Plant lactobacillus and application thereof

Abstract

The invention relates to plant lactobacillus and application thereof. Wherein the gene of the molecular marker for the plant lactobacillus has a nucleotide sequence selected from one of the following sequences: (1) a nucleotide sequence as shown in SEQ ID NO: 24; (2) a nucleotide sequence having at least 97%, 98%, 99% or higher homology with the nucleotide sequence as shown in SEQ ID NO: 24; and (3) a nucleotide sequence with one or more nucleotide sequences, such as 1, 2, 3, 4, 5 or more nucleotide substitutions, deletion or insertion, in the nucleotide sequence as shown in SEQ ID NO: 24. The plant lactobacillus provided by the invention is non-toxic, has good biological characteristics, has a good curative effect on prevention and / or treatment of vaginal infection and related diseases, and avoids the problems of increased drug resistance, high recurrence rate and the like caused by use of antibiotic and other antibacterial drugs during treatment of vaginal infection and related diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and specifically relates to a Lactobacillus plantarum and its use in preparing a medicament for preventing and / or treating vaginal infection and related diseases. Background Art

[0002] With economic development and improvement of living standards, female reproductive tract diseases are becoming more and more common, and vaginitis is even more common, which has a serious impact on women's quality of life and physical and mental health.

[0003] Among vaginal infections, bacterial vaginosis (BV) is the most common. Epidemiological surveys show that the incidence of BV in my country ranges from 4.96% to 36.00%. The typical clinical features of BV are foul-smelling, watery, or gray vaginal discharge. Patients may also develop various complications due to ascending infection, such as chronic cervicitis, pelvic inflammatory disease, endometritis, and even infertility.

[0004] Vulvovaginal candidiasis (VVC) is the second most common vaginal infection after bacterial vaginosis. Studies have found that approximately 78% of women experience at least one episode of VVC caused by Candida albicans in their lifetime, with 65% experiencing three or fewer annual episodes and 35% experiencing four or more. VVC typically presents with vulvar itching, burning, painful urination, and a dreg-like vaginal discharge, often impairing patients' quality of life.

[0005] Currently, the treatment of BV is primarily based on Western medicine, often using antimicrobial agents such as antibiotics, including metronidazole, clindamycin, and tinidazole. However, the formation and persistence of pathogenic biofilms can lead to recurrence. Reports indicate that recurrence rates in BV patients treated with oral metronidazole are 23%, 49%, 59%, and 68% at 1, 3, 6, and 12 months, respectively. At 12 months after treatment, 84% of patients still experience abnormal vaginal flora. Furthermore, long-term, high-dose antibiotic use not only increases pathogen resistance but also inhibits the growth of some vaginal flora, allowing the previously limited Candida albicans to proliferate, leading to vaginal flora disturbance. The role of live bacterial preparations in the treatment of BV remains controversial. Clinical studies have shown that the use of live bacterial preparations is primarily divided into two categories: conventional antibiotic treatment followed by probiotics or probiotics alone. The results of several clinical trials examining combination therapy have been inconsistent, making it uncertain whether the combined use of antibiotics and probiotics is effective for the treatment of bacterial vaginosis. Other studies have shown that some patients fail to achieve satisfactory results with probiotic preparations alone. Therefore, the effectiveness of probiotics alone in treating BV remains controversial, and extensive research is needed. Currently, the primary probiotic marketed for the treatment of bacterial vaginosis is Dingjunsheng (live lactobacillus capsules for vaginal use), developed by Inner Mongolia Shuangqi Pharmaceutical Co., Ltd.

[0006] Currently, azoles and polyenes, such as diazepam suppositories, are commonly used in the treatment of VVC. However, with the long-term use of antifungal drugs in clinical practice, Candida albicans undergoes phenotypic changes and virulence factor mutations, leading to an increase in infections with non-albicans Candida species, posing challenges to the therapeutic efficacy of traditional antifungal drugs. Lactobacilli can be used as an adjunctive therapy in combination with antifungal drugs such as azoles in the treatment of various VVC patients, with some efficacy. However, compared with conventional antifungal drugs, there is currently insufficient evidence to demonstrate that the use of Lactobacillus preparations alone is effective in the treatment of VVC. Summary of the Invention

[0007] In view of the above problems, the present invention aims to provide a Lactobacillus plantarum strain and its use in the preparation of a medicament for preventing and / or treating vaginal infection and its related diseases. The Lactobacillus plantarum strain is a new strain screened by the inventors, has good biological properties, and has good therapeutic effects for preventing and / or treating vaginal infection and its related diseases.

[0008] The above object of the present invention is achieved by providing the following technical solutions:

[0009] In a first aspect, the present invention provides a gene (or DNA molecule) for molecular marker of Lactobacillus plantarum, which has a nucleotide sequence selected from one of the following:

[0010] (1) the nucleotide sequence shown in SEQ ID NO: 24;

[0011] (2) a nucleotide sequence having at least 97%, 98%, 99% or higher homology to the nucleotide sequence shown in SEQ ID NO: 24;

[0012] (3) A nucleotide sequence having one or more, for example, 1, 2, 3, 4, 5 or more, nucleotide substitutions, deletions or insertions in the nucleotide sequence shown in SEQ ID NO: 24.

[0013] Preferably, the molecular marker gene for Lactobacillus plantarum has a nucleotide sequence as shown in SEQ ID NO: 24, or its nucleotide sequence is as shown in SEQ ID NO: 24.

[0014] In a second aspect, the present invention provides a Lactobacillus plantarum comprising the gene for molecular marking according to the present invention.

[0015] In a third aspect, the present invention provides a primer set for identifying the plant lactobacillus according to the second aspect of the present invention, comprising a forward primer and a reverse primer, wherein:

[0016] The nucleotide sequence of the forward primer is shown in SEQ ID NO: 1;

[0017] The nucleotide sequence of the reverse primer is shown in SEQ ID NO: 2.

[0018] In a fourth aspect, the present invention provides a method for identifying the Lactobacillus plantarum according to the second aspect of the present invention, comprising: amplifying the genomic DNA of the strain to be identified using the primer set according to the present invention, and then detecting the amplified product.

[0019] Preferably, the method comprises the following steps: extracting a genomic DNA sample from the strain to be identified, performing PCR amplification on the genomic DNA sample using the primer set described in the present invention, and then comparing the amplified product with the gene for molecular marker according to the present invention; if the amplified product contains the gene for molecular marker according to the present invention, the strain to be identified is the Lactobacillus plantarum according to the second aspect of the present invention.

[0020] Preferably, if the amplified product comprises the nucleotide sequence shown in SEQ ID NO: 24, the strain to be identified is the Lactobacillus plantarum according to the second aspect of the present invention.

[0021] In a fifth aspect, the present invention provides a use of the gene for molecular marker according to the present invention in identifying the Lactobacillus plantarum according to the second aspect of the present invention.

[0022] The term "molecular marker" as used in the present invention refers to a specific DNA fragment that can reflect certain differences in the genome between biological individuals or populations.

[0023] In a sixth aspect, the present invention provides a plant lactobacillus comprising a nucleotide sequence selected from one of the following:

[0024] (1) the nucleotide sequence shown in SEQ ID NO: 23;

[0025] (2) a nucleotide sequence having at least 99.8%, 99.9% or higher homology to the nucleotide sequence shown in SEQ ID NO: 23;

[0026] (3) a nucleotide sequence having one or more, for example, 1, 2, 3, 4, 5 or more, nucleotide substitutions, deletions or insertions in the nucleotide sequence shown in SEQ ID NO: 23;

[0027] Preferably, the genome of the Lactobacillus plantarum comprises the nucleotide sequence shown in SEQ ID NO: 23.

[0028] Preferably, the 16S rDNA gene sequence of the Lactobacillus plantarum is shown as SEQ ID NO: 23.

[0029] Preferably, the plant lactobacillus according to the second aspect or the sixth aspect of the present invention is a plant lactobacillus having a deposit number of CGMCC No. 26502. Specifically, the plant lactobacillus (HY2946) provided by the present invention has been deposited in the General Microbiology Center of the China Culture Collection of Microorganisms (CGMCC for short), the deposit address of which is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with a deposit number of CGMCC No. 26502 and a deposit date of February 7, 2023.

[0030] This strain has the following properties:

[0031] 1. Colony morphological characteristics:

[0032] The strain forms round, smooth-edged, milky white colonies with a protrusion in the middle after being cultured on MRS plates.

[0033] 2. Morphological characteristics of strains:

[0034] After staining, the strain was observed under an optical microscope, and it was determined that the bacteria were Gram-positive and had a short rod shape.

[0035] 3. Physiological and biochemical characteristics:

[0036] The culture temperature of this strain is 35-38℃, and the optimal growth temperature is 37℃.

[0037] The carbon sources that can be utilized by the strain include esculin, cellobiose, maltose, mannitol, salicin, sorbitol, sucrose, raffinose, inulin and lactose.

[0038] 4. Nutritional characteristics:

[0039] The strain does not require any special nutrients for cultivation and is cultured in a basic medium, which is MRS medium, in a facultative anaerobic manner.

[0040] Preferably, the Lactobacillus plantarum is isolated from naturally fermented food or human samples, wherein the naturally fermented food is a naturally fermented food with regional characteristics; and the human sample is selected from one or more of vaginal secretions, feces and breast milk.

[0041] In a seventh aspect, the present invention provides a microbial preparation for preventing and / or treating vaginal infection and related diseases, comprising the Lactobacillus plantarum according to the second aspect or the sixth aspect of the present invention.

[0042] Preferably, the Lactobacillus plantarum is the only probiotic active ingredient in the microbial preparation.

[0043] Preferably, the total number of viable Lactobacillus plantarum in the microbial preparation is not less than 1×10 5 CFU / g, preferably 1×10 5 to 1×10 10 CFU / g.

[0044] Preferably, the microbial preparation further comprises a pharmaceutically acceptable excipient.

[0045] Further preferably, the pharmaceutically acceptable excipient is selected from one or more of a surfactant, a preservative, an antioxidant, a hardener, a thickener and an absorption enhancer.

[0046] Preferably, the microbial preparation is a suppository.

[0047] In an eighth aspect, the present invention provides a combination drug for preventing and / or treating vaginal infections and related diseases, wherein the combination drug comprises the Lactobacillus plantarum described in the second aspect or the sixth aspect of the present invention or the microbial preparation described in the present invention, and other antibacterial drugs.

[0048] In a ninth aspect, the present invention provides use of the Lactobacillus plantarum according to the second aspect or the sixth aspect of the present invention or the microbial preparation according to the present invention in the preparation of a medicament for preventing and / or treating vaginal infection and related diseases.

[0049] Preferably, the vaginal infection and related diseases are bacterial vaginosis and / or vulvovaginal candidiasis.

[0050] Further preferably, the pathogenic bacteria of bacterial vaginosis are selected from one or more of Gardnerella vaginalis, Escherichia coli, Staphylococcus aureus, Fannyhessea vaginae and Prevotella bivia.

[0051] More preferably, the Gardnerella vaginalis is the Gardnerella vaginalis with a deposit number of ATCC 14018, which can be purchased.

[0052] More preferably, the Escherichia coli is the Escherichia coli with a deposit number of ATCC 25922, which can be purchased.

[0053] More preferably, the Staphylococcus aureus is Staphylococcus aureus with a deposit number of ATCC 25923, which can be purchased.

[0054] More preferably, the Atopobium vaginalis is Atopobium vaginalis with a deposit number of CCUG 38953, which can be purchased.

[0055] More preferably, the Prevotella diversa is the Prevotella diversa with a deposit number of NCTC 11156, which can be purchased.

[0056] The present invention has at least the following beneficial effects:

[0057] The specific primers for Lactobacillus plantarum provided by the present invention can highly match the DNA sequence of Lactobacillus plantarum of the present invention and can generate specific amplification only for the DNA sequence of Lactobacillus plantarum of the present invention. The method of the present invention can efficiently and conveniently identify and detect Lactobacillus plantarum of the present invention.

[0058] The present invention provides a novel plant lactobacillus, which is non-toxic and has good biological properties, and has a good therapeutic effect for preventing and / or treating vaginal infection and related diseases thereof. Specifically, the plant lactobacillus of the present invention can produce organic acids with antibacterial effects. The plant lactobacillus of the present invention can inhibit the biofilm formation of pathogenic bacteria that cause vaginal infection and related diseases thereof, thereby having a strong inhibitory effect on pathogenic bacteria that cause vaginal infection and related diseases thereof, such as Gardnerella vaginalis (GV), Candida albicans (CA), Escherichia coli (EC), Staphylococcus aureus (SA), etc.

[0059] The present invention demonstrates the therapeutic potential of the strain for bacterial vaginosis caused by GV and vulvovaginal candidiasis caused by CA through conventional antibacterial experiments, co-culture antibacterial experiments and in vivo animal efficacy experiments.

[0060] The plant lactobacillus strain screened by the present invention avoids the problems of increased drug resistance and high recurrence rate caused by the use of antibacterial drugs such as antibiotics in the treatment of vaginal infection and related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, in which:

[0062] Figure 1 These are colony morphology diagrams of Lactobacillus plantarum of the present invention, wherein the left diagram is a colony diagram using the three-zone line method, and the right diagram is a colony diagram using the plate spreading method.

[0063] Figure 2 This is a diagram of the bacterial morphology of Lactobacillus plantarum of the present invention.

[0064] Figure 3 The electrophoresis patterns of PCR amplification of specific nucleotide sequences of four Lactobacillus plantarum strains by primer 3-p3 according to an embodiment of the present invention are shown, wherein M is a marker, 1 uses the supernatant after lysis of HY2946 as a template, 2 uses the supernatant after lysis of HY02938 as a template, 3 uses the supernatant after lysis of HY00854 as a template, and 4 uses the supernatant after lysis of HY12772 as a template.

[0065] Figure 4 The electrophoresis patterns of PCR amplification of specific nucleotide sequences of four Lactobacillus plantarum strains using primers 2-p3 according to an embodiment of the present invention are shown, wherein M is a marker, 1 uses the supernatant after lysis of HY2946 as a template, 2 uses the supernatant after lysis of HY02938 as a template, 3 uses the supernatant after lysis of HY00854 as a template, and 4 uses the supernatant after lysis of HY12772 as a template.

[0066] Figure 5 The figure is the result of hemolysis experiment using Lactobacillus plantarum of the present invention.

[0067] Figure 6 These are the results of efficacy tests in BV animal models.

[0068] Figure 7 These are the results of efficacy tests in VVC animal models. DETAILED DESCRIPTION

[0069] The present invention will be further described in detail below in conjunction with specific embodiments. The examples given are only for illustrating the present invention, not for limiting the scope of the present invention.

[0070] Example

[0071] 1.1 Experimental methods

[0072] 1.1.1 Screening and Isolation of Lactobacillus plantarum

[0073] (1) Collect samples

[0074] The samples were collected from vaginal secretion samples of volunteers who met the inclusion criteria (healthy, disease-free, no use of antibiotics or other drugs in the past month, and no oral probiotic products) by gynecologists at the Yuhang First People's Hospital in Hangzhou.

[0075] (2) Strain isolation

[0076] The sample was diluted tenfold with physiological saline, and the dilution solution with appropriate dilution gradient was spread on MPYG plates and anaerobic blood plates (purchased from Huankai Microorganisms), and cultured at 37°C in an anaerobic workstation for 48-72 hours. Single colonies with different morphologies were picked and streaked on the blood plates for purification, and culture was continued. Pure cultures were picked for strain identification (16S rDNA sequencing).

[0077] After the strain species is determined, the pure culture is inoculated into MPYG liquid medium for expansion. When the strain grows to an appropriate concentration, sterile 50% (v / v) glycerol solution is used to mix evenly with the liquid culture of the same volume and stored in a -80°C strain bank.

[0078] 1.1.2 Identification of Lactobacillus plantarum

[0079] (1) Colony characteristics

[0080] Use an inoculating loop to dip the bacterial solution in the culture tube, streak inoculate on the MRS plate, and culture anaerobically at 37℃ for 48h to observe the morphology of the colonies (such as Figure 1 The bacterial solution was diluted and spread, and cultured anaerobically at 37°C for 48 hours. The morphology of the colonies on the plate was observed (as shown in the figure on the left). Figure 1 shown on the right).

[0081] (2) Staining microscopy

[0082] Use an inoculating loop to transfer one loop of sterile distilled water onto a clean glass slide. Pick a single colony from the MRS plate, mix it with distilled water, and evenly spread it onto the glass slide. Stain according to the instructions of the Gram stain kit (purchased from Qingdao Haibo Biotechnology Co., Ltd.). Then observe the bacterial morphology.

[0083] (3) Biochemical identification and analysis

[0084] ① Cultivation of Lactobacillus plantarum: The target strain was inoculated into MRS broth medium (purchased from Qingdao Haibo Biotechnology Co., Ltd.), placed in an anaerobic incubator, and cultured at 37°C for 24 hours.

[0085] ② Preparation of bacterial suspension: centrifuge the fermentation liquid of the strain at 4000 rpm for 5 min, remove the supernatant, wash the bacterial mud with physiological saline, centrifuge at 4000 rpm for 5 min, remove the supernatant, wash the bacterial mud with physiological saline again, centrifuge at 4000 rpm for 5 min, remove the supernatant, add physiological saline and mix evenly with the bacterial mud for later use.

[0086] ③1% sodium hippurate identification test: aspirate 50 μL of bacterial suspension, add it to 1% sodium hippurate identification tube, seal it with sealing film and incubate it in a 37°C water bath for 2 hours, then slowly add 200 μL of ninhydrin solution (3.5% ninhydrin solution: 0.175 g of hydrated ninhydrin, 2.5 mL of acetone, and 2.5 mL of butanol) along the wall of the tube. Do not shake it, place it in a 37°C water bath for 10 minutes and then read the results.

[0087] ④ Other identification experiments: aspirate 50 μL of bacterial suspension and add it to the biochemical identification tube (Note: after adding the bacterial solution to the aesculin identification tube, the liquid surface needs to be covered with sterile liquid paraffin), seal it with sealing film, place it in an anaerobic incubator, and incubate it at 37°C for 48 hours before interpreting the results.

[0088] (4) 16S rDNA identification

[0089] The above-mentioned Lactobacillus plantarum was amplified and sequenced by 16S rDNA, and the obtained sequence was then compared with BLAST in the NCBI database.

[0090] (5) Specific fragment molecular markers

[0091] ①Specific primer design

[0092] a. Specific nucleotide sequence screening: The whole genome of Lactobacillus plantarum HY2946 strain was sequenced and analyzed, and the genome sequence was compared with the genome sequence of Lactobacillus plantarum strains included in the NCBI database to screen out the specific nucleotide sequence of Lactobacillus plantarum HY2946.

[0093] b. Primer Design: Design primers for the specific nucleotide sequences screened in step a. Design primer fragments with predicted product lengths of approximately 200 to 800 bp for these specific nucleotide sequences, as shown in Table 1. Also design 10 primer pairs for other nucleotide sequences across the entire genome as controls. See Table 1 for primer sequences.

[0094] Table 1 Primer list

[0095]

[0096]

[0097] c. Primer Screening: Template preparation was performed using Lactobacillus plantarum HY2946 and three strains of the same species as controls (see Table 2 for specific strain information). Single colonies were picked from each strain in 50 μL of lysis buffer (TaKaRa). After brief centrifugation, the lysate was lysed at 80°C for 15 minutes and then centrifuged at 4000 rpm for 5 minutes. The supernatant was used as the template. Primer screening selected 3Dp3 for PCR amplification. The PCR amplification system consisted of 12.5 μL of Taq enzyme, 1 μL of F, 1 μL of R, 1.5 μL of template, and ddHO to 25 μL. PCR reaction conditions are shown in Table 3. After the PCR experiment, 1.5 g of agarose was added to 100 mL of 1× TAE buffer and thoroughly heated to melt. 5 μL of Gel Red dye was added and the sample was poured onto a gel plate to solidify. 4 μL of the PCR product was subjected to agarose gel electrophoresis under the following conditions: voltage 130 V, current 400 mA, and time 35 minutes.

[0098] Table 2 Strain information

[0099] strain number Identification name HY2946 Lactiplantibacillus plantarum HY02938 Lactiplantibacillus plantarum HY00854 Lactiplantibacillus plantarum HY12772 Lactiplantibacillus plantarum

[0100] Note: The strains numbered HY02938, HY00854, and HY12772 in the above table are Lactobacillus plantarum screened by the inventors from the samples collected in Example 1. In order to verify the specificity of the above-mentioned specific nucleotide sequence to Lactobacillus plantarum HY2946, the above-mentioned primers were used to perform PCR amplification on Lactobacillus plantarum HY2946 and three strains of the same species.

[0101] Table 3 PCR reaction conditions

[0102]

[0103] 1.1.3 Characteristics of the strain

[0104] (1) Antibiotic sensitivity test

[0105] Lactobacillus plantarum HY2946 was cultured in MRS broth. The culture solution was then evenly spread on an MRS plate. After the solution was absorbed and dried, antibiotic susceptibility paper was applied. The plate was incubated anaerobically at 37°C for 48 hours. The diameter of the inhibition zone was measured with a vernier caliper. The inhibition zone diameter was used to determine the strain's antibiotic sensitivity.

[0106] (2) Toxicity test

[0107] ①Hemolysis test

[0108] Dip the bacterial solution in the frozen tube of the culture and streak inoculate it on the anaerobic blood plate. Incubate it anaerobically at 37℃ for 48 hours and observe the color change of the blood plate around the colony.

[0109] ②Toxicity test in mice

[0110] Five mice weighing 18-22 g were used, and each mouse was orally gavaged with 0.5 mL of fresh bacterial solution (no less than 1.0 × 10 9 CFU / 0.5ml), once a day for 3 consecutive days, and the mice were observed continuously from the first day of gavage to the seventh day, and the survival of the mice was observed and weighed.

[0111] (3) Determination of metabolite content

[0112] ①D-lactic acid detection

[0113] The D-lactic acid production of the supernatant of Lactobacillus plantarum was detected using a D-lactic acid detection kit (purchased from Sigma-Aldrich). The principle used in this kit is that D-lactic acid is oxidized by a specific D-lactate hydrogenase, producing a color reaction that is proportional to the D-lactic acid concentration, and the absorbance at 450 nm is measured.

[0114] ②L-lactic acid detection

[0115] The supernatant was filtered through a 0.22 μm sterile filter membrane and the L-lactic acid concentration was measured using a biosensor.

[0116] 1.1.4 Application Function Analysis

[0117] (1) Antibacterial experiment

[0118] 1. working bacterial liquid prepares: by 0.5% inoculum size, plant lactobacillus is inoculated into MM medium (MRS broth modified medium, component composition is: peptone 10g / L, beef extract 5.0g / L, yeast extract 4.0g / L, glucose 15g / L, K2HPo42.0g / L, triammonium citrate 1.0g / L, sodium acetate 2.5g / L, magnesium sulfate 0.2g / L, manganese sulfate 0.05g / L) in, cultivate in anaerobic workstation. The following six pathogenic bacteria (Gardnerella vaginalis ATCC 14018, GV; Escherichia coli ATCC 25922, EC; Staphylococcus aureus ATCC 25923, SA; Fannyhessea vaginae CCUG 38953, FV; Prevotella bivia NCTC 11156, PB; Canidia albicans ATCC 10231, CA) were cultured using adapted culture medium. After culturing the target strain, the supernatant was centrifuged and filtered through 0.22 μm to obtain a cell-free supernatant. The supernatant was used immediately or stored in a -80°C freezer. OD values ​​were measured after culturing the pathogenic bacteria. 600 value, diluted to about OD 600 The value is 0.005 (the number of viable bacteria is maintained at 5.0×10 5 CFU / mL to 5.0×10 6 CFU / mL) (WS / T650-2019 Antibacterial and antibacterial effect evaluation method).

[0119] ② Interaction: Take the same volume of supernatant and pathogenic bacteria solution and mix them evenly. Immediately take 100 μL of the mixed solution and place it into a blank 96-well plate to measure OD 600 The remaining culture medium was placed at 37°C and cultured anaerobically or aerobically according to the culture conditions of the pathogen. After 48 hours of culture, 100 μL of the mixed bacterial solution was taken and placed into a blank 96-well plate to measure the OD 600 A blank control group was set up, with two replicate wells for each sample, and the inhibition rate of Lactobacillus plantarum against pathogenic bacteria was calculated according to the following formula.

[0120] Antibacterial rate = (AB) / A*100%

[0121] A: OD increase in the positive control group (blank culture medium) within 48 hours 600 value;

[0122] B: OD increase in the experimental group within 48 hours 600 value.

[0123] (2) Co-culture antibacterial experiment

[0124] ① Interaction and co-culture experiment of Lactobacillus plantarum and CA

[0125] Lactobacillus plantarum culture: Take a glycerol tube of Lactobacillus plantarum and inoculate it into MM medium (MRS broth modified medium, the composition is: peptone 10g / L, beef extract 5.0g / L, yeast extract 4.0g / L, glucose 15g / L, K2HPO42.0g / L, ammonium citrate tribasic 1.0g / L, sodium acetate 2.5g / L, magnesium sulfate 0.2g / L, manganese sulfate 0.05g / L), and concentrate or dilute the cultured bacterial liquid to about 1×10 9 CFU / mL, used as the working bacterial solution of Lactobacillus plantarum.

[0126] CA culture: Inoculate the CA glycerol tube into Sabouraud medium and culture aerobically at 37°C. Centrifuge the culture solution to remove the supernatant, and then adjust the concentration of the culture solution to about 1.0 × 10 7 CFU / mL, used as CA working bacterial solution.

[0127] 400 μL of each Lactobacillus plantarum and CA working culture solution were inoculated into 40 mL of MM liquid medium. CA alone was inoculated as a positive control. Two replicates were used for each group. The culture solution was gently shaken and incubated at 37°C in an anaerobic workbench. Samples were taken 20 hours after incubation, and viable CA bacteria were counted using Candida albicans chromogenic medium (purchased from CHROMagar, France).

[0128] ② Interaction and co-culture experiment of Lactobacillus plantarum and GV

[0129] Lactobacillus plantarum culture: Take a glycerol tube of Lactobacillus plantarum and inoculate it into MM medium (MRS broth modified medium, the composition is: peptone 10g / L, beef extract 5.0g / L, yeast extract 4.0g / L, glucose 15g / L, K2HPO4 2.0g / L, ammonium citrate tribasic 1.0g / L, sodium acetate 2.5g / L, magnesium sulfate 0.2g / L, manganese sulfate 0.05g / L), and concentrate or dilute the cultured bacterial solution to about 1×10 9 CFU / mL concentration is used as the working bacterial solution of Lactobacillus plantarum.

[0130] GV culture: GV glycerol tubes were inoculated into BHI liquid medium containing 10% fetal bovine serum and cultured anaerobically at 37°C. The cultured bacterial solution was centrifuged to remove the supernatant, and then the bacterial solution concentration was adjusted to about 1.0 × 10 7 CFU / mL, used as GV working bacterial solution.

[0131] 400 μL of Lactobacillus plantarum and GV working bacterial solution were respectively inoculated into 40 mL of BHI liquid culture medium containing 10% fetal bovine serum. GV was inoculated alone as a positive control group. Two replicates were placed in an anaerobic workstation for culture. Samples were taken 27 hours after culture, and the number of viable GV bacteria was detected by fluorescent quantitative qPCR using GV-specific probe primers.

[0132] (3) Biofilm removal experiment

[0133] ① Experiment on the removal of GV biofilm by Lactobacillus plantarum

[0134] After GV culture, the bacterial solution was adjusted to 1.0×10 7 CFU / mL were inoculated into 96-well plates, with two groups of four replicates each. The two groups were supplemented with equal volumes of blank MM liquid medium (MRS broth modified medium, consisting of: peptone 10 g / L, beef extract 5.0 g / L, yeast extract 4.0 g / L, glucose 15 g / L, K2HPO4 2.0 g / L, ammonium citrate tribasic 1.0 g / L, sodium acetate 2.5 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L) and supernatant from Lactobacillus plantarum HY2946. The plates were incubated at 37°C in an anaerobic workstation for 24 hours.

[0135] After the culture, the inhibitory effect of Lactobacillus plantarum HY2946 on GV biofilm formation was determined by microplate crystal violet staining.

[0136] ②Experiment on the removal of CA biofilm by Lactobacillus plantarum

[0137] Inhibition of biofilm removal experiment: The bacterial solution after CA culture was adjusted to 1.0×10 7 CFU / mL were inoculated into 96-well plates in two replicate groups. Equal volumes of Sabouraud dextrose broth and Lactobacillus plantarum supernatant were added to each group. The plates were incubated at 37°C in an anaerobic workstation for 24 hours. After incubation, the inhibitory effect of Lactobacillus plantarum on CA biofilm formation was determined using crystal violet staining in microplates.

[0138] Biofilm removal experiment: adjust the bacterial solution after CA culture to 1.0×10 7CFU / mL, 100 μL was inoculated into 96-well plates, with two groups of four replicates each. The plates were incubated at 37°C in an anaerobic workstation for 24 hours. After 24 hours of incubation, the mixture was discarded and the wells were washed with sterile PBS. An equal volume of MM medium (MRS broth modified medium, consisting of: 10 g / L peptone, 5.0 g / L beef extract, 4.0 g / L yeast extract, 15 g / L glucose, 2.0 g / L K₂HPO₄, 1.0 g / L triammonium citrate, 2.5 g / L sodium acetate, 0.2 g / L magnesium sulfate, and 0.05 g / L manganese sulfate) and Lactobacillus plantarum supernatant were added to each group and incubated at 37°C for 24 hours. After incubation, the effect of the Lactobacillus plantarum supernatant on the removal of mature CA biofilms was determined using a microplate crystal violet staining method.

[0139] (4) Cell adhesion assay

[0140] ① Preparation of working bacterial solution: culture Lactobacillus plantarum and test the OD of bacterial solution 600 The culture solution of Lactobacillus plantarum was centrifuged at 4000 rpm for 5 min at 4°C, the supernatant was discarded, and the solution was washed three times with PBS. Finally, Lactobacillus plantarum was resuspended in MEM complete medium (purchased from Zhongqiao Xinzhou). An appropriate amount of the resuspended solution was used to detect the number of viable bacteria by plate coating, which was L1.

[0141] ②Cell culture: HeLa cells were seeded in 24-well plates, with 1.5×10 cells per well. 5 The HeLa cells cultured in 24-well plates were washed with serum-free MEM medium (purchased from Zhongqiao Xinzhou) and then counted, recording C1.

[0142] ③ Interaction: Lactobacillus plantarum was inoculated into the cells at a bacteria-to-cell ratio of 100:1 and incubated at 37°C in a 5% CO2 environment for 1.5 hours. The supernatant after centrifugation was added to the cells as a blank control. After 1.5 hours, the culture medium in the wells was collected, and the cells in the wells were washed with MEM medium. Trypsin was then added to each well for digestion, and the reaction was terminated by adding MEM complete medium. The suspension was collected, and a portion of the cell count (C2) was taken. The number of viable Lactobacillus plantarum cells adhering to the cells was determined by plate spreading (L2).

[0143] ④ Calculate the number of adhesions and adhesion rate: average number of Lactobacillus plantarum cells adhered = L2 / C2, adhesion rate (%) = 100*L2 / L1.

[0144] (5) BV animal model efficacy test

[0145] Healthy SPF-grade Balb / c female mice, 6-8 weeks old, were used for modeling. After adaptive culture, the animals were randomly divided into 8 groups: a model group (M), an experimental group (Lactobacillus plantarum HY2946), and a positive control group (DJS). Before inoculation with pathogenic bacteria, each group of animals received a subcutaneous injection of estradiol benzoate injection, followed by vaginal administration of the same concentration of GV (20 μL) to establish the BV pathogen model. After successful modeling, the experimental group received vaginal administration of the corresponding Lactobacillus plantarum solution (1×10 10 CFU / mL, 20 μL), and the positive control group was given vaginally an equal amount of Lactobacillus delbrueckii solution (Dingjunshengzhong strain, 1×10 10 CFU / mL, 20 μL). The model group received an equal volume of normal saline. After treatment, vaginal lavage was performed four times per animal with 50 μL of PBS. The fluid was collected, placed in a 1.5 mL Eppendorf tube, and frozen at -80°C. The GV load in the lavage fluid was determined by qPCR, and the differences in GV load in the lavage fluid were compared between the groups.

[0146] (6) VVC animal model efficacy test

[0147] Healthy SPF-grade Balb / c female mice, 6-8 weeks old, were used for modeling. After adaptive culture, the animals were randomly divided into 8 groups: a model group (M), an experimental group (Lactobacillus plantarum HY2946), and a positive control group (Dapoxetine suppository). Each group received a subcutaneous injection of estradiol benzoate injection for pretreatment. CA (15 μL / mouse) was continuously administered from D0 to D2, and the experimental group received vaginal administration of Lactobacillus plantarum HY2946 (5×10 β-lactamase) daily from D3 to D7. 9 CFU / mL, 20 μL). A positive control group was given 30 mg of bisoprolol suppositories, and a model group was given an equal volume of normal saline (20 μL). Vaginal lavage was performed on day 8. The lavage fluid was diluted and spread on a Candida albicans identification medium (chromogenic medium). After incubation at 37°C for 48 hours, the number of green colonies on the culture plate was observed and counted. One-way analysis of variance was performed using GraphPad Prism 5 software, and P < 0.05 was considered statistically significant.

[0148] 1.2 Experimental Results

[0149] 1.2.1 Screening and Isolation of Lactobacillus plantarum

[0150] Through identification and screening, a strain of Lactobacillus plantarum was obtained and named Lactobacillus plantarum HY2946.

[0151] 1.2.2 Identification of Lactobacillus plantarum

[0152] (1) Colony characteristics

[0153] like Figure 1 As shown, the strain forms round, smooth-edged, milky white colonies with a protrusion in the middle after being cultured on MRS plates.

[0154] (2) Staining microscopy / electron microscopy

[0155] like Figure 2 As shown, after the strain was stained and observed under an optical microscope, it can be determined that the bacteria were Gram-positive and had a short rod shape.

[0156] (3) Biochemical identification and analysis

[0157] The results of the target Lactobacillus plantarum biochemical identification tube were interpreted according to the instructions for use of the lactic acid bacteria biochemical identification tube. The interpretation results are detailed in Table 4.

[0158] Table 4 Interpretation results of the Lactobacillus plantarum identification tube

[0159]

[0160] Note: + represents positive; - represents negative; +w represents weak positive.

[0161] The results of biochemical identification showed that except for 1% sodium hippurate, Lactobacillus plantarum could utilize the other 10 carbon sources (esculin, cellobiose, maltose, mannitol, salicin, sorbitol, sucrose, raffinose, inulin and lactose).

[0162] (4) 16S rDNA identification

[0163] 16S rDNA comparison results showed that the strain belongs to Lactobacillus plantarum. The 16S rDNA gene sequence (SEQ ID NO: 23) is shown below:

[0164]

[0165] (5) Specific fragment molecular labeling results

[0166] Among the primers designed for specific nucleotide sequences, PCR amplification experiments were performed using 3-p3 as primer. Only HY2946 had a specific amplification band. The band was clear and high in concentration, and the primer dimer was relatively small. However, HY02938, HY00854, and HY12772 had no amplification band. The agarose gel electrophoresis detection pattern is shown in Figure 3 When PCR amplification experiments were performed with primers designed using other nucleotide sequences, the control bacteria group also amplified bands (taking 2-p3 as an example, the agarose gel electrophoresis detection pattern is shown in Figure 4 ). Therefore, primer 3-p3 is a molecular marker primer for HY2946, and the amplification product generated by this primer is a molecular marker for Lactobacillus plantarum HY2946. The amplification product of primer 3-p3 is 578 bp in size, and the nucleotide sequence is shown in SEQ ID NO: 24. This confirms that the nucleotide sequence shown in SEQ ID NO: 24 is part of the specific nucleotide sequence screened in step a, further verifying that primer 3-p3 can serve as a specific primer for Lactobacillus plantarum HY2946, and that the nucleotide sequence shown in SEQ ID NO: 24 is a specific nucleotide sequence marker for Lactobacillus plantarum HY2946. This provides a reliable tool and basis for identifying and detecting Lactobacillus plantarum HY2946.

[0167] (SEQ ID NO: 24).

[0168] In addition, during the experiment, the inventors found that when using the 3-p3 primer to sequence multiple Lactobacillus plantarum HY2946 samples, the amplified product sequences were found to differ from the nucleotide sequence shown in SEQ ID NO: 24 in individual bases. However, compared with the control bacteria, they all had specific amplification bands, and the bands were clear and high in concentration, and the primer dimers were relatively few.

[0169] In one experiment, it was found that the nucleotide sequence of the product amplified from the Lactobacillus plantarum HY2946 sample using the 3-p3 primer was shown in SEQ ID NO: 25, which had a 99.65% identity with the nucleotide sequence shown in SEQ ID NO: 24.

[0170] (SEQ ID NO: 25)

[0171] Therefore, after multiple sequencing and alignment, the homology range between the molecular marker of Lactobacillus plantarum HY2946 and the nucleotide sequence shown in SEQ ID NO: 24 was determined, that is, the nucleotide sequence of the molecular marker of Lactobacillus plantarum HY2946 has at least 97%, 98%, 99% or higher homology with the nucleotide sequence shown in SEQ ID NO: 24.

[0172] 1.2.3 Characteristics of the strain

[0173] (1) Antibiotic sensitivity test

[0174] The antibiotic sensitivity of the strain was determined by the diameter of the bacterial ring on the antibiotic sensitivity paper. The results are shown in the table below. The strain was resistant to metronidazole, norfloxacin, ciprofloxacin and kanamycin, moderately sensitive to ofloxacin, and sensitive to clindamycin and cefuroxime.

[0175] Table 5 Antibiotic sensitivity test results

[0176]

[0177] Note: S: sensitive (15-20mm highly sensitive; >20mm extremely sensitive); I: intermediate (10-14mm moderately sensitive); R: resistant (<10mm insensitive)

[0178] (2) Toxicity test

[0179] ① Hemolytic

[0180] Hemolysis test results Figure 5 As shown, gray-white tiny colonies appeared in the culture medium around the colonies, and no hemolytic ring appeared around the colonies, indicating that Lactobacillus plantarum HY2946 was γ-hemolytic, that is, non-hemolytic.

[0181] ② After oral gavage with fresh bacterial liquid of Lactobacillus plantarum HY2946, all mice survived healthily and gained weight.

[0182] (3) Determination of metabolite content

[0183] Table 6 Metabolite content determination results

[0184]

[0185] Lactobacillus plantarum HY2946 was cultured in MRS broth for 24 hours. After testing, the D-lactic acid production was 13.23 g / L, the L-lactic acid production was 7.01 g / L, and the total acid production was 20.24 g / L, which was higher than the 5.48 g / L of DJS-Lactobacillus delbrueckii. These organic acids are antibacterial substances that can compete with pathogenic bacteria for nutrients and adhesion sites, thereby improving the host's mucosal immunity and anti-infection ability.

[0186] 1.2.4 Application Function Analysis

[0187] (1) Antibacterial experiment

[0188] Two strains of the same species, the positive drug DJS and the screened Lactobacillus plantarum HY2946 were selected to test their antibacterial properties.

[0189] Table 7 Antibacterial test results

[0190]

[0191] The experimental results are shown in the table above. HY2946 has a good inhibitory effect on GV, PB, EC, and CA. Regarding CA, the strain has a strong inhibitory effect, compared to the positive drug's 48.74% essentially no inhibitory effect.

[0192] (2) Results of co-culture antibacterial experiment

[0193] ① Results of antibacterial experiments on co-culture of Lactobacillus plantarum and CA

[0194] The number of viable CA cells was detected after Lactobacillus plantarum and CA were co-cultured for 20 h.

[0195] Table 8 Antibacterial test results of co-culture of Lactobacillus plantarum and CA

[0196]

[0197] The experimental results are shown in the table above. After Lactobacillus plantarum and CA were co-cultured for 20 hours, the inhibition rate reached 80.3%, indicating that the strain had a good inhibitory effect on the growth of CA.

[0198] ② Antibacterial experiment of co-culture of Lactobacillus plantarum and GV

[0199] After Lactobacillus plantarum and GV were co-cultured for 27 h, the number of viable GV cells was detected.

[0200] Table 9 Antibacterial experiment of co-culture of Lactobacillus plantarum and GV

[0201]

[0202] The experimental results are shown in the table above. After Lactobacillus plantarum and GV were co-cultured for 27 hours, the inhibition rate reached 86%, indicating that the strain had a strong inhibitory effect on the growth of GV.

[0203] (3) Biofilm removal test results

[0204] ① Experimental results of Lactobacillus plantarum on the removal of GV biofilm

[0205] The effect of Lactobacillus plantarum on GV biofilm formation was investigated.

[0206] Table 10 Experimental results of the removal of GV biofilm by Lactobacillus plantarum

[0207] strain Inhibitory rate of Lactobacillus plantarum supernatant on GV biofilm formation Lactobacillus plantarum HY2946 29.4％±20.7％

[0208] The experimental results are shown in the table above. The supernatant of Lactobacillus plantarum has a certain inhibitory effect on the formation of GV biofilm.

[0209] ② Experimental results of Lactobacillus plantarum removing CA biofilm

[0210] The effects of Lactobacillus plantarum on the formation of CA biofilm and the removal of formed biofilm were investigated.

[0211] Table 11 Results of the experiment on the removal of CA biofilm by Lactobacillus plantarum

[0212]

[0213] The experimental results are shown in the table above. The strain not only has a strong inhibitory effect on the formation of CA biofilm, but also has a strong ability to destroy the formed CA biofilm.

[0214] (4) Cell adhesion assay

[0215] The number of single-cell adhesion of Lactobacillus plantarum HY2946 to Hela cells was 5.05 CFU, indicating that this strain has good adhesion or colonization characteristics to vaginal epithelial cells.

[0216] (5) BV animal model efficacy test

[0217] The model group (M), experimental group (HY2946) and positive control group (DJS) were respectively subjected to BV pathogenic bacteria modeling. After successful modeling, the experimental group was given corresponding Lactobacillus plantarum solution (1×10 10 CFU / mL, 20 μL), and the positive control group was given vaginally an equal amount of Lactobacillus delbrueckii solution (Dingjunshengzhong strain, 1×10 10 CFU / mL, 20μL), and the model group was treated with an equal volume of normal saline. Finally, the differences in the GV load in the lavage fluid between the groups were detected and compared. The experimental results are as follows Figure 6 shown.

[0218] The experimental results showed that using HY2946 to treat BV-infected animal models significantly reduced the GV content in their vaginas, achieving a certain therapeutic effect, while positive drugs did not show any therapeutic effect.

[0219] (6) VVC animal model efficacy test

[0220] The model group (M), experimental group (HY2946) and positive control group (Dapoxetine suppository) were used to establish VVC models. After successful modeling, the experimental group was given Lactobacillus plantarum HY2946 (5×10 9 CFU / mL, 20μL), the positive control group was given bisoprolol suppository (30mg), and the model group was given an equal volume of normal saline (20μL). Finally, the content of Candida albicans in the vaginal lavage fluid was detected. The experimental results are as follows Figure 7 shown.

[0221] The experimental results showed that compared with the model group, the content of pathogenic bacteria in the vaginal lavage fluid of the HY2946 group was significantly reduced after treatment, indicating that this strain has a good therapeutic effect on VVC in mice.

Claims

1. A gene for molecular marker of Lactobacillus plantarum, having a nucleotide sequence selected from one of the following: (1) the nucleotide sequence shown in SEQ ID NO: 24; (2) a nucleotide sequence having at least 97%, 98%, 99% or higher homology to the nucleotide sequence shown in SEQ ID NO: 24; (3) a nucleotide sequence having one or more, for example, 1, 2, 3, 4, 5 or more, nucleotide substitutions, deletions or insertions in the nucleotide sequence shown in SEQ ID NO: 24; Preferably, the gene for the molecular marker of Lactobacillus plantarum has a nucleotide sequence as shown in SEQ ID NO: 24, or its nucleotide sequence is as shown in SEQ ID NO:

24.

2. A Lactobacillus plantarum comprising the gene for molecular marking according to claim 1.

3. A primer set for identifying the plant lactobacillus according to claim 2, comprising a forward primer and a reverse primer, wherein: The nucleotide sequence of the forward primer is shown in SEQ ID NO: 1; The nucleotide sequence of the reverse primer is shown in SEQ ID NO:

2.

4. A method for identifying the plant lactobacillus according to claim 2, comprising: The genomic DNA of the strain to be identified is amplified using the primer set according to claim 3, and then the amplified product is detected.

5. The method according to claim 4, comprising the steps of: extracting a genomic DNA sample from the strain to be identified, performing PCR amplification on the genomic DNA sample using the primer set according to claim 3, and then comparing the amplified product with the gene for molecular marker according to claim 1; if the amplified product contains the gene for molecular marker according to claim 1, then the strain to be identified is the Lactobacillus plantarum according to claim 2; Preferably, if the amplification product comprises the nucleotide sequence shown in SEQ ID NO: 24, the strain to be identified is the Lactobacillus plantarum according to claim 2.

6. Use of the gene for molecular marker according to claim 1 in identifying the Lactobacillus plantarum according to claim 2.

7. A Lactobacillus plantarum comprising a nucleotide sequence selected from one of the following: (1) the nucleotide sequence shown in SEQ ID NO: 23; (2) a nucleotide sequence having at least 99.8%, 99.9% or higher homology to the nucleotide sequence shown in SEQ ID NO: 23; (3) a nucleotide sequence having one or more, for example, 1, 2, 3, 4, 5 or more, nucleotide substitutions, deletions or insertions in the nucleotide sequence shown in SEQ ID NO: 23; Preferably, the genome of the Lactobacillus plantarum comprises the nucleotide sequence shown in SEQ ID NO: 23; Preferably, the 16S rDNA gene sequence of the Lactobacillus plantarum is shown as SEQ ID NO:

23.

8. The Lactobacillus plantarum according to claim 2 or 7, wherein The Lactobacillus plantarum is a Lactobacillus plantarum with a preservation number of CGMCC No. 26502.

9. A microbial preparation for preventing and / or treating vaginal infection and related diseases, comprising the Lactobacillus plantarum according to claim 2 or 7.

10. The microbial preparation according to claim 9, wherein The Lactobacillus plantarum is the only probiotic active ingredient in the microbial preparation.

11. The microbial preparation according to claim 9 or 10, wherein The total number of viable Lactobacillus plantarum in the microbial preparation is not less than 1×10 5 CFU / g, preferably 1×10 5 to 1×10 10 CFU / g.

12. The microbial preparation according to any one of claims 9 to 11, wherein The microbial preparation further comprises a pharmaceutically acceptable excipient; Preferably, the pharmaceutically acceptable excipient is selected from one or more of a surfactant, a preservative, an antioxidant, a hardener, a thickener and an absorption enhancer.

13. A combined drug for preventing and / or treating vaginal infection and related diseases, comprising the Lactobacillus plantarum according to claim 2 or 7 or the microbial preparation according to any one of claims 9 to 12, and other antibacterial drugs.

14. Use of the Lactobacillus plantarum according to claim 2 or 7 or the microbial preparation according to any one of claims 9 to 12 in the preparation of a medicament for preventing and / or treating vaginal infection and related diseases; Preferably, the vaginal infection and related diseases are bacterial vaginosis and / or vulvovaginal candidiasis.

15. The use according to claim 14, wherein The pathogenic bacteria of bacterial vaginosis are selected from one or more of Gardnerella vaginalis, Escherichia coli, Staphylococcus aureus, Atopobium vaginalis and Prevotella diversa.

Images