Preparation method of mixed bacteria competent cells, coprophilous fungus transplantation flora, separation method and application

Abstract

The invention provides a preparation method of mixed bacteria competent cells, a coprophilous fungus transplantation flora, a separation method and application, and belongs to the technical field of coprophilous fungus transplantation. The invention develops a method for integrally preparing competent and transforming intestinal flora through low-temperature treatment and glycerol resuspension research, the method does not need to separate a single bacterial strain, can perform efficient and comprehensive gene modification on the intestinal flora, expresses a corresponding product in the intestinal tract to treat diseases, and has a good application prospect. The method has a huge application prospect. Meanwhile, a new strategy for comprehensively modifying the microbiota in a complex system is also developed, after the whole intestinal microbiota is prepared into competent cells, the competent cells are subjected to gene modification through plasmid electrotransfection and then transplanted to the intestinal tract, and multiple strains which are successfully modified can be screened out from the intestinal tract content. The method is simple, time-saving, labor-saving and high in transformation efficiency, and can be used for carrying out gene modification on a plurality of strains in the intestinal microbiome at one time.

Description

Technical Field

[0001] The present invention relates to the technical field of fecal microbiota transplantation, and in particular to a method for preparing mixed bacterial competent cells, a fecal microbiota transplantation flora, a separation method and applications. Background Art

[0002] Fecal microbiota transplantation is a method of restoring the patient's intestinal microecological balance and correcting diseases caused by dysbiosis by transplanting the intestinal flora of a healthy donor. However, this technology can only obtain normal intestinal flora from a healthy donor and cannot transform the intestinal flora.

[0003] Currently, research on the preparation and transformation of competent cells primarily focuses on single strains. Traditional methods for engineering intestinal bacteria require repeated isolation of single colonies from complex fecal samples, followed by transformation, and then the engineered bacteria are grown on resistant plates. This process takes weeks and can only be performed on one bacterial species at a time, preventing the simultaneous engineering of functionally complementary bacterial communities, making it difficult to meet the needs of treating complex diseases. To circumvent this problem, engineering the entire intestinal microbiome is an alternative approach. However, the main obstacle to achieving holistic microbiome engineering is that most gut-associated microbes are difficult to genetically engineer, and there are no available tools for simultaneously engineering multiple organisms. Current bacterial bioengineering must first identify a suitable host. However, isolating the most suitable host bacterium is often challenging, particularly considering whether it is easy to grow, genetically manipulate, compatible with the environment, and well-suited to expressing the target protein.

[0004] Therefore, developing a method to prepare and transform the intestinal flora as a whole is of great significance for the efficient transformation of the intestinal microbiome. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for preparing mixed bacterial competent cells, a fecal microbiota transplantation flora, an isolation method and an application, which can prepare the intestinal flora as a whole and transform it, without the need for complex single colony isolation, avoiding the steps of multiple plate cultures in vitro, and greatly simplifying the screening method.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides a method for preparing mixed bacterial competent cells, comprising the following steps: (1) Filter the feces, centrifuge, and resuspend the precipitate in glycerol to obtain a bacterial suspension; (2) Cool the bacterial suspension on ice, centrifuge at low temperature, take the precipitate and resuspend it with glycerol, repeat the cooling and centrifugation steps after resuspension, take the precipitate after centrifugation and resuspend it with glycerol to obtain competent cells.

[0007] Preferably, in step (1), the pore size of the filter membrane used in the filtration is 4-6 µm; and the glycerol is 5-15% glycerol.

[0008] Preferably, in step (2), the ice cooling time is 8 to 12 minutes, the low-temperature centrifugation temperature is 4°C; the number of repetitions is 2 to 4 times; and the glycerol is 5 to 15% glycerol.

[0009] The present invention also provides a method for isolating fecal microbiota for preventing or treating alcoholic liver disease, comprising the following steps: (1) Plasmid construction The ampicillin resistance gene promoter and ampicillin resistance gene were inserted into the pT5-sfFGFP plasmid to obtain the empty vector plasmid pT5-AG. The ADH and ALDH gene fragments were added to the plasmid pT5-AG. The promoter J23119 was introduced before each enzyme gene fragment to construct the functional plasmid pT5-AA that co-expresses ADH and ALDH. (2) Preparation of mixed bacterial competent cells Prepare mixed bacterial competent cells by the method for preparing mixed bacterial competent cells according to any one of claims 1 to 3; (3) Take the mixed bacteria competent cells prepared in step (2), add the plasmid pT5-AA prepared in step (1), mix well, electrotransform, resuspend, and centrifuge at low temperature to obtain a mixed bacteria introduced with the plasmid pT5-AA; (4) Alcohol model mice were gavaged with mixed bacteria containing plasmid pT5-AA. After gavage, fecal microbiota that can prevent or treat alcoholic liver disease were isolated from the intestines of the mice.

[0010] Preferably, in step (3), the concentration of the competent cells is OD 600 The value is 0.4~0.6; the volume ratio of the competent cells to the plasmid is 20~30:0.8~1.2, the concentration of the plasmid is 90~110ng / μL, the output voltage of the electroporation is 2~3kV, and the number of electric pulses is 1.

[0011] Preferably, in step (3), the mixed bacteria into which the plasmid pT5-AA is introduced include one or more of Pseudomonas aeruginosa, Chryseobacterium indophylogenes, Alcaligenes faecalis, Klebsiella, Stenotrophomonas maltophilia, and Cellulomonas finkelii.

[0012] Preferably, in step (4), the alcohol model mice are gavaged for 7 to 10 days.

[0013] Preferably, in step (4), the alcohol model mice are also required to consume sterile drinking water containing two antibiotics, ampicillin and kanamycin, during the gavage period.

[0014] The present invention also provides a method for isolating a fecal microbiota transplantation flora that can prevent or treat alcoholic liver disease, and obtains a fecal microbiota transplantation flora that can prevent or treat alcoholic liver disease.

[0015] The present invention also provides a method for using a fecal microbiota transplantation flora that can prevent or treat alcoholic liver disease in the preparation of a drug for preventing or treating alcoholic liver disease. The beneficial effects of the present invention compared with the prior art are: (1) The present invention develops a method for preparing competent cells and transforming intestinal flora as a whole through low-temperature treatment and glycerol resuspension. After glycerol treatment, the competent cells are generally dispersed, with local aggregation. The cells lose water and shrink, with large depressions appearing on the surface, cavitation and wrinkling, and the cell membrane edges become blurred. The permeability changes, allowing for successful plasmid introduction. This method does not require the isolation of individual bacterial strains and can efficiently and comprehensively genetically modify intestinal microbial communities, expressing corresponding products in the intestine to treat diseases, which has great application prospects.

[0016] (2) The present invention uses the original intestinal microbiome extracted from mice as a sample to develop a new strategy for comprehensively transforming the microbiome in a complex system. After the entire intestinal microbiome is prepared into competent cells, these competent cells are genetically modified by plasmid electroporation and then transplanted into the mouse intestine. Multiple successfully transformed strains can be screened from the intestinal contents. This method does not require the isolation of individual bacterial strains and can comprehensively genetically modify the microbial community. The method is simple, time-saving, and labor-saving, with high transformation efficiency. It does not require the selection of a single host, and many strains in the intestinal microbiome can be genetically modified at one time. Finally, multiple successfully transformed strains can be screened from the intestinal contents. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0018] Figure 1 A comparison chart of the traditional intestinal flora modification method in Example 1 of the present invention and the new method developed in this study; Figure 2 This is a diagram showing the construction of the plasmid pT5-AA for co-expressing ADH and ALDH in Example 1 of the present invention; Figure 3This is the competent cell preparation device for the mixed bacteria in Example 1 of the present invention, wherein A is a filtration device, B is the bacterial liquid obtained by filtering feces, and C is the competent cells prepared from the mixed bacteria; Figure 4 is the effect of plasmid transformation in Experimental Example 1 of the present invention, wherein A is the effect of plasmid size on electroporation efficiency, B is the effect of plasmid concentration on electroporation efficiency, C is the PCR result of the electroporated strain, D is a scanning electron micrograph of the surface of mouse feces, E is a scanning electron micrograph of fecal bacterial competent cells, F is a scanning microscope image of fecal bacterial competent cells after electroporation, G is a close-up image of a single cell in D, H is a close-up image of a single cell in E, I is a close-up image of a single cell in F, J is a transmission electron micrograph of the surface of mouse feces, K is a transmission electron micrograph of intestinal microbial competent cells, L is a transmission electron micrograph of intestinal microbial competent cells after electroporation, M is a close-up image of a single cell in J, N is a close-up image of a single cell in K, and O is a close-up image of a single cell in L; Figure 5 Figures 2 and 3 show the treatment results of different treatment groups in Experimental Example 2 of the present invention, wherein A shows the changing trend of NADH content in the ADH reaction system; B shows the spectral scan of the ADH reaction system; C shows the changing trend of NADH content in the ALDH reaction system; D shows the spectral scan of the ALDH reaction system; E shows the gas chromatogram of a standard system containing certain amounts of ethanol, acetaldehyde, and acetic acid; F shows the gas chromatogram of the reaction system at 0 hours of reaction; and G shows the gas chromatogram of the reaction system at 3 hours of reaction. Figure 6 This is a graph showing the enzyme activity results of mixed bacteria transformed with different plasmids in the in vitro reaction system of Experimental Example 2 of the present invention, wherein A is a trend of change in NADH content in the ADH reaction system of the intestinal microbiome of mice administered with three bacterial solutions by oral gavage; B is a trend of change in NADH content in the ALDH reaction system of the intestinal microbiome of mice administered with three bacterial solutions by oral gavage; C is a spectral scan of the ADH reaction system of the intestinal microbiome of mice in the pT5-AA-Mix group, D is a spectral scan of the ADH reaction system of the intestinal microbiome of mice in the pT5-AA-DH5α group, E is a spectral scan of the ADH reaction system of the intestinal microbiome of mice in the pT5-AG-Mix group, F is a spectral scan of the ALDH reaction system of the intestinal microbiome of mice in the pT5-AA-Mix group, G is a spectral scan of the ALDH reaction system of the intestinal microbiome of mice in the pT5-AA-DH5α group, and H is a spectral scan of the ALDH reaction system of the intestinal microbiome of mice with mixed bacteria in the pT5-AG-Mix group; Figure 7: The graph shows the results of mice gavage in Experimental Example 2 of the present invention, wherein A is the experimental mouse grouping; B is the weight of each group of mice; C is the survival percentage of each group of mice; D is the liver index of each group of mice; E is the blood ethanol content of each group of mice; F is the effect of the intestinal microbiota co-expressing ADH and ALDH on the ALT activity of mouse serum; G is the effect of the intestinal microbiota co-expressing ADH and ALDH on the AST activity of mouse serum; H is the effect of the intestinal microbiota co-expressing ADH and ALDH on the AKP activity of mouse serum; I is the effect of the intestinal microbiota co-expressing ADH and ALDH on the TG level of mouse serum, J is the effect of the intestinal microbiota co-expressing ADH and ALDH on the TG level of mouse liver, and K is the effect of the intestinal microbiota co-expressing ADH and ALDH on the TG level of mouse serum Effect of co-expression of ADH and ALDH on TC level, L is the effect of intestinal microbiota co-expressing ADH and ALDH on TC level in mouse liver; M is the effect of intestinal microbiota co-expressing ADH and ALDH on serum HDL-C level in mouse, N is the effect of intestinal microbiota co-expressing ADH and ALDH on serum LDL-C level in mouse, O is the effect of intestinal microbiota co-expressing ADH and ALDH on LDL-C level in mouse liver; Effect of co-expression of ADH and ALDH on lipid peroxidation and antioxidant levels in mouse liver, P is the effect of co-expression of ADH and ALDH on lipid MDA level in mouse liver, Q is the effect of liver T-AOC level, R is the effect of co-expression of ADH and ALDH on lipid SOD in mouse liver, S is the effect of co-expression of ADH and ALDH on lipid T-GSH level in mouse liver; Figure 8 These are stained sections of mouse liver tissue and small intestinal tissue observed under a 40x optical microscope in Experimental Example 2 of the present invention, wherein A is a HE stained section of the liver tissue of mice in the control group C, B is a HE stained section of the liver tissue of mice in the alcohol group E, C is a HE stained section of the liver tissue of mice in the AA group, and D is a HE stained section of the liver tissue of mice in the AG group; E is an Oil Red O stained section of the liver tissue of mice in the control group C, F is an Oil Red O stained section of the liver tissue of mice in the alcohol group E, G is an Oil Red O stained section of the liver tissue of mice in the AA group, and H is an Oil Red O stained section of the liver tissue of mice in the AG group; I is a HE stained section of the small intestinal tissue of mice in the control group C, J is a HE stained section of the small intestinal tissue of mice in the alcohol group E, K is a HE stained section of the small intestinal tissue of mice in the AA group, and L is a HE stained section of the small intestinal tissue of mice in the AG group. DETAILED DESCRIPTION

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0021] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0022] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0023] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0024] Example 1 Embodiment 1 of the present invention is based on Figure 1 The next part of the process is to extract the fecal microbiota for transplantation. The specific steps are as follows: (1) Plasmid construction: The mouse intestinal flora was isolated and inoculated on ampicillin and kanamycin resistance plates. It was found that the mouse intestinal flora did not grow on the ampicillin and kanamycin resistance plates, that is, the mouse intestinal flora itself did not have both ampicillin and kanamycin resistance.

[0025] pT5-sfFGFP itself has kanamycin resistance gene and sfFGFP gene. Using pT5-sfFGFP as plasmid vector, the ampicillin resistance gene promoter and ampicillin resistance gene are inserted after the kanamycin resistance gene of pT5-sfFGFP to obtain the empty vector plasmid pT5-AG. Then, ADH and ALDH gene fragments are added to the plasmid pT5-AG. Both enzyme genes are induced by promoter J23119 before constructing the functional plasmid pT5-AA that co-expresses ADH and ALDH. Figure 2 shown.

[0026] (2) Preparation of mixed bacterial competent cells: 1) Thaw frozen healthy mouse feces, crush and mix thoroughly, filter through a 70 µm filter to remove impurities, filter the filtrate through a 5 µm filter, centrifuge at 6000 × g for 10 min, discard the supernatant, and resuspend the bacterial pellet in 10% glycerol. 2) Concentrate the bacterial solution to an OD600 value of 0.4-0.6. Immediately place the bacterial solution on ice. Always perform the following steps in a clean bench and on ice, moving as quickly and steadily as possible. 3) Pipette 1 mL of bacterial solution into a 1.5 mL centrifuge tube and cool on ice for 10 min. 4) Centrifuge at 6000 × g for 10 min at 4°C, discard the supernatant, gently resuspend the cells in 1 mL of ice-cold 10% glycerol, and chill on ice for 30 min. Repeat this step twice. 5) Centrifuge at 6000×g for 10 min at 4°C and gently suspend the cells in 200µL of ice-cold 10% glycerol to obtain competent cells. The concentration of competent cells is OD 600 The value is 0.4~0.6, such as Figure 3 As shown; 6) Use immediately or store immediately at -80℃.

[0027] (3) Plasmid electroporation into mixed bacterial competent cells: 1) Remove the prepared competent cells and thaw them on ice. Add 2 µL (100 ng / µL) of plasmid per 50 µL of competent cells, pipette gently to mix evenly, and place on ice. 2) Add the mixture to be transformed into a pre-cooled electroporation cuvette. Select 2.5 kV as the output voltage of the electroporation instrument and apply a single electric pulse (the duration of the electric pulse is approximately 5 ms). 3) Immediately add 1 mL of LB medium to the transformation cup to resuspend the cells. Centrifuge at 6000 × g for 10 minutes at 4°C. Discard 900 µL of supernatant and resuspend the cells in the remaining 100 µL of supernatant to obtain a mixed bacterial strain introduced with the plasmid pT5-AA. 16S rRNA sequencing of the bacteria grown on the plate revealed that six bacteria were successfully transformed with the plasmid using the new method: Pseudomonas aeruginosa, Chryseobacterium indologenes, Alcaligenes faecalis, Klebsiella pneumoniae, Stenotrophomonas maltophilia, and Cellulomicrobium finkelii. 4) Mice were gavaged with 50% alcohol (5.6 g / kg body weight) and 100 µL of a mixed bacterial strain containing the plasmid pT5-AA was also gavaged into C57 male mice. Ampicillin and kanamycin were added to the sterile drinking water (final concentration: 25 µL / mL). Sterile mouse standard chow and sterile drinking water were available ad libitum. This gavage was repeated for 7 consecutive days. After the final gavage, the mice were deprived of food but not water. One hour later, blood samples were collected from the tail vein for alcohol content. Samples were collected 16 hours later, and the mice were sacrificed.

[0028] Cecal contents: The cecum was removed and its contents were squeezed out aseptically. After crushing and mixing, impurities were filtered out using a 70µm filter membrane. The filtrate was then filtered using a 5µm filter membrane and centrifuged at 6000×g for 10 min. The supernatant was discarded and the bacterial pellet was resuspended in 10% glycerol to obtain the fecal microbiota.

[0029] Since mouse intestinal bacteria do not have resistance to ampicillin and kanamycin, adding two antibiotics, ampicillin and kanamycin, to the mouse drinking water can minimize the impact of the original bacterial flora on the modified bacterial flora. The mixed bacterial flora with successful plasmid transformation can grow under the pressure of these two antibiotics, while the original intestinal flora will die.

[0030] Test Example 1 Experimental Example 1 of the present invention measured the effect of plasmids of different sizes constructed in Example 1 on electroporation efficiency. The specific steps are as follows: (1) Using the method of Example 1, the ADH gene fragment was added to the plasmid pT5-AG on the basis of pT5-AG to construct the pT5-ADH plasmid. Under aerobic and anaerobic conditions, the plasmids of different concentrations and sizes were transferred into the mixed bacterial competent cells. The results are as follows: Figure 4 As shown in A, B, and C.

[0031] Figure 4 A, B, and C show that plasmids of three sizes can be successfully electroporated into the mixed bacterial population.

[0032] (2) The mouse feces, the mixed bacteria competent cells, and the mixed bacteria competent cells after electroporation in Example 1 were analyzed by scanning electron microscopy and transmission electron microscopy respectively. Figure 4 As shown in D~O.

[0033] Depend on Figure 4 It can be seen that the cells on the untreated feces are clustered, the cells are adhered and have clear edges, and there are tiny protrusions on the surface ( Figure 4 D, G); After glycerol treatment, the competent cells were dispersed as a whole, with some aggregation occurring locally. The cells lost water and shrank, with large depressions appearing on the surface, cavitation, and wrinkles. ( Figure 4E, H); After electroporation, competent cells have integrated exogenous DNA and are in an independent state, with unusually clear edges, raised surfaces and tiny bright spots, plump cells, improved surface smoothness, and enhanced brightness ( Figure 4 F, I). Transmission electron microscopy images show that the cell membrane edges of cells in untreated feces are clear, the internal structure of the cells is intact, and typical bacterial structures such as cell wall, cytoplasm and nuclear region are observed. Organic matter or particles are attached to the surface of the cell membrane ( Figure 4 I, M); After glycerol treatment, the cell membrane edge of competent cells became blurred, the permeability changed significantly, cytoplasm concentration caused by dehydration appeared inside the cells, and the gap between the cell wall and the cell membrane increased. These changes are related to cell dehydration and membrane structure damage caused by glycerol treatment ( Figure 4 K, N); After electroporation, the cell membrane of competent cells has a fine light on its surface, the internal structure of the cells is more uniform, the cytoplasm density increases, and the gap between the cell wall and the cell membrane decreases. These characteristics reflect the repair and adaptation process of the cells after the successful introduction of foreign DNA. ( Figure 4 L, O).

[0034] Test Example 2 Experimental Example 2 of the present invention tested the effects of pT5-AA and fecal microbiota transplantation prepared in Example 1, and the specific steps were as follows: (1) Enzyme induced expression and activity detection: Referring to the steps of Example 1, pT5-AA was introduced into Escherichia coli DH5α to induce the expression of ADH and ALDH. If normal expression was achieved, E. coli could grow normally on ampicillin and kanamycin dual-resistance plates, thereby obtaining pT5-AA-DH5α.

[0035] 1) Induction of enzyme expression: Pick a normally growing E. coli DH5ɑ colony and place it in 5 mL of Amp+Kan LB liquid and shake it at 180 rpm at 37°C overnight. Add the overnight culture liquid to three 100 mL Amp+Kan LB (100µM Zn 2+ ) at 37°C with shaking at 180 rpm until OD 0.4, then add 100 µL 1 M IPTG (final concentration 1 mM), and culture at 30°C with shaking at 80 rpm overnight.

[0036] 2) Preparation of crude enzyme solution: ① Collect 100 mL of induced expression pT5-AA culture medium and let it stand at 4°C for 20 minutes. ② Centrifuge at 5000 × g at 4°C for 20 minutes, then collect the cells and supernatant. ③ Resuspend and wash the cells in PBS buffer (pH 7.4), then centrifuge at 5000 × g at 4°C for 20 minutes. ④ Collect the washed cells and resuspend them in 10 mL of lysis buffer. Add lysozyme to a final concentration of 3 mg / mL and incubate in a shaking water bath at 37°C for 30 minutes. ⑤ Place the treated cells in an ice-water bath and disrupt them using an ultrasonic disruptor. The following conditions are: 30 minutes total treatment time, 400 W ultrasonic power, 1 second on, 2 seconds off. ⑥ After sufficient disruption, centrifuge at 8000 × g for 20 minutes. Collect the supernatant to obtain the crude enzyme solution. Enzyme activity is measured in an in vitro reaction system using a spectrophotometer to measure the absorbance change of NADH at 340 nm.

[0037] 3) Alcohol dehydrogenase activity assay: The reaction system is as follows: the total volume of the system is 3 mL, containing 50 mmol / L PB buffer (pH 9.0), 40 mmol / L ethanol, 0.5 mmol / L NAD + Finally, 0.1 mL of the enzyme solution to be tested was added to initiate the enzyme activity assay. The absorbance change was measured at 340 nm at 25°C. One unit (U) of enzyme activity was defined as the amount of enzyme required to generate 1 mol of NADH per minute. The enzyme activity assay was performed in triplicate, and the results are presented as the mean and standard deviation.

[0038] 4) Acetaldehyde dehydrogenase activity detection: The reaction system is as follows: the total volume of the system is 3 ml, containing 2 mmol / L acetaldehyde, 0.5 mmol / L NAD + , 0.1 mol / L Tris-HCl (pH = 8.0), 10 mmol / L β-mercaptoethanol, and 0.1 mol / L KCl. Finally, 0.1 mL of the enzyme solution to be tested is added to initiate the enzyme activity assay. The absorbance change is measured at 340 nm at 25°C. One unit (U) of enzyme activity is defined as the amount of enzyme required to generate 1 μmol of NADH per minute.

[0039] 5) Calculate enzyme activity: The final calculation of fermentation broth activity is: absorbance change per minute / 0.00622 * 10 / 10 (where " / 0.00622" is the molar extinction coefficient of NAD+, "*10" is the amount of enzyme added (0.1 mL), and " / 10" is the concentration factor of the crude enzyme used in fermentation broth preparation). The enzyme activity assay was repeated three times, and the results are presented as the mean and standard deviation.

[0040] The above test results are as follows Figure 5 As shown in A, B, C, D, E, and F.

[0041] At the beginning of the reaction, there is only ethanol and a small amount of acetaldehyde in the system. After the reaction is completed, both ethanol and acetaldehyde in the system decrease, and a large amount of acetic acid is generated. Figure 5 A, B, C, D, E, and F show that the NADH content gradually increases as the reaction proceeds.

[0042] (2) First oral administration of mice In a control experiment, 24 6-8-week-old male C57 mice were randomly divided into three groups of 8 mice each: the pT5-AA-Mix group, the pT5-AA-DH5α group, and the pT5-AG-Mix group. Each group was gavaged with a different bacterial solution: the pT5-AA-Mix group was gavaged with a mixture of bacteria electroporated with the pT5-AA plasmid; the pT5-AA-DH5α group was gavaged with Escherichia coli DH5α electroporated with the pT5-AA plasmid; and the pT5-AG-Mix group was gavaged with a mixture of bacteria electroporated with the pT5-AG plasmid. Ampicillin and kanamycin were added to the sterile drinking water of each group (final concentration of 25 µL / mL). Sterile mouse standard chow and sterile drinking water were available ad libitum. After 7 consecutive days of gavage, the mice were euthanized and autopsied. The intestinal contents of each group were collected, added to PBS buffer, and mixed evenly with magnetic beads. Then, the fecal homogenate was filtered through a 70µm filter membrane to remove impurities and obtain a bacterial solution. The obtained bacterial solution was used to prepare a crude enzyme solution, and the enzyme activity was tested in an in vitro reaction system. The enzyme activity can be characterized by measuring the absorbance change of NADH at 340nm using a spectrophotometer. The results are shown in Figure 2. Figure 6 As shown in A, B, C, D, E, F, G, and H.

[0043] Second oral administration of mice: Twenty 6-8 week old C57 male mice were randomly divided into four groups, with 5 mice in each group, namely, a blank control group (C), an alcohol model group (E), a fecal microbiota transplantation gavage group (AA) according to Example 1, and a mixed microbiota gavage group transformed with the empty vector plasmid pT5-AG (transformed according to the method of Example 1, AG).

[0044] The alcohol model group was gavaged with 50% alcohol (5.6 g / kg body weight) and 100 µL of normal saline. The blank control group was gavaged with normal saline (the same amount as the alcohol group). The AA group was gavaged with the same dose of 50% alcohol as the alcohol group and 100 µL of the fecal microbiota transplanted in Example 1. The AG group was gavaged with the same dose of 50% alcohol as the alcohol group and 100 µL of mixed bacteria transformed with the plasmid pT5-AG. The pT5-AA-DH5α gavage group was gavaged with the same dose of 50% alcohol as the alcohol group and 100 µL of pT5-AA-DH5α. Ampicillin and kanamycin, two antibiotics, were added to the sterile drinking water of each group (final concentration of 25 µL / mL). Sterile mouse standard feed and sterile drinking water were available ad libitum. Gavage was continued for 7 consecutive days. During the experiment, the body weight, mental state, activity, diet, hair gloss, and death of mice in each group were observed and recorded (see [ 15 ]). Figure 7 B and C) and other routine conditions. After the final oral gavage treatment, mice in each group were fasted but not watered. One hour later, blood samples were collected from the tail vein and tested for alcohol content. Samples were collected and mice were sacrificed 16 hours later.

[0045] 1) Serum: Blood was collected from the retroorbital vein of mice in a sterile centrifuge tube. The blood was allowed to stand at room temperature until it coagulated and concentrated. Serum was obtained by centrifugation at 3500×g for 10 min at 4°C. The serum samples were stored at -80°C for subsequent biochemical analysis. Figure 7 E, F, G, H, I, K, M, N.

[0046] 2) Liver: After blood collection, mice were killed by cervical dislocation and autopsied. The liver was excised and weighed, and the liver index (liver weight / body weight) was calculated. Liver lipid peroxidation and liver antioxidant levels were measured. A portion of the liver was fixed in formalin and used for pathological section analysis, while the remaining portion was quickly frozen in liquid nitrogen and stored at -80°C for subsequent biochemical analysis and real-time PCR analysis. The results are shown in [ 1 ]. Figure 7 D, J, L, O, P, Q, R, S, and Figure 8 .

[0047] 3) Cecal contents: The cecum was removed and its contents were aseptically squeezed out, collected in a sterile centrifuge tube, quickly frozen in liquid nitrogen, and stored in a -80°C freezer for subsequent enzyme activity analysis, fecal genomic analysis, and microbial analysis.

[0048] Figure 5The plasmid pT5-AA was electroporated into Escherichia coli DH5α to induce the expression of ADH and ALDH. The bacteria were then disrupted to prepare a crude enzyme solution, and the enzyme activity was measured in an in vitro reaction system. Using a spectrophotometer to measure the absorbance change of NADH at 340 nm, the enzyme activity of ADH and ALDH in the reaction system was calculated to be 0.065 U and 0.072 U, respectively. The ALDH enzyme activity is stronger than that of ADH, thus preventing the accumulation of the intermediate acetaldehyde. Spectral scans of the reaction system every 15 minutes revealed a continuous increase in the NADH content in the ADH reaction system and in the ALDH reaction system. Gas chromatography analysis of the components before and after the reaction was performed allowed the peak areas to be used to calculate the concentrations of the components in the reaction system. It can be seen that at the beginning of the reaction, only ethanol and a small amount of acetaldehyde were present in the system. After the reaction was completed, both ethanol and acetaldehyde decreased, and a large amount of acetic acid was produced.

[0049] Figure 6 Calculated ALDH activity in the crude enzyme solution from the pT5-AA-Mix group was 0.056 U, and ADH activity was 0.028 U. The crude enzyme solution from the pT5-AA-DH5α group had 0.056 U, and ADH activity was 0.013 U. The pT5-AG-Mix group had no ADH or ALDH activity. The ALDH activity of the crude enzyme solutions from the pT5-AA-Mix and pT5-AA-DH5α groups was stronger than their respective ADH activities, enabling complete degradation of ingested ethanol into acetic acid without the accumulation of the intermediate acetaldehyde, thus preventing its toxicity. More importantly, it can be seen that the ADH enzyme activity of the pT5-AA-Mix group is stronger than that of the pT5-AA-DH5α group, and the ALDH enzyme activity of the pT5-AA-Mix group is equivalent to that of the pT5-AA-DH5α group, indicating that the mixed bacteria containing plasmid pT5-AA have a stronger alcohol-degrading effect in the mouse intestine than the Escherichia coli DH5α containing plasmid pT5-AA in the mouse intestine. In other words, the modified mixed bacteria can more effectively prevent mice from liver damage and intestinal damage caused by alcohol intake than single colonies.

[0050] Depend on Figure 7 It can be seen that the mice were killed and samples were taken 7 days after gavage. Statistically, the weight of the mice in the alcohol group decreased rapidly, while there was no significant difference in the other three groups ( Figure 7 B). On the third day after gavage, one mouse died in each of the alcohol group and the empty vehicle group. On the sixth day, another mouse died in the alcohol group. No mouse died in either the experimental group or the control group ( Figure 7 C). The liver index of each group of mice increased compared with the control group, but there was no significant difference ( Figure 7D). One hour after the last oral gavage, the ethanol content in the mouse serum was measured and it was found that the experimental group had a significantly lower ethanol content than the alcohol group ( Figure 7 E).

[0051] Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (AKP) activities are commonly used indicators for evaluating liver function. After 7 consecutive days of gavage, the serum ALT, AST, and AKP levels in the alcohol group and the empty vehicle group increased significantly compared with the control group, while those in the experimental group were significantly lower than those in the alcohol group. There was no significant difference compared with the control group, and the levels returned to normal ( Figure 7 F, G, H).

[0052] Alcohol can cause fatty degeneration of mouse liver cells, so the fat levels in the mouse serum and liver were further tested. In the serum, the triglyceride (TG) content in the alcohol group and the empty vehicle group was significantly reduced compared with the control group, while the difference in the experimental group was not significant ( Figure 7 I). In the liver, triglyceride (TG) levels were significantly increased in the alcohol group and the empty vehicle group compared with the control group, while there was no significant difference between the experimental group and the control group ( Figure 7 J). There was no significant difference in total cholesterol (TC) between serum and liver, a phenomenon similar to that reported in previous studies ( Figure 7 K, L). In serum, there were no significant differences in high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) levels among the groups, a phenomenon similar to that reported in previous studies ( Figure 7 M, N). In the liver, compared with the control group, the low-density lipoprotein cholesterol (LDL-C) content in the alcohol group and the empty vector group was significantly reduced, while the HDL-C level in the experimental group was increased, but the difference was not significant compared with the control group ( Figure 7 O).

[0053] Oxidative stress is one of the main pathogenic mechanisms of alcoholic liver disease, so the liver lipid peroxidation and liver antioxidant levels of mice were measured. The results showed that compared with the control group, the liver malondialdehyde (MDA) level in the alcohol group and the empty vector group was significantly increased ( Figure 7 P), while the levels of total antioxidant capacity T-AOC, superoxide dismutase SOD and total glutathione T-GSH were significantly decreased ( Figure 7 Q, R, S). This suggests that alcohol exacerbates lipid peroxidation in mouse liver cells and weakens their antioxidant capacity. However, after the experimental group mice were gavaged with functional bacterial flora, oxidative stress levels in their liver cells essentially returned to normal.

[0054] Figure 8In the experiment, the liver tissues of mice in each group were fixed, dehydrated and waxed, and then embedded in an embedding machine. The wax blocks were then sliced in a paraffin slicer and stained with HE. The results were observed under a microscope. The nuclei appeared blue, while the cytoplasm appeared red. The HE staining results of liver tissue sections showed that the control group C ( Figure 8 A) Hepatic cords are arranged regularly and neatly, hepatocytes are of normal size, uniform in shape, and neatly arranged, with uniform cytoplasm, round nuclei located in the center, uniform in size, and nucleoli clearly visible. Compared with the control group C, the alcohol group E ( Figure 8 B) Liver tissue showed extensive hydropic degeneration of hepatocytes, more hepatocytes with ballooning, cell swelling, loose and lightly stained cytoplasm, increased fat vesicles in the hepatocyte cytoplasm, multiple vacuoles, disordered and irregular arrangement of hepatic cords, a small amount of venous congestion, multiple cell membrane ruptures near the central vein, disappearance of cell nuclei, and a small amount of granulation tissue formation, indicating mild fibrosis. Compared with the control group C, the experimental group AA ( Figure 8 C) The liver tissue section is closest to the control group results. The hepatic cords are arranged in a regular radial pattern, the cells are neatly arranged, the cell size and morphology are normal, the cytoplasm is uniform and the nucleus is uniform in size. The overall structure is intact and there is no obvious pathological change. Compared with the control group C, the empty vector control group AG ( Figure 8 D) Multiple areas of hydropic degeneration were observed, along with hepatocyte swelling, sparse cytoplasm with vacuoles, and inflammatory infiltration. Hepatic cords were disorganized and irregularly arranged, and multiple cell membrane ruptures were observed near the central vein. Liver tissue sections resembled those in the alcohol group. Overall, the intestinal microbiota co-expressing ADH and ALDH in the AA group of mice demonstrated a certain degree of alcohol degradation, thereby exhibiting a protective effect on liver tissue. The results of hematological examination (HE) staining of liver tissue sections were most similar to those in the control group (C). However, HE staining of liver tissue sections from the empty vector control group (AG) and the alcohol group (E) showed similar results, indicating that alcohol ingestion failed to eliminate the liver's damaging effects, resulting in liver manifestations typical of alcoholic liver disease.

[0055] The liver tissues of mice in each group were quickly frozen in liquid nitrogen for 15 seconds and then stored in a -80℃ freezer. They were directly frozen and sectioned and embedded with OCT embedding medium. The embedding platform was then fixed on a microtome and cut into tissue slices with a thickness of 8-10μm. The tissue slices were then stained with Oil Red O and observed under a microscope. The lipid droplets appeared orange to bright red, and the cell nuclei appeared blue. The results of Oil Red O staining of liver tissue sections showed that the control group C ( Figure 8 E) Only a small amount of lipid droplets were visible in the liver cells. The lipid droplets were evenly distributed and small in size. They were mainly located around the central vein of the liver lobule. The liver cells were neatly arranged and structurally intact. There was no obvious lipid accumulation. Compared with the control group C, the alcohol group E ( Figure 8F), a large number of lipid droplets stained with Oil Red O can be seen in the liver cells. The volume of lipid droplets has increased significantly and they are widely distributed, especially around the central vein and in the portal area. The lipid droplets are orange-red, clustered and even fused into large lipid vacuoles, filling the cytoplasm of liver cells. In severe cases, they even squeeze the nucleus, causing changes in the morphology of liver cells; the structure of liver lobules is disordered, the liver sinusoids are compressed and deformed, and there are signs of inflammatory cell infiltration or fibrosis in the portal area. These changes indicate that the lipid metabolism in the liver is seriously disordered, the fatty degeneration is significant, and fatty liver may have developed, reflecting the toxic effect of alcohol on liver lipid metabolism and the pathological damage caused by it. Compared with the control group C, the experimental group AA ( Figure 8 G) Oil red O stained sections of liver tissue were closest to the control group. Lipid droplets were orange-red, evenly distributed in the cytoplasm of hepatocytes, with a small number and uniform size, indicating that liver lipid metabolism was normal. There were no abnormalities in the hepatic sinusoids, portal tracts, or vascular structures. Overall liver tissue morphology was normal, with no fatty degeneration or other pathological changes. Compared with control group C, empty vector control group AG ( Figure 8 In the Oil Red O-stained sections of the mouse liver (H), a large number of stained lipid droplets were visible in the hepatocytes. The lipid droplets were widely distributed and significantly larger in size than those in the control group. These orange-red lipid droplets aggregated into clusters or fused into larger lipid vacuoles, filling the cytoplasm of the hepatocytes and changing the hepatocyte morphology. These characteristics were similar to those in the alcohol group E, indicating that the empty vector group AG was unable to avoid the liver damage caused by alcohol intake in mice, resulting in disordered liver lipid metabolism, significant fatty degeneration, and structural pathological damage to the liver.

[0056] The small intestinal tissues of each group of mice were fixed, dehydrated and waxed, and then embedded in an embedding machine. The wax blocks were then placed on a paraffin slicer and sliced into cross sections. HE staining was performed and the results were observed under a microscope. The nuclei of the cells appeared blue, while the cytoplasm appeared red. The HE staining results of the small intestinal tissue sections showed that the control group C ( Figure 8 In the mucosal layer of the control group (I), the intestinal villi were densely arranged, with intact structure and neat arrangement. The epithelial cells had normal morphology, with nuclei located at the base, without abnormal proliferation or shedding. The columnar and goblet cells had intact morphology, were evenly distributed, and had normal numbers. In the lamina propria of the control group (C), the glandular structure was normal, without expansion or atrophy, with a small number of inflammatory cells and no significant infiltration. In the muscular layer of the control group (C), the smooth muscles were neatly arranged and of uniform thickness, without hypertrophy or atrophy. In the serous layer of the control group (C), the mesothelial cells were completely covered, without thickening or fibrosis. Compared with the control group (C), the alcohol group (E) had normal glandular structure, without expansion or atrophy, with a small number of inflammatory cells and no significant infiltration. Figure 8 J) showed shortening, fusion and shedding of intestinal villi in the mucosal layer, disordered arrangement, degeneration, necrosis and shedding of epithelial cells, abnormal nuclear position, and a significant decrease in the number of columnar and goblet cells and uneven distribution. In the alcohol group E, the glands in the lamina propria showed structural destruction and increased inflammatory cell infiltration, which may indicate chronic inflammation. Compared with the control group C, the experimental group AA ( Figure 8 K) showed slightly disordered intestinal villi, normal epithelial cell morphology, and a small number of detached cells. Overall, the overall morphology of the intestinal tract of the experimental group AA was similar to that of the control group C, with no obvious lesions, indicating that the intestinal microbiota co-expressing ADH and ALDH played a certain protective role in the mouse intestine under alcohol stress. Compared with the control group C, the empty vector group AG ( Figure 8 Intestinal epithelial cells in the small intestine (L) shed, the villus structure disappeared, and the intestinal glands in the lamina propria became necrotic and disappeared, replaced by proliferating connective tissue. Compared with the alcohol group (E), the empty vector group (AG) showed significant localized lymphoid tissue hyperplasia in the submucosal layer, disorganized smooth muscle in the muscularis, hypertrophy, or atrophy, and thickening and fibrosis of the mesothelial cells in the serosal layer. Overall, the small intestinal lesions in the empty vector group (AG) were more severe than those in the alcohol group (E). This may be because the combined effects of alcohol stress and the influx of foreign bacteria led to significant changes in the intestinal flora composition in the empty vector group (AG), exacerbating the damage to intestinal cells caused by alcohol in these mice.

[0057] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for preparing mixed bacterial competent cells, characterized in that: The steps include: (1) Filter the feces, centrifuge, and resuspend the precipitate in glycerol to obtain a bacterial suspension; (2) Cool the bacterial suspension on ice, centrifuge at low temperature, take the precipitate and resuspend it with glycerol, repeat the cooling and centrifugation steps after resuspension, take the precipitate after centrifugation and resuspend it with glycerol to obtain competent cells.

2. The method for preparing the mixed bacterial competent cells according to claim 1, characterized in that: In step (1), the pore size of the filter membrane used in the filtration is 4-6 μm; the glycerol is 5-15% glycerol.

3. The method for preparing the mixed bacterial competent cells according to claim 1, characterized in that: In step (2), the ice cooling time is 8 to 12 minutes, the low-temperature centrifugation temperature is 4°C; the number of repetitions is 2 to 4 times; and the glycerol is 5 to 15% glycerol.

4. A method for isolating fecal microbiota for preventing or treating alcoholic liver disease, characterized in that: The steps include: (1) Construction of plasmid The ampicillin resistance gene promoter and ampicillin resistance gene were inserted into the pT5-sfFGFP plasmid to obtain the empty vector plasmid pT5-AG. The ADH and ALDH gene fragments were added to the plasmid pT5-AG. The promoter J23119 was introduced before each enzyme gene fragment to construct the functional plasmid pT5-AA that co-expresses ADH and ALDH. (2) Preparation of mixed bacterial competent cells Prepare mixed bacterial competent cells by the method for preparing mixed bacterial competent cells according to any one of claims 1 to 3; (3) Take the mixed bacteria competent cells prepared in step (2), add the plasmid pT5-AA prepared in step (1), mix well, electrotransform, resuspend, and centrifuge at low temperature to obtain a mixed bacteria introduced with the plasmid pT5-AA; (4) Alcohol model mice were gavaged with mixed bacteria containing plasmid pT5-AA. After gavage, fecal microbiota that can prevent or treat alcoholic liver disease were isolated from the intestines of the mice.

5. The method for isolating fecal microbiota for preventing or treating alcoholic liver disease according to claim 4, characterized in that: In step (3), the OD of the competent cell concentration is 600 The value is 0.4~0.6; the volume ratio of the competent cells to the plasmid is 20~30:0.8~1.2, the concentration of the plasmid is 90~110ng / μL, the output voltage of the electroporation is 2~3kV, and the number of electric pulses is 1.

6. The method for isolating fecal microbiota for preventing or treating alcoholic liver disease according to claim 4, characterized in that: In step (3), the mixed bacteria into which the plasmid pT5-AA is introduced include one or more of Pseudomonas aeruginosa, Chryseobacterium indolegenes, Alcaligenes faecalis, Klebsiella, Stenotrophomonas maltophilia, and Cellulomonas finkelii.

7. The method for isolating fecal microbiota for preventing or treating alcoholic liver disease according to claim 4, characterized in that: In step (4), the alcohol model mice are gavaged for 7 to 10 days.

8. The method for isolating fecal microbiota for preventing or treating alcoholic liver disease according to claim 4, characterized in that: In step (4), the alcohol model mice also need to consume sterile drinking water containing two antibiotics, ampicillin and kanamycin, during the gavage period.

9. A fecal microbiota transplantation flora for preventing or treating alcoholic liver disease obtained by the method for isolating a fecal microbiota transplantation flora for preventing or treating alcoholic liver disease according to any one of claims 4 to 8.

10. Use of the fecal microbiota transplantation flora capable of preventing or treating alcoholic liver disease according to claim 9 in the preparation of a drug for preventing or treating alcoholic liver disease.

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