the figure. Usp45 is a signal peptide in Lactococcus lactis responsible for secreting a variety of functional proteins, which can promote the extracellular secretion of AcmA, which has poor transmembrane transport activity; Spycatcher is a passenger protein used for subsequent connection with Spytag; AcmA provides anchoring units, which are fixed to the cell wall during transmembrane transport and are the main structures for constructing the Lactococcus lactis surface display system.

[0190] Usp45 (SEQ ID NO: 16):

[0191] AAAAAAAAGATTATCTCAGCTATTTTAATGTCTACAGTGATACTTTCTGCTGCAGCCCCGTTGTCAGGTGTTTACGCT

[0192] SpyCatcher (SEQ ID NO: 15):

[0193] GGATCCGTGACCACCCTGAGCGGCCTGAGCGGCGAACAAGGCCCGAGCGGCGATATGACCACCGAAGAAGATAGCGCGACCCATATTAAATTTAGCAAACGCGATGAAGATGGCCGCGAACTGGCGGGCGCGACCATGGAACTGCGCGATAGCAGCGGCAAAACCATTAGCACCTGGATTAGCGATGGCCATGTGAAAGATTTTTATCTGTATCCGGGCAAATATACCTTTGTGGAAACCGCGGCGCCGGATGGCTATGAAGTGGCGACCCCGATTGAATTTACCGTGAACGAAGATGGCCAAGTGACCGTGGATGGCGAAGCGACCGAAGGCGATGCGCATACC

[0194] AcmA(SEQ ID NO:14):

[0195] Agttatggcgcttcactgaatcgcattatttctcaatataatttgactcgttttgacggagcttcttcagctggaaatactaattctggtggctcgacaaccacaattacgaataataattctggaaccaatagcagttcaactacttataccgtcaaatctggtgatactctttggggaatctcacaaagatatggaattagtgtcgctcaaattcaaagtgcgaataatcttaaaagtaccattatctacattggtcaaaaacttgtactgacaggttcagcttcttctacaaattcaggtggttcaaacaattccgcaagcactactccaaccacttctgtgacacctgcaaaaccaact tcacaaacaactgttaaggttaaatccggagataccctttgggcgctatcagtaaaatataaaactagtattgctcaattgaaaagttggaatcatttaagttcagataccatttatattggtcaaaatcttattgtttcacaatctgctgctgcttcaaatccttcgacaggttcaggctcaactgctaccaataactcaaactcgacttcttctaactcaaatgcctcaattcataaggtcgttaaaggagatactctctggggactttcgcaaaaatctggcagcccaattgcttcaatcaaggcttggaatcatttatctagcgatactattttaattggtcagtatctacgaataaaa

[0196] Usp45-SpyCatcher-AcmA(SEQ ID NO:18):

[0197]

[0198] The construction process of SC-LAB genetically engineered bacteria is as follows: Figure 3 As shown in the diagram. Specifically, the target gene fragment and linearized plasmid were first obtained from the genome of *Lactococcus lactis* NZ9000, pET28a-SC-sfGFP (SEQ ID NO: 19), and pNZ8148 (laboratory deposit) using PCR and agarose gel electrophoresis, respectively. The fragment and vector were then ligated and transformed into *Escherichia coli* TG1 strain. Positive transformants were selected and amplified, and the plasmid was extracted for gene sequencing. The results showed that the obtained sequence matched the theoretical sequence by 100%. Finally, the correctly sequenced plasmid was electroporated into competent *Lactococcus lactis* NZ9000 cells, and SC-LAB genetically engineered bacteria were obtained after PCR verification.

[0199] pET28a-SC-sfGFP (SEQ ID NO: 19):

[0200] HHHHHHVTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTISTWISDG

[0201] HVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGEATEGDAHTGSSGSRKGEELFTGV

[0202] VPILFEDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFARYPDHM

[0203] KQHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFN

[0204] SHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNE

[0205] KRDHMVLLEFVTAAGITHGMDELYK

[0206] The specific steps are as follows:

[0207] (1) Preparation of Lactococcus lactis NZ9000 electrocompetent cells

[0208] 50 μL of frozen Lactococcus lactis NZ9000 glycerol was inoculated into 5 mL of antibiotic-free GM17 medium and incubated overnight at 30°C. Then, a 1% inoculum was transferred to 100 mL of GSGM medium and incubated at 30°C until OD600 = 0.4. The cells were then collected by centrifugation, washed with 100 mL of pre-chilled electroporation wash buffer I, centrifuged again, washed with 25 mL of pre-chilled electroporation wash buffer II, and placed on ice for 15 min. The cells were centrifuged again at low temperature and washed with 25 mL of pre-chilled electroporation wash buffer I. Finally, the cells were resuspended in 1 mL of electroporation wash buffer I, aliquoted into 42 μL tubes, and stored at -80°C.

[0209] (2) Extraction of Lactococcus lactis NZ9000 genome

[0210] 50 μL of frozen Lactococcus lactis NZ9000 glycerol was inoculated into 5 mL of antibiotic-free GM17 medium and incubated statically overnight at 30°C. The bacterial cells were collected by centrifugation, resuspended in lysozyme solution to a final concentration of 25 mg / mL, and incubated in a 37°C water bath for 30 min. The cells were then centrifuged again to obtain the bacterial cells. Genomic DNA was extracted according to the instructions of the genomic DNA extraction kit, and after concentration determination, stored at -20°C.

[0211] (3) Construction of plasmids and screening of transformants

[0212] Taking pNZ8148-AcmASC as an example, the construction of plasmids and the screening of transformants are briefly described. Primers AcmA-F / R, UspSC-F / SC-R, and pNZ8148-Linear-F / R were designed and used as templates with NZ9000 genome, pET28a-SC-sfGFP, and pNZ8148-GFP, respectively. PCR amplification was performed according to the instructions of the DNA polymerase Phanta Max Master Mix to obtain the AcmA gene fragment with homologous arms, the Spycathcer gene fragment, and the pNZ8148 linearized vector.

[0213] PCR products were separated by 1% agarose gel electrophoresis. The corresponding DNA bands were excised, and the genes were recovered using an agarose gel DNA recovery kit. After the concentration was determined, the DNA was stored at -20°C.

[0214] Following the instructions of the ClonExpress Ultra One Step Cloning Kit, the AcmA gene fragment, SC gene fragment, pNZ8148 linearized vector, and cloning enzyme were mixed and placed in a 50℃ PCR instrument for 20 min for seamless cloning.

[0215] 5 μL of seamless cloning mixture was used to chemically transform 50 μL of *E. coli* TG1 competent cells. The transformed cells were plated and incubated overnight at 37°C. Eight single colonies from each plate were randomly selected and resuspended in 10 μL of sterile ddH2O. 7 μL of this resuspended colony was transferred to 100 μL of antibiotic-free LB broth, and the remaining 3 μL was infused with LAmp Master Mix (2×) DNA polymerase and pNZ8148 verification primers for colony PCR. The PCR products were analyzed by agarose gel electrophoresis. Positive transformants matching the target band size were transferred to 5 mL of LB broth containing the corresponding antibiotic and incubated overnight at 37°C and 150 rpm. The following day, 1 mL of the bacterial culture was immediately frozen at -80°C, and plasmids were extracted from the remaining culture and sent to Chengdu Youkang Biotechnology Co., Ltd. for sequencing. The correctly sequenced plasmids were stored at -20°C.

[0216] Using sfGFP-F / R, ST-28aLinear-F, and 28aLinear-R as primers, and pET28a-SC-sfGFP as a template, PCR amplification was performed to obtain the ST-sfGFP gene fragment with homologous arms and the pET28a linearized vector. pET28a-ST-sfGFP was then constructed following the steps described above.

[0217] (4) Electroporation and induced expression of Lactococcus lactis NZ9000

[0218] Take 42 μL of NZ9000 competent cells, add 5 μL of correctly sequenced pNZ8148-AcmASC, mix gently, and place on ice for 10 min. Then transfer to an electroporation cuvette for electroporation. Add 1 mL of recovery medium, incubate at 30°C for 3 h, centrifuge, remove 900 μL of supernatant, resuspend the remaining portion, spread it on GM17 solid plates containing chloramphenicol (CM), and incubate at 30°C for 24-36 h.

[0219] Electro-rotation conditions: voltage 2.4KV, electro-rotation time 5.2-5.8ms.

[0220] After single colonies grew on the plate, 8 colonies were randomly selected for PCR colony verification and agarose gel electrophoresis. Positive transformants that matched the size of the target band were selected and inoculated into 5 mL of GM17 liquid medium containing CM. The culture was carried out overnight at 30°C to obtain NZ9000 / pNZ8148-AcmASC. 1 mL of the culture was added to glycerol and stored at -80°C.

[0221] To induce expression of the plasmid in NZ9000 / pNZ8148-AcmASC, it was transferred to 50 mL of GM17 liquid medium containing CM at an inoculum of 1%, and cultured at 30°C until OD600 = 0.4. Then, nisin (10 ng / mL) was added and induced overnight.

[0222] (5) Fractional purification and SDS-PAGE of NZ9000 / pNZ8148-AcmASC

[0223] (5.1) Isolation of periplasmic proteins: Take 2 mL of bacterial culture before and after induction of NZ9000 / pNZ8148-AcmASC, centrifuge, wash the bacterial cells with Tris-HCl (20 mM, pH 8) and resuspend them in 0.5 mL of Tris-HCl containing 20% ​​sucrose and 1 mM Na2EDTA, and incubate at room temperature for 10 min. After centrifugation at 12000 r / min for 5 min, resuspend the bacterial cells in 0.5 mL of MgCl2 (0.5 mM) and incubate on ice for 10 min. Centrifuge again, transfer the supernatant to a new EP tube, and store at -20℃ to obtain periplasmic proteins.

[0224] (5.2) Separation of cytoplasmic proteins and membrane proteins: The precipitate was resuspended in 0.5 mL Tris-HCl, sonicated for 5-10 min (200 W, sonication for 2 s, pause for 5 s), centrifuged at 5000 r / min for 10 min to remove incompletely lysed cells, and the supernatant was centrifuged at 12000 r / min for 40 min to obtain cytoplasmic proteins (supernatant) and membrane proteins (precipitate).

[0225] (5.3) Separation of inner and outer membranes: Add 0.5 mL of Tris-HCl containing 0.01 mM MgCl2 and 2% Triton-X-100 to the membrane protein. After incubation at room temperature for 30 min, centrifuge at 12000 r / min for 40 min to separate the inner membrane (supernatant) and the outer membrane (precipitate). Resuspend the outer membrane in 0.2 mL of Tris-HCl and store both at -20℃.

[0226] Take 10 μL each of periplasmic proteins, cytoplasmic proteins, inner cell membrane proteins, and outer cell membrane proteins, add 2.5 μL of SDS-PAGE protein loading buffer, and separate them by electrophoresis on a 12% polyacrylamide gel at 120V. The gels are then stained with Coomassie Brilliant Blue using a rapid protein staining system. The SDS-PAGE results show (…). Figure 4 After induction, new protein bands appeared in the outer membrane of SC-LAB, with a size consistent with the protein (~50kDa).

[0227] To further verify the surface display function of SC-LAB, a Spytag-fused green fluorescent protein sfGFP-ST was constructed and co-incubated with induced SC-LAB. The cells were then washed with PBS solution containing surfactant to remove non-specifically linked sfGFP-ST. The specific steps are as follows:

[0228] (6) Coupling of NZ9000 / pNZ8148-AcmASC with sfGFP-ST

[0229] Collect 1 mL of NZ9000 / pNZ8148-AcmASC that had been induced overnight by centrifugation. Wash three times with PBS, then add 50 μL of sfGFP-ST (2 mg / mL) to the cells, mix well, and incubate at 30℃ and 150 r / min for different times (0.5 h, 1 h, 2 h, 4 h, 6 h, 9 h). After coupling, centrifuge and wash the cells three times with PBS solution containing 0.1% Triton-X-100. Finally, resuspend in PBS and measure the fluorescence intensity (ex / em = 460 / 515 nm).

[0230] Take 0.625 mL, 1.25 mL, 2.5 mL, and 5 mL of the sample and conjugate them with 10 μL of sfGFP-ST for 1 h using the same method. Centrifuge and collect the supernatant. Take 12 μL of each sample for SDS-PAGE at a gel concentration of 12% and stain with Coomassie Brilliant Blue.

[0231] Figure 5 The fluorescence emission spectrum of the ligation product is shown. It can be seen that the emission intensity of SC-LAB at 518 nm gradually increases with increasing co-incubation time, with the largest change observed in the first hour, suggesting that coupling mainly occurs within the first hour. A fixed amount of sfGFP-ST was co-incubated with different amounts of SC-LAB, and the residual sfGFP-ST in the supernatant was detected by SDS-PAGE. Figure 6 As shown, with the increase of SC-LAB dosage, the content of sfGFP-ST (~33kDa) in the supernatant gradually decreased.

[0232] Example 2: Synthesis and Characterization of ST-PDA

[0233] Dopamine is an essential endogenous neurotransmitter in the human body, possessing rich biological activity and participating in the regulation of various physiological activities. Furthermore, dopamine's typical catechol structure endows it with strong adhesive properties, making it suitable as a monomer for synthesizing various adhesives. In this invention, polydopamine is prepared using the ATRP method as an intestinal adhesive material. Figure 7 ).

[0234] First, using dopamine, acryloyl chloride, and tert-butyldimethylchlorosilane as raw materials, a dopamine monomer DOPMS with protected phenolic hydroxyl groups was synthesized in two steps. Each step of the reaction proceeded smoothly, with a final product yield of approximately 47%, and its structural characterization was consistent with expectations. The tert-butyldimethylchlorosilane was introduced as a protecting group to the catechol structure to prevent it from capturing free radicals and hindering ATRP formation.

[0235] Subsequently, DOPMS was polymerized at 60°C via ATRP bulk polymerization using ST-Br as the initiator and CuBr and Me6TREN as catalysts. To improve the yield and water solubility of the polymer, OEGMA (2.0 eq.) was introduced as a comonomer. The resulting polymer was then deprotected by acid to obtain ST-PDA, with a final yield of approximately 70%.

[0236] (1) The specific steps for ST-PDA synthesis are as follows:

[0237] (1.1) Synthesis of dopamine monomer DOPM

[0238] First, dopamine (DOP, 20 mmol) and sodium borohydride (NaBH4, 20 mmol) were dissolved in 200 mL ddH2O in a 500 mL three-necked flask. After purging with nitrogen for 10 min, anhydrous sodium carbonate (Na2CO3) was added under nitrogen protection to adjust the pH to 9-10. Then, the reaction flask was transferred to an ice-water bath, and acryloyl chloride (26 mmol, 1.3 eq.) was slowly added dropwise. Finally, anhydrous NaCO3 was added to bring the pH of the reaction solution to 9-10, and nitrogen purging was continued for another 10 min. The ice-water bath was then removed, and the reaction was allowed to proceed overnight at room temperature. After the reaction was complete, the pH of the reaction solution was adjusted to 1-2 with concentrated HCl. The product was then extracted with ethyl acetate. The organic phase was dried over anhydrous MgSO4, and the solvent was removed by vacuum distillation. The product was then dried to constant weight in a vacuum drying oven, yielding the dopamine monomer DOPM (3.15 g light brown oily liquid, yield: 76%). 1 H NMR spectrum as follows Figure 8 As shown.

[0239] (1.2) Protection of dopamine monomers

[0240] tert-Butyl dimethylchlorosilane (TBDMS-Cl, 45 mmol, 3 eq.) was dissolved in 40 mL of anhydrous acetonitrile, and DOPM (15 mmol) was added with stirring. After complete dissolution, the reaction flask was transferred to an ice-water bath, and then 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 45 mmol, 3 eq.) dissolved in 10 mL of anhydrous acetonitrile was added dropwise. The reaction was carried out at room temperature for 24 h. After the reaction was completed, the solvent was removed from the reaction solution by vacuum distillation. The crude product was purified by column chromatography using silica gel as the stationary phase and methanol / dichloromethane (75:1, v:v) as the eluent. The eluent was collected and the solvent was removed by vacuum distillation. The eluent was dried in a vacuum drying oven to constant weight to obtain the purified product DOPMS (3.07 g yellow solid, yield: 47%). 1 H NMR spectrum as follows Figure 9 As shown.

[0241] (1.3) Synthesis of initiator ST-Br The initiator ST-Br was synthesized using the Fmoc solid-phase polypeptide synthesis method.

[0242] (1.3.1) The resin connects the first amino acid.

[0243] Weigh 3g of Wang resin (degree of substitution SD1 = 1.4mmol / g) and place it in a solid-phase synthesis tube. Add an appropriate amount of N,N-dimethylformamide (DMF) to swell for 30 min, then filter to remove the DMF. Then, dissolve the first amino acid at the C-terminus of the polypeptide chain (Fmoc-Lys(Boc)-OH, 12.6mmol, 3eq.), N-hydroxybenzotriazole (HOBt, 12.6mmol, 3eq.), benzotriazole-N,N,N,N-tetramethylurea hexafluorophosphate (HBTU, 12.6mmol, 3eq.), and N,N-diisopropylethylamine (DIEA, 16.8mmol, 4eq.) in 30ml of anhydrous DMF and add this solution to the resin. After reacting overnight, filter to remove the reaction solution. Wash the resin 2-3 times alternately with dichloromethane (DCM) and anhydrous ethanol (15-20mL each time). The resin was then dried overnight in a vacuum drying oven, and its weight was measured as W2. The new degree of substitution, SD2, was calculated using the following formula:

[0244]

[0245] Among them, M w It is the molecular weight of Fmoc-Lys(Boc)-OH.

[0246] (1.3.2) Blocking unreacted hydroxyl groups on the resin

[0247] Swell the resin again, add acetic anhydride (10 mL), pyridine (10 mL), DMF (10 mL) and 4-dimethylaminopyridine (DMAP, 1 mmol), react at room temperature for 3-4 h, dry the reaction solution and wash the resin 2-3 times.

[0248] (1.3.3) Deprotection of Fmoc group

[0249] Add 20 mL of DMF solution containing 20% ​​piperidine to the resin and react for 30 min. Drain the reaction solution and wash the resin 2-3 times.

[0250] (1.3.4) Connect the second amino acid

[0251] The amounts of the second amino acid and various condensing agents were calculated based on the new degree of substitution. These were dissolved in anhydrous DMF and added to the resin, then reacted at room temperature for 1-2 hours. The reaction progress was monitored using the Kaiser assay: a small amount of resin was placed in a 1.5 mL EP tube, washed with DCM or anhydrous ethanol, and then 10 μL of 5% ninhydrin ethanol solution, 10 μL of 80% phenol ethanol solution, and 10 μL of pyridine were added. The mixture was then heated in a 120 °C metal bath for 3 minutes. If the resin turned blue, it indicated the presence of exposed amino groups, and the reaction was not yet complete. If the resin was colorless, the reaction was complete.

[0252] (1.3.5) Connecting subsequent amino acids

[0253] The process of deprotection and amino acid condensation is repeated, and the resin is washed and Kaiser tests are performed in each cycle.

[0254] (1.3.6) Connecting Initiator

[0255] The resin after Fmoc removal was dried overnight in a vacuum drying oven and weighed (W3). The degree of substitution SD3 was calculated according to the following formula:

[0256]

[0257] Among them, M w It is the sum of the molecular weights of 21 Fmoc-amino acids, M Fmoc It is the sum of the molecular weights of the Fmoc groups on each amino acid, M. H2O It is the total molecular weight of the water molecules removed during the condensation reaction.

[0258] The resin was swollen and dried under vacuum. 50 mL of anhydrous DMF and triethylamine (TEA, 1.2 eq) were added, and 2-bromoisobutyryl bromide (BIBB, 1.0 eq) was slowly added dropwise to the resin suspension under ice bath conditions. The reaction progress was monitored using the Kaiser assay. After the reaction was complete, the resin was washed and dried under vacuum.

[0259] (1.3.7) Chopping, precipitation and purification of crude peptides

[0260] Five volumes of cleavage solution (TFA:ddH2O:methyl phenyl sulfide:phenol:ethylene dithiol = 95:1.25:1.25:1.25:1.25) were added to the dried resin, and the reaction was carried out at 25°C for 3 hours. The mixture was filtered, and the resin was washed with trifluoroacetic acid. The filtrate was precipitated dropwise in ice-cold ether and centrifuged to obtain the crude peptide. The crude peptide was separated and purified by HPLC, and the different components were qualitatively analyzed and their purity was determined by LC-MS. Components with a purity of 90-100% were selected and freeze-dried to obtain the initiator ST-Br.

[0261] The amino acid sequence of Spytag(ST) is SEQ ID NO: 17: GGGGSRGVPHIVMVDAYKRYK.

[0262] (1.4) Synthesis of ST-PDA

[0263] The polymerization of ST-PDA was carried out using ATRP, and the specific experimental steps are as follows: First, CuBr (0.08 mmol) catalyst, ST-Br (0.06 mmol) initiator, OEGMA (2 mmol) monomer, and DOPMS (1 mmol) were added to a 10 mL Schlenk tube, sealed, and purged with nitrogen for 20 min. Then, Me6TREN (0.16 mmol) complexing agent was added using a deoxygenated microsyringe, and nitrogen purging continued for 30 min. Finally, the nitrogen purging was removed, and the Schlenk tube was placed in a 60 °C oil bath for 24 h. After the reaction, the Schlenk tube was opened, and 5 mL of HCl solution (2 M) was added. The reaction was carried out at room temperature for 6 h to remove the dopamine protecting group. Subsequently, the reaction solution was dialyzed against methanol and ddH2O for 1 day each to remove unreacted monomer and initiator, with the dialysate changed every 6 h. Finally, the purified polymerization product ST-PDA (yield: 70%) was obtained by freeze-drying.

[0264] from 1 The 1H NMR results show that the signal of unsaturated olefins located at δ = 5.5-6.5 has disappeared. Figure 10 A new NMR peak appears at δ = 1.7-2.0, belonging to protonated hydrogen atoms on the copolymer backbone. Simultaneously, the NMR signal of the protonated hydrogen atoms on the benzene ring changes from a sharp peak to a broad peak, consistent with the polymer... 1 The characteristics of H NMR were observed. Furthermore, signals from the PEG side chains in OEGMA (b, c, d) could be observed at δ = 3.2–4.2. These results confirm the synthesis of the dopamine-based copolymer. By comparison... 1The proton hydrogen integrals of the initiator -CH3 (j, δ = 1.25), benzene ring (a, δ = 6.5-6.8), and -COOCH2- (b, δ = 4.1) in the HNMR spectrum indicate that the molar ratio of DOPM to OEGMA in ST-PDA is approximately 1:4, and the calculated molecular weight is approximately 17.6 kDa.

[0265] All other monomers were polymerized using the method described above.

[0266] (2) Through 1 The stability of ST-PDA in simulated gastrointestinal fluid environments (SGF, SIG, bile salt solution, and pH 2 / 8 aqueous solution) was evaluated by 1H NMR and GPC, and the specific steps are as follows:

[0267] A certain amount of ST-PDA was dissolved in a small amount of ddH2O, and then diluted to a concentration of 5 mg / mL in aqueous solution at pH 2 / 8, simulated gastric juice (SGF, 20 mg NaCl, 3.2 mg pepsin, 1 mL ddH2O, pH 1.2), simulated intestinal juice (SIF, 6.8 mg KH2PO4, 10 mg trypsin, pH 6.8), and bile salt solution (0.3 mg / mL). The concentrations were measured at specified times. 1 H NMR spectra and GPC curves. For example... Figure 11 and 12 As shown, after 20 hours of co-culture, ST-PDA's... 1 The H NMR and GPC curves showed no significant changes, indicating that the polymer has good structural stability in a simulated gastrointestinal environment.

[0268] (3) The coupling function and microbial safety of ST-PDA were verified by SDS-PAGE and antibacterial assay, respectively.

[0269] (3.1) Verify the coupling function of ST-PDA using SDS-PAGE experiments.

[0270] After co-incubating excess ST-PDA with SC-sfGFP for 1 hour, electrophoresis was performed. Figure 13 As shown, no protein band of SC-sfGFP was observed in the lane of the conjugate PDA-Spy-sfGFP, indicating that the two were successfully conjugated, and also proving that the chemical synthesis process did not affect the inherent function of Spytag.

[0271] (3.2) Antibacterial properties of ST-PDA

[0272] Activate the *Micrococcus luteus* ATCC10240 culture medium preserved on the slant, and prepare a bacterial suspension by adding 100 μL to 6 mL of sterile physiological saline. Separately, take 100 mL of NB solid medium, heat to melt, add 1 mL of Tween 20 and 1 mL of *Micrococcus luteus*, mix well, and pour 15 mL into a petri dish, allowing it to stand. After complete solidification, use a 7 mm diameter punch to evenly punch 5 wells on the plate.

[0273] Weigh ST-PDA and add PBS to prepare a stock solution of 100 mg / mL. Filter the solution through a 0.22 μm filter for sterilization. The stock solution was then diluted sequentially to 10, 20, 40, and 80 mg / mL, and 100 μL of each solution was added to each well. 100 μL of Nisin solution (10 μg / mL) was used as a positive control. The plates were incubated at 37°C for approximately 24 hours until a clear inhibition zone was observed. The results were photographed and recorded. Three parallel groups were also included.

[0274] Antimicrobial experiments showed that ST-PDA has good microbial safety, exhibiting no inhibitory effect on microorganisms at concentrations up to 40 mg / mL. Figure 14 These results establish the feasibility for subsequent coupling of ST-PDA with genetically engineered bacteria and its in vivo evaluation.

[0275] Example 3: Preparation and characterization of Lactococcus lactis-polydopamine conjugate LAB-Spy-PDA

[0276] (1) Preparation of LAB-Spy-PDA

[0277] LAB-Spy-PDA was prepared using a co-incubation method. First, the OD600 of the overnight induced NZ9000 / pNZ8148-AcmASC culture was measured, and 2 mL was centrifuged. The precipitate was collected and washed three times with PBS (10 mM, pH 7.4). The precipitate was then resuspended in PBS and divided into two groups. The first group was treated with PBS, and the second group was treated with ST-PDA in PBS solution (10 mg / mL, pre-filtered through a 0.45 μm membrane) (the final ST-PDA concentration in the mixture was 5 mg / mL). Finally, the mixture was incubated at 30°C and 150 rpm for 2 hours. After co-incubation, the bacterial cells were collected by centrifugation, washed three times, and resuspended in 1 mL of PBS to obtain the desired product. LAB and LAB-Spy-PDA.

[0278] The same method was used to prepare LAB-Spy-FLPDA.

[0279] (2) Fluorescence characterization of LAB-Spy-FLPDA

[0280] To more intuitively determine the coupling between the two, SC-LAB was first labeled with the red fluorescent probe TAMRA-NHS, and then FITC-O-methacrylate monomer was introduced into the ATRP polymerization system to obtain the fluorescent copolymer ST-FLPDA with Spytag terminus. Figure 15 The two were then co-incubated, and the coupling product was washed with 0.1% Triton-X100 and centrifuged before being used for fluorescence emission spectroscopy and LSCM characterization. The specific steps are as follows:

[0281] Red fluorescent labeling of cells was achieved through a nucleophilic reaction between an active ester and an amine compound. Specifically, 5-carboxytetramethylrhodamine succinimide ester (TAMRA-NHS, 1 mg / mL) was added to a PBS suspension of LAB-Spy-FLPDA, and the cells were incubated at 30°C and 150 rpm for 30 min. After centrifugation, the cells were collected, fixed with 4% paraformaldehyde at room temperature for 20 min, and finally washed three times with PBS. The cells were then spread onto glass slides for laser scanning confocal microscopy (LSCM). The remaining suspension was diluted to an appropriate concentration, and its emission range and intensity were measured at different excitation wavelengths (ex = 450 / 530, em = 515 / 590).

[0282] The results are as follows Figure 16 As shown, under excitation wavelengths of 450 nm and 530 nm, TAMRA-LAB-Spy-FLPDA exhibits two different emission wavelengths of 520 nm and 590 nm, originating from FITC in the polymer and the engineered bacterial probe TAMRA, respectively. High colocalization between the red SC-LAB cells and the green ST-PDFL cells can also be observed in the fluorescence confocal imaging results. Figure 17 These results demonstrate that polymers obtained via ATRP using ST-Br as an initiator can be successfully conjugated with the genetically engineered probiotic SC-LAB.

[0283] Subsequently, LAB-Spy-PDA was prepared using ST-PDA and a series of characterizations were performed. Figure 18 The changes in particle size and surface potential of SC-LAB before and after ST-PDA linkage were revealed. It can be observed that, compared with the original lactic acid bacteria... Compared to SC-LAB, with prolonged co-incubation time, the hydration kinetic diameter of LAB-Spy-PDA gradually increased (from 1991.3±121.3 nm to 2143.0±44.5 nm), while the surface negative charge gradually decreased (from -37.36±0.85 mV to -22.9±1.77 mV), tending towards the surface potential of ST-PDA (-6.54±0.97 mV). Figure 19Clearly, this change is caused by the coupling of the ST-PDA.

[0284] TEM characterization of the morphology of the polymer ST-PDA SC-LAB and its coupling compound LAB-Spy-PDA are described intuitively. For example... Figure 20 As shown in a) and b), ST-PDA exhibits two morphologies: spherical particles with a diameter of approximately 300-1000 nm and dendritic clusters of varying sizes. This is presumably the result of the combined effect of polymer self-assembly and the adhesive properties of polydopamine. SC-LAB has a regular shape and a flat surface. In contrast, LAB-Spy-PDA shows a significant increase in size (1.2 μm to 1.45 μm), and its surface and surrounding area are covered with nanoparticles with a diameter of about 300-500 nm and dendritic polymer agglomerates, which is consistent with the morphology of ST-PDA described above.

[0285] Finally, the surface hydrophilicity and hydrophobicity of LAB-Spy-PDA were evaluated using contact angle testing. Figure 21 The display shows that for The water contact angle of SC-LAB was 2.485°, while that of LAB-Spy-PDA was 48.915°, indicating that the hydrophobicity of SC-LAB was improved after coupling with the polymer. This is likely due to the abundant alkane chains on the polymer backbone. This result further confirms the successful coupling of ST-PDA onto SC-LAB. It is worth noting that hydrophobic surfaces are more conducive to cell adhesion because, in an aqueous body fluid environment, hydrophobic surfaces are more likely to interact with hydrophobic groups on the substrate and thus be "adsorbed".

[0286] Example 4: In vitro bioactivity of LAB-Spy-PDA

[0287] As a live bacteria preparation, LAB-Spy-PDA's excellent bioactivity is an important prerequisite for it to exert its probiotic effects in vivo.

[0288] (1) Growth curves and glucose consumption rates of SC-LAB and LAB-Spy-PDA

[0289] First, SC-LAB and ST-PDA were co-incubated for 1 h, 2 h, and 4 h, respectively. After centrifugation and purification, the bacterial cells were diluted to an appropriate fold, and 100 μL was plated onto an agar plate. After incubation at 30°C for 24 h, cell counts were performed to assess cell viability. Figure 22 As shown in Figure a, SC-LAB maintained its coupling with ST-PDA for 2 hours. SC-LAB exhibited comparable growth activity. Further monitoring of the growth curves of both was conducted over 24 hours as a control. Simultaneously, SC-LAB coated with a polydopamine PDA layer (LAB+DA-1h) was prepared using a self-polymerization method. Figure 22 As shown in Figure b, it is clear that, with Compared to SC-LAB, the growth curve of LAB-Spy-PDA did not change significantly, but under the same conditions, SC-LAB with PDA coating showed obvious growth lag, with a lag period of up to 4 hours.

[0290] For measurement The growth curves and glucose consumption rates of SC-LAB and LAB-Spy-PDA were determined by inoculating both at an initial DO600 concentration of 0.1 into 10 mL of GM17 medium containing chloramphenicol and incubating at 30°C. Small amounts of the bacterial suspension were taken at fixed time intervals, their OD600 was measured, and then the cells were removed by centrifugation. The glucose concentration of the supernatant was then measured. The remaining... LAB and LAB-Spy-PDA were diluted 1×10⁻⁶ with PBS. 6 After double-distillation, 100 μL of the solution was spread onto a GM17 solid plate containing chloramphenicol, incubated overnight at 30°C, and colony counts were performed. Each group was repeated 3 times.

[0291] like Figure 23 As shown, for Both SC-LAB and LAB-Spy-PDA consumed all their glucose within 8 hours, and the downward trend was consistent. However, the PDA-coated SC-LAB consumed only about 20% of its glucose within 8 hours.

[0292] These results indicate that the coupling of SC-LAB and ST-PDA did not affect cell growth. This is because Spy-mediated coupling achieves point-to-point chemical bonding, which facilitates cell surface exposure. Therefore, during cell growth, it does not hinder the cell's access to and uptake of nutrients, nor does it affect intercellular communication. Conversely, dopamine tends to form a relatively dense coating layer during self-polymerization, and the mechanism of this reaction is not yet fully understood. In-situ polymerization may cause unknown damage to cells, thereby affecting cell growth.

[0293] Considering the results of cell viability and growth curves, LAB-Spy-PDA obtained after 1 hour of coupling was used for subsequent research.

[0294] (2) Detection Tolerance of LAB and LAB-Spy-PDA to simulated solutions

[0295] The unique environment of the gastrointestinal tract is one of the main factors contributing to its poor colonization in vivo. This is because H+ ions in gastric acid alter the zeta potential of cell surfaces, thereby affecting permeability and ultimately leading to cell death. Furthermore, high concentrations of trypsin and bile salts in the intestine can digest various proteins and lipids on the cell surface, resulting in decreased cell viability and cell death. Therefore, the tolerance of LAB-Spy-PDA to SGF, SIF, and bile salt solutions was further investigated. The specific steps are as follows:

[0296] First, centrifuge the PBS suspensions of both bacteria, collect the precipitates, and resuspend them in 1 mL of SGF, SIF, and bile salt solutions, respectively, and incubate at 37°C for 0.5 h, 1 h, and 1.5 h. Then wash the bacterial cells and dilute with PBS to 1×10⁻⁶. 6 After doubling, take 100 μL and spread it on a GM17 solid plate containing chloramphenicol. Incubate overnight at 30°C and then count the colonies. Repeat each group 3 times.

[0297] Cell plate count results showed ( Figure 24 After co-culturing with SGF for 0.5 h and 1 h, the survival rate of LAB-Spy-PDA was [missing information]. The survival rate of LAB-Spy-PDA was approximately four times that of SC-LAB, indicating that polymer coupling effectively protected cells from damage by gastric acid and pepsin. Similarly, higher survival rates were observed in SIF and bile salt solutions compared to SC-LAB. Based on SC-LAB, it can be concluded that LAB-Spy-PDA has significantly enhanced tolerance to the gastrointestinal environment.

[0298] TEM images provide even more direct evidence for this conclusion, such as... Figure 25 As shown, after exposure to SGF and SIF, LAB-Spy-PDA largely maintained its intact cell morphology, while SC-LAB showed significant deformation and breakage, creating a stark contrast. Further analysis revealed a significant reduction in polymers in LAB-Spy-PDA at this stage. This was attributed to the disruption of peptide bonds in the polypeptide and protein structures by pepsin and trypsin, allowing cells to be released from the encapsulation layer. This release process may have provided a "survival window" for the cells, increasing their survival rate in the gastrointestinal environment, and also facilitating the beneficial activity of lactic acid bacteria.

[0299] (3) In vitro adhesion of LAB-Spy-PDA

[0300] for In vitro adhesion experiments of LAB and LAB-Spy-PDA to small intestinal tissue require first obtaining fresh small intestinal tissue from ICR mice and washing away the intestinal contents with PBS. Then, [the following steps are performed]... LAB and LAB-Spy-PDA were instilled into the small intestine, sealed tightly, and incubated together with PBS for 1 hour. Finally, the small intestine was rinsed with PBS, and the unadhered contents were collected. LAB and LAB-Spy-PDA, diluted to a certain factor, were spread in 100 μL onto GM17 solid plates containing chloramphenicol. After incubation at 30°C overnight, colony counting was performed. Each group was repeated three times. Results are as follows: Figure 26 The remaining in the incubation solution SC-LAB is approximately twice the size of LAB-Spy-PDA, providing preliminary evidence that polymer coupling enhances the interaction between cells and the intestinal wall, making them more likely to adhere.

[0301] Example 5: Colonization of LAB-Spy-PDA in the gastrointestinal tract of mice

[0302] Six- to eight-week-old ICR mice were randomly divided into two groups and fasted for 12 hours. Subsequently, each group was administered 200 μl of Rh-NHS-labeled [a specific ingredient / product] via gavage. LAB and LAB-Spy-PDA (2×10) 8 CFU / mL). Animals were sacrificed at specified times (4h, 8h, 14h, 24h), and then dissected. Gastrointestinal tissues were collected and placed in a small animal in vivo optical three-dimensional imaging system for fluorescence imaging. The fluorescence intensity of different sites was analyzed using Living Image 4.4 software, and each group was repeated at least 5 times.

[0303] In order to treat different parts of the gastrointestinal tract Quantitative analysis was performed using LAB and LAB-Spy-PDA to separate the stomach, small intestine, cecum, and colon. The contents were collected by washing with 5 mL PBS, homogenized, and diluted 1×10⁻⁶. 3 Take 100 μL of the solution and spread it on a GM17 solid plate containing chloramphenicol. Incubate overnight at 30°C and then count the colonies. Repeat each group three times.

[0304] Add 5-carboxyfluorescein succinimide ester (FITC-NHS, 1 mg / mL) to the remaining diluent, incubate at 30°C and 150 r / min for 30 min, centrifuge to collect the precipitate, add 4% paraformaldehyde to fix at room temperature for 20 min, finally wash three times with PBS, spread on a glass slide for LSCM.

[0305] according to Figure 27 and Figure 28 It can be observed that 4 hours after gavage, SC-LAB rapidly reached the ileum, cecum, and colon. In contrast, LAB-Spy-PDA was largely concentrated in the stomach and jejunum. After 24 hours, only small amounts were observed in the stomach and cecum. While SC-LAB was observed, LAB-Spy-PDA still had a significant presence, with colonization primarily occurring in the small and large intestines, where probiotics ultimately exert their effects. Further analysis of fluorescence intensity throughout the gastrointestinal tract over 24 hours after gavage revealed that LAB-Spy-PDA colonization remained consistently higher than SC-LAB after 4 hours. SC-LAB reached three times the level of the latter after 24 hours. Figure 29 ).

[0306] Intestinal contents were collected 24 hours after gavage and imaged using LSCM. Figure 30 As shown, LAB-Spy-PDA was significantly more abundant than SC-LAB in the small intestine, cecum, and colon. This result provides a more intuitive understanding of the relative abundance of LAB-Spy-PDA compared to SC-LAB at the cellular level. SC-LAB enhances adhesion.

[0307] However, for lactic acid bacteria to exert their beneficial activity in vivo, they not only need sufficient adhesion to the intestinal wall but also a certain survival rate to gain a competitive advantage in the complex gastrointestinal environment. Therefore, plate counts were performed on contents collected at different time points to examine the survival rate of LAB-Spy-PDA in the intestine. Figure 31 and 32 As shown, within 24 hours after gavage, The relative viable counts of SC-LAB and LAB-Spy-PDA at various sites showed a pattern almost identical to that of fluorescence intensity, indicating that lactic acid bacteria using Spy chemically linked polymers can achieve enhanced adhesion and colonization in the gastrointestinal tract.

[0308] Example 6: Therapeutic effect of LAB-Spy-PDA on DSS-induced mouse colitis model

[0309] ICR mice aged 6-8 weeks were acclimatized for one week before the experiment was conducted.

[0310] The process of establishing and treating colitis in mice is as follows: Figure 33 As shown, the specific steps include:

[0311] Mice were randomly divided into four groups of ten each. Group 1 was given distilled water, while groups 2, 3, and 4 were given a 3.5% sodium dextran sulfate solution (Mn = 40 kDa). All mice were weighed on days 1, 3, 5, and 7. After seven days, mice in groups 2, 3, and 4 all exhibited bloody stools and weight loss, confirming the successful establishment of the colitis model.

[0312] Starting from day 8, mice in groups 2, 3, and 4 were treated by gavage with 200 μL of PBS daily. LAB and LAB-Spy-PDA (2×10)8 Mice were administered the drug (CFU / mL) for five consecutive days, with daily monitoring of body weight. On day 13, fresh feces were collected, immediately flash-frozen in liquid nitrogen, and then stored at -80°C for 16S rRNA sequencing. All mice were then euthanized, and cardiac blood was immediately collected. After being kept at room temperature for 2 hours, the blood was centrifuged at 3000 rpm for 15 minutes at 4°C. The supernatant was collected and stored at -80°C for the detection of TNF-α and interleukin-6 (IL-6). A healthy control group (Control) was set up as a negative control, and the PBS group and... The SC-LAB group served as a positive control.

[0313] Mouse colon tissue was collected and its length measured. It was then preserved in 4% paraformaldehyde fixative and sent to Wuhan Sewell Biotechnology Co., Ltd. for hematoxylin-eosin staining (H&E), periodic acid-Schiff staining (PAS), and immunofluorescence staining.

[0314] GraphPad Prism 8 was used for significant difference analysis; CaseViewer was used to process the stained images; Image-Pro Plus 6.0 was used for statistical analysis of goblet cells; and ImageJ was used for statistical analysis of fluorescence intensity in immunofluorescence staining. At least three 200× fields of view were randomly selected from each group of slides. Weight monitoring results showed ( Figure 34 Compared to the healthy group, the DSS-induced mice showed a significant decrease in body weight. After two days of treatment, the PBS group continued to experience a decrease in body weight. The SC-LAB and LAB-Spy-PDA groups recovered rapidly, reaching 93-94% of the level of the healthy group by day 13. Analysis of colon tissue collected from mice revealed that the LAB-Spy-PDA group had a colon length similar to that of healthy mice and was significantly longer than the two positive groups. Statistical analysis also showed no significant difference between the LAB-Spy-PDA group and the healthy group, but a significant difference compared to the positive groups (p < 0.0001).

[0315] Further evaluation was conducted on the expression levels of pro-inflammatory factors IL-6 and TNF-α in mouse blood. Figure 35 Obviously, Both the SC-LAB and LAB-Spy-PDA groups showed reductions, indicating that both improved the inflammatory response, but the LAB-Spy-PDA group was closer to the level of the healthy group.

[0316] In addition, H&E and PAS staining were performed on the colon tissue to assess pathological changes. Figure 36As shown, the colon tissue in the PBS group was severely damaged, with a significant reduction in the number of mucosal crypts and the presence of a large amount of fibrous tissue and inflammatory cell proliferation (green arrows), some epithelial cells eroded and sloughed off (black arrows), submucosal edema, and increased interstitial spaces (yellow arrows). The SC-LAB group showed significant improvement compared to the PBS group, with more orderly arrangement of mucosal crypts, an increased number of goblet cells (red arrows), and a reduced number of inflammatory cells, although some mucosal epithelial cell erosion and shedding were still observed. The LAB-Spy-PDA treatment group showed even more significant improvement, with tightly packed mucosal epithelial cells, no obvious degeneration or shedding, a greater number of crypts, abundant goblet cells, and a significantly reduced number of inflammatory cells.

[0317] Based on the PAS staining results, the number of goblet cells was further counted to evaluate the degree of recovery of the colonic mucosa. As expected, the number of goblet cells in the LAB-Spy-PDA group was comparable to that in the healthy group, and five times that of the PBS group. The SC-LAB group showed a 2-fold increase, indicating that LAB-Spy-PDA can significantly repair the mucus layer, thereby preventing probiotics from crossing the intestinal barrier into the bloodstream and causing side effects such as cell translocation. Figure 37 ).

[0318] Tight junctions are an important component of the intestinal epithelial barrier, with Claudin-1 and ZO-1 being key members. Their interaction links transmembrane proteins and the cytoskeleton, facilitating the maintenance of tight junction integrity and playing a crucial role in mucosal healing. Immunofluorescence staining images and fluorescence intensity statistics were used to quantify Claudin-1 and ZO-1 in colonic tissue. Figure 38 and 39 As shown, Both SC-LAB and LAB-Spy-PDA can increase the expression levels of Claudin-1 and ZO-1. Comparatively, LAB-Spy-PDA showed a more significant therapeutic effect, especially on Claudin-1, whose expression level clearly tended to normalize.

[0319] Finally, the in vivo systemic toxicity of LAB-Spy-PDA was evaluated. Heart, liver, spleen, lung, and kidney tissues were collected from each group of mice and subjected to H&E staining. Figure 40 Using the healthy group as a reference, no obvious organ lesions, inflammation or abnormalities were observed in any organ of the LAB-Spy-PDA group, indicating that it has good biocompatibility.

[0320] In conclusion, displaying the Spycathcer protein domain on the surface of Lactococcus lactis NZ9000 through genetic engineering did not affect the inherent beneficial viability of the cells. Furthermore, conjugation with polydopamine could further enhance the colonization of Lactococcus lactis NZ9000 in the intestine, thereby improving its therapeutic effects.

[0321] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0322] The foregoing embodiments and methods described in this invention may vary based on the capabilities, experience, and preferences of those skilled in the art.

[0323] Listing the steps of the method in a certain order in this invention does not constitute any restriction on the order of the method steps.

Claims

1. An engineered probiotic, characterized in that, The probiotics exhibit SpyCatcher protein on their surface; Preferably, the engineered probiotics are obtained by transforming probiotics with a recombinant expression vector containing nucleotides encoding AcmA, SpyCatcher, and a signal peptide.

2. The probiotic according to claim 2, characterized in that, The signal peptide is selected from Usp45, ATPγ, or Rf1b, preferably Usp45.

3. The probiotic according to claim 1, characterized in that, The probiotics are selected from one or more of lactic acid bacteria, bifidobacteria, or Escherichia coli; preferably, the lactic acid bacteria are selected from Streptococcus, Enterococcus, Lactococcus, Pediococcus, or Lactobacillus; more preferably, the Lactococcus is selected from Lactococcus lactis, Lacococcus garvieae, or Lactococcus cremoris.

4. A recombinant expression vector comprising the AcmA gene, the Spycathcer gene, and nucleotides encoding a signal peptide; Preferably, the recombinant expression vector contains nucleotides encoding the signal peptide-SpyCatcher-AcmA; Preferably, the signal peptide is selected from Usp45, ATPγ, or Rf1b, with Usp45 being the most preferred. Preferably, the expression vector is selected from pNZ8148, pET28a, pNZ8048, and pNZ8150.

5. A polymer containing SpyTag residues, comprising the following structure: in, ST stands for SpyTag residues; and Where m is an integer from 1 to 50; Preferably, the polymer further comprises one or more of the following structural portions: Where n is an integer from 1 to 50; p is an integer from 1 to 20; R1 is selected from: H, C1-C10 alkyl groups; Preferably, the polymer has a molecular weight of 1-25 kDa.

6. An engineered probiotic-polymer conjugate, wherein the probiotic surface displays SpyCatcher protein, the polymer comprises SpyTag residues and a dopamine structural moiety, and the SpyTag is coupled to SpyCatcher via isopeptide bonds.

7. The coupling as described in claim 6, characterized in that, The polymer is as described in claim 4.

8. The coupling as described in claim 6, characterized in that, The modified probiotics are as described in any one of claims 1-3.

9. The use of a probiotic according to any one of claims 1-3, a recombinant expression vector according to claim 4, or a polymer according to claim 5 in the preparation of a reagent to enhance the colonization ability of probiotics in the gut.

10. The use of the conjugate according to any one of claims 6-8 in the preparation of a medicament for treating and / or preventing gastrointestinal diseases; Preferably, the gastrointestinal disease is selected from one or more of diarrhea, gastritis, enteritis, colitis, Crohn's disease, appendicitis, peptic ulcer, gastric cancer, or intestinal cancer.

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