Construction of gene engineering lactic acid bacteria with high expression of IL22 and protection effect of gene engineering lactic acid bacteria on alcoholic liver injury

Abstract

The invention provides construction of gene engineering lactic acid bacteria with high expression of IL22 and a protective effect of the gene engineering lactic acid bacteria on alcoholic liver injury. The gene engineering lactic acid bacteria are lactobacillus reuteri DSM17938-IL22. The invention further provides a preparation method of the gene engineering lactic acid bacteria. The lactobacillus reuteri is obtained by electrically transforming a recombinant plasmid pSET4s-himar1-cat-IL22 into a competent cell of a probiotic lactobacillus reuteri DSM17938, and the lactobacillus reuteri is preserved in the China Center for Type Culture Collection in March 2023. The IL22 and the lactobacillus reuteri can effectively improve intestinal epithelium injury and relieve liver inflammation, the lactobacillus reuteri can further induce generation of the IL22, and the combination of the IL22 and the lactobacillus reuteri can obviously improve intestinal microbiota. Therefore, the lactobacillus reuteri can give full play to the synergistic effect of the lactobacillus reuteri and the lactobacillus reuteri, and a better intestinal liver axis immunoregulation effect is achieved.

Description

Technical Field

[0001] The present application belongs to the field of microbial technology, and specifically relates to the application of Lactobacillus reuteri and its preparation in preventing and / or treating alcoholic liver injury. Background Art

[0002] Long-term heavy drinking can damage the liver and cause a series of liver diseases, which initially manifest as alcoholic fatty liver, and further develop into alcoholic liver damage (ALD) such as alcoholic hepatitis and alcoholic liver fibrosis. Severe cases can also develop into cirrhosis or even liver cancer. Alcoholic liver disease (ALD) has serious intestinal flora disorders and mucosal barrier damage, leading to immune imbalance of the "gut-liver axis" and aggravating inflammatory damage in the liver. Lactobacillus reuteri is a probiotic present in the digestive tract of humans and animals. Animal studies have shown that Lactobacillus reuteri can significantly improve the intestinal mucosal barrier function of alcoholic liver damage, while correcting the immune imbalance of the "gut-liver axis", inhibiting inflammation and damage in the liver, etc.

[0003] Traditional treatments for alcoholic liver injury mainly include alcohol abstinence, drug therapy, nutritional support, liver transplantation, etc. Although alcohol abstinence is the best treatment for patients with alcoholic liver injury, more than half of patients may continue to drink, and patients with alcohol use disorder often experience alcohol withdrawal symptoms such as insomnia, agitation, and delirium during abstinence. Studies have shown that almost all patients with alcoholic liver injury are accompanied by varying degrees of malnutrition. Timely nutritional supplementation can partially improve patients' liver function indicators, but cannot improve their survival rate and the incidence of complications. In addition, although there are some liver-protecting and enzyme-lowering drugs for the treatment of alcoholic liver injury, such as silymarin and glycyrrhizic acid glycosides, they cannot effectively improve the intestinal microecological disorder and intestinal mucosal barrier destruction in patients with alcoholic liver injury, especially alcoholic cirrhosis, so that the immune imbalance of the "gut-liver axis" persists, and the incidence of intestinal infections such as spontaneous peritonitis remains high.

[0004] Probiotics are defined as "live microorganisms that are beneficial to the health of the host when ingested in adequate doses". Lactic acid bacteria, as a common probiotic, are natural and safe. Lactic acid bacteria have the effects of maintaining the microecological balance of intestinal flora, protecting the gastrointestinal mucosal barrier, relieving inflammation, strengthening the body's immune function, lowering cholesterol, delaying the aging of the body, and improving depression. Therefore, lactic acid bacteria have good application prospects and utilization value in improving alcoholic liver damage. Interleukin-22 (IL22) is an intestinal protective factor that plays an important role in maintaining the balance of intestinal flora and the integrity of the mucosal barrier. IL22 in the intestine can reduce the migration of bacteria to the liver and alleviate alcohol-induced liver damage by inducing the expression of intestinal regenerating islet-derived protein 3 gamma (REG3G). It has been verified that the expression of IL22 in the intestine of mice with alcoholic liver disease is significantly decreased, and the expression of IL22 in the probiotic (Lactobacillus) preparation treatment group is significantly higher than that in the model group, suggesting that probiotics can induce IL22 production and is beneficial to the protection of the intestinal mucosa in alcoholic liver disease.

[0005] Therefore, by combining the intestinal protective functions of probiotics and IL22, a genetically engineered lactic acid bacteria that can highly express IL22 was constructed, the intestinal microecological intervention technology for alcoholic liver damage was optimized, the balance of the "gut-liver axis" was accurately regulated, and the progression of ALD intrahepatic inflammation and fibrosis was effectively improved and blocked, which has very important clinical significance. Summary of the invention

[0006] To achieve the above objectives, this study adopts the following technical solutions: In a first aspect of the present application, a genetically engineered lactic acid bacteria for preventing and / or treating alcoholic liver damage is provided.

[0007] Furthermore, the genetically engineered lactic acid bacteria is Lactobacillus reuteri DSM17938-IL22.

[0008] Furthermore, the DSM17938-IL22 was deposited in the China Center for Type Culture Collection in March 2024, with the deposit number being CCTCC NO: M2024364.

[0009] Furthermore, the genetically engineered lactic acid bacteria are prepared by electro-transforming a recombinant plasmid pSET4s-himar1-cat-IL22 containing an IL22 gene sequence, wherein the recombinant plasmid pSET4s-himar1-cat-IL22 uses pMG36e as a vector and can achieve stable expression of IL22 in a lactic acid bacteria expression system, wherein the IL22 gene sequence is shown in SEQ ID No. 1.

[0010] The second aspect of the present application provides a method for preparing the genetically engineered lactic acid bacteria.

[0011] Further, the method comprises: 1) Preparation of competent cells of Lactobacillus reuteri DSM17938; 2) Electroporate IL22 into the DSM17938 competent cells obtained in 1); 3) Screen the culture medium for the strains successfully transformed in 2).

[0012] Furthermore, in the step 2), the electroporation parameters are high voltage pulse 2.5 kV / cm, capacitance 25 uF, resistance 200 Ω, and electric shock 5 ms.

[0013] Furthermore, the culture medium in step 3) is an MRS solid culture medium containing 5ug / ml chloramphenicol.

[0014] The third aspect of the present application provides an active lactic acid bacteria preparation.

[0015] Furthermore, the active lactic acid bacteria preparation comprises the genetically engineered lactic acid bacteria DSM17938-IL22 or its fermentation broth, wherein the number of viable bacteria is not less than 10 8 cfu / ml.

[0016] Furthermore, the active lactic acid bacteria preparation is in the form of capsules, tablets, liquid beverages or powders.

[0017] Furthermore, the active lactic acid bacteria preparation also includes a functional additive.

[0018] Preferably, the functional auxiliary agent includes any one of fructooligosaccharides, galacto-oligosaccharides, xylooligosaccharides, isomaltooligosaccharides, soybean oligosaccharides, inulin, spirulina, arthrospira, versicolor polysaccharide, stachyose, polydextrose, α-lactalbumin or lactoferrin, or a combination of at least two thereof.

[0019] Furthermore, the active lactic acid bacteria preparation acts directly on the intestines through oral administration, and is used to improve the intestinal microecology and regulate the immune response of the gut-liver axis.

[0020] The fourth aspect of the present application provides a method for preparing the active lactic acid bacteria preparation.

[0021] Furthermore, the method comprises: 1) Cultivating the genetically engineered lactic acid bacteria DSM17938-IL22 in a culture medium; 2) Select the IL22-expressing single clone cultured in 1).

[0022] Furthermore, in step 1), the culture medium is a lactic acid bacteria culture medium (MRS) containing 5 ug / ml chloramphenicol and 1% xylose.

[0023] The fifth aspect of the present application provides the use of the genetically engineered lactic acid bacteria or the active lactic acid bacteria preparation in the preparation of a product for preventing and / or treating alcoholic liver injury.

[0024] Furthermore, the application includes: 1) The product is used to prevent and / or treat alcoholic liver damage; 2) The product is used to prevent and / or treat alcohol-induced intestinal inflammation and intestinal damage; 3) The product is used as a functional probiotic in food, food additives, health products, medicines or feed.

[0025] Compared with the prior art, this application has the following technical effects: 1. This application constructs a new genetically engineered lactic acid bacteria DSM17938-IL22 for the first time. The lactic acid bacteria is successfully prepared by integrating the IL22 gene sequence into Lactobacillus reuteri DSM17938. The lactic acid bacteria can achieve stable expression of IL22 in the lactic acid bacteria expression system. Given that both IL22 and probiotics can effectively improve intestinal epithelial damage and reduce liver inflammation, and probiotics can further induce IL22 production, the combination of the two can effectively enhance the anti-inflammatory effect. Compared with existing exogenous recombinant IL22 products, the combination of IL22 and probiotics overcomes the shortcomings of exogenous recombinant IL22 targeted intestinal oral administration, easy degradation, and low utilization rate, and Lactobacillus reuteri is resistant to gastric acid, which significantly improves the intestinal barrier function and microecological disorders of alcoholic liver damage. Lactobacillus reuteri DSM17938-IL22 enables IL22 and probiotic preparations to fully synergize, and then explores the immunomodulatory effect of genetically engineered lactic acid bacteria on the "intestinal liver axis".

[0026] 2. This application also provides an active lactic acid bacteria preparation containing the genetically engineered lactic acid bacteria DSM17938-IL22 or its fermentation broth, which can be directly taken orally and act on the intestine. The development of this preparation provides a possible new treatment method for patients with alcoholic liver damage (ALD), helps to improve the intestinal microbiome, regulate the balance of the intestinal liver axis, avoids the complex and high-cost later extraction process of general genetically engineered bacteria, and provides a new idea for the production of safe and cheap biological products by genetic engineering; it also provides a theoretical basis and technical support for the feasibility study of the later probiotic immune preparations for the treatment of alcoholic liver damage.

[0027] In summary, this application provides a new treatment approach for diseases such as alcoholic liver damage, and shows good therapeutic effects and clinical application potential by improving intestinal microecology and regulating the immune response of the gut-liver axis. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The PCR experiment diagram of the pMG36e-Cat-IL22 plasmid electroporation results, where lane 1 is pMG36e-Cat-IL22 Trans2K plusII DNA marker, lanes 2 to 7 are PCR results of the genome of IL-22 transformants 1 to 6 (lane 3 is considered a false negative, and positive bands can be seen in the remaining lanes), and lane 8 is the PCR result of the wild-type genome. The electrophoresis results showed that pMG36e-Cat-IL22 was successfully transformed, that is, the Lactobacillus reuteri IL22 overexpression strain was successfully constructed.

[0029] Figure 2 The results of HE staining and Oil Red O staining show that in the context of mice fed with the same alcohol liquid feed, the hepatocyte degeneration, necrosis and fat deposition in the LR-IL22 group were significantly improved compared with the model group and LR group.

[0030] Figure 3 Schematic diagram of the expression levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lipopolysaccharide (LPS) in the serum of mice in the control group (Normal group), model group (Model group), LR group and LR-IL22 group.

[0031] Figure 4 Schematic diagram of the expression levels of liver-related genes IL-6, IL-1β and TNF-α in mice in the control group (Normal group), model group (Model group), LR group and LR-IL22 group.

[0032] Figure 5The following are the results of HE staining of the intestine of mice in the control group (Normal group), model group (Model group), LR group and LR-IL22 group. It shows that under the background of mice fed with alcohol liquid feed, compared with the Model group and LR group, the alcohol-induced intestinal villus-crypt structure destruction in the LR-IL22 group mice was significantly improved.

[0033] Figure 6 These are the results of determination of the expression levels of intestinal tight junction proteins Occludina, Claudin-1, and ZO-1 in the control group (Normal group), model group (Model group), LR group, and LR-IL22 group of mice. DETAILED DESCRIPTION

[0034] In this application, the control group (Normal group) represents the liquid control feed + normal saline gavage group; The model group (Model group) represents the alcohol liquid feed + saline gavage group; LR group represents alcohol liquid feed + DMS17938 bacterial solution gavage; The LR-IL22 group was fed with alcohol liquid feed + DMS17938-IL22 bacterial solution by intragastric administration.

[0035] The transformation, identification and screening methods of Lactobacillus reuteri that highly expresses IL22 are as follows: 1. Preparation method of Lactobacillus reuteri competent cells: 1) Inoculate a single colony from a freshly grown plate into 3 ml of MRS liquid medium; 2) Oscillate and mix at 200 rpm in an anaerobic incubator at 37°C (10% CO 2 , 10%H 2 , 80%N 2 ) Culture overnight; 3) Inoculate 1.5 ml of overnight cultured seeds into 150 ml (in a 500 ml Erlenmeyer flask) of fresh MRS medium, shake at 200 rpm to mix, and culture anaerobically at 37 °C until the bacterial count reaches about 1 × 10 8 cells / ml; 4) Transfer the bacterial solution to a 50 ml centrifuge tube, place on ice for 15 min, and then centrifuge at 4°C, 6000 rpm, 1 min to collect the bacteria; 5) Discard the supernatant and use 30 ml of ice-cold competent preparation buffer (5% sucrose, 1 mM MgSO 4 , 2mMKH 2 PO 4 ) Resuspend the cells, place on ice for 15 min, then centrifuge at 4°C, 6000 rpm, 1 min to collect the cells; 6) Discard the supernatant, resuspend the cells with 15 ml of ice-cold competent preparation buffer, place on ice for 15 min, then centrifuge at 4°C, 6000 rpm, 1 min to collect the cells; 7) Resuspend the cells in 2 ml of pre-cooled competent preparation buffer to a cell concentration of 1×10 10 cells / ml; 8) Aliquot the competent cells into 1.5 ml centrifuge tubes, 50 ul each, freeze them at ultra-low temperature, and store them at -80℃.

[0036] 2. The steps of the electrotransformation method of Lactobacillus reuteri are as follows: 1) Take 200ul of competent Lactobacillus reuteri bacterial solution, mix it with 5ug of pMG36e-Cat-IL22 knockout plasmid, add it into the electroporation transformation cup pre-cooled to 0.2cm electrode gap, and place it on ice for 5min; 2) Perform electroporation according to the parameters of the electroporator, the specific parameters are high voltage pulse 2.5kV / cm, capacitance 25uF, resistance 200Ω, and electric shock 5ms; 3) After the electroporation, remove the cup and immediately add 500 ul of MRS solution to elute the competent cells. Add the solution to a 1.5 ml centrifuge tube and incubate at 37°C, 250 rpm for anaerobic recovery for 1 hour. 4) Spread on MRS screening plates (containing 5ug / ml chloramphenicol) and culture at 37°C until obvious clones grow (about 20 days); 5) Pick a single clone into liquid MRS screening medium (containing 5ug / ml chloramphenicol) and culture at 37℃ with shaking; 6) Take 1 ml of bacterial solution and extract the genome according to the instructions of the Bacterial Genomic DNA Extraction Kit (Vazyme #DC103).

[0037] 3. PCR verification process During the PCR verification process, the design of primer sequences, PCR amplification system and reaction procedures are shown in Tables 1, 2 and 3.

[0038] Table 1 Primer sequences for PCR amplification

[0039] Table 2 PCR amplification system

[0040] Table 3 PCR reaction procedure

[0041] The electrophoresis identification results are shown in the attached Figure 1, where lane 1 is Trans2K plusII DNA marker; lanes 2-7 represent the genomic PCR results of pMG36e-Cat-IL22 transformants 1-6; and lane 8 represents the genomic PCR results of the wild-type strain. Figure 1 The electrophoresis results showed that pMG36e-Cat-IL22 was successfully transformed, that is, the Lactobacillus reuteri IL22 overexpression strain was successfully constructed.

[0042] Example 1 This example is used to demonstrate that recombinant IL22 Lactobacillus reuteri can reduce liver pathological damage and lipid deposition in mice fed with alcohol liquid feed.

[0043] 1. Recombinant IL22 Lactobacillus reuteri DMS17938-IL22 was fed to mice on an alcohol liquid diet background A single clone of recombinant IL22 Lactobacillus reuteri DMS17938-IL22 was selected and inoculated into MRS solid medium containing 5ug / ml chloramphenicol. Similarly, Lactobacillus reuteri DMS17938 was selected as a control and inoculated into MRS solid medium. The culture was carried out for 3-5 days to obtain DMS17938-IL22 bacterial solution (10 8 CFU / ml) and DMS17938 bacterial solution (10 8 CFU / ml).

[0044] Table 4 Feed formula

[0045] Two groups of feeds were prepared in sequence according to the feed formula in Table 4: 1) liquid control feed (hereinafter referred to as control feed for comparison with alcohol liquid feed; 2) alcohol liquid feed.

[0046] C57BL / 6 mice aged 6-8 weeks were divided into four groups: ①Liquid control feed + normal saline gavage group (hereinafter referred to as Normal group); ②Alcohol liquid feed + saline gavage group (hereinafter referred to as Model group); ③ Alcohol liquid feed + DMS17938 bacterial solution gavage (hereinafter referred to as LR group); ④ The mice were fed with alcohol liquid feed + DMS17938-IL22 bacterial solution by gavage (hereinafter referred to as LR-IL22 group).

[0047] 2. Recombinant IL22 Lactobacillus reuteri DMS17938-IL22 reduces alcohol-induced liver fat accumulation and inflammatory damage in mice HE staining: The livers of the mice in the Normal group, Model group, LR group, and LR-IL22 group in Example 1 were respectively fixed with 4% paraformaldehyde for more than 24 hours, washed with xylene, 95% ethanol, 95% ethanol, 80% ethanol, and distilled water in sequence, stained with hematoxylin-eosin (H&E), and then washed with distilled water, 80% ethanol, 95% ethanol, 95% ethanol, and xylene in sequence, and sealed with neutral gum. Observe and take pictures under a microscope (see Figure 1 ).

[0048] Oil Red O staining: fix fresh tissue in 4% paraformaldehyde for more than 24 h. Dehydrate with 15% sucrose and 30% sucrose, embed in OCT, and slice with a thickness of 8-10 µm. Rewarm the frozen sections and dry them for 10 minutes, fix them with 4% paraformaldehyde, and rinse them with PBS solution three times. Stain with Oil Red O working solution, differentiate with 75% ethanol, rinse with distilled water, counterstain with Harris hematoxylin, rinse with distilled water, differentiate with 1% hydrochloric acid alcohol, rinse with distilled water, blue with ammonia, rinse with distilled water, and seal with glycerol gelatin. Observe and take pictures of the lipid deposition in the liver under a microscope (see Figure 2 ).

[0049] The results are as follows Figure 2 As shown, in the context of mice fed with alcohol liquid feed, the hepatocyte degeneration, necrosis and fat deposition in the LR-IL22 group were significantly improved compared with the Model group and LR group.

[0050] Example 2 This example is used to prove that the recombinant IL22 Lactobacillus reuteri DMS17938-IL22 can improve alcohol-induced liver dysfunction and alleviate liver endotoxemia.

[0051] In this example, the serum of the mice in the Normal group, Model group, LR group and LR-IL22 group in Example 1 was taken to detect the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lipopolysaccharide (LPS) in the serum. The detection method was carried out according to the instructions for AST and ALT of Nanjing Jiancheng (Cat. No.: C010-2-1, C009-2-1) and the instructions for LPS enzyme-linked immunosorbent assay kit of Huamei Biotechnology (Cat. No.: CSB-E13066m).

[0052] The results are as follows Figure 3 As shown, under the background of mice fed with alcohol liquid feed, compared with the model group (Model group) and LR group, the ALT, AST and LPS levels in the serum of mice in the LR-IL22 group were significantly reduced (p<0.05), which indicates that recombinant IL22 Lactobacillus reuteri DMS17938-IL22 can significantly inhibit liver function damage induced by alcohol liquid feed.

[0053] Example 3 This example is used to demonstrate that recombinant IL22 Lactobacillus reuteri DMS17938-IL22 can significantly reduce the level of alcohol-induced inflammatory factors in the liver of mice.

[0054] In this example, the livers of the Normal group, Model group, LR group and LR-IL22 group of mice in Example 1 were respectively taken, RNA was extracted, and reverse transcribed to obtain cDNA of each group of mice; cDNA was used as a template, and the expression levels of inflammation-related genes IL-6, IL-1β and TNF-α were detected by fluorescent quantitative PCR, GAPDH was used as an internal reference gene, and the primer sequences were shown in Table 5.

[0055] like Figure 4 As shown, in the context of mice fed with alcohol liquid feed, the expression levels of inflammation-related genes IL-6, TNF-α and IL-1β in the liver of mice in the LR-IL22 group were significantly decreased compared with the Model group and the LR group (p<0.05). This shows that recombinant IL22 Lactobacillus reuteri DMS17938-IL22 can significantly inhibit the expression of inflammation-related genes IL-6, IL-1β and TNF-α in mice induced by alcohol liquid feed (p<0.05). It can be seen that when feeding alcohol liquid feed, recombinant IL22 Lactobacillus reuteri DMS17938-IL22 can reduce inflammation in the liver of mice induced by alcohol liquid feed.

[0056] Table 5 Primers used for fluorescence quantitative PCR detection of inflammation-related genes

[0057] Example 4 This example is used to demonstrate that recombinant IL22 Lactobacillus reuteri DMS17938-IL22 has a protective effect on intestinal mucosal damage induced by alcohol liquid feed in mice, and the method is as follows: 1. HE staining: The intestines of the mice in the Normal group, Model group, LR group and LR-IL22 group in Example 1 were taken respectively, fixed with 4% paraformaldehyde for more than 24 hours, washed with xylene, 95% ethanol, 95% ethanol, 80% ethanol and distilled water in sequence, stained with hematoxylin-eosin (H&E), washed with distilled water, 80% ethanol, 95% ethanol, 95% ethanol and xylene in sequence, and sealed with neutral gum. Observed under a microscope and photographed.

[0058] The results are as follows Figure 5As shown, in the context of mice fed with alcohol liquid feed, the alcohol-induced intestinal villus-crypt structure destruction in the LR-IL22 group was significantly improved compared with the Model group and LR group (p<0.05).

[0059] 2. Determination of the expression of tight junction proteins Occludina, Claudin-1, and ZO-1 The intestines of the Normal group, Model group, LR group and LR-IL22 group of mice in Example 1 were respectively taken, RNA was extracted, and reverse transcription was performed to obtain intestinal cDNA of each group of mice; using intestinal cDNA as a template, fluorescent quantitative PCR was used to detect the expression levels of tight junction proteins Occludina, Claudin-1, and ZO-1, and GAPPDH was used as an internal reference gene (detection primers are shown in Table 6).

[0060] Table 6 Primers used for fluorescence quantitative PCR detection of tight junction protein genes

[0061] The results are as follows Figure 6 As shown in the figure, under the background of mice fed with alcohol liquid feed, the expression levels of tight junction protein genes Occludina, Claudin-1, and ZO-1 in the intestine of mice fed with LR-IL22 group were significantly increased compared with those in Model group and LR group (p<0.05). This indicates that recombinant IL22 Lactobacillus reuteri DMS17938-IL22 can significantly increase the expression of tight junction protein genes in the intestine of mice. The above results suggest that recombinant IL22 Lactobacillus reuteri DMS17938-IL22 can improve the intestinal mucosal barrier damage of ALD.

[0062] The present application has been described in detail above. For those skilled in the art, the present application can be implemented in a wider range under equivalent parameters, concentrations and conditions without departing from the purpose and scope of the present application and without unnecessary experimentation. Although the present application provides specific embodiments, it should be understood that further improvements can be made to the present application. In short, according to the principles of the present application, the present application is intended to include any changes, uses or improvements to the present application, including changes that are out of the scope of the disclosure in the present application and are made with conventional techniques known in the art. According to the scope of the following claims, some basic features can be applied.

Claims

1. A genetically engineered lactic acid bacterium for preventing and / or treating alcoholic liver damage, characterized in that: The genetically engineered lactic acid bacteria is Lactobacillus reuteri DSM17938-IL22, which is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: M2024364.

2. The genetically engineered lactic acid bacteria according to claim 1, characterized in that The genetically engineered lactic acid bacteria are prepared by electrotransforming a recombinant plasmid pSET4s-himar1-cat-IL22 containing an IL22 gene sequence; the recombinant plasmid pSET4s-himar1-cat-IL22 uses pMG36e as a vector and can achieve intracellular expression of IL22 in a lactic acid bacteria expression system.

3. A method for preparing the genetically engineered lactic acid bacteria according to any one of claims 1 or 2, characterized in that: The method comprises: 1) Prepare competent cells of Lactobacillus reuteri DSM17938; 2) Electroporate IL22 into the DSM17938 competent cells obtained in 1); 3) Screen the successfully transformed strains in 2) in a medium containing chloramphenicol.

4. An active lactic acid bacteria preparation, characterized in that The active lactic acid bacteria preparation comprises the genetically engineered lactic acid bacteria DSM17938-IL22 or its fermentation broth as claimed in claim 1, wherein the number of viable bacteria is not less than 10 8 cfu / ml.

5. The active lactic acid bacteria preparation according to claim 4, characterized in that The active lactic acid bacteria preparation is in the form of capsules, tablets, liquid beverages or powders.

6. The active lactic acid bacteria preparation according to claim 4, characterized in that The active lactic acid bacteria preparation acts directly on the intestine through oral administration, and is used to improve intestinal microecology and regulate the immune response of the intestinal liver axis.

7. A method for preparing an active lactic acid bacteria preparation as claimed in claim 4, characterized in that: The method comprises: 1) Cultivating the genetically engineered lactic acid bacteria DSM17938-IL22 according to claim 1 in a culture medium; 2) Select the IL22-expressing monoclone obtained by culture in 1).

8. The preparation method according to claim 7, characterized in that: In step 1), the culture medium is a lactic acid bacteria culture medium (MRS) containing 5 ug / ml chloramphenicol and 1% xylose.

9. Use of the genetically engineered lactic acid bacteria according to claim 1 or 2 or the active lactic acid bacteria preparation according to any one of claims 4 to 6 in the preparation of a product for preventing and / or treating alcoholic liver injury.

10. The use according to claim 9, characterized in that: The applications include: 1) The product is used to prevent and / or treat alcoholic liver damage; 2) The product is used to prevent and / or treat alcohol-induced intestinal inflammation and intestinal damage; 3) As functional probiotics, added to food, food additives, health products, medicines or feed.

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