A probiotic composition and its use in improving immunity and treating diseases
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- GUANGZHOU QIFENG BIOMEDICAL TECHNOLOGY CO LTD
- Filing Date
- 2025-10-23
- Publication Date
- 2026-07-24
Smart Images
Figure CN121609806B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedicine, specifically to a probiotic composition and its application in enhancing immunity and treating diseases. Background Technology
[0002] In recent years, probiotic-based immunomodulatory therapy has shown unique advantages in the intervention of intestinal diseases and systemic immune-related diseases, but its clinical application is still limited by three core technological bottlenecks. First, the ability to target and colonize is lacking. During the transport of conventional probiotics through the gastrointestinal tract, due to chemical damage from gastric acid and bile and physical clearance by intestinal peristalsis, the survival rate is less than 1%. Furthermore, it lacks a specific recognition mechanism for receptors highly expressed at inflammatory sites, resulting in the concentration of live bacteria in the lesion area being only 1 / 20 of that in non-target sites, and the effective dose is seriously insufficient.
[0003] Secondly, the limited range of immune regulatory mechanisms restricts the depth of therapeutic efficacy. Currently available probiotics mostly rely on a single metabolite (such as short-chain fatty acids) or surface antigen to trigger an immune response, which can only achieve limited cytokine balance regulation and cannot cope with the multi-cellular subset imbalance in the complex immune microenvironment (such as Th1 / Th2 / Th17 polarization disorder). As a result, the clinical remission rate in diseases such as inflammatory bowel disease has consistently been below 40%.
[0004] Furthermore, the delivery efficiency of small molecule active substances is low. Key immunomodulatory molecules such as sodium butyrate have a half-life of only 5-10 minutes, and their bioavailability is less than 2.3% after oral administration due to rapid intestinal metabolism. Existing carrier systems (such as liposomes and polymer nanoparticles) have poor mucus penetration ability (only 10%-15%) and lack of targeting, making it difficult to achieve sustained drug release at the lesion site.
[0005] Existing improvement schemes all have significant limitations: although genetically engineered probiotics can express antibody fragments, the secretion level is generally lower than 50 ng / mL, and they lack the effector function mediated by the Fc fragment, with bioactivity only 10%-15% of that of intact antibodies; multi-strain combination strategies are affected by individual differences in gut microbiota baseline, with efficacy fluctuations exceeding 45%; and novel vector systems face industrialization obstacles such as high preparation costs and insufficient biocompatibility.
[0006] To address the aforementioned issues, there is an urgent need to construct an innovative technology system that combines targeted colonization, multidimensional immune regulation, and efficient drug delivery. This system should overcome existing technological barriers through multi-mechanism synergy, providing a novel solution for the clinical translation of probiotic therapies. Summary of the Invention
[0007] In the field of intestinal inflammation treatment, existing technologies face numerous challenges. Regarding fusion peptides, issues include structural conflicts leading to impaired biological activity, insufficient targeting efficiency in intestinal inflammatory areas, and poor resistance to enzymatic degradation. For example, when YIGSR is directly fused with LL37, the α-helixity of LL37 significantly decreases, reducing antibacterial activity; single YIGSR targeting systems have low colonization efficiency in inflammatory areas; and natural LL37 has a short half-life in the intestinal environment and is easily degraded. In antibody applications, there is a lack of highly efficient and specific monoclonal antibodies against *E. coli* O157:H7, making it difficult to effectively block the adhesion and invasion of this bacterium, thus affecting the treatment efficacy for related intestinal infections. While curcumin possesses good anti-inflammatory, antioxidant, and immunomodulatory effects, its poor water solubility and low bioavailability greatly limit its application in intestinal inflammation treatment. Regarding probiotic preparations, existing formulations suffer from weak colonization ability, poor synergistic effects among functional components, susceptibility to degradation in the acidic environment of the stomach, and unsatisfactory therapeutic effects on intestinal inflammation, failing to fully realize the therapeutic potential of probiotics.
[0008] Based on this, the present invention provides a dual-targeting adhesion-immune activation fusion peptide: a specific fusion peptide sequence is designed, comprising a targeting domain, an anti-enzymatic and structural stabilization domain, and an LL37 variant functional domain. The targeting domain incorporates YIGSR and PTPSFTT to achieve dual targeting, improving targeting efficiency to different regions of the intestine; the anti-enzymatic and structural stabilization domain is inserted into the GPGP buffer domain and the EAAAK tetramer stabilization domain, while LL37 is mutated with R23Q, K25P, and V32P to resolve structural conflicts and enhance anti-enzymatic properties. This fusion peptide is prepared through gene synthesis and cloning, induced expression, and purification. Gene synthesis is optimized for codon bias specific to *Lactobacillus plantarum* LP-28, and induced expression is performed at a low temperature of 25°C to improve expression levels and purity.
[0009] This invention also provides a monoclonal antibody against *Escherichia coli* O157:H7, GS2-D3: Six- to eight-week-old Balb / c female mice were immunized with formaldehyde-inactivated *Escherichia coli* O157:H7 whole-cell antigen via intraperitoneal injection four times (including one booster immunization). The initial immunization was emulsified with Freund's complete adjuvant, followed by Freund's incomplete adjuvant. High-titer mice were selected, and spleen cells were prepared and fused with SP2 / 0 myeloma cells. Stable antibody-secreting cell lines were obtained through screening and subcloning, followed by humanization optimization to obtain monoclonal antibodies with specific sequences in the heavy and light chain variable regions.
[0010] This invention also provides curcumin nanoparticles: prepared by an ultrasonic-solvent evaporation method using zein as a carrier and tea saponin as an emulsifier. Zein, curcumin, and tea saponin are dissolved in an 80% ethanol aqueous solution, followed by ultrasonic treatment, rotary evaporation concentration, and freeze-drying to obtain the nanoparticles, thereby improving the stability and bioavailability of curcumin.
[0011] This invention also provides a probiotic composition comprising: *Lactobacillus plantarum* LP-28 engineered bacteria capable of expressing the aforementioned fusion peptide, a monoclonal antibody, and curcumin nanoparticles. Enteric-coated capsule formulation is prepared through steps including the cultivation and pretreatment of the engineered bacteria, the combination and processing of the composite components, freeze-drying protection and molding, and the preparation of enteric-coated capsules. The enteric-coated capsules use a specific coating solution formulation and are coated using a fluidized bed coating machine to ensure minimal release in the gastric acid environment and rapid release in the intestinal fluid environment.
[0012] This invention achieves synergistic effects through multi-dimensional technological innovation: the dual-target adhesion-immune activation fusion peptide, through structural optimization, resolves the conformational conflict between YIGSR and LL37, restoring α-helixity to natural levels while fully preserving antibacterial activity. The dual-target system increases the binding efficiency to healthy intestinal epithelium to over 90% and improves the capture rate in inflammatory areas by 2.8%. The drug exhibits a 16.4-fold increase in anti-enzymatic half-life through sequence mutation and structural design. The anti-Escherichia coli O157:H7 monoclonal antibody GS2-D3 blocks pathogenic bacterial adhesion with high specificity (no cross-reactivity) and high titer (1:128000). Curcumin nanoparticles achieve efficient curcumin delivery thanks to their excellent dispersion stability. The probiotic composition, through the synergistic effects of engineered bacterial colonization, antibody antibacterial activity, and curcumin anti-inflammatory effects, increases the Escherichia coli O157:H7 inhibition rate by 2-3 times, achieves a colitis relief rate of 86.7%, and regulates intestinal flora balance (restoring the Bacteroidetes / Firmwallis ratio). The enteric-coated capsule formulation is stably retained in gastric acid (2h release rate ≤5%), rapidly released into the intestine (4h release rate ≥90%), and has a freeze-dried survival rate of 85.2%. Furthermore, its cost is only 1 / 50th that of antibody drugs, and its safety has been verified by hemolysis experiments (hemolysis rate 3.1%). This combination achieves the integrated functions of targeted therapy, immune regulation, and barrier repair.
[0013] This technology system has broad application prospects in the treatment of intestinal diseases: it can be directly used in the clinical treatment of inflammatory bowel diseases such as ulcerative colitis and Crohn's disease, achieving non-invasive intervention through oral enteric-coated preparations; its dual-targeting and anti-enzymatic design provides a general technical template for local intestinal drug delivery systems, which can be extended to targeted drug delivery for diseases such as intestinal tumors and infectious diarrhea; the low-cost, large-scale preparation advantage of probiotic compositions (Lactobacillus plantarum expression system) is suitable for primary healthcare and chronic disease management, and is expected to replace some high-priced biological agents, promoting the technological upgrading of the biopharmaceutical and health industries. Attached Figure Description
[0014] Figure 1 Schematic diagram of the "dual-target synergy" system.
[0015] Figure 2 Schematic diagram of the mechanism of action of dual-target adhesion-immune activation fusion peptide.
[0016] Figure 3 SDS-PAGE analysis results of dual-target adhesion-immune activation fusion peptide, where 1 is the dual-target adhesion-immune activation fusion peptide.
[0017] Figure 4 The results of SDS-PAGE analysis of monoclonal antibody GS2-D3, where 1 represents monoclonal antibody GS2-D3. Detailed Implementation
[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.
[0019] Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.
[0020] Example 1: Design, preparation and validation of dual-targeted adhesion-immune activation fusion peptide
[0021] I. Core Challenges and Solutions in the Design Process
[0022] The design of fusion peptides must simultaneously meet four core requirements: targeting, structural stability, resistance to enzymatic degradation, and bioactivity. Addressing key technological bottlenecks encountered during the research and development process, this embodiment achieves breakthroughs through multi-dimensional technological innovation, as detailed below:
[0023] 1. Structural conflict between YIGSR (adhesion peptide) and LL37 variant (immunomodulatory peptide)
[0024] (1) Technical problem: The function of the targeting domain YIGSR depends on its rigid β-turn conformation, while the immunoactivating activity of the functional domain LL37 requires the maintenance of an amphiphilic α-helix structure; when the two are directly fused, the spatial conformational conflict between the β-turn and the α-helix leads to a significant decrease in the α-helix degree of LL37. Circular dichroism (CD) analysis showed that the intensity of the α-helix characteristic peak of the direct fusion product (YIGSR-LL37) at 222 nm was only 28% of that of the natural LL37, and its minimum inhibitory concentration (MIC) against Escherichia coli increased to 32 μg / mL, which is 75% lower than that of the natural LL37 (MIC about 8 μg / mL). The structural conflict led to a serious impairment of biological activity.
[0025] (2) Solution: Use a dual strategy of “structural isolation-spiral stability” to resolve conformational conflicts.
[0026] Insertion of α-helical stability domain (EAAAK) 4: This sequence consists of 4 repeating EAAAK units, which can form rigid spacers through the charge interaction between glutamic acid (E) and lysine (K), providing LL37 with an independent helical formation space;
[0027] Introducing the flexible buffer domain GPGP: The GPGP sequence is inserted between YIGSR and the stable domain, where the rigid loop structure of proline (P) can block the conformational transfer of β-turn to the LL37 region, disrupt the continuity of the β chain, and avoid interference with the α helix.
[0028] (3) Verification results: The CD spectrum of the optimized fusion peptide showed that its α-helicity at 222 nm was restored to 94%, close to the level of natural LL37; the MIC against E. coli was reduced to 8 μg / mL, and the antibacterial activity was completely restored, confirming that the structural compatibility problem was solved.
[0029] 2. The Challenge and Solutions to Insufficient Targeting Efficiency in Intestinal Inflammation Zones
[0030] (1) Technical problem: In the state of intestinal inflammation, the expression level of laminin receptor on the surface of healthy intestinal epithelial cells is downregulated by about 50% (confirmed by Western Blot detection of mouse colitis model), which leads to a significant reduction in the colonization efficiency of the YIGSR-dependent targeting system in the inflammatory area, and it is impossible to achieve effective enrichment in the lesion area.
[0031] (2) Solution: Construct a "dual-target synergy" system ( Figure 1 As shown in the figure, intestinal colonization is achieved through multi-domain collaboration.
[0032] Introducing the MUC2 mucin-binding domain PTPSFTT: This sequence is derived from a mucus-layer-specific binding peptide and can specifically bind to the gel network of MUC2 mucin, which is highly expressed in the inflammatory area (secretion increases by about 2-3 times under inflammatory conditions);
[0033] The design of the gradient targeting logic is as follows: the healthy intestinal epithelial region achieves basic colonization by binding YIGSR to the laminin receptor, while the inflammatory and damaged region completes specific anchoring by using PTPSFTT with high affinity to MUC2, forming a full-domain coverage of "healthy area - inflammatory area".
[0034] (3) Validation results: The dual-targeting system can maintain the binding efficiency of the fusion peptide in healthy intestinal epithelium at over 90% (flow cytometry detection), while the capture rate in the inflammatory mucus layer is 2.8 times higher than that of the single YIGSR system (fluorescent labeling tracking), achieving efficient colonization in different pathological regions of the intestine.
[0035] 3. The challenge and solution of LL37's sensitivity to protease degradation.
[0036] (1) Technical issues: Natural LL37 (amino acid sequence LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES) is easily degraded by trypsin, elastase, etc. in the intestinal environment, with a half-life of only 0.5 h (confirmed by in vitro simulated intestinal fluid experiments). Sequence analysis shows that arginine at position 23 (R23) and lysine at position 25 (K25) are the main cleavage sites of trypsin, and the flexible conformation of valine at position 32 (V32) makes the C-terminal helix easily destroyed by enzymatic digestion.
[0037] (2) Solution: Improve resistance to enzymatic degradation through a dual strategy of "site mutation-domain optimization".
[0038] Key amino acid mutations:
[0039] R23Q: Replaces positively charged arginine (R) with glutamine (Q), eliminating the recognition site of basic amino acids by trypsin;
[0040] K25P: Lysine (K) is replaced with proline (P). The rigid ring structure of proline is used to disrupt the spatial conformation required for enzyme cleavage, thereby reducing cleavage efficiency.
[0041] V32P: By introducing a stable α-helix inflection point through proline, the structural stability of the C-terminal helix is enhanced, and the exposure of enzyme-sensitive regions is reduced.
[0042] Buffer domain design: A GPGP sequence is inserted between LL37 and the target domain to block the diffusion of protease within the peptide chain by utilizing the steric hindrance effect of proline, thereby slowing down the degradation process.
[0043] (3) Validation results: In in vitro simulated intestinal fluid (containing 1 mg / mL trypsin), the half-life of the optimized LL37 variant was extended to 8.2 h, which was 16.4 times longer than that of natural LL37 (detected by high performance liquid chromatography), confirming that the anti-enzymatic performance was significantly enhanced.
[0044] II. Final sequence of dual-target adhesion-immune activation fusion peptide (as shown in SEQ ID NO:1)
[0045] Based on the above technical solution, the fusion peptide sequence designed in this embodiment consists of a functional domain, a linker domain, and a mutation region. The structure and function of each part are as follows:
[0046] 1. Target domain
[0047] (1) The N-terminal 1-5 positions are YIGSR sequences, which achieve basic colonization by recognizing laminin receptors on the surface of healthy intestinal epithelial cells;
[0048] (2) Positions 6-10 are GGGGS flexible linkers, composed of glycine (G) and serine (S), which provide structural flexibility between the target domain and other functional domains;
[0049] (3) Positions 11-17 are PTPSFTT sequences, which can achieve targeting of the inflammatory area by binding to the proline-rich region of MUC2 mucin.
[0050] 2. Anti-enzymatic degradation and structural stability domains
[0051] (1) Positions 18-21 are GPGP buffer domains, which block protease diffusion through the rigid structure of proline;
[0052] (2) Positions 22-41 are (EAAAK)4 stable regions (EAAAK repeated 4 times), which maintain the α-helical conformation of LL37 through charge interaction.
[0053] 3. Functional Domain (LL37 Variant)
[0054] (1) Positions 42-78 are optimized variants of the LL37 core sequence (LLGDFFRKSK EKIGKEFKRIVQ) Q I P DFLRNL P PRTES), and contains 3 key mutations (bold underlined):
[0055] R23Q (corresponding to position 23 in the sequence): Eliminates the trypsin cleavage site;
[0056] K25P (corresponding to position 25 in the sequence): reduces enzyme digestion efficiency through conformational interference;
[0057] V32P (32nd position in the sequence): stable C-terminal α-helix structure.
[0058] The above sequence achieves integrated functions of dual-target colonization, anti-enzymatic degradation, and immune activation through the synergistic effect of its various functional domains. A schematic diagram of its mechanism of action is shown below. Figure 2 As shown.
[0059] III. Preparation of Fusion Peptides
[0060] 1. Gene synthesis and cloning: Construct recombinant expression vectors containing genes encoding fusion peptides to achieve efficient expression in Lactobacillus plantarum.
[0061] (1) Gene sequence design and synthesis: Based on the amino acid sequence of the fusion peptide (SEQ ID NO:1), the codons of Lactobacillus plantarum LP-28 were optimized, with a focus on adjusting rare codons. The optimized gene sequence is shown in SEQ ID NO:2 (if purification is required, a 6His tag is added to the N-terminus; otherwise, the 6His tag is not needed). Gene synthesis was commissioned to Nanjing GenScript, and sequencing verification showed a 100% accuracy rate.
[0062] (2) Vector digestion and ligation: pSIP409 (a lactic acid bacteria-specific inducible vector containing an erythromycin resistance gene and a SppIP inducible promoter) was selected and double-digested with restriction endonucleases NcoI and XhoI (37℃, 2 h, enzyme amount 10 U / μg vector). The digestion products were recovered by 1% agarose gel electrophoresis. NcoI / XhoI restriction sites were introduced at both ends of the synthesized target gene, and it was mixed with the digested vector at a molar ratio of 3:1. T4 DNA ligase was added (20℃, 16 h, enzyme amount 5 U / reaction) to construct the recombinant vector pSIP409-fusion peptide.
[0063] (3) Preparation of Lactobacillus plantarum LP-28 competent cells: single colonies were picked and inoculated into MRS medium (containing 0.5 M sucrose) and cultured statically at 37°C until OD. 500 Centrifuge at 0.6, 4℃, 6000×g for 10 min, wash three times with pre-cooled electroconversion buffer (0.5 M sucrose + 10% glycerol), and resuspend in 100 μL buffer.
[0064] (4) Electroporation: Take 50 μL competent cells + 1 μg recombinant plasmid, add to a 0.2 cm electroporation cup, set the voltage to 2.5 kV and the pulse time to 4 ms, and immediately add 1 mL of MRS medium after transformation.
[0065] (5) Screening: 100 μL of resuscitated bacterial culture was spread on MRS solid plates containing 10 μg / mL erythromycin and incubated at 37℃ for 48 h. Single colonies were picked for PCR verification, and the positive clone rate was 38%.
[0066] 2. Induction and purification: Optimize induction conditions to improve the soluble expression of the fusion peptide, and obtain high-purity products through affinity purification.
[0067] (1) Seed culture and induction: Positive clones were selected and inoculated into 5 mL of MRS liquid medium (containing 10 μg / mL erythromycin) and incubated at 37℃ for 12 h (OD). 600 =1.2), transfer to 200 mL MRS medium at a 1% inoculum rate, and incubate at 37°C until OD.600 =0.6 (approximately 3.5 h). Add the inducer SppIP (final concentration 20 ng / mL, dissolved in methanol, storage concentration 1 mg / mL), and incubate at 25°C for 12 h (a 37°C induction group was set up as a control, and each group was repeated 3 times).
[0068] (2) Collection and disruption of bacterial cells: After induction, the bacterial cells were collected by centrifugation at 4°C and 8000×g for 10 min. The cells were washed twice with PBS (pH 7.4) and resuspended in 20 mL of lysis buffer (50 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole, pH 8.0).
[0069] (3) Ultrasonic disruption: Under ice bath conditions, power 300 W, working time 3 s, interval 5 s, total time 15 min (ensure the bacterial solution is clear and no intact bacterial cells are found under microscopic examination), centrifuge at 4℃ and 12000×g for 20 min, the supernatant is the crude extract.
[0070] (4) Nickel column affinity purification: A HisTrap HP column of 5 mL was purified and equilibrated with lysis buffer (flow rate 1 mL / min, equilibration for 5 column volumes). The crude extract was filtered through a 0.22 μm filter and loaded onto the column at a flow rate of 0.5 mL / min. Elution was performed sequentially with elution buffers containing 20 mM, 50 mM, 100 mM, and 250 mM imidazole (50 mM Tris-HCl, 300 mM NaCl, pH 8.0), and the 250 mM imidazole elution peak was collected.
[0071] (5) Dialysis and purity verification: The eluent was placed in a dialysis bag with a molecular weight cutoff of 3.5 kDa and dialyzed in PBS (pH 7.4) at 4°C for 24 h (changing the medium every 8 h) to remove imidazole and salt ions. A 15% separating gel was prepared. 10 μL of the dialyzed sample was mixed with 5× loading buffer (containing β-mercaptoethanol), denatured at 95°C for 5 min, and electrophoresed at a constant voltage of 80 V until bromophenol blue was expelled from the gel. Coomassie Brilliant Blue R-250 was stained for 30 min, and destaining was performed until the bands were clear.
[0072] 3. Experimental Results
[0073] SDS-PAGE results show ( Figure 3 The purified product showed a single protein band at approximately 9.2 kDa with no obvious impurities, and the purity was > 95%. The soluble expression level in the 25℃ induced group was approximately 6.2 times higher than that in the 37℃ group (22.3±1.5 mg / L), confirming that low temperature induction can significantly reduce inclusion body formation.
[0074] The above results indicate that a high-purity fusion peptide was successfully obtained through codon optimization, low-temperature induction, and nickel column affinity purification, with an expression level of 138.2 mg / L, which meets the requirements for subsequent experiments.
[0075] Table 1. Detection results of fusion peptide purification
[0076]
[0077] 4. It should be noted that the *Lactobacillus plantarum* LP-28 strain expressing the fusion peptide selected in this step can also be cultured on a large scale. In this case, the N-terminus of the fusion peptide in *Lactobacillus plantarum* LP-28 may not contain a 6His tag to reduce non-specific reactions in vivo. The *Lactobacillus plantarum* LP-28 strain expressing the fusion peptide prepared in this way is named *Lactobacillus plantarum* LP-28 engineered strain (hereinafter referred to as engineered strain in subsequent examples), and is lyophilized and stored at -80℃ for later use.
[0078] IV. In vitro testing
[0079] 1. Adhesion test to verify the effect of the dual-targeting system of fusion peptide (YIGSR+PTPSFTT) on enhancing the ability of engineered bacteria to adhere to intestinal epithelial cells.
[0080] (1) Caco-2 cell culture: Human colon adenocarcinoma cells Caco-2 were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C in a 5% CO2 incubator. The medium was changed every 2 days and passaged to the 20th-30th generation (to ensure differentiation and maturity).
[0081] (2) Monolayer preparation: Caco-2 cells were prepared at a ratio of 1×10⁻⁶ 5 Seeds were inoculated per well in a 24-well plate and cultured for 14 days (medium changed daily). Transepithelial resistance (TEER > 500 Ω·cm) was measured. 2 Confirm the integrity of a single layer.
[0082] (3) Bacterial fluorescent labeling: Log-phase engineered bacteria (expressing fusion peptide) and wild-type Lactobacillus plantarum LP-28 were washed twice with PBS, resuspended in PBS containing 5 μM CFSE (fluorescent probe, excitation wavelength 492 nm, emission wavelength 517 nm), and incubated at 37℃ in the dark for 30 min. The bacteria were washed three times with PBS to remove free CFSE, and the bacterial concentration was adjusted to 1×10⁻⁶. 8 CFU / mL.
[0083] (4) Adhesion experiment: Discard the culture medium in the Caco-2 cell wells, add 500 μL of fluorescently labeled bacterial solution (MOI=100:1) to each well, and incubate at 37℃ and 5% CO2 for 2 h.
[0084] (5) Termination of reaction: Discard the bacterial solution and gently rinse 3 times with PBS pre-warmed to 37°C (500 μL each time, slowly added along the well wall to avoid dispersing the bacteria that have adhered).
[0085] (6) Counting: Remove the coverslip, fix it with 4% paraformaldehyde for 15 min, stain the nuclei with DAPI (5 μg / mL, 10 min), observe under a fluorescence microscope with excitation light 492 nm, emission light 517 nm, and magnification of 200×. Randomly select 5 fields of view in each group to count the number of adhering bacteria.
[0086] (7) Experimental results (Table 2): The average number of adherent bacteria in the engineered bacteria group (63.7 bacteria / field) was significantly higher than that in the wild-type bacteria group (15.3 bacteria / field), and the relative adhesion rate increased by 4.2 times (relative adhesion rate 320%) (p<0.01). Further observation using laser confocal microscopy revealed that the engineered bacteria mainly aggregated on the apical membrane of Caco-2 cells (consistent with the distribution area of laminin receptors), confirming the synergistic targeting effect of YIGSR and PTPSFTT. These results indicate that the dual-targeting system can significantly enhance the adhesion ability of engineered bacteria to intestinal epithelial cells, laying the foundation for in vivo colonization.
[0087] Table 2 Adhesion test results
[0088]
[0089] 2. Immune activation assay (HT-29 cell model): to detect the activation of the immune pathway of intestinal epithelial cells and the regulation of anti-inflammatory factors by the fusion peptide.
[0090] (1) HT-29 cell culture: Human colon cancer cells HT-29 were cultured in McCoy's 5A medium containing 10% FBS and 1% penicillin-streptomycin at 37°C and 5% CO2 until confluence reached 80%, and then seeded into 6-well plates (5×10⁻⁶ cells / wells). 5 (each well), incubate for 24 h.
[0091] (2) Bacterial-cell co-culture: Engineered and wild-type bacteria were washed twice with McCoy's 5A medium and the concentration was adjusted to 5×10⁻⁶. 5 CFU / mL was added to the wells of HT-29 cells at an MOI of 10:1 (bacteria:cells = 10:1) and incubated at 37°C and 5% CO2 for 6 h (the cell group without bacteria was set as the blank control).
[0092] (3) RNA extraction and qPCR: Total RNA was extracted from cells using TRIzol reagent, as follows: (discard the culture medium, add 1 mL of TRIzol to each well, incubate at room temperature for 5 min, add 200 μL of chloroform, shake for 15 s, centrifuge at 12000×g for 15 min at 4℃, take the upper aqueous phase, add 500 μL of isopropanol to precipitate RNA, wash with 75% ethanol, dissolve in DEPC water, and detect RNA concentration using NanoDrop (A260 / A280=1.8-2.0)). Take 1 μg of RNA and reverse transcribe it using PrimeScript RT reagent Kit (reaction conditions: 37℃ for 15 min, 85℃ for 5 s). The reaction was performed using SYBR Green Premix Ex Taq on a Roche LightCycler 480. The reaction mixture (20 μL) consisted of 2 μL cDNA, 0.8 μL each of forward and reverse primers, 10 μL SYBR Green, and 6.4 μL ddH2O. Cycling conditions were 95℃ for 30 s, for 40 cycles (95℃ for 5 s, 60℃ for 30 s). Melting curves were used to verify primer specificity. Relative expression levels were calculated using the 2^(-ΔΔCt) method, with the blank control group used as a calibrator.
[0093] (4) The experimental results are shown in Table 3. Compared with the wild-type fungus group, the expression of TLR4 (toll-like receptor 4, a key molecule in the immune activation pathway) mRNA in the engineered fungus group was upregulated by 6.1-fold, the expression of antimicrobial peptide BD2 (β-defensin 2) mRNA was upregulated by 17.3-fold, and the expression of pro-inflammatory factor IL-1β mRNA was downregulated by 7.2-fold (p < 0.01). This confirms that the fusion peptide can enhance antimicrobial immunity by activating the TLR4 pathway, while inhibiting the inflammatory response. The above results indicate that the fusion peptide has both immune activation and anti-inflammatory functions, providing a molecular basis for intestinal inflammation repair.
[0094] Table 3 Results of Immune Activation Test
[0095]
[0096] V. Internal Examination
[0097] 1. Animal and Model Establishment
[0098] (1) Animals: C57BL / 6 male mice (6-8 weeks old, weight 20±2 g, SPF grade), acclimatized for 1 week, with free access to food and water.
[0099] (2) Grouping: Randomly divide into 3 groups (n=6):
[0100] PBS group: PBS was administered by gavage (0.2 mL / animal / day);
[0101] Wild-type fungus group: wild-type Lactobacillus plantarum LP-28 (1×10⁻⁶) administered by gavage 9 CFU / 0.2 mL / animal / day);
[0102] Engineered bacterial group: engineered bacteria expressing fusion peptides via gavage (1×10⁻⁶) 9 CFU / 0.2 mL / animal / day).
[0103] (3) DSS induction: From day 1 to day 7 of modeling, all mice were allowed to drink 3% (w / v) sodium dextran sulfate (DSS) solution freely. From day 8 onwards, normal drinking water was restored and intervention was started at the same time (lasting for 14 days).
[0104] 2. Sample Collection and Testing
[0105] (1) Colony establishment detection: On day 7 of intervention, fresh feces (approximately 0.1 g) were collected from each mouse and added to 0.9 mL of PBS homogenate, then serially diluted (10 g / mL). -1 Up to 10 -8 Take 100 μL and spread it on an MRS plate containing 10 μg / mL erythromycin (engineered bacteria containing resistance genes), incubate at 37℃ for 48 h to count CFU, and calculate the colonization amount per gram of feces.
[0106] (2) Inflammation score: On the 14th day of intervention, mice were sacrificed and colon tissue (2 cm from the anus) was taken, fixed with 4% paraformaldehyde, embedded in paraffin and sectioned (4 μm), stained with HE, and scored according to the following criteria (0-12): Mucosal damage (0 = intact, 1 = slight erosion, 2 = local ulcer, 3 = extensive ulcer, 4 = full-thickness damage); Inflammatory infiltration (0 = no infiltration, 1 = small amount in mucosal layer, 2 = large amount in mucosal layer, 3 = mucosa + submucosa, 4 = full-thickness infiltration); Crypt destruction (0 = intact, 1 = partial destruction, 2 = most destruction, 3 = complete destruction, 4 = crypt disappearance).
[0107] (3) Immunohistochemistry of ZO-1: After dewaxing and antigen retrieval, the sections were incubated with rabbit anti-human ZO-1 primary antibody (diluted 1:200) overnight at 4°C, incubated with HRP-labeled secondary antibody for 30 min, DAB staining, hematoxylin counterstaining, and Image-Pro Plus 6.0 was used to quantitatively analyze the average optical density (IOD / Area), which represents the relative expression level of ZO-1.
[0108] 3. The experimental results are shown in Table 4.
[0109] Statistical analysis showed that the colonization rate of the engineered bacteria group was 843 times higher than that of the wild bacteria group (p<0.001), the inflammation score was significantly reduced (2.4 vs 6.1, p<0.001), and the ZO-1 expression level was close to that of normal mice (0.90±0.06), confirming that the engineered bacteria can repair the intestinal barrier and alleviate inflammation through efficient colonization.
[0110] The above results indicate that engineered bacteria modified with fusion peptides can achieve efficient colonization in vivo, significantly reduce the degree of inflammation in colitis, promote the expression of tight junction proteins, and have a better therapeutic effect than wild-type bacteria.
[0111] Table 4 Results of DSS-induced mouse colitis model
[0112]
[0113] VI. Safety Inspection
[0114] 1. Evaluate the hemolytic toxicity of the fusion peptide and verify the role of mutation in reducing the toxicity of natural LL37.
[0115] 2. Red blood cell preparation: Take whole blood from healthy New Zealand rabbits (containing heparin anticoagulation), centrifuge at 1500×g for 10 min, discard the plasma, wash 3 times with PBS (centrifuge at 1500×g for 5 min each time), resuspend in PBS and adjust the concentration to 2% (v / v).
[0116] 3. Hemolysis test
[0117] (1) Sample preparation: Natural LL37 (GlpBio, USA) and fusion peptide were diluted to 10 μM with PBS. PBS was set as a negative control (0% hemolysis) and 0.1% Triton X-100 was set as a positive control (100% hemolysis).
[0118] (2) Reaction system: Add 100 μL of 2% red blood cell suspension + 100 μL of sample to a 96-well plate, incubate at 37℃ for 1 h, centrifuge at 4℃ and 3000×g for 10 min, take 100 μL of supernatant to a new plate, and detect the absorbance at 540 nm (A540) using an ELISA reader.
[0119] (3) Calculation of hemolysis rate: Hemolysis rate (%) = (sample A540 - negative control A540) / (positive control A540 - negative control A540) × 100%, with 3 replicates per group.
[0120] 4. The experimental results are shown in Table 5.
[0121] The results showed that the hemolysis rate of the 10 μM fusion peptide was only 3.1%, significantly lower than that of natural LL37 (48.3%), and the toxicity was reduced to 1 / 15 (p<0.001), confirming that the mutation can effectively eliminate the hemolytic toxicity of natural LL37.
[0122] The above results indicate that the fusion peptide exhibits significantly reduced hemolytic toxicity and good biocompatibility.
[0123] Table 5 Results of hemolytic activity test
[0124]
[0125] VII. Conclusion
[0126] 1. Dual-targeting synergistic mechanism: An innovative dual-targeting system combining YIGSR (lamin receptor targeting) and PTPSFTT (MUC2 mucin targeting) was designed. In vitro adhesion experiments showed that the adhesion efficiency of the engineered bacteria to Caco-2 cells was 320% higher than that of the single YIGSR design (4.2× vs 1.0×), and the in vivo colonization reached 2.7×10⁻⁶. 7 CFU / g feces (wild mushroom group: only 3.2×10) 4 (CFU / g) solves the problem of colonization blind spots for single targets in intestinal pathological states.
[0127] 2. Breakthrough in stability: By blocking protease diffusion through the GPGP buffer domain, stabilizing the α-helix with (EAAAK)4, and eliminating cleavage sites through R23Q / K25P / V32P mutations, the half-life of the fusion peptide in simulated intestinal fluid is extended from 0.5 h of natural LL37 to >8 h, and the anti-enzymatic properties are improved by 16 times, ensuring its activity in the intestinal environment.
[0128] 3. Significant therapeutic advantages: The remission rate of colitis in the engineered bacteria group reached 86.7% (based on the inflammation score decreasing from 8.9 to 2.4), which was significantly higher than that in the wild-type bacteria group (32.1%). At the same time, it promoted the expression of tight junction protein ZO-1 (0.85 vs. 0.42 in the wild-type bacteria group) and the secretion of anti-inflammatory factor IL-10 (210 pg / mL vs. 45 pg / mL in the wild-type bacteria group), achieving synergistic treatment of "targeted colonization-immune regulation-barrier repair", and the cost is only 1 / 50 of that of antibody drugs, which has the potential for clinical translation.
[0129] In summary, this invention, through multi-dimensional technological innovation, solves key challenges in the targeting, stability, safety, and efficacy of fusion peptides, providing a novel technical solution for the treatment of intestinal inflammatory diseases.
[0130] Example 2: Preparation of anti-Escherichia coli O157:H7 monoclonal antibody GS2-D3
[0131] Escherichia coli O157:H7 is a common foodborne pathogen that can cause serious intestinal infections and complications. Anti-Escherichia coli O157:H7 monoclonal antibodies can specifically recognize and bind to the surface antigens of this bacterium, blocking its adhesion and invasion, while simultaneously activating the immune system to clear the pathogen.
[0132] Six- to eight-week-old female Balb / c mice were immunized with formaldehyde-inactivated Escherichia coli O157:H7 whole-cell antigen. The immunization program consisted of four intraperitoneal injections (including one booster). The first injection was emulsified with Freund's complete adjuvant, followed by Freund's incomplete adjuvant. Seven days after the final immunization, the serum titer reached 1:64000. High-titer mice were selected, and spleen cells were aseptically harvested to prepare splenocytes. These splenocytes were fused with logarithmically growing SP2 / 0 myeloma cells at a ratio of 5:1 using 50% PEG 1500. The fused cells were seeded on HAT selective medium, achieving a fusion rate of 48.7%. Positive clones were screened by indirect ELISA, yielding 45 hybridoma cell lines secreting specific antibodies, including 6 strongly positive clones. The strongly positive clone GS2-D3 was subjected to three limiting dilutions to obtain stable antibody-secreting cell lines. The antibody secretion capacity showed no significant decrease after 15 passages and resuscitation (OD). 450 The value was stable at 1.6-1.8. Meanwhile, to reduce the rejection response to exogenous proteins, the screened monoclonal antibody was humanized and optimized, ultimately obtaining a CHO cell line expressing a relatively good monoclonal antibody. The resulting monoclonal antibody was named anti-Escherichia coli O157:H7 monoclonal antibody GS2-D3, and its amino acid sequences of the heavy chain variable region and light chain variable region are shown in SEQ ID NO:3 and SEQ ID NO:4, respectively.
[0133] The CHO cells expressing the monoclonal antibody GS2-D3 obtained through the above screening were fermented and cultured, and the supernatant was collected by centrifugation. The supernatant was purified by Protein G affinity chromatography. The purified antibody, after membrane exchange buffer and filtration sterilization, achieved a purity of 96.3% (SDS-PAGE results are shown below). Figure 4 As shown in the figure, the yield is approximately 3.2 ± 1.1 mg / mL. After purification, the antibody ELISA titer is increased to 1:128000 (which is higher than the titer of commercial monoclonal antibodies, whose ELISA titer is ≤1:32000). It also shows no cross-reactivity with Escherichia coli O111, O127 and Salmonella, and has good specificity.
[0134] Example 3: Preparation and Characterization of Curcumin Nanoparticles
[0135] Curcumin possesses broad-spectrum anti-inflammatory, antioxidant, and immunomodulatory effects, but its poor water solubility and low bioavailability limit its applications. By using nano-encapsulation technology, curcumin is encapsulated within a nanocarrier composed of zein and tea saponin, significantly improving its stability and bioavailability.
[0136] Curcumin nanoparticles were prepared using zein as a carrier and tea saponin as an emulsifier via an ultrasonic-solvent evaporation method: 30.6 mg of zein, 7.5 mg of curcumin, and 61.9 mg of tea saponin were weighed and placed in a 50 mL beaker. 10 mL of 80% ethanol aqueous solution (V / V) was added, and the mixture was magnetically stirred (300 rpm) for 2 h (25℃) until completely dissolved (the solution was orange-yellow and transparent). The solution was transferred to 10 mL centrifuge tubes and treated with an ultrasonic cell disruptor under ice bath conditions. The parameters were set as follows: power 200 W, 3 seconds on, 3 seconds off, total processing time 15 minutes (avoiding the solution temperature from exceeding 30℃). After ultrasonication, the solution was transferred to a rotary evaporator, concentrated to approximately 2 mL (approximately 30 minutes) in a 40℃ water bath under a vacuum of 0.08 MPa and a rotation speed of 100 rpm, and the ethanol was removed. The concentrate was transferred to a freeze-drying bottle, pre-frozen at -80℃ for 2 h, placed in a freeze dryer, set to -50℃ and vacuum degree 0.01 MPa, and freeze-dried for 48 h to obtain an orange-yellow loose powder (curcumin nanoparticles), which was then sealed and stored at 4℃.
[0137] Characterization results showed that the nanoparticles had an average particle size of 189.5±2.9 nm, a polydispersity index (PDI) of 0.19±0.01, and a zeta potential of -33.2±0.6 mV, exhibiting good dispersibility and high electrostatic stability (particle size change <5% after 30 days at 4℃). Scanning electron microscopy revealed that the nanoparticles were spherical with a smooth surface and no obvious agglomeration. The encapsulation efficiency was 90.3±1.2%, and the drug loading was 6.9±0.2%, providing an ideal carrier for the efficient delivery of curcumin.
[0138] Example 4: Preparation of Probiotic Composition
[0139] I. Preparation Steps
[0140] 1. Cultivation and pretreatment of engineered Lactobacillus plantarum LP-28 (wild-type bacteria can also be cultured following the same steps)
[0141] Seed culture: Take freeze-dried powder of engineered Lactobacillus plantarum LP-28 bacteria (viable count ≥1×10⁻⁶). 11 CFU / g was inoculated into 5 mL of MRS liquid medium (containing 2% glucose, pH 6.2) and anaerobically cultured at 37°C (anaerobic bag, containing 10% CO2) for 12 h. After activation, it was transferred to 200 mL of MRS medium at a 1% inoculation rate and anaerobically cultured at 37°C for 18 h. OD was measured every 6 h during this period.600 Value, endpoint OD 600 =1.8±0.1, the viable count determined by plate counting method was 3.2×10. 10 CFU / mL.
[0142] Bacterial cell collection and washing: 4 ℃ Collect bacterial cells by centrifugation at 8000×g for 10 min, discard the supernatant, resuspend in pre-cooled PBS (pH 7.4), centrifuge at 8000×g for 10 min, and wash twice (to remove culture medium residue). Finally, adjust the bacterial concentration to 2×10⁻⁶ cells / mL with PBS. 9 CFU / mL (bacterial suspension volume 50 mL).
[0143] 2. Combination and processing of composite components
[0144] Component preparation: Monoclonal antibody GS2-D3 was diluted with PBS to 1 mg / mL; curcumin nanoparticles were dispersed with PBS to 10 mg / mL.
[0145] Mixing and incubation: Add the above components to 50 mL of bacterial suspension in proportion to make the final concentrations as follows: GS2-D3 10 μg / mL and curcumin nanoparticles 100 μg / mL. After gently inverting and mixing, incubate at 4°C in the dark for 1 h (mix gently once every 15 min to avoid bacterial precipitation).
[0146] Binding efficiency detection: After incubation, 1 mL of the mixture was taken, centrifuged at 4℃ and 10000×g for 5 min, and the supernatant was collected. The content of free GS2-D3 was detected by ELISA (binding rate = (initial amount - free amount) / initial amount × 100%), and the content of free curcumin nanoparticles was detected by fluorescence spectrophotometry (excitation wavelength 428 nm). The results showed that the binding rate of GS2-D3 was 92.3±2.1%, and the binding rate of curcumin nanoparticles was 88.7±1.8%, confirming that each component effectively bound to the bacteria.
[0147] 3. Freeze-drying protection and molding
[0148] Preparation of the preservative: Weigh 10 g of trehalose (purity ≥99%) and 5 g of skim milk (containing 3.2% protein), add ultrapure water to dissolve to 100 mL, filter through a 0.22 μm filter membrane for sterilization, and store at 4℃.
[0149] Mixing and Dispensing: Gently mix 50 mL of the bacterial-component mixture with 50 mL of the preservative (1:1 volume ratio), and dispense into 2 mL vials (each vial contains 1 mL of engineered live bacteria, 1×10⁶). 9 CFU, GS2-D3 5 μg, curcumin nanoparticles 50 μg).
[0150] Freeze-drying: Hold at -40℃ for 2 h, sublime dry under vacuum of 0.01 MPa for 12 h (cold trap temperature -55℃), and desorption dry (30℃) for 4 h. The freeze-dried sample is a light yellow loose powder with a moisture content ≤3%.
[0151] 4. Preparation of enteric-coated capsules
[0152] Contents filling: Take the lyophilized powder and fill it into empty capsules using a semi-automatic capsule filling machine. Each capsule fills one vial of the above-mentioned lyophilized powder (approximately 55 mg dry weight). Each capsule contains 1 × 10⁶ engineered Lactobacillus plantarum LP-28 bacteria. 9 CFU, GS2-D3 5 μg, curcumin nanoparticles 50 μg.
[0153] Enteric coating: Coating solution formulation (10% hydroxypropyl methylcellulose phthalate + 3% glycerol + 87% ethanol aqueous solution, stirred and dissolved, then passed through a 100-mesh sieve); fluidized bed coating machine was used with the following parameters: inlet air temperature 40℃, outlet air temperature 30℃, spray rate 2 mL / min, coating weight gain 5% (10 mg weight gain per capsule), and drying at 40℃ for 2 h after coating to solidify the enteric coating film.
[0154] Table 6. Preparation and testing results of probiotic compositions
[0155]
[0156] II. Summary
[0157] This embodiment utilizes optimized culture, binding, and freeze-drying processes to prepare a composite formulation containing *Lactobacillus plantarum* LP-28 (engineered bacteria), monoclonal antibody GS2-D3, and curcumin nanoparticles, which is then encapsulated using HPMCP enteric coating. Testing showed that the freeze-dried formulation achieved a survival rate of 85.2%, with each functional component exhibiting a binding rate ≥88%. The enteric-coated capsules showed a release rate ≤5% in simulated gastric juice after 2 hours (avoiding gastric acid degradation) and a release rate ≥90% in simulated intestinal juice after 4 hours (ensuring effective intestinal colonization and functional expression). This provides a stable and efficient formulation for the oral delivery of probiotics and functional components.
[0158] Example 5: Testing of the probiotic composition
[0159] Experiment 1: Antipathogenic activity test. This experiment aims to verify the inhibitory effect of the probiotic composition (containing Lactobacillus plantarum LP-28, monoclonal antibody GS2-D3 and curcumin nanoparticles) on Escherichia coli O157:H7, and to analyze the synergistic effect of each component.
[0160] 1. Experimental materials: Escherichia coli O157:H7; probiotic composition (prepared as needed, the viable count of engineered bacteria in this experiment is 1×10⁻⁶).9 CFU / mL, GS2-D3 5 μg / mL, curcumin nanoparticles 50 μg / mL; Control group 1 (without GS2-D3, otherwise the same as the composition), Control group 2 (without curcumin nanoparticles, otherwise the same as the composition), Control group 3 (without GS2-D3 and curcumin nanoparticles, otherwise the same as the composition); LB medium (tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, pH 7.0); SS agar plates (selective culture of enteropathogenic bacteria, containing bile salts to inhibit other bacteria); 96-well cell culture plates.
[0161] 2. Experimental Procedure
[0162] Activation of *E. coli*: *E. coli* O157:H7 stored at -80℃ was streaked onto LB agar plates and incubated at 37℃ for 24 h. A single colony was picked and inoculated into 5 mL of LB liquid medium, and cultured at 37℃ with shaking at 200 rpm for 12 h until the logarithmic growth phase (OD200) was reached. 600 =0.6-0.8), adjust the bacterial concentration to 1×10 using the plate count method. 5 CFU / mL.
[0163] Construction of co-culture system: Add 100 μL of Escherichia coli culture (1×10⁻⁶) to each well of a 96-well plate. 5 (CFU / mL); 100 μL of probiotic composition was added to the experimental group, the corresponding composition was added to the control groups 1-3 respectively, and 100 μL of PBS was added to the blank control group. Each group had 6 replicates, and the experiment was repeated 3 times.
[0164] Culture and Counting: After co-culturing at 37℃ for 4 h, 100 μL of bacterial culture from each well was taken and serially diluted with PBS (10⁻⁶ ppm). -1 Up to 10 -5 Take 100 μL of the diluted solution and spread it on an SS plate. Incubate at 37°C for 24 h and count the number of colonies (only Escherichia coli O157:H7 forms colorless colonies with a black center on the SS plate).
[0165] 3. Experimental Results
[0166] After 4 hours of co-culturing, the viable Escherichia coli count in the blank control group was (8.5±0.6)×10⁻⁶. 5 CFU / mL indicates vigorous natural bacterial proliferation; the experimental group had the lowest viable bacterial count, at (1.2±0.3)×10⁻⁶. 4 The CFU / mL count was 98.6% lower than that of the blank control group and significantly lower than that of all control groups (P<0.001). The viable count of control group 1 (without GS2-D3) was (2.6±0.2)×10⁻⁶. 4The CFU / mL concentration was 117% higher than that of the experimental group; the control group 2 (without curcumin nanoparticles) had a concentration of (3.9±0.3)×10⁻⁶. 4 The CFU / mL concentration was 225% higher than that of the experimental group; the control group 3 (without GS2-D3 and curcumin nanoparticles) had a concentration of (5.3±0.3)×10⁻⁶. 4 The CFU / mL level was 342% higher than that of the experimental group.
[0167] The results showed that the antibacterial effect of each control group decreased sequentially, indicating that engineered bacteria, GS2-D3, and curcumin nanoparticles were all key antibacterial components: engineered bacteria enhanced the competitive advantage by strengthening the intestinal colonization ability of probiotics, GS2-D3 blocked the adhesion of Escherichia coli O157:H7 by specifically binding to the surface antigen of Escherichia coli O157:H7, and curcumin nanoparticles directly inhibited bacterial proliferation through antibacterial activity. The synergistic effect of the three made the antibacterial effect of the experimental group significantly better than that of the control group lacking a single component.
[0168] 4. Summary: The probiotic composition has a strong inhibitory effect on Escherichia coli O157:H7. The synergistic effect of engineered bacteria, monoclonal antibody GS2-D3 and curcumin nanoparticles is the core mechanism of its high-efficiency antibacterial effect, which increases the antibacterial rate by 2-3 times compared with the single component missing group.
[0169] Experiment 2: Verification of anti-inflammatory effects. This experiment established an inflammation model by inducing RAW264.7 cells with LPS to verify the anti-inflammatory activity of the probiotic composition, focusing on the regulatory effect of curcumin nanoparticles on the secretion of inflammatory factors.
[0170] 1. Experimental materials: RAW264.7 cells (mouse mononuclear macrophage cell line); LPS (E. coli O111:B4, purity ≥99%); probiotic composition (same as Experiment 1); control group (without curcumin nanoparticles, otherwise the same as the composition); DMEM medium (containing 10% FBS, 1% penicillin-streptomycin); TNF-α ELISA kit, IL-10 ELISA kit.
[0171] 2. Experimental Procedure
[0172] Cell culture: RAW264.7 cells were seeded in 24-well plates (5 × 10⁶ cells / well). 5 Add 1 mL of DMEM medium to each well (each cell / well) and incubate at 37°C and 5% CO2 for 24 h until the confluence reaches 80%.
[0173] Drug pretreatment: 100 μL of probiotic composition (final concentration 100 μg / mL) was added to each well of the experimental group, 100 μL of composition without curcumin nanoparticles was added to the control group, and 100 μL of PBS was added to the blank control group. The culture was continued for 2 h. The normal control group (without LPS) was maintained only by basic culture.
[0174] Inflammation induction: Except for the normal control group, LPS (final concentration 1 μg / mL) was added to each well of the other groups and cultured at 37℃ and 5% CO2 for 24 h.
[0175] Factor detection: Collect the supernatant from each well and follow the instructions of the ELISA kit to calculate the concentrations of TNF-α (pro-inflammatory factor) and IL-10 (anti-inflammatory factor) using a standard curve.
[0176] 3. Experimental Results
[0177] In the normal control group, the TNF-α concentration was 23±5 pg / mL and the IL-10 concentration was 48±6 pg / mL, indicating a basal secretion state. In the LPS control group, TNF-α was significantly increased to 487±23 pg / mL and IL-10 was slightly decreased to 52±4 pg / mL, indicating that the inflammation model was successfully constructed.
[0178] Compared with the LPS control group, the experimental group showed a decrease in TNF-α concentration to 198±15 pg / mL (a decrease of 59.3%) and an increase in IL-10 to 189±12 pg / mL (an increase of 263.5%). In the control group, TNF-α was 297±21 pg / mL (a decrease of 39.0%) and IL-10 was 125±9 pg / mL (an increase of 140.4%). Statistical analysis showed that the experimental group exhibited significantly greater reductions in TNF-α and increases in IL-10 compared to the control group (P<0.01).
[0179] The results showed that the probiotic composition exerted its anti-inflammatory effect by downregulating pro-inflammatory factors and upregulating anti-inflammatory factors, while curcumin nanoparticles were the key synergistic ingredient—they reduced the release of pro-inflammatory factors such as TNF-α by inhibiting the activation of the NF-κB signaling pathway, while promoting the secretion of IL-10 and enhancing the anti-inflammatory effect.
[0180] 4. Summary: The probiotic composition has significant anti-inflammatory activity. Curcumin nanoparticles can enhance the anti-inflammatory effect by regulating the balance of inflammatory factors. Compared with the control group without curcumin, the inhibition rate of TNF-α was increased by 20.3%, and the promotion rate of IL-10 was increased by 123.1%.
[0181] Experiment 3: Animal Model Evaluation
[0182] 1. Experimental Materials: SPF-grade C57BL / 6 female mice (6-8 weeks old, weight 20±2 g); DSS (molecular weight 36,000-50,000, Sigma); probiotic composition (containing Lactobacillus plantarum LP-28 engineered bacteria, GS2-D3 and curcumin nanoparticles, wherein the concentration of Lactobacillus plantarum LP-28 engineered bacteria is 1×10⁻⁶. 8CFU / mL and 1×10 9 (CFU / mL, GS2-D3 5 μg / mL, curcumin nanoparticles 50 μg / mL remained unchanged); HE staining kit; 16S rRNA gene sequencing kit.
[0183] 2. Experimental Procedure
[0184] (1) Animal grouping and adaptation: 40 mice were acclimatized for 1 week (free access to food and water) and randomly divided into 4 groups (10 mice in each group): normal control group, DSS model group, low-dose experimental group, and high-dose experimental group. The grouping was done using a random number table to ensure baseline consistency.
[0185] (2) Model building and intervention
[0186] Normal control group: drank distilled water throughout the process, and were given 0.2 mL of physiological saline by gavage at 9:00 AM every day;
[0187] DSS model group: For the first 7 days, the group drank 3% DSS aqueous solution (with fresh solution changed daily) and was given 0.2 mL of physiological saline by gavage daily;
[0188] Low / high dose experimental groups: For the first 7 days, participants drank 3% DSS aqueous solution and were given 0.2 mL of solution containing 1×10⁻⁶ DSS by gavage daily. 8 CFU / mL or 1×10 9 A probiotic composition of CFU / mL.
[0189] (3) Indicator monitoring
[0190] Record weight (accurate to 0.1 g), diarrhea status (0 points: normal; 1 point: mild loose stool; 2 points: loose stool; 3 points: watery stool) and rectal bleeding status (0 points: none; 1 point: positive occult blood; 2 points: significant rectal bleeding) daily, and calculate the Disease Activity Index (DAI) using the formula (weight loss rate score + diarrhea score + rectal bleeding score) / 3;
[0191] On day 7, the mice were euthanized by cervical dislocation, and the colon (from the ileocecal junction to the anus) was aseptically removed and its length was measured with a ruler (accurate to 0.1 cm).
[0192] Gut microbiota analysis: DNA from colon contents was extracted using the CTAB method, the V4-V5 region of the 16S rRNA gene was amplified, and sequencing was performed. The α-diversity and phylum-level composition of the microbiota were analyzed using QIIME2.
[0193] 3. Experimental Results
[0194] (1) General condition and DAI: The weight of mice in the normal control group continued to increase, with a weight change rate of +6.3±2.1% on day 7, and no diarrhea or bloody stools (DAI=0). The weight of mice in the DSS model group decreased significantly (-16.9±3.2%), with severe diarrhea (2-3 points) and bloody stools (2 points), and the DAI reached 2.8±0.3. The weight loss rate in the low-dose experimental group was -8.3±2.5%, and the DAI decreased to 1.5±0.2; the weight loss in the high-dose experimental group was only -3.4±1.8%, and the DAI was 0.6±0.1, with diarrhea and bloody stools basically relieved, showing dose-dependent improvement. As shown in Table 7.
[0195] Table 7 Statistical Results of Weight Change
[0196]
[0197] (2) Colon length: The colon length in the normal control group was 8.5±0.3 cm, while that in the DSS model group it was shortened to 5.1±0.3 cm (P<0.001). The colon length in the low-dose experimental group recovered to 6.3±0.4 cm, and that in the high-dose group it reached 7.2±0.4 cm, which is close to the normal level (P<0.01).
[0198] (3) Intestinal flora balance, as shown in Table 8.
[0199] The microbial community structure of the DSS model group was significantly disordered: the proportion of Bacteroidetes decreased from 58.2±3.5% in the normal group to 32.1±4.2%, Firmicutes increased from 35.6±2.8% to 58.7±4.5%, and Proteobacteria (pro-inflammatory bacteria) increased from 2.1±0.5% to 7.3±1.2%.
[0200] The bacterial community structure in the high-dose experimental group was significantly restored: Bacteroidetes rose to 51.3±3.8%, Firmicutes dropped to 42.5±3.6%, Proteobacteria dropped to 3.2±0.7%, close to the level of the normal control group, and Actinobacteria (beneficial bacteria) rose from 1.9±0.4% to 3.0±0.5%.
[0201] Table 8. Composition of Gut Microbiota
[0202]
[0203] 4. Summary
[0204] This experiment demonstrated that the probiotic composition could dose-dependently improve DSS-induced colitis in mice: the high-dose group showed an 80.6% reduction in body weight loss and a 41.2% increase in colon length compared to the model group, and effectively restored intestinal flora balance (the Bacteroidetes / Firmwallis ratio increased, and pro-inflammatory bacteria decreased). Its mechanism of action is related to the synergistic effects of the probiotics in the composition in enhancing colonization, the antibodies blocking pathogen adhesion, the anti-inflammatory effects of curcumin, and the regulation of the gut microbiota, providing a holistic solution for the intervention of colitis.
[0205] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.
Claims
1. A dual-targeting adhesion-immune activation fusion peptide, characterized in that, The fusion peptide comprises a targeting domain, an anti-enzymatic and structurally stable domain, and an LL37 variant functional domain, wherein the amino acid sequence of the fusion peptide is shown in SEQ ID NO:
1.
2. The fusion peptide according to claim 1, characterized in that, The optimized gene sequence of the fusion peptide codon is shown in SEQ ID NO:
2.
3. The fusion peptide according to claim 1, characterized in that, The target domains include YIGSR and PTPSFTT, which are connected by a GGGGS flexible linker to achieve dual targeting. YIGSR can bind to laminin receptors on the surface of healthy intestinal epithelial cells, while PTPSFTT can bind to MUC2 mucin highly expressed in inflammatory areas, thus achieving colonization throughout the intestinal tract.
4. The fusion peptide according to claim 1, characterized in that, The anti-enzymatic and structurally stable domains include a GPGP buffer domain and an EAAAK tetramer stable domain; wherein the GPGP buffer domain blocks protease diffusion through the rigid structure of proline, and the EAAAK tetramer stable domain maintains the α-helical conformation of LL37 through charge interactions.
5. The fusion peptide according to claim 1, characterized in that, The LL37 variant functional domain contains R23Q, K25P, and V32P mutations, and the amino acid sequence of the LL37 variant functional domain is LLGDFFRKSK EKIGKEFKRIVQQIPDFLRNLPPRTES.
6. A probiotic composition, characterized in that, The composition comprises an effective amount of *Lactobacillus plantarum* LP-28 engineered bacteria, an effective amount of anti-*Escherichia coli* O157:H7 monoclonal antibody GS2-D3, and an effective amount of curcumin nanoparticles, wherein the *Lactobacillus plantarum* LP-28 engineered bacteria is *Lactobacillus plantarum* LP-28 capable of expressing the dual-targeting adhesion-immune activation fusion peptide of claim 1; the amino acid sequence of the heavy chain variable region of the anti-*Escherichia coli* O157:H7 monoclonal antibody GS2-D3 is shown in SEQ ID NO:3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:
4.
7. The composition according to claim 6, characterized in that, The curcumin nanoparticles were prepared by an ultrasonic-solvent evaporation method using zein as a carrier and tea saponin as an emulsifier. The average particle size was 189.5±2.9 nm, the polydispersity index was 0.19±0.01, the zeta potential was -33.2±0.6 mV, the encapsulation efficiency was 90.3±1.2%, and the drug loading was 6.9±0.2%.
8. The composition according to claim 6, characterized in that, The composition is formulated into enteric-coated capsules, each containing 1 × 10⁶ engineered Lactobacillus plantarum LP-28 bacteria. 9 CFU, anti-Escherichia coli O157:H7 monoclonal antibody GS2-D3 5 μg, curcumin nanoparticles 50 μg.
9. The use of the dual-targeting adhesion-immune activation fusion peptide as described in claim 1 in the preparation of probiotic compositions.