Composition containing probiotics and application thereof in treating gastrointestinal diseases

Abstract

The invention discloses a composition containing probiotics and fusion polypeptide and application of the composition in treatment of intestinal inflammatory diseases. The composition is prepared from lactobacillus plantarum (Lactobacillus plantarum CCFM8661) and a functional fusion polypeptide, wherein the amino acid sequence of the fusion polypeptide is as shown in SEQ ID NO. 1. The invention also discloses a preparation method of the composition. The fusion polypeptide can be used for directly inhibiting the growth of pathogenic bacteria and neutralizing toxins by enhancing the intestinal colonization capability of probiotics, so that the triple synergistic effect of bacteriostasis, repair and immunoregulation is realized. Experiments show that the composition significantly reduces the disease activity index in a DSS-induced mouse colitis model, recovers the colon length, and inhibits serum inflammatory factors IL-6 and TNF-alpha. In addition, the survival rate of the freeze-dried microcapsule preparation in simulated gastric juice is gt; the colon targeted release characteristic is realized. The invention provides an efficient and stable new treatment strategy for intestinal inflammatory diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to a composition comprising probiotics and use thereof in treating gastrointestinal diseases. Background Art

[0002] Inflammatory Bowel Disease (IBD) is a type of disease characterized by chronic, recurrent intestinal inflammation, mainly including ulcerative colitis and Crohn's disease. Its pathogenesis is complex, and current studies have shown that the occurrence of IBD is closely related to factors such as intestinal flora imbalance, pathogen invasion, and abnormal immune system. Specifically, the number of beneficial bacteria (such as Bacteroides and Lactobacillus) in the intestines of IBD patients decreases significantly, while opportunistic pathogens (such as Clostridium difficile and Escherichia coli) proliferate abnormally, inducing damage to the intestinal barrier function, thereby activating excessive immune responses and causing persistent inflammation.

[0003] Although existing treatment methods are constantly developing, there are still many technical bottlenecks. First of all, probiotic therapy, as a common intervention method, can regulate the intestinal microecology to a certain extent, but its application is obviously limited. Traditional probiotics such as Lactobacillus and Bifidobacterium are easily destroyed by factors such as gastric acid and bile salts in the gastrointestinal environment, resulting in their actual colonization rate in the intestine usually being less than 20%; at the same time, this type of probiotics has a weak direct inhibitory effect on specific pathogens such as pathogenic Escherichia coli and Clostridium difficile, and its anti-infection effect is limited.

[0004] Secondly, while biological agents targeting inflammatory responses, such as anti-TNF-α monoclonal antibodies (e.g., infliximab and vedolizumab), can block key immune pathways, these drugs are expensive, carry risks of adverse reactions such as immunosuppression and susceptibility to infection, and cannot directly eliminate pathogenic bacteria. Furthermore, while antimicrobial peptides (e.g., human defensins) have broad-spectrum antimicrobial effects, they have a short half-life in the body, are easily hydrolyzed by enzymes, and lack targeting, limiting their effectiveness in local intestinal applications.

[0005] More importantly, there is currently a lack of integrated treatment strategies that can simultaneously achieve the multiple functions of "antibacterial-repair-immunomodulation." Simple combinations of probiotics with antimicrobial ingredients such as bacteriophages and antimicrobial peptides suffer from low synergistic efficiency, poor component compatibility, and poor stability in the complex intestinal environment, making it difficult to achieve multi-mechanism synergistic intervention in the pathological process of IBD. Therefore, there is an urgent need to develop a new intestinal intervention technology that combines targeted antimicrobial, repair-promoting, and immune-modulating functions to achieve more effective and long-lasting treatment for IBD. Summary of the Invention

[0006] The present invention provides a composition comprising probiotics and functional fusion polypeptides, which is used to treat intestinal inflammatory diseases by enhancing probiotic colonization, inhibiting pathogens and neutralizing toxins.

[0007] Therefore, in one aspect, the present invention discloses a high-affinity single-chain antibody against Clostridium difficile toxin B, the amino acid sequence of which is shown in SEQ ID NO.2.

[0008] In one aspect, the present invention further discloses a fusion polypeptide, the amino acid sequence of the fusion polypeptide is shown as SEQ ID NO.1.

[0009] Preferably, the codon-optimized nucleotide sequence of the fusion polypeptide of the present invention is shown in SEQ ID NO.3.

[0010] In one aspect, the present invention further discloses a composition comprising probiotics, the composition comprising the following components:

[0011] (1) Probiotics: Lactobacillus plantarum CCFM8661, concentration ≥1×10 9 CFU / g;

[0012] (2) Fusion polypeptide: The fusion polypeptide is mixed with probiotics in a mass ratio of 1:0.5 in the composition.

[0013] Preferably, the composition of the present invention is an enteric-coated microcapsule prepared by a freeze-drying process, the coating material of the enteric-coated microcapsule is hypromellose, and the particle size of the enteric-coated microcapsule is 300-500 μm.

[0014] In one aspect, the present invention also discloses a use of the fusion polypeptide in preparing a composition containing probiotics.

[0015] In one aspect, the present invention further discloses a use of the composition comprising probiotics in the preparation of a medicine for treating gastrointestinal diseases.

[0016] The composition of the present invention has the following beneficial effects:

[0017] 1. Synergistic effect: The fusion peptide synergistically reduces intestinal inflammation with probiotics through the triple mechanisms of adhesion, antibacterial and toxin neutralization (the efficacy of the combination group was significantly better than that of the single component, P < 0.001);

[0018] 2. Targeted delivery: Enteric-coated microcapsules protect the active ingredients, with a gastric acid survival rate of >90% and a colon-targeted release rate of >80%;

[0019] 3. Clinical translation advantages: The preparation process is stable (SDS-PAGE purity >95%), and the viable bacterial retention rate of the lyophilized preparation is ≥80% after 3 months of storage at room temperature;

[0020] 4. Safety: No hepatotoxicity, renal toxicity or immune overactivation was observed in animal experiments.

[0021] The above results indicate that the composition comprising probiotics and functional fusion polypeptides of the present invention can be widely used in multiple fields such as probiotic preparations, antibiotic substitutes, enteritis treatment, and bioprotection preparations, and has good commercial application prospects and market promotion potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Fusion protein SDS-PAGE detection results, where 1 is the fusion protein.

[0023] Figure 2 Western blot detection results of fusion proteins, where 1 is the fusion protein. DETAILED DESCRIPTION

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. The terms used herein in the specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0025] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0026] Example 1: Construction, preparation and testing of fusion polypeptides

[0027] 1. Fusion peptide design

[0028] This example constructs a fusion polypeptide with the triple functions of intestinal localization, antibacterial and toxin neutralization. Its full-length amino acid sequence is shown in SEQ ID NO.1 and contains four functional segments. The details are as follows:

[0029] 1. Adhesion domain: Derived from the surface protein of Lactobacillus plantarum SlpA. This fragment is located in the N-terminal domain of SlpA and has the ability to bind to the GPI-anchor protein of intestinal epithelial cells, conferring intestinal targeting to the fusion protein. Its amino acid sequence is: MKLFIAVLSLLLQSVASTQKQKVTAAQNPAANKTVKQTLDKVKEIADKVQKTVTQAT.

[0030] 2. Flexible linker peptide: Derived from the commonly used linker peptide Gly-Ser repeating structure, it can avoid spatial interference between functional regions and ensure protein folding freedom. The specific amino acid sequence is: GGGGSGGGGSGGGGS.

[0031] 3. Antimicrobial peptide domain: Derived from human β-defensin-3 (hBD-3), it possesses broad-spectrum antimicrobial activity against Gram-positive and Gram-negative bacteria, including Clostridium difficile, Escherichia coli, and Staphylococcus aureus. The specific amino acid sequence is: GIINTLQKYYCRVRGGRCAVLSCLPKEEQIGKCSTRGRKCCRRKK.

[0032] 4. Toxin binding domain: The inventors prepared a high-affinity single-chain antibody against Clostridium difficile toxin B (as shown in Example 3). This scFv can effectively neutralize TcdB toxin and prevent it from binding to host cells. Its specific amino acid sequence is shown in SEQ ID NO.2.

[0033] 2. Construction of Fusion Peptide Gene

[0034] 1. Fusion polypeptide sequence design and codon optimization: The full-length fusion polypeptide DNA sequence was designed using SnapGene v6.0 or Geneious Prime, and codon optimization was performed using the JCat online tool for E. coli-preferred codons, with a target CAI (Codon Adaptation Index) > 0.9 and a GC content range of 30% to 70%. Restriction enzyme recognition sites were added at both ends of the sequence: NdeI upstream and XhoI downstream. A 6×His tag was also introduced downstream for purification and detection. The nucleotide sequence of the codon-optimized fusion polypeptide is shown in SEQ ID NO. 3.

[0035] 2. Vector Construction: The codon-optimized fusion polypeptide gene fragment was fully synthesized and constructed into pET-28a(+). The pET-28a(+) vector and the fusion gene insert were double-digested with NdeI and XhoI, respectively (37°C, 2 h). The fragments were recovered by agarose gel electrophoresis. Ligation was performed using T4 DNA ligase at a molar ratio of insert:vector = 3:1. The specific reaction system is shown in Table 1. The ligation reaction was incubated at 4°C overnight. The ligation product was transformed into DH5α competent Escherichia coli, and single colonies were picked from the plates for plasmid amplification.

[0036] Table 1 Ligation reaction system

[0037]

[0038] 3. Plasmid extraction and sequence verification: Use a plasmid miniprep kit to extract the recombinant plasmid in the DH5α strain; perform Sanger sequencing with T7 forward / reverse primers or specific internal primers to confirm that the fusion fragment sequence has no base mutations, no frameshift mutations, and the restriction enzyme cutting sites are intact.

[0039] 4. Construction of expression strain: The verified recombinant expression plasmid was transformed into BL21 (DE3) competent cells; positive strains were screened by coating LB+Kan plates; PCR or colony PCR was used to preliminarily confirm the presence of the fusion gene; and the cells were stored in glycerol (30%)-LB liquid culture medium (-80°C for long-term storage).

[0040] 3. Expression and purification of fusion polypeptides

[0041] 1. Optimization of expression conditions and induced expression

[0042] 1.1 Screening of optimal induction conditions: BL21 (DE3) engineered bacteria were inoculated into LB liquid medium containing kanamycin (50 μg / mL), and the following conditions (Table 2) were compared to optimize the induced expression. After screening and testing, group B was the best.

[0043] Table 2 Screening of different conditions

[0044]

[0045] 1.2 Expression operation steps: Pick a single positive clone and inoculate it into 5 mL LB-Kan culture medium, pre-culture at 37°C and 220 rpm for 8 h; take 1% of the inoculum (such as 1 mL of seed solution) and inoculate it into 100 mL LB-Kan culture medium; culture until OD 600 ≈0.6; IPTG was added to a final concentration of 0.5 mM for induction, and the cells were cultured at 16°C with shaking for 16 h; after the induction, the cells were collected by centrifugation (5000×g, 10 min, 4°C) for subsequent lysis and purification.

[0046] 2. Bacterial Lysis and Soluble Protein Detection

[0047] 2.1 Ultrasonic disruption: Resuspend the cells in pre-chilled lysis buffer (50 mM Tris-HCl, 300 mM NaCl, 10 mM Mimidazole, 1 mM PMSF, 1 mg / mL Lysozyme, pH 8.0) (10 mL / 100 mL original culture volume); ultrasonically disrupt the culture in an ice bath (e.g., 10 s working / 20 s rest × 15 min, total energy approximately 200 W); centrifuge at 12,000 × g for 20 min at 4°C. The supernatant is used as the soluble protein, and the precipitate is used to determine whether inclusion bodies have formed. The fusion protein is detected as a soluble supernatant protein.

[0048] 3.Ni-NTA affinity chromatography purification

[0049] 3.1 Materials and Equipment: Ni-NTA prepacked column (Qiagen 1 mL); elution buffer was prepared as follows:

[0050] Table 3 Buffer configuration table

[0051]

[0052] 3.2 Operation steps: Load the supernatant onto a Ni-NTA column (pre-equilibrated); wash nonspecifically bound proteins with 10 column volumes of wash buffer; collect the elution peak with elution buffer (gradient or one-time wash); filter and concentrate using a 0.22 μm filter at 4°C and exchange the solution into PBS buffer; add glycerol to a final concentration of 10% and store frozen at -80°C.

[0053] 4. Expression and Purification Verification

[0054] 4.1 SDS-PAGE detection: Take 20 μL of the purified fusion protein and separate it using 12% SDS-PAGE gel, and then stain it with Coomassie brilliant blue. The results show ( Figure 1 ), the molecular weight of the fusion protein is approximately 41.7 kDa, and the degree should be >95%.

[0055] 4.2 Western Blot Verification: Take 20 μL of the purified fusion protein and separate it using 12% SDS-PAGE gel. After transfer to the membrane, incubate with the antibody. The antibody is HRP-labeled Mouse anti-His tag (1:5000). ECL chemiluminescence is used for color development. The results show ( Figure 2 ), with a specific band at a molecular weight of approximately 41.7 kDa.

[0056] 5. Expression Statistics: The protein concentration was determined using the BCA assay (Thermo Scientific kit). The results showed that the concentration of the fusion protein was 3.56 mg / mL. After calculation, the expression level after purification was approximately 1.2 g / L culture medium. Therefore, the fusion protein has a relatively high expression level, laying the foundation for its large-scale application.

[0057] Example 2: Functional verification experiment of fusion polypeptide

[0058] This example verifies the triple function of the fusion polypeptide prepared in Example 1 in promoting the adhesion of probiotics, inhibiting the growth of Clostridium difficile and neutralizing its toxins.

[0059] 1. Verification of adhesion promotion function

[0060] 1. Experimental steps: Human intestinal epithelial Caco-2 cells were seeded in 24-well plates (1×10 5 cells / well) and cultured to a monolayer in DMEM medium containing 10% FBS; DiO fluorescent probe was used to label Lactobacillus plantarum (1×10 7 CFU / mL); the fusion polypeptide was diluted to 0 (blank), 5 μg / mL, and 10 μg / mL, respectively, and preincubated with Lactobacillus for 30 min; the mixture was added to Caco-2 cell wells and incubated at 37°C for 2 hours; the cells were washed three times with PBS to remove unbound bacteria; the fluorescence intensity (Ex / Em=484 / 501 nm) was measured using a multifunctional microplate reader, and the relative adhesion rate was calculated.

[0061] 2. The results (Table 4) show that the fusion peptide significantly enhances the adhesion of probiotics to Caco-2 cells in a concentration-dependent manner. This effect is attributed to the ability of its N-terminal SlpA-derived domain to recognize and bind to the intestinal GPI-anchor structure.

[0062] Table 4 Adhesion promotion function verification results

[0063]

[0064] 2. Antibacterial activity test (against Clostridium difficile)

[0065] 1. Experimental procedures: Prepare BHIS agar plates and inoculate a suspension of Clostridium difficile (ATCC 43255) on the plate surface. Add the following: fusion peptide solution (10 μg), vancomycin (10 μg, positive control), and PBS buffer (negative control) to sterile filter paper. Attach the filter paper to the plate surface and incubate anaerobically at 37°C for 24 hours. Measure the diameter (mm) of the inhibition zone as an indicator of antibacterial efficacy.

[0066] 2. The results showed (Table 5) that the fusion polypeptide exhibited an inhibition zone diameter superior to that of vancomycin, indicating that the antimicrobial peptide HBD-3 synergistically inhibited the growth of Clostridium difficile by destroying the pathogen membrane structure.

[0067] Table 5 Antibacterial activity test results

[0068]

[0069] 3. Toxin neutralization function test (for TcdB)

[0070] 1. Experimental procedures: Vero cells (African green monkey kidney cells, TcdB-sensitive) were seeded in 96-well plates and cultured to 90% confluency. TcdB toxin (final concentration 50 ng / mL), TcdB + fusion peptide (5 μg / mL or 10 μg / mL), TcdB + known anti-TcdB scFv positive control (10 μg / mL), and PBS control were added. Incubation was continued for 24 hours. Cell viability was measured using the CCK-8 assay (450 nm absorbance), and toxin neutralization efficiency was calculated. Calculation formula: Toxin neutralization efficiency = [(OD experimental group - OD toxin group) / (OD control group - OD toxin group)] × 100%.

[0071] 2. The results (Table 6) show that the fusion peptide significantly neutralized TcdB toxin and restored Vero cell viability, with efficacy comparable to or better than that of the positive scFv antibody. This suggests that the C-terminal antitoxin domain retains biological activity in the fusion configuration and may be a candidate for antitoxin therapy.

[0072] Table 6 Toxin neutralization function test results

[0073]

[0074] Example 3: Preparation of high-affinity single-chain antibody (scFv) against Clostridium difficile toxin B

[0075] 1. Immunization and antibody library construction: First, mice were immunized intraperitoneally with TcdB (Sino Biological, 50 μg) combined with Freund's complete adjuvant, and two booster immunizations were performed with incomplete adjuvant on the 7th and 14th days respectively. Blood was collected from the tail vein on the 4th week and the anti-TcdB antibody titer was detected by ELISA. Three mice with higher antibody titers were screened, and their spleens were taken for subsequent antibody library construction. Subsequently, total spleen RNA was extracted using TRIzol reagent, and reverse transcription reaction was performed to obtain cDNA, which was used as a template for subsequent PCR amplification of antibody variable region sequences. Universal primers were used to amplify the VH and VL regions, respectively, and Overlap PCR technology was used to connect them into a VH-(G4S)3-VL scFv structure. At the same time, an SfiI restriction site was introduced to facilitate subsequent cloning. The constructed scFv fragment was inserted into the pComb3XSS phage display vector and introduced into E. coliTG1 competent cells by electroporation to form a phage display library. The library capacity was estimated to be greater than 1×10 8 CFU, with an insertion rate of about 90%.Finally, the cells were infected with M13KO7 helper phage to obtain a highly efficient display scFv phage library.

[0076] 2. scFv screening and validation: First, three rounds of solid-phase panning screening were performed using TcdB-coated 96-well plates. The phage library was added to the coated wells and incubated at room temperature. Nonspecific binding was removed by increasing the salt concentration and wash intensity in successive rounds, with the most stringent wash conditions used in the third round to enrich for high-affinity phage. The eluted phage were infected with the E. coli TG1 strain, and positive clones were selected for subsequent analysis. ELISA validation was then performed. The supernatant of the monoclonal culture was added to a TcdB-coated 96-well plate, and the color reaction was developed using an HRP-labeled anti-M13 antibody. Positive clones with strong binding ability were selected by OD value. Ultimately, 10 scFv clones with high affinity were screened and sequenced.

[0077] 3. scFv gene subcloning and protein expression

[0078] (1) To achieve recombinant expression of a high-affinity single-chain antibody (scFv) against Clostridium difficile toxin B, the screened scFv gene was subcloned into the pET-28a(+) expression vector. First, NcoI and XhoI restriction sites were introduced at both ends of the scFv to ensure smooth insertion into the vector's multiple cloning site, and the 6×His tag at the C-terminus was retained for subsequent purification. After double enzyme digestion, the PCR product was ligated to the linearized pET-28a(+) vector using T4 ligase at 16°C, transformed into the competent DH5α strain for amplification, and after Sanger sequencing confirmed that the inserted sequence had no mutations, it was transformed into Escherichia coli BL21(DE3) for protein expression.

[0079] (2) During the protein induction expression stage, BL21 (DE3) containing the recombinant plasmid was inoculated into LB medium containing kanamycin (Kan) and cultured at 37°C until OD 600 When the p-value was approximately 0.6, 0.5 mM IPTG was added for induction, and expression was allowed to proceed overnight at 16°C. The next day, the cells were harvested, lysed by ultrasonication, and centrifuged. The supernatant was then purified for protein purification. The His-tagged scFv in the supernatant was purified using Ni-NTA affinity chromatography resin and dialyzed against PBS buffer to remove excess salt, yielding a highly pure, functional recombinant scFv protein.

[0080] 4. Functional testing and affinity evaluation: In order to verify the binding ability and neutralization effect of the screened scFv antibodies on Clostridium difficile toxin B (TcdB), a series of functional testing and affinity evaluation experiments were carried out.

[0081] (1) First, the affinity was evaluated by ELISA. After TcdB was coated on the surface of a 96-well plate, scFv antibodies with different concentration gradient dilutions were added, and the color reaction was performed with HRP-labeled anti-His secondary antibody. The half effective concentration (EC50) was calculated based on the change in absorbance. 50 The results showed that the EC of scFv antibody 50 The affinity of the antibody was 2.7 nM, which was significantly better than that of the commercial antibody (ab270452, 15.8 nM) tested simultaneously, indicating that it has stronger affinity binding ability.

[0082] (2) Subsequently, surface plasmon resonance (SPR) technology was used on the Biacore T200 platform to further accurately determine its affinity constant (KD). In the experiment, TcdB was coupled to the surface of a CM5 chip, and different concentrations of scFv antibodies were injected to analyze the binding kinetic parameters. The results showed that the KD value of the scFv antibody was 1.1nM, while the KD value of the commercial antibody (ab270452) was 15.9nM; therefore, the dissociation rate of the scFv antibody was significantly lower than that of the commercial antibody, indicating that it binds to TcdB more stably and has an affinity increased by about 14.5 times.

[0083] (3) Finally, the neutralization activity was verified using a Vero cell cytotoxicity protection assay. The scFv antibody was pre-incubated with TcdB at different concentrations and then added to the Vero cell culture system. After 24 hours, the cell activity was detected using the MTT assay. The results (Table 7) showed that when the scFv concentration was 100 nM, the cell survival rate reached 79.7%, while the toxin-treated control group was only 21% and the commercial antibody (ab270452) was 58.6%. This indicates that the scFv has good toxin neutralization activity and potential protective effects, which is better than the commercial antibody.

[0084] Table 7 Comparison of neutralization activity verification results

[0085]

[0086] 4. scFv antibody amino acid sequence: The sequence is a VH-(G4S)3-VL structure, where VH and VL are derived from positive clones screened from immunized mice and have undergone humanized modification and optimization of the amino acid sequence. Therefore, the amino acid sequence of the scFv antibody is shown in SEQ ID NO.2.

[0087] Example 4: Composition and preparation of a composition containing probiotics

[0088] 1. Composition of the composition containing probiotics

[0089] 1.1 Probiotics: Lactobacillus plantarum CCFM8661 was selected. The source of the strain is detailed in Chinese patent application publication number CN111869735A. This strain has excellent intestinal adhesion and antibacterial ability.

[0090] (1) Content standard: ≥1×10 9 CFU / g (viable bacteria count after freeze-drying);

[0091] (2) Culture conditions: MRS liquid medium, anaerobic culture at 37°C for 18 hours;

[0092] (3) Harvesting method: Centrifuge at 8000 rpm for 10 minutes, discard the supernatant, resuspend in sterile PBS and use for premixing before lyophilization.

[0093] 1.2 Fusion polypeptide: the fusion polypeptide prepared in Example 1.

[0094] (1) Purity requirement: >90% (SDS-PAGE);

[0095] (2) Addition ratio: Mix with probiotics at a mass ratio of 1:0.5 (i.e., 1g bacterial powder + 0.5g fusion polypeptide).

[0096] 2. Preparation process of composition containing probiotics

[0097] 2.1 Mixing and pre-lyophilization treatment: The probiotics were concentrated by centrifugation and resuspended, and then mixed with the fusion polypeptide under sterile conditions; a freeze-drying protectant system (10% trehalose, 5% skim milk) was added to protect the cell membrane structure and protein conformation, and to improve the survival rate and stability of the freeze-dried product; the mixture was divided into freeze-drying bottles (≤10mL / bottle), pre-frozen at -80℃ for 4 hours, and then entered the freeze-drying process.

[0098] 2.2 Freeze-drying program parameter setting (freeze drying):

[0099] (1) Pre-freezing temperature: -80°C, time: 4 hours;

[0100] (2) Initial drying stage: -40℃ to -10℃, vacuum degree <50Pa, for 18 hours;

[0101] (3) Secondary drying stage: raise the temperature to 25°C and maintain for 10 hours, with the moisture content <3%.

[0102] 2.3 Enteric Coating and Microencapsulation

[0103] (1) Use of Hydroxypropyl Methylcellulose (HPMC) as an enteric coating material (soluble in pH > 6.0 to protect the active ingredient from being destroyed by gastric acid);

[0104] (2) Coating method: suspend the freeze-dried powder in an ethanol / water mixture and add 2% HPMC solution; use spray drying or fluidized bed coating technology, set the inlet air temperature to 60-70°C and the outlet air temperature to no higher than 40°C; control the final microcapsule particle size to 300-500 μm; after drying, seal and store in a light-proof, dry, low-temperature (4°C) environment.

[0105] 3. Product stability verification

[0106] 3.1 First, a systematic test was conducted on the number of viable probiotics. After Lactobacillus plantarum CCFM8661 and the fusion polypeptide were freeze-dried and coated, they were stored under refrigeration (4°C), room temperature (25°C) and accelerated aging (40°C) conditions, and the changes in their viable counts were continuously monitored. The results showed that after 6 months of refrigeration, more than 90% of the bacterial activity could still be maintained, while the activity retention rate dropped slightly to about 80% after 3 months of storage at room temperature. The activity decreased rapidly at 40°C, but it still had acceptable stability, indicating that the composition has good viable bacterial stability under reasonable storage conditions.

[0107] (2) To evaluate the functional retention of the fusion polypeptide during the preparation process, ELISA and cell neutralization experiments were used for verification. The results showed that the OD value of the polypeptide binding activity before and after freeze-drying decreased slightly from 1.280 to 1.192, with a retention rate of approximately 93.1%; in the TcdB toxin neutralization experiment, after Vero cells were treated with 100nM concentration of fusion polypeptide, the cell survival rate increased significantly from 22.6% in the untreated group to 78.5%, which was basically consistent with 81.2% before freeze-drying, indicating that the freeze-drying and microencapsulation process did not significantly affect the function of the polypeptide.

[0108] (3) To evaluate the enteric coating effect of the microcapsules, in vitro release experiments were further conducted in simulated gastric fluid at pH 1.2 and simulated intestinal fluid at pH 6.8. The results showed that in the gastric fluid environment, the release rates of the fusion peptide and probiotics were both less than 10%, while the release efficiency in the intestinal fluid was significantly improved, reaching more than 80% and more than 85%, respectively. This fully verified that the HPMC coating used has good pH-dependent release ability and can achieve effective gastrointestinal targeted release.

[0109] Comprehensive analysis shows that the fusion polypeptide-probiotic composition has good freeze-drying stability, environmental tolerance and targeted release performance while ensuring biological activity, and is suitable for promotion as a functional biological preparation in oral applications.

[0110] Example 5: Study on the therapeutic effect of the composition in a DSS-induced colitis model in mice

[0111] 1. Experimental Animals and Grouping: 40 SPF-grade C57BL / 6 male mice (20-22 g) aged 6-8 weeks were randomly divided into 4 groups, with 10 mice in each group.

[0112] (1) Control group: 0.2 mL of normal saline was gavaged daily.

[0113] (2) Probiotic group: daily gavage of Lactobacillus plantarum CCFM8661, dose 1×10 9 CFU.

[0114] (3) Fusion polypeptide group: Fusion polypeptide (20 mg / kg) dissolved in PBS was administered orally daily.

[0115] (4) Combination group: daily gavage of probiotics + fusion polypeptide, the dosage is the same as above.

[0116] 2. Model establishment and intervention plan

[0117] (1) Starting from day 0, mice were allowed to freely drink drinking water containing 3% (w / v) dextran sodium sulfate (DSS) for 7 consecutive days to establish an acute colitis model.

[0118] (2) From day 1, except for the control group, the other groups began to be gavaged with the corresponding treatment substances every day for 14 days (including the DSS modeling period and the recovery period).

[0119] 3.Indicator detection and methods:

[0120] (1) Disease Activity Index (DAI) score (recorded daily): weight change score (0-4 points), stool morphology score (0-4 points), and stool blood score (0-4 points); DAI = the average of the three scores, and the data on day 14 were finally used for comparison.

[0121] (2) Colon length measurement: After the animals were sacrificed, the colon was removed and the length from the cecum to the anus was measured as an indirect reflection of the degree of inflammation.

[0122] (3) Histopathological analysis (HE staining): The distal colon was paraffin-embedded, sectioned, and stained with HE. Crypt structure, epithelial integrity, goblet cell count, and inflammatory cell infiltration were observed. The degree of inflammation was analyzed using a blinded semiquantitative scoring system (0-4 points).

[0123] (4) Serum inflammatory factor detection (ELISA): Orbital blood was collected to separate serum, and the concentrations of IL-6 and TNF-α were detected using the BioLegend mouse ELISA kit.

[0124] 4. Experimental results are shown in Table 8.

[0125] (1) DAI score and colon length: The average DAI of the control group was 8.2, the colon was significantly shortened (6.1 cm), and there was obvious diarrhea and weight loss; the DAI of the combination group decreased significantly to 2.3, the colon length recovered to 9.8 cm, and inflammation was significantly relieved; the use of probiotics or peptides alone was also effective, but lower than the combination group (P < 0.01).

[0126] (2) Changes in inflammatory factors: In the control group, IL-6 and TNF-α were significantly increased, reaching 245 pg / mL and 180 pg / mL, respectively. In the combination group, IL-6 decreased to 68 pg / mL and TNF-α to 45 pg / mL after treatment, representing decreases of 72% and 75%, respectively. The peptide group and the probiotic group also showed a downward trend, but the combination group had a better effect (P < 0.001).

[0127] (3) Histological results: In the control group, the crypt structure was extensively destroyed, with more than 80% goblet cell loss and a large number of neutrophil infiltration; in the combination group, the number of goblet cells recovered to 70% of normal, the mucosal structure was reconstructed, and the inflammation score decreased by 85%; the polypeptide group and the probiotic group also showed some improvement, but not as good as the combination group.

[0128] Table 8 Summary of experimental results

[0129]

[0130] Note: *P<0.05, **P<0.01, ***P<0.001, all were statistically significant compared with the control group.

[0131] 5. Mechanism discussion and summary: The probiotic + fusion peptide combination showed significant synergistic effects:

[0132] (1) Colonization promotion: The fusion peptide enhanced the adhesion of probiotics to the intestinal mucosa of mice. Fluorescence quantitative analysis showed that the retention time of the bacteria in the combination group was prolonged by about 3.5 times.

[0133] (2) Dual anti-pathogen ability: HBD-3 peptide directly kills conditional pathogens. Western blot showed that the binding rate of antibody scFv to Clostridium difficile toxin B exceeded 90%, significantly neutralizing its toxicity.

[0134] (3) Immune regulatory effect: The combination group significantly reduced the level of serum pro-inflammatory factors, restored the integrity of the intestinal epithelial barrier, increased the distribution of goblet cells, and effectively alleviated colitis.

[0135] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A high-affinity single-chain antibody against Clostridium difficile toxin B, characterized in that The amino acid sequence of the single-chain antibody is shown in SEQ ID NO.

2.

2. A fusion polypeptide comprising the single-chain antibody according to claim 1, characterized in that: The amino acid sequence of the fusion polypeptide is shown in SEQ ID NO.

1.

3. The fusion polypeptide according to claim 2, characterized in that The nucleotide sequence of the fusion polypeptide after codon optimization is shown in SEQ ID NO.

3.

4. A composition comprising probiotics, characterized in that The composition comprises the following components: (1) Probiotics: Lactobacillus plantarum CCFM8661, concentration ≥1×10 9 CFU / g; (2) Fusion polypeptide: The fusion polypeptide according to claim 2, wherein the fusion polypeptide in the composition is mixed with the probiotics at a mass ratio of 1:0.

5.

5. The composition according to claim 4, characterized in that The composition is an enteric-coated microcapsule prepared by a freeze-drying process. The coating material of the enteric-coated microcapsule is hypromellose, and the particle size of the enteric-coated microcapsule is 300-500 μm.

6. Use of the fusion polypeptide according to claim 2 in preparing a composition containing probiotics.

7. Use of the composition comprising probiotics according to claim 4 in the preparation of a medicament for treating gastrointestinal diseases.

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