WO2023116283A1 - 一种抗结直肠肿瘤的乳酸肠球菌及其应用 - Google Patents

一种抗结直肠肿瘤的乳酸肠球菌及其应用 Download PDF

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WO2023116283A1
WO2023116283A1 PCT/CN2022/132525 CN2022132525W WO2023116283A1 WO 2023116283 A1 WO2023116283 A1 WO 2023116283A1 CN 2022132525 W CN2022132525 W CN 2022132525W WO 2023116283 A1 WO2023116283 A1 WO 2023116283A1
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tumor
enterococcus
lactic acid
preparation
tumors
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French (fr)
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朱永亮
穆晓静
刘丹
朱蒙蒙
马梦楠
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苏州普瑞森生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57419—Specifically defined cancers of colon

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  • the invention belongs to the field of microorganisms, and in particular relates to a strain of lactic acid enterococcus against colorectal tumors and its application.
  • Colorectal cancer is a common malignant tumor of the digestive system with an insidious onset and a high mortality rate.
  • the treatment of advanced colorectal cancer is mainly based on 5FU or capecitabine plus oxaliplatin or irinotecan chemotherapy combined with targeted drugs such as bevacizumab, cetuximab, panitumumab, etc. .
  • targeted drugs such as bevacizumab, cetuximab, panitumumab, etc.
  • chemotherapy combined with targeted therapy has improved the overall survival rate of patients with advanced colorectal cancer, few drugs can be used for colorectal cancer patients who have progressed after multiple lines of chemotherapy, and chemotherapy-induced gastrointestinal reactions, bone marrow suppression , neurotoxicity and other adverse reactions often occur.
  • Microbes are involved in the regulation of intestinal homeostasis, and some commensal and pathogenic organisms of the human gut microbiome are required in the pathogenesis of colorectal cancer. Therefore, manipulation of gut bacterial composition and local metabolites through the use of probiotics was demonstrated for therapeutic intervention in colorectal cancer.
  • Probiotics are live microbial feed additives that beneficially affect the health of the host.
  • Chinese patent 201910909859.7 discloses a probiotic formula and screening method for inhibiting colorectal cancer pathogenic bacteria.
  • the probiotic formula is: Clostridium butyricum 1.3 ⁇ 10 10 CFU/mL, Enterococcus faecalis 1 ⁇ 10 9 CFU/mL mL, Lactobacillus brevis 1 ⁇ 10 9 CFU/mL, Lactobacillus plantarum 1 ⁇ 10 9 CFU/mL, Lactobacillus rhamnosus 1 ⁇ 10 9 CFU/mL, Lactobacillus sake 1 ⁇ 10 9 CFU/mL, mesenteric Contodia 1 ⁇ 10 9 CFU/mL.
  • the probiotic formula of the invention does not produce drug resistance, is safe, effective, and has excellent performance, but involves many strains and the scheme is complicated.
  • Chinese patent 201480080834.6 discloses the application of microorganisms, especially fungal strains, in the treatment and prevention of diseases related to colorectal cancer.
  • Compositions comprising fungal strains are contemplated.
  • the results of animal experiments proved the ability of Eubacterium ventriosum and Eubacterium eligens to effectively prevent and treat colitis and colorectal cancer.
  • Enriching microbial germplasm resources for the treatment of colorectal cancer is very beneficial to the treatment and prognosis of colorectal cancer.
  • the present invention isolates a strain of Enterococcus lactis against colorectal tumors from the fecal liquid of healthy people, and applies it to the treatment of colorectal tumors, in order to provide a way to obtain it for the treatment of colorectal tumors Drugs that are easier and more effective.
  • the present invention provides a strain of Enterococcus lactic acid.
  • the 16S rRNA sequence of Enterococcus lactis is a sequence having more than 99.93% identity with SEQ ID NO.1.
  • the 16S rRNA sequence of Enterococcus lactis is SEQ ID NO.1, and the preservation number is CCTCC NO: M20211220, which is preserved in the China Center for Type Culture Collection, and the preservation date is September 26, 2021.
  • the present invention provides the use of Enterococcus lactis in the preparation of products or medicines for diagnosing, preventing and/or treating tumors.
  • the lactic acid enterococcus is the aforementioned lactic acid enterococcus.
  • the tumors include, but are not limited to: tumors of the digestive system, tumors of the respiratory system, tumors of the reproductive system, tumors of the motor system, tumors of the nervous system, tumors of the endocrine system, tumors of the circulatory system, tumors of the urinary system, and tumors of the reproductive system.
  • the tumor is a tumor of the digestive system
  • the tumor of the digestive system is one or more of esophageal tumors, stomach tumors, cardiac tumors, intestinal tumors, liver tumors, and gallbladder tumors.
  • the tumor is a colorectal tumor
  • the colorectal tumor includes one or more of polypoid, stenotic, and ulcerative types.
  • the present invention provides a formulation for the aforementioned use.
  • the preparation includes the aforementioned Enterococcus lactis and/or its preparations.
  • the preparations are dead bacteria, broken cells, fermentation supernatant, fermentation precipitate, modified bacteria, mutant bacteria and/or mutagenic bacteria of Enterococcus lactis.
  • the preparation method of the fermentation supernatant of described Enterococcus lactis comprises:
  • the preparation method of the cell disruptor of Enterococcus lactic acid comprises:
  • the conditioned culture includes but not limited to: anaerobic culture, aerobic culture, and facultative anaerobic culture.
  • the preparation is a liquid preparation, and the amount of Enterococcus lactis contained in the liquid preparation is 5 ⁇ 10 9 CFU/mL.
  • the preparation is a solid preparation or a semi-solid preparation, and the amount of Enterococcus lactis in the preparation is 5 ⁇ 10 9 CFU/g.
  • the preparation is freeze-dried powder.
  • the present invention provides a gene encoding product.
  • the gene encoding product is encoded by the gene on the genome of the aforementioned Enterococcus lactis, and can diagnose, prevent and/or treat colorectal tumors.
  • the present invention provides the aforementioned cultivation method of Enterococcus lactic acid, characterized in that it comprises the following steps:
  • the conditioned culture includes but not limited to: anaerobic culture, aerobic culture, and facultative anaerobic culture.
  • the present invention provides the application of the aforementioned preparation in the preparation of products or medicines for diagnosing, preventing and/or treating colorectal tumors.
  • the present invention provides a medicament for the aforementioned applications.
  • the medicine includes the aforementioned Enterococcus lactis or its preparation.
  • the medicament includes the aforementioned preparations.
  • the amount of enterococcus lactic acid included in the medicine is 5 ⁇ 10 9 CFU/mL.
  • the drug also includes one or more of 5FU, capecitabine, oxaliplatin, irinotecan, bevacizumab, cetuximab, and panitumumab.
  • the medicine also includes other pharmaceutically acceptable carriers or excipients.
  • the drug is an oral drug.
  • Enterococcus lactic acid CCTCC NO:M 20211220 provided by the present invention can still ensure a survival rate of 77.60% when incubated in an artificial gastric juice environment with pH 4.0 for 3 hours. In the mouse MC38 model, this Enterococcus lactis was significantly effective in suppressing tumor volume.
  • Bacteria name Enterococcus lactic acid PRS-15;
  • CCTCC CCTCC
  • Registration number of the collection center CCTCC NO:M 20211220.
  • Figure 1 is the curve of tumor size change in the MC38 mouse model after administration.
  • Figure 2 is the body weight change curve of the MC38 mouse model after administration.
  • Example 1 Isolation of an anti-colorectal tumor-resistant Enterococcus lactic acid
  • the prepared healthy human feces bacteria liquid was diluted 10 times, and three concentration gradients were selected, streaked and inoculated on NA and MRS medium, and three replicate plates were placed in a constant temperature incubator at 37°C. Oxygen culture was carried out for 72 hours, and then colonies of different forms were picked for Gram staining and microscopic examination, and streaked and purified twice in MRS medium, and then stained for microscopic examination to observe the bacterial morphology and whether it was purified, and then 16srRNA sequencing was performed using The primer sequences are:
  • the PCR reaction system is as follows:
  • the PCR reaction conditions are as follows:
  • the amplified PCR products were subjected to 16srRNA sequencing and compared at NCBI.
  • the sequencing result is shown in SEQ ID NO.1.
  • Embodiment 2 drug resistance detection
  • the drug-sensitive disc diffusion method was used to carry out the bacterial liquid cultured in MRS liquid medium (purchased from Qingdao Haibo Biotechnology Co., Ltd., article number: HB0384-1), and centrifuged at 4000rpm for 15min, removed the supernatant, and carried out with PBS.
  • MRS liquid medium purchased from Qingdao Haibo Biotechnology Co., Ltd., article number: HB0384-1
  • This strain is mainly resistant to quinolones, nitrofurans, polymyxin B, lincomycins, cotrimoxazole and gentamicin; Lipids, tetracyclines, cephalosporins, and glycopeptide antibiotics showed susceptibility.
  • Embodiment 3 Resistance to artificial gastrointestinal fluid detection
  • MTT solution Weigh 0.5g MTT (purchased from Beijing Suo Laibao, article number: M8180-250), add 100mL PBS to dissolve (final concentration is 5mg/mL), filter and sterilize, and distribute in 15mL centrifuge tubes, Freeze at -20°C for later use, valid for half a year.
  • Bacteria collection the strain was cultured statically at 37°C for 8 hours, and reached the logarithmic growth phase. The bacterial solution was divided into 50mL sterile EP tubes, centrifuged at 4000rpm at room temperature for 15min, the supernatant was discarded, and the bacterial cells were resuspended with PBS and adjusted.
  • MTT detection Take 3mL of the bacterial solution of the control group and the bacterial solution incubated for different pHs and different times into new centrifuge tubes, and add 1mL of MTT reagent to incubate overnight (in this step, the bacterial solution can be centrifuged at 3500rpm for 8min , remove the supernatant artificial gastric juice, then add MTT reagent, MTT reagent can be reduced to 500ul, and the incubation time can also be shortened), after incubation, then centrifuge (3500rpm, 8min), remove the supernatant, add 600 ⁇ L DMSO ( Sinopharm Analytical Pure 98.5%), after pipetting and dissolving, add to deep-well plate respectively, then transfer to 96-well sterile culture plate with row gun, add 150 ⁇ L to each well (and mark clearly the order and name of each well), and then use Use a microplate reader to detect, select a wavelength of 570nm or 630nm to obtain the absorbance value, and
  • the bacteria collection method is the same as the Simulated Gastric Fluid (SGF) experiment;
  • MTT detection the same as above simulated gastric fluid (Simulated Gastric Fluid, SGF) experiment;
  • Bile preparation add ox gall powder to the target bacteria culture medium (MRS or BHI) solution, set three final concentrations, respectively 10g/L (1% ox gall powder), 20g/L (2% ox gall powder ) and 40g/L (4% ox gall powder), standby after autoclaving, meanwhile, without adding the culture medium solution 0h of gall powder as contrast.
  • MRS medium was selected.
  • strain collection is the same as above.
  • Cultivation of ox gall powder add 10 mL of MRS or BHI medium containing 0%, 1%, 2% and 4% ox gall powder (purchased from Hefei BASF Biotechnology Co., Ltd., article number: N0101-100g) to After washing, 20 15mL centrifuge tubes were used to incubate and cultivate 0h, 1h, 2h, 3h and 4h of bacterial precipitates collected, and 0% ox gall powder medium solution 0h was used as the control group, and MRS medium was selected in this embodiment.
  • Enterococcus lactic acid CCTCC NO:M20211220 it can still maintain a good survival rate in the gastric juice environment with a low pH value; it can still guarantee a survival rate of 77.60% when incubated in an artificial gastric juice environment with a pH of 4.0 for 3 hours; It can grow normally in 1%-4% ox bile powder medium, and as the incubation time increases, the number of viable bacteria also increases, indicating that the strain has a certain degree of tolerance to ox bile salt and can grow normally reproduce.
  • mice used in this example are female C57BL/6 or mice, with an age of 6-7 weeks (the estimated age of the mice at the time of tumor cell inoculation), and the weight range of the mice at the time of inoculation of cells is 18-22g, purchased from Junke Biotechnology Co., Ltd.
  • the mouse MC38 model is used for drug efficacy verification, and the model construction method is as follows:
  • MC38 cells (Shanghai Fuheng Biotechnology Co., Ltd., product number: FH-0125) were cultured in RPMI1640 medium containing 10% fetal bovine serum. MC38 cells in the exponential growth phase were collected and resuspended to 1 ⁇ 107 /mL in PBS for subcutaneous tumor inoculation in mice. 0.1 mL of MC38 cells were inoculated subcutaneously on the right back of the experimental mice, and the tumor growth was observed regularly. When the tumor grew to an average volume of 96 mm 3 , the mice were randomly divided into groups according to the tumor size and body weight, and the tumor inoculation was recorded as the 0th day. sky.
  • the experimental groups are as follows:
  • the preparation method of live Enterococcus lactis is: Enterococcus lactis suspended in PBS+0.25% glycerol.

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Abstract

提供一株乳酸肠球菌，保藏编号为CCTCC NO:M20211220，保存于中国典型培养物保藏中心。提供的乳酸肠球菌CCTCC NO:M20211220在pH4.0的人工胃液环境中孵育3h仍能保证77.60％的存活率。在小鼠MC38模型中，该乳酸肠球菌在抑制肿瘤体积方面有显著效果。

Description

一种抗结直肠肿瘤的乳酸肠球菌及其应用 技术领域
本发明属于微生物领域，具体涉及一株抗结直肠肿瘤的乳酸肠球菌及其应用。
背景技术
结直肠癌是常见的消化系统恶性肿瘤，其发病隐匿，死亡率较高。目前对于晚期结直肠癌的治疗，以5FU或卡培他滨加奥沙利铂或者伊立替康方案化疗联合靶向药物例如贝伐单抗、西妥昔单抗、帕尼单抗等为主。尽管化疗联合靶向治疗使晚期结直肠癌患者总的生存率得到了提高，但是对于多线化疗进展后的结直肠癌患者可以使用的药物很少，且化疗引起的胃肠道反应、骨髓抑制、神经毒性等不良反应经常发生。
微生物涉及肠内稳态的调节，人肠微生物组的一些共生和致病生物在结直肠癌发病机理中是必需的。因此，通过使用益生菌操纵肠细菌组成和局部代谢物被证实可以用于结直肠癌治疗干预。益生菌是活的微生物饲养添加剂，有利地影响宿主的健康。
中国专利201910909859.7中公开了一种抑制结直肠癌致病菌的益生菌配方及其筛选方法，益生菌配方为：丁酸梭菌1.3×10 10CFU/mL，粪肠球菌1×10 9CFU/mL，短乳杆菌1×10 9CFU/mL，植物乳杆菌1×10 9CFU/mL，鼠李糖乳杆菌1×10 9CFU/mL，清酒乳杆菌1×10 9CFU/mL，肠系膜明串珠菌1×10 9CFU/mL。该发明益生菌配方不会产生耐药性，并且安全、有效，性能优良，但涉及的菌种较多，方案复杂。
中国专利201480080834.6公开了微生物特别是真杆菌菌株在治疗和预防结直肠癌相关的疾病中的应用。涉及了包含真杆菌菌株的组合物。动物实验的结果证明了凸腹真杆菌(Eubacterium ventriosum)和挑剔真杆菌(Eubacteriumeligens)有效预防和治疗结肠炎和结直肠癌的能力。
丰富治疗结直肠癌的微生物种质资源对于结直肠癌的治疗和预后都是十分有利的。
发明内容
为了解决上述问题，本发明自健康人粪便菌液中分离出一株抗结直肠肿瘤的乳酸肠球菌，将其应用于结直肠肿瘤的治疗中，以期为结直肠肿瘤的治疗提供一种获得途径更为便捷且效果更好的药物。
一方面，本发明提供了一株乳酸肠球菌。
所述的乳酸肠球菌16S rRNA序列为与SEQ ID NO.1具有99.93％以上同一性的序列。
所述的乳酸肠球菌16S rRNA序列为SEQ ID NO.1，保藏编号为CCTCC NO:M20211220，保存于中国典型培养物保藏中心，保藏日期为2021年9月26日。
另一方面，本发明提供了乳酸肠球菌在制备用于诊断、预防和/或治疗肿瘤的产品或药物中的应用。
所述的乳酸肠球菌为前述的乳酸肠球菌。
所述的肿瘤包括但不限于：消化系统肿瘤、呼吸系统肿瘤、生殖系统肿瘤、运动系统肿瘤、神经系统肿瘤、内分泌系统肿瘤、循环系统肿瘤、泌尿系统肿瘤、生殖系统肿瘤。
优选地，所述的肿瘤为消化系统肿瘤，所述的消化系统肿瘤为食道肿瘤、胃肿瘤、贲门肿瘤、肠肿瘤、肝肿瘤、胆囊肿瘤中的一种或多种。
进一步优选地，所述的肿瘤为结直肠肿瘤，所述的结直肠肿瘤包括息肉样型、狭窄型和溃疡型中的一种或多种。
再一方面，本发明提供了一种用于前述应用的制剂。
所述的制剂中包括前述的乳酸肠球菌和/或其制备物。
所述的制备物为乳酸肠球菌的死菌、细胞破碎物、发酵上清液、发酵沉淀、改造菌、突变菌和/或诱变菌。
所述的乳酸肠球菌的发酵上清液的制备方法中包括：
S1、接种乳酸肠球菌CCTCC NO:M20211220；
S2、条件培养；
S3、去除菌体，即得。
所述的乳酸肠球菌的细胞破碎物的制备方法中包括：
S1、接种乳酸肠球菌CCTCC NO:M20211220；
S2、条件培养；
S3、菌体细胞破碎。
所述的条件培养包括但不限于：厌氧培养、好氧培养、兼性厌氧培养。
优选地，所述的制剂为液体制剂，所述的液体制剂中包括乳酸肠球菌的量为5×10 9CFU/mL。
优选地，所述的制剂为固体制剂或半固体制剂，所述的制剂中包括乳酸肠球菌的量为5×10 9CFU/g。
优选地，所述的制剂为冻干粉。
又一方面，本发明提供了一种基因编码产物。
所述的基因编码产物由前述的乳酸肠球菌的基因组上的基因编码，且能够诊断、预防和/或治疗结直肠肿瘤。
又一方面，本发明提供了前述的乳酸肠球菌的培养方法，其特征在于，包括以下步骤：
S1、接种前述的乳酸肠球菌；
S2、条件培养。
所述的条件培养包括但不限于：厌氧培养、好氧培养、兼性厌氧培养。
又一方面，本发明提供了前述的制剂在制备诊断、预防和/或治疗结直肠肿瘤的产品或药物中的应用。
又一方面，本发明提供了一种用于前述应用的药物。
所述的药物中包括前述的乳酸肠球菌或其制备物。
或者，所述的药物中包括前述的制剂。
所述的药物中包括乳酸肠球菌的量为5×10 9CFU/mL。
所述的药物中还包括5FU、卡培他滨、奥沙利铂、伊立替康、贝伐单抗、西妥昔单抗、帕尼单抗中的一种或多种。
所述的药物中还包括其他药学上可接受的载体或赋形剂。
优选地，所述的药物为口服药物。
本发明的有益效果：
本发明提供的乳酸肠球菌CCTCC NO:M 20211220在pH4.0的人工胃液环境中孵育3h仍能保证77.60％的存活率。在小鼠MC38模型中，该乳酸肠球菌在抑制肿瘤体积方面有显著效果。
保藏说明
保藏地址：中国，武汉，武汉大学；
保藏日期：2021年09月26日；
菌种名称：乳酸肠球菌PRS-15；
拉丁名：Enterococcus Faecium PRS-15；
保藏机构：中国典型培养物保藏中心；
保藏机构简称：CCTCC；
保藏中心登记入册编号：CCTCC NO:M 20211220。
附图说明
图1为给药后MC38小鼠模型的肿瘤大小变化曲线。
图2为给药后MC38小鼠模型的体重变化曲线。
具体实施方式
下面结合具体实施例，对本发明作进一步详细的阐述，下述实施例不用于限制本发明，仅用于说明本发明。以下实施例中所使用的实验方法如无特殊说明，实施例中未注明具体条件的实验方法，通常按照常规条件，下述实施例中所使用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1一株抗结直肠肿瘤的乳酸肠球菌的分离
在无菌条件下，制备好的健康人粪便菌液进行10倍梯度稀释，选取三个浓度梯度，划线接种于NA及MRS培养基上，三个重复平板，置37℃恒温培养箱中厌氧培养72h，随后挑取不同形态的菌落进行革兰氏染色镜检，并于MRS培养基中进行2次划线纯化，再染色镜检，观察细菌形态及是否纯化，再进行16srRNA测序，使用的引物序列为：
27F：SEQ ID NO.2；
1429R：SEQ ID NO.3。
PCR反应体系如下：
Figure PCTCN2022132525-appb-000001
PCR反应条件如下：
Figure PCTCN2022132525-appb-000002
扩增得到的PCR产物进行16srRNA测序后在NCBI进行比对。
测序结果如SEQ ID NO.1所示。
实施例2耐药性检测
采用药敏纸片扩散法进行，将MRS液体培养基(购自青岛海博生物科技有限公司，货号：HB0384-1)培养好的菌液，进行4000rpm离心15min，去掉上清液，用PBS进行悬浮，吹打均匀，将OD值调整为0.6左右，均匀涂布于含有MRS固体培养基的培养皿中，待菌液完全吸收后，放置药敏纸片(含有抗菌药物或抗生素)，一块平板放置一种药敏纸片(3片)和空白对照(1片)，倒置培养于37℃培养箱，24h用直尺测量抑菌环直径，记录数据，根据纸片法抗菌药物试验标准(WS/T125—1999)判读结果。结果如下：
Figure PCTCN2022132525-appb-000003
备注：S＝敏感，I＝中介，R＝耐药，/为无抑菌圈，—为剔除抑菌圈。
Figure PCTCN2022132525-appb-000004
Figure PCTCN2022132525-appb-000005
备注：S＝敏感，I＝中介，R＝耐药，/为无抑菌圈，—为剔除抑菌圈。
本株菌主要对喹诺酮类、硝基呋喃类抗菌药、多粘菌素B、林克霉素类、复方新诺明和庆大霉素耐药；对于青霉素类、氯霉素类、大环内脂类、四环素类、头孢菌素类和糖肽类抗生素表现为敏感。
实施例3耐人工胃肠液检测
(1)MTT溶液配制：称0.5gMTT(购自北京索莱宝，货号：M8180-250)，加入100mL PBS溶解(终浓度为5mg/mL)，过滤除菌，分装于15mL离心管中，-20℃冻存备用，有效期半年。
(2)模拟胃液(Simulated Gastric Fluid，SGF)实验
1、取2.0g NaCl和3.2g胃蛋白酶(索莱宝胃蛋白酶，1:3000，标识为每mg中含800-2500个活度单位)，加7.0mL 37％稀盐酸和水使溶解至1000mL，溶液pH值为1.2。
2、调pH分别至1.2(模拟人空腹肠液pH)、2.0、3.0(模拟人饭后肠液pH及小鼠空腹肠液pH)和4.0，过滤除菌；
3、集菌：菌株于37℃静止培养8h，达到对数生长期，将菌液分装于50mL无菌EP管，4000rpm室温离心15min后，弃上清，用PBS重悬菌体，并调取菌液OD 600值，得到细菌数约为2×10 9CFU/mL，随后取17个15mL无菌EP管，分别加入调好OD值的6mL菌液，进行离心(条件同上)，16个离心管(实验组)去除PBS，收集菌沉淀待用，1个吹打重悬(对照组)；
4、人工胃液培养：将上述不同pH的人工胃液分别加入6mL至洗涤后收集的菌沉淀16个15mL离心管中，并吹打混匀，分别孵育培养1h、2h和3h；
5、MTT检测：先对照组菌液、孵育不同pH不同时间的菌液分别取3mL于新的离心管中，并分别加入1mL MTT试剂进行孵育过夜(这一步可将菌液通过3500rpm，8min离心，去掉上清液人工胃液，再加入MTT试剂，MTT试剂可降低至500ul，且孵育时间也可缩短)，孵育过后，再进行离心处理(3500rpm，8min)，去掉上清液，加入600μL DMSO(国药分析纯98.5％)，吹打溶解后，分别加入深孔板中，而后用排枪转移至96孔无菌培养板中，每 孔加入150μL(并标记清楚每孔加样顺序和名称)，然后用酶标仪进行检测，选取波长570nm或630nm获取吸光值，推算出活细菌的比例；
6、进行数据处理，计算对应的存活率，其中细菌存活率＝实验组OD值/对照组OD值×100％，得到菌株模拟人工胃液作用不同时间后的耐受程度。
(3)模拟肠液(SIF)-SimulatedIntestinalFluid(SIF)实验
1、取磷酸二氢钾6.8g，加水500mL使溶解，用0.1mol/L氢氧化钠溶液调节pH值至6.8；另取胰酶1g(索莱宝胰蛋白酶，1:250)，加水适量使溶解，将两液混合后，加水稀释至1000mL，过滤除菌；
2、集菌方法同上模拟胃液(SimulatedGastricFluid，SGF)实验；
3、人工肠液培养：将上述pH6.8的人工肠液分别加入8mL至洗涤后收集的菌沉淀4个15mL离心管中，并吹打混匀，孵育培养0h、1h、2h和3h；
4、MTT检测：同上模拟胃液(SimulatedGastricFluid，SGF)实验；
5、进行数据处理，计算对应的存活率，其中细菌存活率＝实验组OD值/对照组OD值×100％，得到菌株模拟人工肠液作用不同时间后的耐受程度。
(4)模拟人体内胆盐环境实验
1、胆汁配制：在目标菌培养基(MRS或BHI)溶液中加入牛胆粉，设置三个终浓度，分别为10g/L(1％牛胆粉)、20g/L(2％牛胆粉)和40g/L(4％牛胆粉)，高压灭菌后备用，同时，以不加胆粉的培养基溶液0h作为对照。本实施例中选用MRS培养基。
2、菌株收集同上。
3、牛胆粉培养：将配置含有0％、1％、2％和4％牛胆粉(购自合肥巴斯夫生物科技有限公司，货号：N0101-100g)的MRS或BHI培养基分别加入10mL至洗涤后收集的菌沉淀20个15mL离心管中进行孵育培养0h、1h、2h、3h和4h，以0％牛胆粉培养基溶液0h作为对照组，本实施例选用MRS培养基。
4、倾注培养：同上模拟胃液(SimulatedGastricFluid，SGF)实验。
5、计算细菌耐受胃酸情况：进行平板计数，记录各平板菌落数，进行数据处理，得到菌株对牛胆盐的耐受程度。
结果如下：
Figure PCTCN2022132525-appb-000006
Figure PCTCN2022132525-appb-000007
Figure PCTCN2022132525-appb-000008
Figure PCTCN2022132525-appb-000009
Figure PCTCN2022132525-appb-000010
Figure PCTCN2022132525-appb-000011
Figure PCTCN2022132525-appb-000012
结果表明，乳酸肠球菌CCTCC NO:M20211220：在pH值较低的胃液环境中仍能保持较好的存活率；在pH4.0的人工胃液环境中孵育3h仍能保证77.60％的存活率；在1％-4％牛胆粉培养基中可正常生长，且随着孵育培养的时间增加，其活菌数也在增加，说明该菌株对牛胆盐具备一定的耐受程度，且可正常生长繁殖。
实施例4小鼠模型实验
本实施例中选用的小鼠为雌性C57BL/6或小鼠，周龄6-7周(肿瘤细胞接种时的小鼠预计周龄)，接种细胞时小鼠体重范围为18-22g，购自君科生物技术有限公司。
小鼠MC38模型用于药效验证，模型构建方法如下：
MC38细胞(上海富衡生物科技有限公司，货号为FH-0125)培养在含10％胎牛血清的RPMI1640培养液中。收集指数生长期的MC38细胞，PBS重悬至1×10 7个/mL用于小鼠皮下肿瘤接种。将0.1mL MC38细胞接种于实验小鼠的右侧背部皮下，定期观察肿瘤生长情况，待肿瘤生长至平均体积96mm 3时，根据肿瘤大小和小鼠体重随机分组给药，肿瘤接种记为第0天。
给药开始前，称量动物体重并测量肿瘤体积。
实验分组如下：
Figure PCTCN2022132525-appb-000013
乳酸肠球菌活体菌的配制方式为：PBS+0.25％甘油悬浮的乳酸肠球菌。
肿瘤接种第9天开始给药，并于给药后第6、8、10、12、14、16、19、21、24、27天测量小鼠的体重(g)和肿瘤的大小(mm 3)。肿瘤大小计算公式：肿瘤体积(mm 3)＝0.5×(肿瘤长径×肿瘤短径 2)。
结果如图1-2所示，结果表明：在小鼠MC38模型中，该乳酸肠球菌在抑制肿瘤体积方面有显著效果。

Claims (10)

  1. 一种乳酸肠球菌，其特征在于，其16S rRNA序列为与SEQ ID NO.1具有99.93％以上同一性的序列。
  2. 根据权利要求1所述的乳酸肠球菌，其特征在于，其16S rRNA序列为SEQ ID NO.1，其保藏编号为CCTCC NO:M20211220，保存于中国典型培养物保藏中心，保藏日期为2021年9月26日。
  3. 乳酸肠球菌在制备用于诊断、预防和/或治疗肿瘤的产品或药物中的应用。
  4. 根据权利要求3所述的应用，其特征在于，所述的乳酸肠球菌为权利要求1或2所述的乳酸肠球菌。
  5. 根据权利要求3所述的应用，其特征在于，所述的肿瘤为消化系统肿瘤、呼吸系统肿瘤、生殖系统肿瘤、运动系统肿瘤、神经系统肿瘤、内分泌系统肿瘤、循环系统肿瘤、泌尿系统肿瘤、生殖系统肿瘤中的一种或多种；优选地，所述的肿瘤为消化系统肿瘤；更优选地，所述的消化系统肿瘤为食道肿瘤、胃肿瘤、贲门肿瘤、肠肿瘤、肝肿瘤、胆囊肿瘤中的一种或多种；更优选地，所述的肿瘤为结直肠肿瘤，所述的结直肠肿瘤包括息肉样型、狭窄型和溃疡型中的一种或多种。
  6. 一种用于权利要求3-5任一项所述应用的制剂，其特征在于，所述的制剂中包括权利要求1或2所述的乳酸肠球菌和/或其制备物；所述的制备物为乳酸肠球菌的死菌、细胞破碎物、发酵上清液、发酵沉淀、改造菌、突变菌和/或诱变菌。
  7. 一种基因编码产物，其特征在于，所述的基因编码产物由权利要求1或权利要求2所述的乳酸肠球菌的基因组上的基因编码。
  8. 权利要求1或2所述的乳酸肠球菌的培养方法，其特征在于，包括以下步骤：
    S1、接种权利要求1或2所述的乳酸肠球菌；
    S2、条件培养。
  9. 一种用于权利要求3-5任一项所述应用的药物，其特征在于，所述的药物中包括权利要求1或2所述的乳酸肠球菌和/或其制备物；或者，所述的药物中包括权利要求6所述的制剂。
  10. 根据权利要求9所述的药物，其特征在于，所述的药物中还包括5 FU、卡培他滨、奥沙利铂、伊立替康、贝伐单抗、西妥昔单抗、帕尼单抗中的一种或多种。
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