Fecatobacterium hominis SJ-3 and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and provides a faecium hominis SJ-3, and the collection number of the faecium hominis SJ-3 is CGMCC (China General Microbiological Culture Collection Center) NO.41530. The strain SJ-3 is preserved in the China General Microbiological Culture Collection Center on September 30, 2024, the China General Microbiological Culture Collection Center is called as CGMCC for short, the preservation number is CGMCC NO.41530, and the address of the preservation unit is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The strain provided by the invention has the effect of treating autism spectrum disorders.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine. Background Art

[0002] Autism spectrum disorder (ASD) is a type of neurodevelopmental disease caused by both genetic and environmental factors, with core symptoms of social disorders, repetitive stereotyped behaviors, and language communication disorders. In addition to the core symptoms, some ASD patients also have other symptoms such as anxiety, depression, attention deficit, learning and memory disorders, and sleep disorders. Clinically, the treatment of ASD mainly includes psychotherapy and drug therapy. However, neither of the above two therapies can effectively alleviate the core symptoms of ASD patients. Therefore, it is of great significance to find ways to alleviate the core symptoms of ASD patients.

[0003] In addition to abnormal development of the nervous system, many ASD patients have gastrointestinal symptoms that are highly correlated with the severity of the disease. The human intestine is colonized by a large number of intestinal symbiotic bacteria, called intestinal flora. They participate in host metabolism, regulate host physiological activities, and are closely related to host health. A large number of studies have shown that the composition of the intestinal flora of ASD patients has changed significantly. Therefore, many studies have explored the effects of regulating intestinal flora on the occurrence, development and treatment of ASD. However, in some population experiments, probiotics do not play a role in alleviating ASD symptoms. The types of probiotics in population experiments are mostly aerobic bacteria such as Lactobacillus and Bifidobacterium or facultative anaerobes, while there is still a lack of sufficient research on the effects of strict anaerobic bacteria, such as Faecalibacterium, on ASD. Summary of the invention

[0004] In order to effectively alleviate the clinical symptoms of patients with autism spectrum disorders, the first purpose of the present invention provides Faecalibacterium hominis SJ-3, whose preservation number is CGMCC NO.41530. The strain SJ-3 was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on September 30, 2024, which is referred to as CGMCC, with a preservation number of CGMCC NO.41530, and the address of the preservation unit is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0005] The second objective of the present invention is the application of a live bacterial agent of Faecalibacterium serovar inhabitans, which can alleviate the host's core behavioral symptoms of ASD, such as repetitive stereotyped behaviors and social disorders, as well as ASD-related behavioral symptoms such as anxiety, and play a role in treating ASD by alleviating ASD behavioral symptoms. BRIEF DESCRIPTION OF THE DRAWINGS

[0006] Figure 1 This is the cell morphology and size of strain SJ-3 under transmission electron microscopy. DETAILED DESCRIPTION

[0007] Example 1

[0008] Improved YCFA liquid medium (g / L): Caseinone 10.0 g, Yeast extract 2.5 g, Glucose 2.0 g, Sodium bicarbonate (NaHCO3) 4.0 g, Potassium dihydrogen phosphate (KH2PO4) 0.45 g, Potassium hydrogen phosphate (K2HPO4) 0.45 g, Sodium chloride (NaCl) 0.9 g, Magnesium sulfate heptahydrate (MgSO4 ·7H2O) 0.01g, calcium chloride dihydrate (CaCl2·2H2O) 0.09g and hemin (Haemin) 0.01g (dissolved in sodium hydroxide aqueous solution and adjusted to pH 7.0 with 1M hydrochloric acid), 1.0g cysteine ​​hydrochloride, 1ml resazurin solution (Resazurin, 0.1%), add distilled water to 1L, autoclave at 115℃ for 25 minutes, and add 3ml acetic acid buffer (1M) after sterilization. For solid culture medium, add 15g agar.

[0009] 1. Acquisition, isolation and identification of strains

[0010] The strain was obtained by the patent inventor on June 20, 2019, by anaerobically separating fresh feces from healthy persons provided by the feces donor after the stool donor signed an informed consent form.

[0011] Colony morphology: It grows fast in the modified YCFA medium. After anaerobic culture at 37°C for 48 hours, the colony morphology is milky white, protruding, smooth, neatly edged, and small colonies with a diameter of about 1 mm. Under a transmission electron microscope, the length of the bacteria varies greatly, mostly in the shape of slender rods, with a diameter of 1.1-1.8 μm and a length of 0.5-8.2 μm. No spores, pili or flagella are observed, and there is no motility ( Figure 1 ).

[0012] Molecular identification method: Full sequence analysis of 16S rRNA, identification and classification named Faecalibacteriumhominis, Chinese name human fecal bacillus, strain named SJ-3, the strain has been deposited in the General Microbiological Center of China Microbiological Culture Collection Administration (abbreviated as: CGMCC) on September 30, 2024, with the deposit number: CGMCC NO.41530, and the address of the deposit unit is: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing. That is, the patent strain deposit number is: CGMCCNO.41530; the 16SrRNA sequence homology with the human fecal bacillus standard strain is 99.18%. The sequence of strain SJ-3 is shown in SEQID NO.1 in the sequence table.

[0013] 2. Cultivation and preservation methods:

[0014] Cultivation method of Faecalibacterium hominis SJ-3: Activate once in modified YCFA liquid medium, transfer to modified YCFA liquid medium with the same composition at a 1% inoculum, and culture anaerobically at 37°C for 1-5 days.

[0015] Short-term storage method for Faecalibacterium hominis SJ-3: Use the modified YCFA solid culture medium formula to prepare it in a Hungate tube, sterilize it by high pressure, place it in a slant to cool and solidify it for use. In the anaerobic operation table, use a disposable inoculation loop to inoculate and streak on the slant surface. After sealing the tube mouth, place it in a 37℃ incubator with the tube mouth facing upward and culture it until the colony is clearly visible.

[0016] Long-term storage method of Faecalibacterium hominis SJ-3: a) Freeze-drying storage: After centrifuging the bacterial solution at 5000g for 5 minutes at room temperature, collect the bacteria, wash once with anaerobic PBS buffer solution, resuspend in a solution containing 5% skim milk powder, freeze-dry and seal for storage. b) Anaerobic glycerol cryopreservation: The cryopreservation solution is prepared by adding 0.2ml 80% anaerobic glycerol (containing 5g / L L-cysteine) to every 1.3ml bacterial solution, put it in a cryopreservation box, and store it at -80℃.

[0017] 3. Use and effect of bacteria: Animal experiment on the treatment of ASD with SJ-3 live bacteria

[0018] 3.1 Materials and Methods

[0019] 1) Modified YCFA medium (g / L):

[0020] Improved YCFA liquid medium (g / L): Caseinone 10.0 g, Yeast extract 2.5 g, Glucose 2.0 g, Sodium bicarbonate (NaHCO3) 4.0 g, Potassium dihydrogen phosphate (KH2PO4) 0.45 g, Potassium hydrogen phosphate (K2HPO4) 0.45 g, Sodium chloride (NaCl) 0.9 g, Magnesium sulfate heptahydrate (MgSO4 ·7H2O) 0.01g, calcium chloride dihydrate (CaCl2·2H2O) 0.09g and hemin (Haemin) 0.01g (dissolved in sodium hydroxide aqueous solution and adjusted to pH 7.0 with 1M hydrochloric acid), 1.0g cysteine ​​hydrochloride, 1ml resazurin solution (Resazurin, 0.1%), add distilled water to 1L, autoclave at 115℃ for 25 minutes, and add 3ml acetic acid buffer (1M) after sterilization. For solid culture medium, add 15g agar.

[0021] 2) SJ-3 live bacteria culture method:

[0022] The activated SJ-3 strain was cultured in a modified YCFA medium for 48 hours, and the bacteria on the solid medium were collected by scraping. Before the experiment, the bacteria were washed with anaerobic PBS buffer and then resuspended with anaerobic PBS. The concentration of the resuspended solution was controlled to 2×10 9 Colony-forming units (CFU).

[0023] 3) Animal experiment design:

[0024] Experimental selection BTBR T + Itpr3tf / J mice (hereinafter referred to as BTBR mice) were used as an animal model of ASD. Four-week-old male BTBR mice were selected and divided into a control group and a live bacteria group, with 6 mice in each group. The live bacteria group was gavaged with 200 μL of cells per day, with a cell count of 4×10 8 CFU of SJ-3 live bacteria; the control group was gavaged with the same dose of anaerobic PBS buffer solution every day. Behavioral testing was performed on mice 4 weeks after gavage, and gavage was continued during the behavioral testing. Behavioral testing is used to evaluate the ASD core behaviors of mice, including repetitive stereotyped behaviors and social behaviors, as well as ASD-related behaviors, including anxiety behaviors. The specific contents are as follows: the grooming experiment was selected to evaluate the repetitive stereotyped behaviors of mice, the three-box social experiment was selected to evaluate the social behavior of mice, and the elevated plus maze experiment was selected to evaluate the anxiety behavior of mice.

[0025] 4) Behavioral testing methods:

[0026] Starting from the first 3 days of the behavioral test, the mice to be tested need to be stroked for 3-5 minutes every day to reduce the impact of nonspecific stress on the mice. On the day of the behavioral test, the mice need to be placed in the behavioral test room for more than 1 hour in advance to ensure that the mice are fully adapted to the environment.

[0027] a) Grooming experiment: The test mice were placed in a clean cage and allowed to explore freely for 20 minutes. The first 10 minutes were the adaptation phase, and no data was recorded. During the second 10 minutes, the grooming behavior of the mice was recorded, including: the mice used their mouths to groom their heads, backs, limbs, tails, and perineum.

[0028] b) Three-box social experiment: The apparatus of the three-box social experiment is a white box (60 cm (length) × 40 cm (width) × 30 cm (height)), which is divided into three equal-sized chambers by a transparent movable plastic plate. There is a square gap at the bottom of the partition, and the mice can move freely between the chambers. The three chambers are named "Zone 2", "Zone 1" and "Zone 3" from left to right. Prepare 2 spare tool mice (strain, age, and gender are consistent with the test mice, the weight difference is less than 2g, and they have not been in contact with the test mice before the experiment), 1 odorless object block and 3 inverted cylindrical iron cages as restraint cages (restraint cage 1, restraint cage 2, restraint cage 3). The three-box social experiment is divided into 3 stages. In the first stage, the test mouse is placed in the center of zone 1 and moves freely for 10 minutes. In the second stage (social ability stage), a tool mouse (M1) was placed in restraint cage 1, and restraint cage 1 was placed in the center of zone 2; the object was placed in restraint cage 2, and restraint cage 2 was placed in the center of zone 3; the mouse to be tested was allowed to explore freely for 10 minutes, and the time the mouse stayed in the three chambers was recorded. In the third stage (social novelty preference stage), another tool mouse (M2) was placed in restraint cage 3, and the restraint cage was placed in the center of zone 3; M1 was placed in restraint cage 1, and restraint cage 1 was placed in the center of zone 2; the mouse to be tested was allowed to explore freely for 10 minutes, and the time the mouse stayed in the three chambers was recorded.

[0029] c) Elevated plus maze test: The experimental device is a black plus maze, fixed on an iron frame 1 meter above the ground, consisting of 2 open arms (50 cm (length) × 5 cm (width)) and 2 closed arms (50 cm (length) × 5 cm (width) × 15 cm (height)), and the open arms and closed arms are crossed in a cross shape. At the beginning of the experiment, the mouse to be tested was placed in the center of the plus maze facing the open arm and allowed to explore freely for 10 minutes. The activity of the mouse was recorded by a camera directly above the center of the plus maze. The Xeye Aba software was used to analyze the mouse's stay time in the open arm and the number of times it entered the open arm.

[0030] 3.2 Experimental results:

[0031] 1) SJ-3 live bacteria improves repetitive stereotyped behaviors in BTBR mice:

[0032] The results in Table 1 show that the SJ-3 live bacteria agent can significantly reduce the grooming behavior time of BTBR mice in the grooming experiment, indicating that the SJ-3 live bacteria agent can significantly improve the repetitive stereotyped behavior of BTBR mice.

[0033] Table 1. Grooming behavior time of BTBR mice in the grooming experiment (s)

[0034]

[0035] 2) SJ-3 live bacteria improves social dysfunction behavior in BTBR mice:

[0036] The results in Table 2 show that there was no significant difference in the time the BTBR mice in the control group stayed in Zone 2 and Zone 3 during the social ability phase of the three-box social experiment, indicating that the social ability of the BTBR mice in the control group was impaired, which is a typical manifestation of social disorder.

[0037] Table 2. The duration (s) of BTBR mice in the control group in the sociability phase of the three-box social experiment

[0038]

[0039] The results in Table 3 show that the BTBR mice in the live bacteria group spent significantly more time in zone 2 than in zone 3 during the social ability phase of the three-box social experiment, indicating that the BTBR mice in the live bacteria group had recovered their social ability.

[0040] Table 3. The duration (s) of BTBR mice in the live bacteria group in the sociability phase of the three-box social experiment

[0041]

[0042] The results in Tables 1 and 2 indicate that SJ-3 live bacteria can significantly improve the social disorder behavior of BTBR mice.

[0043] 3) SJ-3 live bacteria improves the anxiety behavior of BTBR mice:

[0044] The results in Tables 4 and 5 show that the SJ-3 live bacteria agent can significantly increase the time that BTBR mice stay in the open arms and the number of times they enter the open arms in the elevated plus maze test, indicating that the SJ-3 live bacteria agent can significantly improve the anxiety behavior of BTBR mice.

[0045] Table 4. The time (s) that BTBR mice stayed in the open arms in the elevated plus maze test

[0046]

[0047] Table 5. Number of times BTBR mice entered the open arms in the elevated plus maze test (times)

[0048]

Claims

1. Faecalibacterium hominis SJ-3, whose accession number is CGMCC NO.41530.

2. A microbial agent, characterized in that: Contains the Faecalibacterium hominis SJ-3 according to claim 1.

3. A composition, characterized in that: Contains the Faecalibacterium hominis SJ-3 described in claim 1.

4. Use of Faecalibacterium hominis SJ-3 according to claim 1 in the preparation of a drug for treating autism spectrum disorders.

5. Use of the Faecalibacterium hominis SJ-3 described in claim 1 in the preparation of a drug for improving repetitive stereotyped behaviors.

6. Use of the Faecalibacterium hominis SJ-3 described in claim 1 in the preparation of a drug for improving social disorder behavior.

7. Use of the Faecalibacterium hominis SJ-3 according to claim 1 in the preparation of a drug for improving anxiety behavior.

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