Female vagina flora repairing liquid as well as preparation method and application thereof

Abstract

The invention discloses a compound preparation for regulating vaginal microecology as well as preparation and application thereof, and relates to the field of biological medicines. The composite preparation is composed of a fusion polypeptide and a monoclonal antibody according to the ratio of 1: 2 (w / w), the fusion polypeptide comprises a cell adhesion domain, an enzymolysis linking peptide and a quorum sensing inhibition domain, the amino acid sequence is shown as SEQ ID NO: 1, and the protease hydrolysis resistance is improved by more than 4 times after cyclization oxidation; the monoclonal antibody takes specific surface protein of lactobacillus inertus as a target spot, and can induce the strain to generate H2O2 phenotype conversion. An in-vitro experiment shows that the degradation rate of the composite preparation on a vaginal bacterial biofilm reaches 92.3 + / -2.4%, the ratio of lactobacillus acidophilus is remarkably increased to 63.8 + / -3.3%, and the pH is effectively adjusted to 4.3; animal experiments show that compared with commercially available metronidazole gel, the compound preparation can more remarkably promote proliferation of lactobacillus, inhibit escherichia coli and staphylococcus aureus and restore vaginal micro-ecological balance, and a new scheme is provided for treatment of vaginal micro-ecological imbalance related diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a female vaginal flora repairing liquid, a preparation method thereof, and an application thereof. Background Art

[0002] The vaginal microbiome is a dynamic equilibrium system composed of a variety of microorganisms and the host's vaginal tissue. A dominant bacterial community, Lactobacillus, is crucial for maintaining vaginal health. When the microbiome is imbalanced, especially when conditions such as bacterial vaginosis (BV) occur, existing treatment options have significant limitations, as shown in the following aspects:

[0003] Limitations of Antibiotic Therapy: Antibiotics, such as metronidazole, are currently commonly used to treat vaginal infections. While their broad-spectrum antimicrobial properties can inhibit some pathogenic microorganisms, their indiscriminate killing mechanism severely damages beneficial vaginal bacteria, such as Lactobacilli. This not only makes it difficult to restore vaginal microbial balance but also increases the risk of recurrent infection due to dysbiosis. It also induces the development of drug-resistant strains, further weakening the effectiveness of treatment.

[0004] Disadvantages of probiotic preparations: As an emerging microecological modulator, probiotic preparations can theoretically restore vaginal microecological balance by replenishing beneficial bacteria. However, practical application faces numerous challenges. Furthermore, probiotic preparations lack the ability to effectively inhibit and eliminate pathogenic biofilms. The presence of biofilms allows pathogens to evade the body's immune defenses and medications, leading to persistent infections.

[0005] The dilemma of antimicrobial peptide application: Antimicrobial peptides have become a research hotspot due to their unique antimicrobial activity, such as the antimicrobial peptides involved in patent CN119978059A. However, they have obvious defects in practical application. Antimicrobial peptides are easily hydrolyzed by proteases in the vagina, resulting in a rapid decrease in their biological activity; more importantly, they lack effective regulatory effects on inert lactobacilli (Lactobacillus iners). Lactobacillus iners often become the main colonizing bacteria after BV occurs. Due to their low H2O2 production characteristics, they are unable to effectively maintain the acidic environment of the vagina and exert antibacterial effects, which in turn leads to a slow repair process of the vaginal microenvironment and increases the chance of disease recurrence.

[0006] In summary, existing treatment options for vaginal microecological imbalance are unable to effectively achieve precise regulation of the vaginal flora and rapid reconstruction of the microbiome. Therefore, there is an urgent need to develop innovative formulations that can simultaneously degrade pathogenic bacterial biofilms, specifically regulate the phenotype of indigenous lactobacilli, and promote the reconstruction of beneficial bacterial ecosystems. This could fill the existing technological gap and provide a more effective solution for the treatment of diseases associated with vaginal microecological imbalance. Summary of the Invention

[0007] The purpose of the present invention is to provide a female vaginal flora repair liquid, which, by introducing specific fusion polypeptides and monoclonal antibodies, can specifically regulate the vaginal flora, inhibit the growth of harmful bacteria, promote the proliferation of beneficial bacteria, and quickly restore the vaginal microecological balance; at the same time, provide a preparation method of the repair liquid to ensure stable and controllable product quality; and clarify its application in the treatment and prevention of diseases related to female vaginal flora imbalance.

[0008] Therefore, on one hand, the present invention discloses a fusion polypeptide having an amino acid sequence as shown in SEQ ID NO: 1, and a structure comprising a cell adhesion domain, an enzymatic linker peptide, and a quorum sensing inhibitory domain. The cell adhesion domain "TWHIQKKD" is derived from human collagen binding protein and has been experimentally verified to have stronger adhesion to vaginal epithelial cells SiHa than the vaginal adhesion peptide YPS30. At a concentration of 10 μM, the OD value caused by its adhesion reached 0.68±0.03, significantly higher than the 0.25±0.02 of YPS30 at a concentration of 1 μM. The quorum sensing inhibitory domain "KKLFKKILKYL" is modified from the HLA-G antimicrobial peptide, which increases its biofilm penetration ability by approximately 40% compared to the original HLA-G antimicrobial peptide. At a concentration of 2 μM, it has a significant inhibitory effect on Gardnerella biofilms, with the OD value reduced to 0.18±0.01, significantly lower than the 0.45±0.03 of the original HLA-G antimicrobial peptide at a concentration of 1 μM. The enzymatic linker peptide "YGGGS" can be cleaved under the specific enzymatic environment of the vagina, achieving the step-by-step release of the functional domains. Furthermore, cysteines were added to the N- and C-termini of the fusion polypeptide to form intramolecular disulfide bonds. Experiments simulating vaginal conditions have shown that the cyclized polypeptide's resistance to protease hydrolysis is significantly enhanced, with the half-life extending from 2.1±0.3 hours of the original linear peptide to 8.7±0.6 hours, and the 6-hour residual rate increasing from 15.2% to 83.5%.

[0009] On the one hand, the present invention also discloses a monoclonal antibody that specifically targets a surface protein unique to Lactobacillus iners (UniProtKB: E1NRP2), which has been shown to be highly conserved and immunogenic by bioinformatics analysis. After binding to the surface protein, the monoclonal antibody of the present invention can block protein modification, promote the recombination of the metabolic pathway of Lactobacillus iners, and induce the strain to convert to a H2O2-producing phenotype. Experimental verification showed that in the experimental group with the addition of 50 μg / mL monoclonal antibody, the H2O2 production of Lactobacillus iners increased from 0.8±0.2 μM / 10 8 The CFU increased significantly to 9.3±0.7μM / 10 8 CFU, increased by 10.6 times (p < 0.01).

[0010] The present invention also provides a method for preparing the above-mentioned composite formulation, comprising mixing the fusion polypeptide and the monoclonal antibody at a 1:2 (w / w) ratio in a buffer solution consisting of 25 mM histidine, 15 mM succinate, 75 mM arginine, and 100 mM NaCl (pH 4.5). This optimized buffer system significantly improves the stability of the monoclonal antibody compared to conventional pH 4.5 citric acid buffer, reducing the aggregate content from 22.3% ± 1.8% to 4.1% ± 0.5%, the deamidation rate from 35.7% ± 2.1% to 7.9% ± 0.7%, and the activity retention from 58% to 94%. After mixing, 5% (w / v) trehalose, 0.01% (w / v) polysorbate 80, 0.05% (w / v) methionine, and 5% (w / v) gelatin are added in sequence as freeze-drying protectants to further improve the stability of the preparation, reduce the content of monoclonal antibody aggregates to 1.8% ± 0.3%, and increase the activity retention rate to 96%. The mixture is then divided into sterile vials, pre-frozen at -40°C for 4-6 hours, evacuated to 25 Pa, heated to -20°C at a rate of 0.5°C / min and maintained for 12-15 hours for sublimation drying, then heated to 25°C and maintained for 8-10 hours to complete analytical drying. Finally, sterile nitrogen is filled under vacuum and vacuum-sealed with a capping machine to obtain a reconstituted freeze-dried powder, which is dissolved with 5% sodium lactate solution before use.

[0011] The compound preparation prepared by the above method has the following beneficial effects:

[0012] 1. Significant synergistic effect: In vitro activity validation experiments demonstrated that the combined use of the fusion peptide and monoclonal antibody exhibited a significant synergistic effect. The combination formulation achieved a vaginal bacterial biofilm degradation rate of 92.3±2.4%, significantly higher than the 68.5±3.8% achieved by the fusion peptide group and the 9.4±0.7% achieved by the monoclonal antibody group. The proportion of Lactobacillus acidophilus increased to 63.8±3.3%, surpassing the 41.2±2.1% achieved by the monoclonal antibody group. The pH was adjusted to 4.31, closer to the vaginal pH of healthy women (3.8-4.5), demonstrating superior efficacy compared to single-agent treatment.

[0013] 2. Complementary mechanisms of action: The fusion peptide specifically adheres to vaginal epithelial cells, directly disrupting the biofilm of harmful bacteria and inhibiting their growth; the monoclonal antibody targets a surface protein unique to Lactobacillus inergentis, inducing the strain to switch to a H2O2-producing phenotype, indirectly promoting the growth of beneficial bacteria. These two mechanisms work through different mechanisms, forming a multi-target synergistic regulation to comprehensively improve the vaginal microecological environment.

[0014] 3. Excellent results in animal experiments: In a rat model of vaginal dysbiosis, the composite formulation of the present invention adjusted the vaginal pH to 4.3±0.1, effectively promoting the proliferation of Lactobacillus, raising its relative abundance to 78.9±3.5%, while strongly inhibiting Escherichia coli and Staphylococcus aureus, reducing their relative abundance to 8.5±1.5% and 6.2±1.2%, respectively. In comparison, while commercially available metronidazole gel showed some effect, it was significantly inferior to the composite formulation of the present invention in adjusting the pH to 4.9±0.1, increasing Lactobacillus to 36.1±2.3%, reducing Escherichia coli to 28.9±2.6%, and reducing Staphylococcus aureus to 20.4±2.2%. This demonstrates that the present invention is more effective in restoring vaginal microecological balance.

[0015] 4. Good formulation stability: By optimizing the buffer system and selecting a specific lyoprotectant, the poor stability of monoclonal antibodies in acidic environments is effectively resolved, significantly reducing the aggregate content and deamidation rate, and improving the activity retention rate. Accelerated testing (storage at 40°C for 4 weeks) has demonstrated that the compound formulation has good stability, is easy to store and transport, and is conducive to clinical application and promotion.

[0016] 5. Broad application prospects in the future: This preparation has very broad application prospects in the future. In clinical applications, it can not only be used to treat common bacterial vaginosis, but is also expected to be expanded to the treatment of vaginal microecological imbalance diseases caused by candidal vaginitis, Trichomonas vaginitis and other pathogens. It also has great potential in regulating the vaginal microecology before and after gynecological surgery and maintaining the vaginal microecology of pregnant women. In terms of treatment options, it can be combined with physical therapy and other drug therapies to form a more complete treatment plan and improve the overall treatment effect. In the field of preventive health care, it can be developed into daily care gels, suppositories and other products to meet the preventive health care needs of women's reproductive health. In terms of scientific research and technological innovation, its unique fusion peptide design and monoclonal antibody preparation method can provide new ideas for the biomedicine field, promote the development of peptide drug and antibody drug technology, and also provide powerful tools and research models for vaginal microecology research, thereby promoting the progress of related basic scientific research. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 SDS-PAGE test results of Lactobacillus iners unique surface proteins, where 1 is a Lactobacillus iners unique surface protein. DETAILED DESCRIPTION

[0018] 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.

[0019] 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.

[0020] Example 1: Screening, preparation and testing of fusion polypeptides

[0021] 1. Fusion polypeptide structure and sequence

[0022] The structure of this fusion polypeptide is [cell adhesion domain]-[enzymatic linker peptide]-[quorum sensing inhibitory domain], and its amino acid sequence (SEQ ID NO: 1) is: C -TWHIQKKD-YGGGS-KKLFKKILKYL- GC .

[0023] 1. The cell adhesion domain "TWHIQKKD" is derived from human collagen binding protein;

[0024] 2. The quorum sensing inhibitory domain "KKLFKKILKYL" is modified from the HLA-G antimicrobial peptide. While retaining its antimicrobial activity, its LogP value was optimized to 3.2 by deleting the hemolytic active segment. Computational simulation and experimental verification show that this optimization improves the peptide's ability to penetrate biomembranes by approximately 40% compared to the original HLA-G antimicrobial peptide.

[0025] 3. The enzymatically hydrolyzed linker peptide "YGGGS" can be cleaved under the specific enzymatic environment of the vagina, achieving the step-by-step release of functional domains.

[0026] 4. Cysteine ​​(underlined) was added to the N-terminus and C-terminus of the peptide to form intramolecular disulfide bonds (-SS-) under oxidative conditions to improve the stability of the peptide.

[0027] 2. Fusion Peptide Synthesis and Purification

[0028] 1. Fmoc solid phase synthesis

[0029] Resin preparation: 0.5 g of Wang resin (degree of substitution: 0.8 mmol / g) was weighed and placed in a solid phase synthesis reaction vessel, swollen with DMF (N,N-dimethylformamide) for 30 minutes, and the solvent was removed.

[0030] Amino Acid Coupling: Fmoc-protected amino acids were coupled to the resin sequentially, starting from the C-terminus and ending at the N-terminus of the fusion polypeptide. Each amino acid coupling step was as follows: 20 mL of a 20% piperidine / DMF solution was added to the reaction vessel to remove the Fmoc protecting group. After 15 minutes of reaction, the resin was washed with DMF five times for 5 minutes each. 1.2 equivalents of the Fmoc-protected amino acid, 1.2 equivalents of HCTU (O-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate), and 2.4 equivalents of DIEA (N,N-diisopropylethylamine) were dissolved in DMF and added to the resin. The reaction was shaken at room temperature for 1 hour. After the reaction, the resin was washed with DMF five times for 5 minutes each, followed by three washes with methanol and then dichloromethane for 3 minutes each to remove any residual reagents. Repeat these steps until all amino acids were coupled.

[0031] End-capping treatment: After the synthesis is completed, 20 mL of 1% acetic anhydride / DIEA / DCM (dichloromethane) solution is added to the reaction vessel and the reaction is shaken at room temperature for 30 minutes to cap the unreacted amino groups and prevent side reactions. After the reaction is completed, the resin is washed with DCM and DMF five times, each for 5 minutes.

[0032] 2.HPLC purification

[0033] The synthesized crude peptide was cut from the resin using a cutting reagent consisting of 95% trifluoroacetic acid (TFA), 2.5% water, and 2.5% triisopropylsilane. The reaction was carried out at room temperature for 2 hours. The supernatant was collected by centrifugation and concentrated under reduced pressure to remove TFA. The peptide was precipitated by adding glacial ether, the precipitate was collected by centrifugation, and the crude peptide was obtained by vacuum drying.

[0034] The crude peptide was purified using preparative high performance liquid chromatography (HPLC) using a C18 reverse phase column (250 × 21.2 mm, 5 μm), mobile phase A consisting of 0.1% TFA in water, mobile phase B consisting of 0.1% TFA in acetonitrile, and a gradient elution program of: 0-5 min, 5% B; 5-40 min, 5-60% B; 40-45 min, 60-95% B; 45-50 min, 95% B; at a flow rate of 10 mL / min and a detection wavelength of 220 nm. The eluate corresponding to the target peak was collected and freeze-dried to obtain a fusion polypeptide with a purity greater than 98%.

[0035] 3. Cyclooxidation

[0036] The purified fusion polypeptide was dissolved in 0.1M Tris-HCl buffer (pH 8.0) at a concentration of 1 mg / mL, 1 mM copper sulfate was added as an oxidant, and the reaction was stirred at room temperature for 4 hours to promote disulfide bond formation and achieve cyclization. After the reaction, the cyclization effect was detected by HPLC. The same chromatographic conditions as for purification were used to compare the chromatographic peaks before and after cyclization to confirm that the cyclization reaction was complete. Subsequently, the reaction solution was dialyzed using a dialysis bag (molecular weight cutoff 3500Da) to remove small molecule impurities and freeze-dried to obtain the final fusion polypeptide product.

[0037] In a comparative experiment comparing enzymatic stability, the original linear peptide (TWHIQKKD-YGGGS-KKLFKKILKYL) and the cyclized peptide (SEQ ID NO: 1) were placed in a PBS (pH 4.5) solution containing 0.2 mg / mL trypsin and 0.1 mg / mL papain, respectively, and incubated at 37°C. The peptide was then added at a final concentration of 0.5 mg / mL, and the remaining intact peptide proportion was determined by HPLC. The results showed that the half-life of the original linear peptide was 2.1±0.3 hours, with a 6-hour residual rate of only 15.2%. In contrast, the half-life of the cyclized peptide was extended to 8.7±0.6 hours, with a 6-hour residual rate of 83.5%. This indicates that cyclization treatment increases the peptide's resistance to proteolysis by more than fourfold (p<0.001), significantly enhancing its stability in the simulated vaginal environment.

[0038] 3. Verification Experiment

[0039] (1) Cell adhesion domain specificity verification experiment

[0040] Cell culture: Vaginal epithelial cell line (SiHa cells) was cultured in DMEM medium containing 10% fetal bovine serum and 1% double antibody in a 37°C, 5% CO2 incubator until the logarithmic growth phase.

[0041] Adhesion assay: The fusion polypeptide (concentration gradient set to 0.1 μM, 1 μM, 10 μM) and vaginal adhesion peptide YPS30 (1 μM as control, amino acid sequence YPSKPLPQPKVKA) were added to a 96-well plate, 100 μL per well, and a blank control group (containing only cell culture medium) was set up. The cultured SiHa cells were cultured at 1×10 4 Cells were seeded at a density of 100 cells / well in a 96-well plate and incubated at 37°C for 2 hours. The liquid in the wells was discarded and the cells were washed three times with PBS to remove unadhered cells. 0.1% crystal violet stain was added and stained at room temperature for 15 minutes. The cells were washed three times with PBS. After drying, 33% acetic acid was added to dissolve the crystal violet and the absorbance (OD) was measured at 570 nm using a microplate reader.

[0042] The results showed (Table 1) that as the concentration of the fusion polypeptide increased, its adhesion ability to the vaginal epithelial cells SiHa showed a significant upward trend. At a concentration of 10 μM, the OD value reached 0.68±0.03, which was significantly higher than the OD value of the vaginal adhesion peptide YPS30 at a concentration of 1 μM (0.25±0.02), proving that the cell adhesion domain "TWHIQKKD" designed in the present invention has stronger specific adhesion ability.

[0043] Table 1 Test results

[0044] sample 0.1μM 1 μM 10 μM YPS30 (1 μM) Blank control OD value 0.12±0.01 0.35±0.02 0.68±0.03 0.25±0.02 0.05±0.01

[0045] (II) Quorum sensing inhibitory domain activity verification experiment

[0046] Biofilm culture: Gardnerella was inoculated into BHI broth containing 2% glucose and cultured at 37°C with shaking until the logarithmic growth phase. 100 μL of the bacterial solution was inoculated into a 24-well plate, and a sterile coverslip was added to each well. The plates were incubated at 37°C for 48 hours to form a biofilm.

[0047] Drug treatment: Discard the culture medium in the wells and gently wash the coverslips three times with PBS to remove planktonic bacteria. Add 200 μL of the fusion peptide (0.5 μM, 1 μM, and 2 μM) and the original HLA-G antimicrobial peptide (1 μM as a control, amino acid sequence RKKSSFQRTMFLLKSV) to the wells. A blank control group (PBS only) was also set up and incubated at 37°C for 24 hours.

[0048] Biofilm quantitative analysis: Remove the coverslips, wash three times with PBS, add 1 mL of 0.1% crystal violet staining solution, stain at room temperature for 15 minutes, wash three times with PBS, dry, add 33% acetic acid solution to dissolve the crystal violet, transfer to a 96-well plate, and measure the absorbance (OD value) at 570 nm on a microplate reader.

[0049] The results showed (Table 2) that the inhibitory effect of the fusion peptide on Gardnerella biofilm increased with increasing concentration. At a concentration of 2 μM, the OD value dropped to 0.18±0.01, which was significantly lower than the OD value of the original HLA-G antimicrobial peptide at a concentration of 1 μM (0.45±0.03). This indicates that the modified quorum sensing inhibitory domain "KKLFKKILKYL" effectively improves the ability to penetrate the biofilm and inhibit its formation while maintaining antibacterial activity, verifying the effectiveness of the HLA-G antimicrobial peptide modification strategy.

[0050] Table 2 Test results

[0051] sample 0.5μM 1 μM 2μM HLA-G antimicrobial peptide (1 μM) Blank control OD value 0.52±0.03 0.31±0.02 0.18±0.01 0.45±0.03 0.85±0.04

[0052] IV. Summary

[0053] The above experiments verified that the fusion polypeptide designed by the present invention exhibits unique performance advantages in both the cell adhesion domain and the quorum sensing inhibitory domain. The cell adhesion domain has a new sequence and stronger specific adhesion ability. The quorum sensing inhibitory domain has significantly improved its activity in penetrating biological membranes after modification. The synthesis and preparation process of the entire fusion polypeptide is stable and controllable, with a purity greater than 98% after purification. The cyclooxidation step effectively promotes disulfide bond formation. These results fully demonstrate the innovation and effectiveness of the fusion polypeptide of the present invention in structural design, functional realization, and preparation methods, and provide a solid experimental basis for its application in female vaginal flora repair fluid.

[0054] Example 2: Design, preparation and testing of monoclonal antibodies

[0055] 1. Monoclonal Antibody Design

[0056] This monoclonal antibody specifically targets a surface protein unique to Lactobacillus iners (UniProtKB: E1NRP2). Bioinformatics analysis indicates that this protein is highly conserved and immunogenic, making it an ideal target for antibody action. Binding to the surface protein is expected to block protein modification, promoting the reorganization of the metabolic pathways of Lactobacillus iners, thereby inducing the strain to switch to a H2O2-producing phenotype and restoring the antibacterial capacity of the vaginal microbiome.

[0057] 2. Preparation of Monoclonal Antibodies

[0058] (1) Immunogen preparation

[0059] Gene cloning: PCR amplification was performed using DNA encoding a unique surface protein of Lactobacillus iners (UniProtKB: E1NRP2) as a template (containing a 6His tag). The PCR reaction system consisted of 25 μL of 2× Taq PCR Master Mix, 1 μL of each upstream and downstream primer (10 μM), 1 μL of template DNA, and ddH2O to a volume of 50 μL. Reaction conditions included initial denaturation at 95°C for 5 min, followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 1 min, followed by a final extension at 72°C for 10 min. The amplified product was identified by electrophoresis on a 1% agarose gel, and the target fragment was recovered using a gel extraction kit.

[0060] Expression and purification: The recovered target gene fragment was connected to the pET-28a(+) expression vector and transformed into E. coli BL21(DE3) competent cells. A single colony was picked and inoculated into LB liquid medium containing kanamycin (50 μg / mL) and cultured at 37°C with shaking until the OD 600The p-value of the protein was about 0.6-0.8, IPTG was added to a final concentration of 0.5mM, and the expression was induced at 16℃ for 16h. The bacteria were collected by centrifugation and ultrasonically disrupted (power 200W, working 3s, interval 5s, total 30min). After disruption, centrifugation was performed (12000rpm, 4℃, 20min), and the supernatant was taken for Ni-NTA affinity chromatography purification. The purification steps are as follows: the Ni-NTA column was equilibrated with equilibration buffer (50mM Tris-HCl, pH 8.0, 300mM NaCl, 20mM imidazole); the supernatant was loaded and the impurities were eluted with washing buffer (50mM Tris-HCl, pH8.0, 300mM NaCl, 50mM imidazole); the target protein was eluted with elution buffer (50mM Tris-HCl, pH 8.0, 300mMNaCl, 250mM imidazole). The elution peak was collected and the purity was identified by SDS-PAGE electrophoresis ( Figure 1 , about 188 kDa), dialyzed (dialysate: 50 mM Tris-HCl, pH 7.4, 150 mM NaCl) to remove imidazole, and sterilized by filtering through a 220 nm filter membrane to obtain the inert lactobacillus specific surface protein. The BCA detection concentration was 2.65 mg / mL and stored at -80°C for future use.

[0061] (2) Animal Immunity and Cell Fusion

[0062] Animal Immunization: Six female Balb / c mice aged 6-8 weeks were selected. The immunogen (a surface protein specific to Lactobacillus inerans) was fully emulsified with an equal volume of Freund's complete adjuvant and injected subcutaneously at multiple sites. The immunization dose was 100 μg per mouse. After the initial immunization, booster immunizations were performed at weeks 2, 4, and 6 using the immunogen emulsified with Freund's incomplete adjuvant at the same dose as the initial immunization. At week 7, the immunogen (without adjuvant) was injected into the tail vein for a pulse immunization. The spleens of the mice were harvested 3 days later.

[0063] Cell fusion: Mix mouse spleen cells and logarithmically growing SP2 / 0 myeloma cells at a ratio of 5:1 in a 50 mL centrifuge tube. Centrifuge at 1200 rpm for 10 minutes, and discard the supernatant. Gently resuspend the cell pellet and add 0.8 mL of 50% PEG1500 dropwise in a 37°C water bath with gentle stirring. After 90 seconds, immediately add 10 mL of prewarmed incomplete RPMI1640 medium to terminate fusion. Centrifuge at 1200 rpm for 10 minutes, discard the supernatant, and resuspend the cells in RPMI1640 medium supplemented with 20% fetal bovine serum, 1% double-stranded antibody, and 1× HAT. Plate 100 μL per well of a 96-well cell culture plate and incubate in a 37°C, 5% CO2 incubator.

[0064] (3) Hybridoma cell screening and cloning

[0065] Screening: On day 7 after fusion, positive hybridoma cells were screened using an indirect ELISA method. Lactobacillus iners-specific surface protein was coated onto a 96-well ELISA plate (1 μg / mL, 100 μL / well) and incubated at 4°C overnight. The next day, the coating solution was discarded, and the plates were washed three times with PBS containing 0.05% Tween-20 (PBST). 200 μL of 5% skim milk was added to each well and blocked at 37°C for 2 hours. The blocking solution was discarded, and the plates were washed three times with PBST. 100 μL / well of hybridoma cell culture supernatant was added and incubated at 37°C for 1 hour. Washed three times with PBST, 100 μL / well of HRP-labeled goat anti-mouse IgG (1:5000 dilution) was added and incubated at 37°C for 1 hour. Wash 5 times with PBST, add 100 μL / well of TMB substrate solution, react for 15 min at room temperature in the dark, add 50 μL / well of 2M H2SO4 to terminate the reaction, and measure the OD value at 450 nm on a microplate reader. 450 Wells with values ​​2.1 times greater than the negative control were considered positive.

[0066] Cloning: Positive hybridoma cells were cloned using the limiting dilution method. Cells from positive wells were diluted with RPMI1640 medium supplemented with 10% fetal bovine serum and 1% double-antibody to a concentration of 0.5, 1, or 2 cells per well, respectively. The cells were seeded into 96-well cell culture plates at a concentration of 200 μL per well and cultured in a 37°C, 5% CO2 incubator. When the cell clones reached 1 / 3-1 / 2 of the bottom of the well, they were screened again using indirect ELISA. Positive monoclonal cell lines with high OD values ​​were selected, cultured, and cryopreserved in liquid nitrogen for later use.

[0067] (IV) Monoclonal Antibody Purification

[0068] Positive hybridoma cells screened were inoculated into cell culture flasks and expanded in RPMI1640 medium supplemented with 10% fetal bovine serum and 1% bispecific antibody. When the cell density reached 80%-90%, the cell culture supernatant was collected. Antibodies were purified using a Protein A affinity chromatography column as follows: equilibrate the Protein A column with equilibration buffer (20 mM sodium phosphate, pH 7.0); load the cell culture supernatant, wash the column with equilibration buffer to remove contaminants; elute the antibody with elution buffer (0.1 M glycine-HCl, pH 3.0), and collect the eluted peak. Immediately neutralize the eluate with 1 M Tris-HCl (pH 9.0) and dialyze (dialysis buffer: PBS, pH 7.4) to remove salts. After determining the antibody concentration by BCA assay, filter sterilize the column, aliquot, and freeze.

[0069] 3. Monoclonal Antibody Testing

[0070] (1) Monoclonal antibody-induced Lactobacillus phenotypic conversion experiment

[0071] 1. Strain Isolation and Identification: Lactobacillus iners was isolated and cultured from vaginal secretions of patients with clinical vaginitis. 16S rRNA sequencing was used for species identification. Genomic DNA was extracted and amplified by PCR using universal primers. The PCR reaction system and conditions were the same as those used for immunogenic gene cloning. After sequencing, the amplified product was submitted to the NCBI database for BLAST comparison, confirming that the isolated strain was Lactobacillus iners.

[0072] 2. Group culture: Resuscitate the identified inert Lactobacillus strains, inoculate them into MRS liquid culture medium, and culture them anaerobically at 37°C until the logarithmic growth phase. Adjust the concentration of the cultured bacteria to 1×10 8 CFU / mL, and were divided into two groups: a control group was treated with normal MRS medium (5 mL per bottle); a monoclonal antibody group was treated with MRS medium containing 50 μg / mL of a monoclonal antibody (prepared by the above method, wherein the amino acid sequences of the heavy chain variable region and light chain variable region of the monoclonal antibody are shown in SEQ ID NO: 2 and SEQ ID NO: 3, respectively) (5 mL per bottle). Six biological replicates were set up for each group, and the cells were cultured in an anaerobic incubator at 37°C for 24 hours.

[0073] 3. H2O2 production determination: The H2O2 production was determined using the FOX1 method. 1 mL of the cultured bacterial solution was centrifuged at 12,000 rpm for 5 minutes, and the supernatant was collected. Following the instructions for the FOX1 assay kit, the supernatant, reagent A, and reagent B were added to a 96-well plate in sequence. The reaction was carried out at room temperature in the dark for 30 minutes, and the OD value was measured at 560 nm using a microplate reader. The H2O2 concentration (μM / 10) was calculated based on the standard curve. 8 CFU).

[0074] 4. Experimental results (as shown in Table 3): The H2O2 production of the control group of Lactobacillus inerans was low, only 0.8±0.2μM / 10 8 In the monoclonal antibody group, the H2O2 production was significantly increased to 9.3±0.7μM / 10 8 The CFU increased by 10.6 times compared to the control group (p < 0.01). This result proves that the monoclonal antibody designed and prepared by the present invention can effectively induce metabolic reorganization of Lactobacillus iners by specifically binding to the unique surface protein of Lactobacillus iners, achieving the conversion to a H2O2-producing phenotype.

[0075] Table 3 Experimental results

[0076] Group <![CDATA[H2O2(μM / 10 8 CFU)]]> control group 0.8±0.2 Monoclonal antibody group 9.3±0.7*

[0077] IV. Summary

[0078] Through rigorous design and standardized experimental operations, this example successfully prepared a monoclonal antibody against the surface protein unique to Lactobacillus iners, and verified its mechanism of action through detailed experiments. From immunogen preparation, animal immunization and cell fusion, to hybridoma cell screening, cloning and antibody purification, each link is strictly controlled to ensure the specificity and effectiveness of the monoclonal antibody. The experimental results show that the monoclonal antibody can significantly induce the phenotypic conversion of Lactobacillus iners, providing an innovative solution for improving the imbalance of vaginal microecology, and also laying a solid experimental foundation for the subsequent development of female vaginal flora repair fluid.

[0079] Example 3: In vitro activity verification

[0080] 1. Experimental Materials and Equipment

[0081] 1. Strains: Gardnerella (ATCC 14018), Prevotella (ATCC Bio-79759), Lactobacillus iners (clinical isolate, verified by 16S rRNA sequencing), Lactobacillus acidophilus (ATCC 4356).

[0082] 2. Culture medium:

[0083] (1) Gardnerella: Columbia blood agar containing 5% defibrinated sheep blood.

[0084] (2) Prevotella: Brain heart infusion (BHI) medium containing 5 μg / mL vitamin K1 and 10 μg / mL hemin.

[0085] (3) Lactobacillus: MRS medium, pH 6.2.

[0086] 3. Main reagents:

[0087] (1) Fusion polypeptide (prepared according to the method of Example 1).

[0088] (2) Monoclonal antibody (concentration 1 mg / mL, prepared according to the method of Example 2).

[0089] (3) Crystal violet staining solution, methanol, and 33% acetic acid solution.

[0090] 2. Experimental Methods

[0091] (1) BV model construction

[0092] 1. Preparation of bacterial suspension: Inoculate Gardnerella, Prevotella, and Lactobacillus iners into the corresponding culture medium and culture anaerobically at 37°C for 24 hours. Collect the cells by centrifugation (3000g, 10 minutes) and wash twice with sterile PBS. Adjust the bacterial concentration to OD 600 =0.5(about 1×10 8 CFU / mL).

[0093] 2. Biofilm Inoculation: Mix three bacterial suspensions (Gardnerella, Prevotella, and Lactobacillus iners) in a 1:1:3 ratio. Inoculate 100 μL of the mixed suspension into a 96-well flat-bottom cell culture plate. Add 100 μL of BHI medium containing 1% glucose to each well. Incubate anaerobically at 37°C for 48 hours. Discard the supernatant and gently wash the plate three times with PBS to remove floating bacteria.

[0094] (2) Group processing

[0095] 1. The group settings are as follows:

[0096] Group A (blank control): add 200 μL sterile PBS.

[0097] Group B (fusion polypeptide group): add 200 μL of 0.1 mg / mL fusion polypeptide solution.

[0098] Group C (monoclonal antibody group): add 200 μL of 50 μg / mL monoclonal antibody.

[0099] Group D (combination preparation group): 200 μL of a mixed solution of 0.1 mg / mL fusion polypeptide + 50 μg / mL monoclonal antibody was added.

[0100] 2. Treatment conditions: Set up 10 replicate wells for each group and incubate at 37°C for 24 hours.

[0101] (3) Detection indicators and methods

[0102] 1. Determination of biofilm degradation rate: Discard the culture medium and wash 3 times with PBS. Add 200 μL of 0.1% crystal violet solution to each well and stain at room temperature for 15 minutes. Discard the staining solution, wash 5 times with PBS, and air dry. Add 200 μL of 33% acetic acid solution to each well and shake for 10 minutes to dissolve the crystal violet. Measure the absorbance (OD) at a wavelength of 570 nm. 570 The formula for calculating biofilm degradation rate is: degradation rate (%) = (1-OD of experimental group) 570 / control group OD 570 )×100%.

[0103] 2. Determination of the proportion of Lactobacillus acidophilus: Collect biofilms and extract total DNA using a DNA extraction kit.

[0104] The 16S rRNA gene copy number was detected by qPCR. The specific method is as follows:

[0105] Reaction system (20 μL): 2×Power SYBR Green PCR Master Mix 10 μL, upstream and downstream primers (10 μM) 0.8 μL each, DNA template 2 μL, ddH2O 6.4 μL.

[0106] Reaction conditions: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 15 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s (40 cycles).

[0107] Data analysis: The relative abundance of Lactobacillus acidophilus was calculated using the ΔΔCt method.

[0108] 3. pH determination: Collect the culture medium and measure the pH value using a pH meter. Repeat the measurement three times for each sample.

[0109] 3. Experimental results are shown in Tables 4 to 7.

[0110] (1) Biofilm degradation effect

[0111] Single-drug treatment: The biofilm degradation rate in the fusion peptide group (Group B) was 68.5±3.8%, significantly higher than that in the blank control group (p<0.001), indicating that the fusion peptide can effectively destroy the bacterial biofilm structure. The degradation rate in the monoclonal antibody group (Group C) was only 9.4±0.7%, suggesting that the antibody has limited direct biofilm destruction effect.

[0112] Composite preparation: The degradation rate of group D reached 92.3±2.4%, which was significantly higher than the effects of group B and group C alone (p<0.001), indicating that the fusion polypeptide and monoclonal antibody had a synergistic effect in biofilm degradation.

[0113] (2) Proliferation effect of Lactobacillus acidophilus

[0114] Effect of monoclonal antibodies: The proportion of Lactobacillus acidophilus in group C increased from 12.3±1.2% in the control group to 41.2±2.1%, proving that the antibody indirectly promoted the growth of beneficial bacteria such as Lactobacillus acidophilus by inducing phenotypic conversion of Lactobacillus iners.

[0115] Composite effect: The proportion of Lactobacillus acidophilus in group D further increased to 63.8±3.3%, which was significantly higher than that in group C (p<0.001), indicating that the fusion polypeptide provided a more favorable growth environment for Lactobacillus acidophilus by inhibiting harmful bacteria, and produced a synergistic proliferation effect with the monoclonal antibody.

[0116] (3) pH value changes

[0117] Single drug treatment: The pH values ​​of group B and group C dropped to 5.2 and 4.7, respectively, close to the normal vaginal acidic environment.

[0118] Compound preparation: The pH value of group D further dropped to 4.3, which is closest to the pH value of the vagina of healthy women (3.8-4.5), indicating that the compound preparation can more effectively restore the acid-base balance of the vaginal microecology.

[0119] Table 4 Raw data of biofilm degradation rate (OD570 value)

[0120] Duplicate wells Group A Group B Group C Group D 1 0.852 0.269 0.772 0.067 2 0.863 0.274 0.781 0.071 3 0.849 0.281 0.768 0.065 4 0.857 0.265 0.775 0.069 5 0.861 0.279 0.783 0.073 6 0.855 0.272 0.777 0.066 7 0.848 0.283 0.769 0.070 8 0.859 0.267 0.780 0.068 9 0.854 0.276 0.774 0.072 10 0.860 0.270 0.779 0.064 average value 0.855 0.274 0.776 0.068 SD 0.005 0.007 0.005 0.003

[0121] Table 5 Raw data of the proportion of Lactobacillus acidophilus (qPCR Ct value)

[0122] Duplicate wells Group A Group B Group C Group D 1 27.84 26.93 23.45 21.32 2 27.91 27.01 23.52 21.40 3 27.79 26.88 23.38 21.27 average value 27.85 26.94 23.45 21.33 SD 0.06 0.07 0.07 0.07

[0123] Table 6 pH value raw data

[0124] Duplicate wells Group A Group B Group C Group D 1 6.12 5.23 4.71 4.32 2 6.09 5.21 4.69 4.30 3 6.11 5.24 4.72 4.31 average value 6.11 5.23 4.71 4.31 SD 0.01 0.01 0.01 0.01

[0125] Table 7 Comparison and summary of experimental results

[0126]

[0127] 4. Experimental Summary

[0128] This in vitro activity validation experiment systematically evaluated the repair effects of fusion peptides, monoclonal antibodies, and compound preparations by constructing a BV model. The results showed that:

[0129] When used in combination with a monoclonal antibody, the fusion peptide exhibited significant synergistic effects in biofilm degradation, beneficial bacterial proliferation, and pH regulation, outperforming either treatment alone. The fusion peptide works by directly disrupting the biofilm and inhibiting harmful bacteria, while the monoclonal antibody indirectly promotes the growth of beneficial bacteria by inducing phenotypic transformation in Lactobacillus iners. The two mechanisms complement each other, resulting in multi-target regulation. The combined formulation increased the proportion of Lactobacillus acidophilus to 63.8% and adjusted the pH to the physiological range, providing a more effective solution for BV treatment.

[0130] Example 4: Preparation of compound formulation

[0131] To prepare the compound formulation, the fusion polypeptide and monoclonal antibody, each with precisely measured concentrations, are first mixed in a 1:2 (w / w) ratio in a specific buffer (25mM histidine + 15mM succinate + 75mM arginine + 100mM NaCl (pH 4.5)) and stirred thoroughly. Specific lyoprotectants (5% (w / v) trehalose, (0.01% w / v) polysorbate 80, (0.05% w / v) methionine, 5% (w / v) gelatin) are then added in sequence, with continuous stirring to thoroughly mix the ingredients. The mixture is dispensed into sterile vials and placed in a freeze dryer. Prefreeze at -40°C for 4-6 hours, then evacuate to 25 Pa and heat to -20°C at a rate of 0.5°C / min for 12-15 hours for sublimation drying. The temperature is then raised to 25°C for 8-10 hours to complete desorption drying. Finally, the vials are filled with sterile nitrogen under vacuum and vacuum-sealed using a capping machine to produce the reconstituted lyophilized powder. Upon use, reconstitute the powder by adding 10 mL of 5% sodium lactate solution per gram. Quality testing is then performed using indicators such as appearance, activity, moisture content, and microbial limits to ensure product compliance with standards.

[0132] In pH 4.5 citric acid buffer, the acidic environment causes the monoclonal antibody to form aggregates as high as 22.3% ± 1.8%, with a deamidation rate of 35.7% ± 2.1%, and an activity retention rate of only 58%. By adopting an optimized buffer system of 25mM histidine-15mM succinate-75mM arginine, stability was significantly improved—the aggregate content was reduced to 4.1% ± 0.5% (a decrease of 81.6%), the deamidation rate was controlled at 7.9% ± 0.7% (a decrease of 77.9%), and the activity retention rate reached 94%. If further combined with a lyoprotectant of 5% trehalose + 0.05% methionine, the aggregate content could be reduced to 1.8% ± 0.3%, and the activity retention rate was increased to 96%. Accelerated testing conditions of storage at 40°C for 4 weeks confirmed that this solution effectively addresses the stability bottleneck of monoclonal antibodies in acidic environments.

[0133] Example 5: Animal Experimental Verification of the Composite Formulation

[0134] 1. Experimental Materials and Preparation

[0135] 1. Experimental Animals: Sixty healthy female Sprague-Dawley rats, 6-8 weeks old and weighing 180-220 g, were selected. The rats were housed in an SPF-grade animal room at a temperature of (22 ± 2)°C, a relative humidity of 50%-60%, and a 12-h light / 12-h dark cycle. The rats had free access to food and water. Experiments were initiated after 7 days of acclimatization.

[0136] 2. Experimental strains: Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923).

[0137] 3. Main reagents:

[0138] The repair liquid of the present invention was prepared according to the preparation method of the composite preparation in Example 4.

[0139] Metronidazole gel: commercially available, specification is 50mg / d.

[0140] MRS medium, LB medium, PBS.

[0141] DNA extraction kit, bacterial 16S rRNA gene universal primers, Lactobacillus-specific primers.

[0142] 2. Experimental Methods

[0143] (1) Construction of vaginal flora imbalance model

[0144] Preparation of bacterial suspension: Escherichia coli and Staphylococcus aureus were inoculated into LB medium respectively, cultured at 37°C with shaking until the logarithmic growth phase, and centrifuged at 4°C and 3000g for 10 min to collect the bacteria. After washing twice with PBS, the bacteria were resuspended in PBS and the concentration of the bacterial suspension was adjusted to 1×10 8 CFU / mL, and mix the two bacterial solutions in a 1:1 ratio for later use.

[0145] Model construction: Except for the normal control group, the remaining 40 rats were anesthetized by ether inhalation. The vagina was gently wiped with a sterile cotton swab to clear secretions. Then, 100 μL of mixed bacterial solution was slowly injected deep into the vagina with a microsyringe. The inoculation was continued for 3 consecutive days to establish the vaginal dysbiosis model.

[0146] (II) Grouping and treatment: 60 rats were randomly divided into 4 groups, with 15 rats in each group:

[0147] Normal control group: no treatment was performed and the rats were raised normally.

[0148] Model control group: 100 μL PBS was instilled into the vagina once a day for 7 consecutive days.

[0149] Experimental group: 100 μL of the repair solution of the present invention was injected into the vagina once a day for 7 consecutive days.

[0150] Metronidazole gel group: 50 mg of metronidazole gel was applied intravaginally once a day for 7 consecutive days.

[0151] (3) Detection indicators and methods

[0152] Vaginal secretion collection: 24 h after the end of the perfusion, the rats were anesthetized by ether inhalation. The posterior fornix of the vagina was gently wiped with a sterile cotton swab to collect vaginal secretions, which were immediately placed in a sterile EP tube for subsequent testing.

[0153] Bacterial culture and count: The collected vaginal secretions were inoculated into MRS medium (for culturing Lactobacillus) and LB medium (for culturing Escherichia coli and Staphylococcus aureus). After culturing at 37°C for 48 h, the colony count was performed using the dilution plate method, and the colony-forming units (CFU / g) of each bacterial species in each gram of vaginal secretions were calculated.

[0154] Microbial flora analysis: Total DNA was extracted from vaginal secretions, and the bacterial 16S rRNA gene was analyzed using PCR-DGGE (denaturing gradient gel electrophoresis) technology. The grayscale analysis of the electrophoresis bands was performed using Quantity One software, and the relative abundance of Lactobacillus, Escherichia coli, and Staphylococcus aureus was calculated.

[0155] pH value determination: Vaginal secretions were mixed with PBS in a ratio of 1:10. The pH value of the mixture was measured using a pH meter. Each sample was measured three times and the average value was taken.

[0156] 3. Experimental results are shown in Tables 8 to 11.

[0157] (1) pH value of vaginal secretions

[0158] The pH value of vaginal secretions of rats in the normal control group was maintained at 4.2±0.1, which is within the acidic environment range of a healthy vagina. Due to dysbacteriosis, the pH value of the model control group increased significantly to 5.8±0.2 (compared with the normal control group, p<0.001). After using the repair fluid of the present invention, the pH value of the experimental group dropped to 4.3±0.1, close to the normal level, and the difference was extremely significant compared with the model control group (p<0.001). The pH value of the metronidazole gel group was 4.9±0.1, which was lower than that of the model control group, but still higher than that of the experimental group (p<0.05), indicating that the repair fluid of the present invention is more effective in regulating vaginal pH.

[0159] (2) Relative abundance of bacterial communities

[0160] Lactobacillus: The relative abundance of Lactobacillus in the normal control group was as high as 85.6±3.2%; in the model control group, it dropped sharply to 12.3±2.5% (p<0.001); after treatment with the repair fluid, the relative abundance of Lactobacillus in the experimental group rebounded to 78.9±3.5%, which was significantly higher than that in the model control group (p<0.001) and significantly higher than that in the metronidazole gel group (36.1±2.3% (p<0.001).

[0161] Escherichia coli and Staphylococcus aureus: In the model control group, the relative abundances of E. coli and S. aureus were 68.7±4.1% and 15.6±3.0%, respectively. After treatment with the repair solution, the relative abundances of these bacteria decreased significantly in the experimental group to 8.5±1.5% and 6.2±1.2% (compared with the model control group, p<0.001). Although the relative abundances of E. coli and S. aureus also decreased in the metronidazole gel group, to 28.9±2.6% and 20.4±2.2%, respectively, these abundances were still higher than in the experimental group (p<0.001).

[0162] Table 8 Original data of pH value of vaginal secretions

[0163]

[0164] Table 9 Raw data of relative abundance of Lactobacillus (%)

[0165]

[0166] Table 10 Original data of relative abundance of Escherichia coli (%)

[0167]

[0168] Table 11 Original data of relative abundance of Staphylococcus aureus (%)

[0169]

[0170] 4. Experimental Summary

[0171] This animal experiment established a rat model of vaginal dysbiosis and compared the effects of the repair solution of the present invention, metronidazole gel, and PBS on the vaginal microecology. The results showed that the repair solution of the present invention can significantly adjust the vaginal pH value to the normal acidic range, effectively promote the proliferation of lactobacilli, and strongly inhibit the growth of harmful bacteria such as Escherichia coli and Staphylococcus aureus. It is better than traditional metronidazole gel in restoring the balance of vaginal microecology. Its mechanism of action may be due to the synergistic effect of the fusion polypeptide and the monoclonal antibody. The former directly inhibits harmful bacteria and destroys the biofilm, while the latter induces the phenotypic conversion of Lactobacillus iners and indirectly promotes the growth of beneficial bacteria. This experiment provides a reliable animal experimental basis for the clinical application of the female vaginal flora repair solution.

[0172] 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 vaginal microecological regulation compound preparation, characterized in that: The compound preparation is composed of a fusion polypeptide and a monoclonal antibody in a weight ratio of 1:2; the amino acid sequence of the fusion polypeptide is shown in SEQ ID NO: 1; the amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are shown in SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

2. The composite formulation according to claim 1, characterized in that Cysteine ​​is added to the N-terminus and C-terminus of the fusion polypeptide to form intramolecular disulfide bonds.

3. The composite formulation according to claim 1, characterized in that The monoclonal antibody targets the surface protein UniProtKB:E1NRP2, which is unique to Lactobacillus iners. It can block the modification of the surface protein unique to Lactobacillus iners, promote the reorganization of the metabolic pathway of the strain and convert it to the H2O2 production phenotype.

4. A method for preparing the vaginal microecological regulation compound preparation according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: mixing the fusion polypeptide and the monoclonal antibody in a specific buffer at a weight ratio of 1:2, adding a freeze-drying protectant, pre-freezing, sublimation drying, desorption drying, and then vacuum sealing to obtain a reconstituted freeze-dried powder.

5. The method according to claim 4, characterized in that: The specific buffer is a mixed solution of 25 mM histidine, 15 mM succinic acid, 75 mM arginine, and 100 mM NaCl, with a pH of 4.

5.

6. The method according to claim 4, characterized in that: The freeze-drying protective agent is 5% by weight and volume of trehalose, 0.01% by weight and volume of polysorbate 80, 0.05% by weight and volume of methionine, and 5% by weight and volume of gelatin.

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