Ackermania muciniphila TMU as well as related biological materials and application of Ackermania muciniphila TMU

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to Ackermania muciniphila TMU as well as a related biological material and application thereof. The invention provides a strain of Ackermania muciniphila TMU, the preservation number of which is CGMCC (China General Microbiological Culture Collection Center) No.30820. According to the embodiment of the invention, DSS is used for treating a mouse to construct an enteritis model, Ackermania muciniphila TMU or a metabolite outer membrane protein Amuc0904 of Ackermania muciniphila TMU is used for treating the model mouse, and the result shows that Ackermania muciniphila TMU or the metabolite outer membrane protein Amuc0904 of Ackermania muciniphila TMU can be used for effectively promoting the quantity of intestinal goblet cells and mucoprotein secretion, improving the intestinal mucosa barrier, and improving the intestinal mucosa barrier. The mouse enteritis is relieved.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and specifically relates to a strain of Akkermansia muciniphila TMU and related biological materials and applications thereof. Background Art

[0002] Inflammatory bowel disease (IBD) is a chronic intestinal inflammatory disease, and patients usually experience symptoms such as diarrhea, abdominal pain, bloody stools, and weight loss. Commonly used anti-IBD drugs are mainly aminosalicylic acid derivatives, including sulfasalazine (SASP) and 5-aminosalicylic acid (also known as mesalazine, 5-ASA), but long-term use has certain side effects.

[0003] Regulating the microbiota by administering live bacteria (probiotics) may be an effective method for preventing or even curing IBD. Some probiotics may have a positive effect on relieving enteritis symptoms, which has attracted the attention of many researchers. Many probiotics exhibit multiple beneficial effects, such as inhibiting the growth of pathogens, increasing the level of beneficial metabolites in the intestine, and regulating the host immune system and intestinal flora. For example, CN116218735A discloses a single Bacteroides strain and its culture method and its application in the preparation of drugs for treating inflammatory bowel disease. The strain is isolated from the healthy human intestine and preserved in the China Center for Typical Microbiological Collection, with a preservation number of CCTCC NO: M20221994. The strain can avoid the integrity of the colorectal mucosa from being destroyed, and repair the existing intestinal mucosal damage, thereby improving the situation of loose stools and bloody stools, alleviating the problem of rapid weight loss caused by colorectal damage, and ultimately achieving the treatment of inflammatory bowel disease, but the efficacy of single Bacteroides strains for inflammatory bowel disease is limited. Therefore, it is particularly important in the art to develop new strains and improve the prevention or treatment effect of inflammatory bowel disease. Summary of the invention

[0004] The purpose of the present invention is to provide a strain of Akkermansia muciniphila TMU and related biological materials and applications thereof, which can effectively promote the number of intestinal goblet cells and mucin secretion, improve the intestinal mucosal barrier, and enhance the prevention or treatment effect of inflammatory bowel disease.

[0005] The invention provides a strain of Akkermansia muciniphila TMU, whose deposit number is CGMCC No.30820.

[0006] The present invention also provides an outer membrane protein Amuc_0904 produced by the metabolism of Akkermansia muciniphila TMU described in the above technical solution, and the amino acid sequence of the outer membrane protein Amuc_0904 is shown in SEQ ID NO.1.

[0007] The present invention also provides a gene encoding the outer membrane protein Amuc_0904 described in the above technical solution, and the nucleotide sequence of the gene is shown in SEQ ID NO.2.

[0008] The present invention also provides a bacterial agent, which includes the Akkermansia muciniphila TMU described in the above technical solution.

[0009] Preferably, the viable bacterial count of Akkermansia muciniphila TMU in the bacterial agent is 5×10 8 ~25×10 8 CFU / mL.

[0010] The present invention also provides a recombinant protein, which comprises the outer membrane protein OmpX of Escherichia coli and the outer membrane protein Amuc_0904 described in the above technical solution;

[0011] The outer membrane protein Amuc_0904 is inserted between 76aa and 77aa of the extracellular region of the Escherichia coli outer membrane protein OmpX.

[0012] The present invention also provides a gene encoding the recombinant protein described in the above technical solution.

[0013] The present invention also provides a reloaded expression vector, which comprises a basic vector and the gene described in the above technical solution inserted into the basic vector.

[0014] The present invention also provides an engineering bacterium, wherein the engineering bacterium comprises the overloaded expression vector described in the above technical solution.

[0015] The present invention also provides the use of Akkermansia muciniphila TMU and / or its metabolites described in the above technical solution or the outer membrane protein Amuc_0904 or the gene or bacterial agent or recombinant protein or overloaded expression vector or engineered bacteria described in the above technical solution in one or more of the following:

[0016] 1) Preparation of drugs for preventing and / or treating inflammatory bowel disease;

[0017] 2) preparing drugs that promote the increase of intestinal goblet cell number and / or mucin secretion;

[0018] 3) Prepare drugs for protecting the intestinal mucosal barrier.

[0019] Beneficial effects:

[0020] The present invention provides a strain of Akkermansia muciniphila TMU, with a deposit number of CGMCC No. 30820. In the present invention, mice were treated with dextran sulfate sodium salt (DSS) to construct an enteritis model, and the model mice were treated with Akkermansia muciniphila TMU or its metabolite outer membrane protein Amuc_0904. The results showed that Akkermansia muciniphila TMU or its metabolite outer membrane protein Amuc_0904 can effectively promote the number of intestinal goblet cells and mucin secretion, improve the intestinal mucosal barrier, and relieve enteritis in mice.

[0021] Biological deposit information

[0022] Akkermansia muciniphila TMU, with the taxonomic name Akkermansia muciniphila, was deposited on May 30, 2024 at the China General Microbiological Culture Collection, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101, with the deposit number CGMCCNo.30820. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0024] Figure 1 is the proportion of Akkermansia muciniphila in different stages of enteritis; A is the percentage of bacterial communities in different treatment groups at the genus level; B is the heat map of the top 50 bacteria in each group at the genus level; C is the linear discriminant analysis Effect Size (LEfSe) at the genus level of each treatment group;

[0025] Figure 2 is the phylogenetic tree of Akkermansia muciniphila TMU;

[0026] Figure 3 To verify the process and results of Akkermansia muciniphila TMU using the DSS-induced enteritis model; A is a flow chart; B is the percentage change in body weight of male mice in each treatment group; C is the disease activity index of male mice in each treatment group; D is the colon length of male mice in each treatment group; E is the HE staining and histological scoring results of male mice in each treatment group;

[0027] Figure 4 This is the experimental flow chart of the protective effect of Akkermansia muciniphila TMU on the intestinal barrier of male mice;

[0028] Figure 5The results are the protective effects of Akkermansia muciniphila TMU on the intestinal barrier of male mice;

[0029] Figure 6 The immunofluorescence results of the engineered bacteria EcN (BAD-wector) and the engineered bacteria EcN (BAD-904);

[0030] Figure 7 Schematic diagram of the engineered bacteria EcN (BAD-904) overexpressing the Akkermansia muciniphila TMU outer membrane protein Amuc_0904;

[0031] Figure 8 The results show the effect of engineered bacteria EcN (BAD-904) on goblet cell differentiation in male mice;

[0032] Fig. 9 The process and results of the preventive effect of engineered bacteria EcN (BAD-904) on DSS enteritis in mice; A is a flow chart; B is the percentage change in body weight of male mice in each treatment group; C is the disease activity index of male mice in each treatment group; D is the colon length of male mice in each treatment group; E is the HE staining and scoring results of male mice in each treatment group; F is the AB-PAS staining and scoring results; G is the immunohistochemical staining and scoring results;

[0033] Fig.10 The process and results of the therapeutic effect of engineered bacteria EcN (BAD-904) on DSS enteritis in mice; wherein, A is a flow chart; B is the percentage change in body weight of male mice in each treatment group; C is the disease activity index of male mice in each treatment group; D is the colon length of male mice in each treatment group; E is the HE staining and scoring results of male mice in each treatment group; F is the AB-PAS staining and scoring results; G is the immunohistochemical staining and scoring results. DETAILED DESCRIPTION

[0034] The present invention provides a strain of Akkermansia muciniphila TMU, with a deposit number of CGMCC No. 30820. The Akkermansia muciniphila TMU provided by the present invention belongs to the AmIII group, and has a distant relationship with A. muciniphila ATCC BAA-835 (AmI) which has been reported more. A enteritis model was constructed by treating mice with DSS, and the model mice were treated with Akkermansia muciniphila TMU or its metabolite outer membrane protein Amuc_0904. Akkermansia muciniphila TMU or its metabolite outer membrane protein Amuc_0904 can effectively promote the number of intestinal goblet cells and mucin secretion, improve the intestinal mucosal barrier, and relieve enteritis in mice.

[0035] The present invention also provides the outer membrane protein Amuc_0904 produced by the metabolism of Akkermansia muciniphila TMU described in the above technical solution, and the amino acid sequence of the outer membrane protein Amuc_0904 is shown in SEQ ID NO. 1. In the present invention, the nucleotide sequence of the gene encoding the outer membrane protein Amuc_0904 is preferably shown in SEQ ID NO.2.

[0036] The present invention also provides a bacterial agent, which includes the Akkermansia muciniphila TMU described in the above technical solution. In the present invention, the viable count of Akkermansia muciniphila TMU in the bacterial agent is preferably 5×10 8 ~25×10 8 CFU / mL, more preferably 6×10 8 ~20×10 8 CFU / mL, more preferably 10×10 8 ~18×10 8 CFU / mL, more preferably 15×10 8 CFU / mL.

[0037]

[0038]

[0039] The present invention also provides a reloaded expression vector, which comprises a basic vector and the gene described in the above technical solution inserted into the basic vector. In the present invention, the gene is preferably inserted behind the promoter of the basic vector.

[0040] The present invention also provides an engineered bacterium, wherein the engineered bacterium comprises the heavy-loaded expression vector described in the above technical solution. In the present invention, the basic strain of the engineered bacterium preferably comprises DH5α or Nissle 1917.

[0041] The present invention also provides the use of Akkermansia muciniphila TMU and / or its metabolites described in the above technical scheme or the outer membrane protein Amuc_0904 or the gene or bacterial agent or recombinant protein or overloaded expression vector or engineered bacteria described in the above technical scheme in one or more of the following: 1) preparing drugs for preventing and / or treating inflammatory bowel disease; 2) preparing drugs for promoting the increase of intestinal goblet cell number and / or mucin secretion; 3) preparing drugs for protecting intestinal mucosal barrier.

[0042] In the present invention, the inflammatory bowel disease preferably includes colitis, and more preferably DSS-induced enteritis. The effective viable count of Akkermansia muciniphila TMU in the drug of the present invention is preferably 5×10 8 CFU / mL; the effective viable count of the engineering in the drug is preferably 5×10 8 The Akkermansia muciniphila TMU and / or the outer membrane protein Amuc_0904 provided by the present invention can improve acute enteritis induced by DSS in mice, and promote the differentiation of colonic goblet cells in mice and increase mucin secretion.

[0043] In order to further illustrate the present invention, a strain of Akkermansia muciniphila TMU and related biological materials and applications provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0044] Example 1

[0045] Isolation of Akkermansia muciniphila TMU

[0046] (1) After C57BL6 / J mice were sterilized with AVMN, feces from healthy individuals were transplanted into C57BL6 / J male mice via fecal microbiota transplantation (FMT). The male mice were then treated with 1% DSS. The bacterial flora characteristics of the DSS-treated group during the acute inflammatory and recovery periods and the untreated group were analyzed. It was found that the proportion of Akkermansia muciniphila increased during the inflammatory recovery period ( Figure 1 ).

[0047] (2) Bacterial liquid was isolated in vitro from the feces of male mice in the recovery period of inflammation (22 days after colonization), and the liquid was aspirated with a sterile pipette tip for streaking. From the streaked culture dish, a single colony was picked up and dissolved with PBS, and then the liquid was injected into an anaerobic culture bottle containing the corresponding liquid culture medium, and then anaerobically cultured in a bacterial incubator at 37°C for 48 to 72 hours to extract bacterial genomic DNA. The extracted bacterial genomic DNA was used as a template to amplify the 16S rDNA fragment of the bacteria by PCR. The PCR product was further purified and sequenced at Shanghai Shenggong Biotechnology Co., Ltd. After sequencing, the 16S rDNA sequence was compared with the sequence in the EzBioCloud16S rDNA database (www.ezbiocloud.net), and a phylogenetic tree was constructed, and a strain of Akkermansia muciniphila and a strain of Bacteroides uniformis were found.

[0048] Among them, Akkermansia muciniphila was named Akkermansia muciniphila strain TMU, referred to as TMU, and the phylogenetic tree is as follows Figure 2

[0049]

[0050] Example 2

[0051] Cultivation of Akkermansia muciniphila TMU

[0052] 10 μL of Akkermansia muciniphila TMU bacterial solution stored in 30 v / v% glycerol was drawn and streaked on a BHI blood plate to separate a single colony. The culture dish with the streaked bacteria was placed in a 2.5L round-bottom vertical anaerobic culture bag (Qingdao Haibo Biological, HBYY007), and a 2.5L anaerobic gas production bag (Qingdao Haibo Biological, HBYY001) was added to simulate the anaerobic growth environment of the intestine. The bag was sealed and placed in a 37°C bacterial incubator for 24 to 48 hours.

[0053] After obvious colonies grow, take out the culture dish, pick up the monoclonal colony of Akkermansia muciniphila TMU and dissolve it in 100 μL sterile PBS, and inject the bacterial solution directly into the vial with a 1 mL sterile syringe, wherein the vial contains 10 mL of sterilized GAM medium (Qingdao Haibo Biological, HB8518) with 0.1% L-cysteine, 0.1% resazurin, 2% vitamin K1 and 0.5% hemin chloride. Place the vial in a bacterial incubator at 37°C and culture it until the logarithmic growth phase, then take it out, detect the turbidity value of the bacterial solution, and calculate the CFU concentration of the bacterial solution; pour the bacterial solution into a 50 mL centrifuge tube, centrifuge at 8000 rpm for 5 minutes, discard the supernatant, wash the bacterial precipitate with PBS, and centrifuge it again. After discarding the supernatant, the bacterial precipitate is resuspended with PBS containing 0.1 wt.% L-cysteine ​​(i.e., anaerobic PBS) to make the bacterial solution concentration reach 25×10 8 CFU / mL.

[0054] Example 3

[0055] according to Figure 3 The process in A uses the DSS-induced enteritis model to clarify the role of Akkermansia muciniphila TMU strain. The specific steps are as follows:

[0056] (1) After 3 days of acclimatization, 6- to 8-week-old C57BL / 6J male mice were randomly divided into a blank control group (con), an experimental group (A. muciniphila), and a control group (B. uniformis), with 9 mice in each group.

[0057] (2) Each male mouse in each treatment group was gavaged with 500 μL AVMN once a day to eliminate intestinal flora;

[0058] Among them, AVMN is a mixture of ampicillin, neomycin, metronidazole and vancomycin; the concentration of ampicillin in the mixture is 1 g / L, purchased from Biochemical Engineering, Shanghai, China, with the catalog number A610028-0025; the concentration of neomycin in the mixture is 1 g / L, purchased from Biochemical Engineering, Shanghai, China, with the catalog number A610366-0025; the concentration of metronidazole in the mixture is 1 g / L, purchased from Biochemical Engineering, Shanghai, China, with the catalog number A600633-0025; the concentration of vancomycin in the mixture is 0.5 g / L, purchased from Biochemical Engineering, Shanghai, China, with the catalog number A600983-0001.

[0059] (3) Five days after intragastric administration of AVMN in step (2), each group was treated as follows:

[0060] Experimental group: 25×10 8 CFU / mL of Akkermansia muciniphila TMU bacterial solution, i.e. 5×10 8 CFU Akkermansia muciniphila TMU / mouse;

[0061] Blank control group: mice were gavaged daily with 200 μL of PBS solution containing 0.1 wt.% L-cysteine.

[0062] Control group: 200 μL 25×10 8 CFU / mL of the Bacteroides uniformis bacterial solution isolated in Example 1, i.e., 5×10 8 CFUB.uniformis / rat.

[0063] (4) After the male mice in each treatment group were treated for 5 days in step (3), DSS was added to the drinking water of the male mice to a final concentration of 2.5 g / 100 mL to induce acute colitis, and LPS (lipopolysaccharide) was gavaged at a dose of 8 mg / kg mouse / d to aggravate the enteritis for 6 days. While DSS and LPS were inducing acute colitis, the experimental group and the control group were gavaged with the corresponding strains in the manner of step (3), and the blank control group was gavaged with sterile water.

[0064] (5) After the male mice in each treatment group were given DSS and LPS, the changes in body weight and feces of the three groups of male mice were recorded every day, and the percentage of body weight change and disease activity index were calculated. Specifically:

[0065] Calculate the weight change of male mice: Starting from the day of DSS administration (day 0), observe and record the weight of male mice every day, and calculate the ratio of the difference between the weight of male mice on each day after DSS administration and the weight on day 0 to the weight on day 0. The results are as follows: Figure 3 As shown in B;

[0066] Calculate the disease activity index (DAI) score: Score the stool characteristics and stool blood every day and calculate the DAI value. DAI is the sum of the stool consistency score, stool bleeding score and weight loss score. Figure 3 As shown in C, the stool consistency score is as follows: 0, normal; 1, soft stool; 2, diarrhea; 3, watery stool. The stool bleeding score is as follows: 0, normal; 1, coffee color; 2, reddish color; 3, blood in the stool. The weight loss score is as follows: 0 (<2%), 1 (2-5%), 2 (5-10%), 3 (10-15%), 4 (≥15%).

[0067] (6) After 6 days of DSS treatment, the male mice in each treatment group were euthanized and the complete colon was removed to measure the length. Figure 3 As shown in D; about 1 cm long colon tissue was cut near the anus, fixed in 4wt.% paraformaldehyde solution for at least 8 hours, and tissue sections, HE staining and histological scoring were performed, as shown in FIG. Figure 3 As shown in E; wherein, the steps of paraffin tissue embedding and sectioning are as follows:

[0068] a. The colon tissue was fixed in 4wt.% paraformaldehyde solution for more than 8h, and then the tissue was removed;

[0069] b. The tissue was placed in the following liquids for dehydration and wax immersion: 70v / v% ethanol for 60min→85v / v% ethanol for 40min→95v / v% ethanol I for 60min→95v / v% ethanol II for 40min→anhydrous ethanol I for 60min→anhydrous ethanol II for 40min→xylene I for 50min→xylene II for 50min→paraffin I for 30min→paraffin II for 60min, and then the tissue was embedded;

[0070] c. Use a paraffin tissue slicer to slice the slices. The slices should be 3 μm thick. After spreading, use tissue anti-shedding slides to mount the slices. Bake the slices at 70°C for more than 20 minutes and they can be stored at room temperature for a long time.

[0071] HE staining:

[0072] Xylene I 10min—Xylene II 8min—Anhydrous ethanol I 2min—Anhydrous ethanol II 1min—95% v / v ethanol 1min—85% ethanol 1min—70% ethanol 1min—Tap water 30s—Hematoxylin 5min—Tap water 30s—0.5% v / v ethanol hydrochloride 20s—Tap water 30s—0.5% v / v ammonia 30s—Tap water 30s—Eosin 30s—Tap water 30s—70% ethanol 5s—85% ethanol 5s—95% ethanol 1min—Anhydrous ethanol I 2min—Anhydrous ethanol II 1min—Xylene I 2min—Xylene II 1min

[0073] Histological scoring: The pathological damage of colon tissue was observed using an ordinary upright microscope, and image information was collected. The histological pathological scoring was performed according to PMID: 23399699. The histological scoring criteria are as follows.

[0074] Table 1 Histological scoring criteria

[0075]

[0076] according to Figure 3 It can be seen that compared with the control group and the blank control group, the experimental group (TMU-treated) male mice showed significant inhibition of enteritis in terms of body weight, DAI score, colon length and histopathological score. These results indicate that Akkermansia muciniphila TMU has a preventive effect on DSS-induced acute colitis in male mice under the condition of clearing intestinal bacteria.

[0077] Example 4

[0078] according to Figure 4 The protective effect of Akkermansia muciniphila TMU on the intestinal barrier of male mice is as follows:

[0079] (1) The male mice in each treatment group were divided into groups according to the method of Example 4, and AVMN was administered intragastrically to eliminate the intestinal flora of the male mice in each treatment group;

[0080] (2) Five days after intragastric administration of AVMN, each group was treated as follows:

[0081] Experimental group: 25×10 8 CFU / mL of Akkermansia muciniphila TMU bacterial solution, i.e. 5×10 8 CFU TMU / mouse;

[0082] Blank control group: mice were gavaged daily with 200 μL of anaerobic PBS containing 0.1 wt.% L-cysteine.

[0083] Control group: 200 μL 25×10 8CFU / mL of the Bacteroides uniformis bacterial solution isolated in Example 1, i.e., 5×10 8 CFUB.uniformis / rat.

[0084] (3) Step (2) After 5 days of treatment, the male mice in each treatment group were euthanized, and the intact colon was removed and the colon tissue of about 1 cm long near the anus was cut and fixed in formalin for at least 8 hours for tissue sectioning and staining.

[0085] (4) Paraffin tissue embedding and sectioning

[0086] a. The colon tissue was fixed in 4wt.% paraformaldehyde solution for more than 8h, and then the tissue was removed;

[0087] b. Place the tissue in the following liquids for dehydration and wax immersion in sequence: 70v / v% ethanol for 60 min → 85v / v% ethanol for 40 min → 95v / v% ethanol I for 60 min → 95v / v% ethanol II for 40 min → anhydrous ethanol I for 60 min → anhydrous ethanol II for 40 min → xylene I for 50 min → xylene II for 50 min → paraffin I for 30 min → paraffin II for 60 min, and embed the tissue.

[0088] c. Use a paraffin tissue slicer to slice the slices. The slices should be 3 μm thick. After spreading, use tissue anti-shedding slides to mount the slices. Bake the slices at 70°C for more than 20 minutes and they can be stored at room temperature for a long time.

[0089] (5) AB-PAS staining

[0090] For goblet cell detection, AB-PAS staining was performed using an AB-PAS staining kit (purchased from Beijing Solebow Technology Co., Ltd.) according to the manufacturer's instructions.

[0091] (6) Immunohistochemical staining

[0092] a. Dewax the paraffin sections in xylene and gradient ethanol: xylene I for 10 min → xylene II for 8 min → anhydrous ethanol I for 2 min → anhydrous ethanol II for 1 min → 95 v / v% ethanol for 1 min → 85 v / v% ethanol for 1 min → 70 v / v% ethanol for 1 min → tap water for 1 min.

[0093] b. Antigen retrieval: Use citrate buffer (pH 6.0) or EDTA antigen retrieval solution (pH 9.0) for high pressure heat retrieval for 3 minutes, return to room temperature naturally, and wash with PBS three times, 3 minutes each time.

[0094] c. Removal of endogenous peroxidase: Incubate with 0.3% v / v H2O2 for 30 min to block endogenous peroxidase activity, and wash with PBS 3 times, 3 min each time.

[0095] d. Blocking: Incubate with 5% v / v goat serum in PBS for 1 hour.

[0096] e. Primary antibody incubation: Incubate with primary antibodies against MUC2, Ki67, Occludin, and Cleaved caspase-3 at 4°C overnight, and wash with PBS five times, 5 minutes each time.

[0097] f. Secondary antibody incubation: stain with horseradish peroxidase (HRP)-labeled secondary antibody for 1 hour, and wash with PBS 5 times, 5 minutes each time.

[0098] g. Use diaminobenzidine (DAB) color development substrate to develop color to an appropriate degree (within 5 minutes), and terminate the color development with tap water.

[0099] h. Stain with hematoxylin for 1 min, rinse with tap water for 30 s, differentiate with 0.5% v / v hydrochloric acid ethanol differentiation solution for 20 s, rinse with tap water for 30 s, debluing with 0.5% v / v ammonia water for 20 s, rinse with tap water for 30 s. After taking out the slides, put them into: 70v / v% ethanol for 2 s → 85v / v% ethanol for 4 s → 95v / v% ethanol for 20 s → anhydrous ethanol I for 1 min → anhydrous ethanol II for 1 min → xylene I for 2 min → xylene II for 2 min, then seal the slides with neutral gum, dry and store.

[0100] (7) Observe the staining of the sections under an ordinary upright microscope and collect image information. Select 5 different high-power fields of view for each tissue to take images. Use Image Pro Plus software to count the number of positive cells and analyze the staining intensity. The results are as follows: Figure 5 shown.

[0101] according to Figure 5 It can be seen that Akkermansia muciniphila TMU colonization can significantly increase the expression of tight junction protein Occludin, the number of Ki67+ proliferating cells, the number of goblet cells and the expression of mucin secreted by them, and the number of cleaved caspase-3+ apoptotic cells in normal male mice, indicating that the intestinal barrier function of male mice is enhanced. The control strain B. uniformis has no such effect, and the number of cleaved caspase-3+ apoptotic cells is significantly increased compared with the control group.

[0102] Example 5

[0103] Construction of engineered bacteria EcN (BAD-904) overexpressing the outer membrane protein Amuc_0904 of Akkermansia muciniphila TMU

[0104] (1) The outer membrane protein Amuc_0904 of Akkermansia muciniphila TMU was added between the 76th and 77th aa of the extracellular region of the outer membrane protein OmpX of Escherichia coli, and a FLAG tag was added to the N-terminus of the Amuc_0904 protein to obtain the pTrc99a-Ompx-Amuc_0904 plasmid; wherein the amino acid sequence of the outer membrane protein Amuc_0904 of Akkermansia muciniphila TMU is as shown in SEQ ID NO.As shown in Figure 1, specifically: MTKIAKSTFTTGAGTPGQFYSLPALAENGYPRLNRLPVSIRIVLESLLRNCDGLKV TEKDVDNLASWNAKNPGSYEIPFTVARIVLQDLTGVPLLVDLAAMRSAVAGLGKDASVIEPLVPVDLVVDHSVQVDWAGRTDALEKNLDIEFQRNAERYEFLKWGQQAFSTFSVVPPSVGIVHQVNLEYLAQGVMEKDGVYFPDTLVGTDSHTTMINGIGVVGWGVGGIEAEAGMLGQPVTFLVPEVVGVHMTGELREGVTATDLALHVTQMLRSHGVVGKFVEFFGDGAAALPLADRATVANMAPEYGATMGFFPMDEHCSEYLRLTGRSEEAITTYENYFKAQDLWGMPRNGELDYTDQLELDLSAIEPAVSGPKRPQDHIPLASIRDSFRKLVSMPAAEGGYGKKESPSVKVAMHSPAGVPVPVEGFSQEAELKDGSILIAAITSCTNTSNPGLMLAAGLLAKKAVALGLQVPPHVKTSIAPGSRIASDYFAANQLQDSLDALGFETVGYGCTTCIGNSGPLNPELEQAVRDNDIVAASVLSGNRNFEARIHASIKTNFLMSPPLVVAFALAGHVDIDFQTEPLGTDRSGNPVFLKDIWPSAPEIAEAVAKSRNPEAYRELYTITPEKNPRWAAVYAPEGSVFQWNENSTYIQNPPFFCGFNSQPRTLADIRNARPLGIFGDSVTTDHISPAGAIRETGPAGLYLQGMGIDRKDFNSFGSRRGNDRVMTRGTFANVRIKNLMVDGTEGGYTLYFGDRSIPAPDKKIAAAAGAPAFIYDAAMAYAQDGVPLIVIGGEDYGMGSSRDWAAKGTNLLGVKAVITKSFERIHRSNLIGMGVLPLNFADKADYDRIAPLKDATFSILGLEGGISPRQQVTLRVQPAGAEAFDVPVIVRIDTPIEKEYYLAGGILQYVLTQILK*;.

[0105]

[0106] (2) Using the pTrc99a-Ompx-Amuc_0904 plasmid obtained in step (1) as a template, PCR amplification was performed using upstream primer F (5'-ATAAA AAATGGAATTCATGAAAAAAATTGCATGTCTTTCAG-3', SEQ ID NO.3) and downstream primer R (5'-ATGCTTGTTATCGTAAAGAGTTTGTAGAAACGCAAAAAGGC-3', SEQ ID NO.4), and the amplified product was recovered by gel amplification; wherein,

[0107] The PCR amplification system was as follows: template 500 ng, upstream primer 0.5 μL, downstream primer 0.5 μL, 5× Buffer 2 μL, 10 mM dNTP 0.2 μL, Q5 enzyme (NEB, M04915) 0.1 μL, Enhancer 2 μL, and ddH2O supplemented to 10 μL;

[0108] The PCR amplification program was pre-denaturation at 98°C for 1 min 30 s; denaturation at 98°C for 10 s, annealing at 60°C for 30 s, extension at 72°C for 2 min, 34 cycles; and final extension at 72°C for 2 min.

[0109] (3) PCR amplification of vector pTOPO-FLC-a (KeMiao Biotechnology) was performed using upstream primer F (5'-GTCGATGTCGCGTCTGCC-3', SEQ ID NO.5) and downstream primer R (5'-TACGATAACAAGCATCAGGACTGAAGC-3', SEQ ID NO.6), and the amplified product was PCR amplified; wherein,

[0110] The PCR amplification system was as follows: template 500 ng, upstream primer 0.5 μL, downstream primer 0.5 μL, 5× Buffer 2 μL, 10 mM dNTP 0.2 μL, Q5 enzyme (NEB, M04915) 0.1 μL, Enhancer 2 μL, and ddH2O supplemented to 10 μL;

[0111] The PCR amplification program was as follows: pre-denaturation at 98°C for 1 min 30 s; denaturation at 98°C for 10 s, annealing at 60°C for 30 s, extension at 72°C for 2 min, 34 cycles; final extension at 72°C for 2 min.

[0112] (4) PCR amplification of vector pKD46 was performed using upstream primer F (5'-AGACGCGACATCGACCACATTTCCCCGAAAAGTGCCAC-3', SEQ ID NO.10) and downstream primer R (5'-AATTCCATTTTTTATAACCTCCTTAGAGCTCGAAT-3', SEQ ID NO.11) to obtain the araBAD promoter and the amplified product; wherein,

[0113] The PCR amplification system was as follows: template 500 ng, upstream primer 0.5 μL, downstream primer 0.5 μL, 5× Buffer 2 μL, 10 mM dNTP 0.2 μL, Q5 enzyme (NEB, M04915) 0.1 μL, Enhancer 2 μL, and ddH2O supplemented to 10 μL;

[0114] The PCR amplification program was as follows: pre-denaturation at 98°C for 1 min 30 s; denaturation at 98°C for 10 s, annealing at 60°C for 30 s, extension at 72°C for 30 s, 34 cycles; and final extension at 72°C for 2 min.

[0115] (5) using seamless cloning technology, the linearized pTOPO-FLC-a in step (3), the araBAD promoter recovered from the gel in step (4), and the amplified product Ompx-Amuc_0904 recovered from the gel in step (2) were fused to obtain the recombinant plasmid pTOPO-araBAD-OmpX-904 (BAD-904);

[0116] (6) The successfully constructed recombinant plasmid was transformed into DH5α bacteria; the successfully constructed recombinant plasmid was transformed into Escherichia coli Nissle 1917, and the positive strains were screened based on the immunofluorescence method, as the engineered bacteria EcN (BAD-904) overexpressing the outer membrane protein Amuc_0904 of Akkermansia muciniphila TMU. The immunofluorescence detection results of the positive strains are shown as follows: Figure 6 As shown, the structural diagram of the engineered bacteria EcN (BAD-904) is as follows Figure 7 shown.

[0117] Comparative Example 1

[0118] pTOPO-araBAD was transformed into Escherichia coli Nissle 1917 to obtain the empty engineered bacteria EcN (BAD-vector), as shown in the schematic diagram Figure 6 shown.

[0119] Construct the engineered bacteria EcN (BAD-904) according to Example 5

[0120] Example 6

[0121] Effects of engineered bacteria EcN (BAD-904) on goblet cell differentiation in male mice

[0122] After 3 days of adaptive feeding, 6-8 week old C57BL / 6J male mice were randomly divided into a blank control group (Control), an experimental group (EcN (BAD-904)) and a blank plasmid control group (EcN (BAD-vector)), with 6 mice in each group. Male mice in each treatment group were treated by gavage, specifically:

[0123] Experimental group: Each male mouse was gavaged with 200 μL of 25×10 8 CFU / mL of Akkermansia muciniphila TMU bacterial solution (solvent is PBS) of the engineered bacteria EcN (BAD-904) of Example 5, i.e., 5×10 8 CFU engineered bacteria EcN (BAD-904) / male mouse;

[0124] Blank control group: 200 μL PBS solution was gavaged every day.

[0125] Blank plasmid control group: Each male mouse was gavaged with 200 μL 25×10 8 CFU / mL Comparative Example 1 The bacterial solution of the engineered bacteria EcN (BAD-vector) (the solvent is PBS), that is, 5×10 8 CFU engineered bacteria EcN (BAD-vector) / male mouse;

[0126] The frequency of gavage in each treatment group was once every two days; 2wt.% arabinose was added to the drinking water of C57BL / 6J male mice in each treatment group to induce the expression of Amuc_0904, and the drinking water was changed every 2-3 days. After gavage for 5 days, colon tissues were collected for AB-PAS, immunohistochemical staining and immunofluorescence staining to analyze goblet cell differentiation-related indicators. The results are as follows Figure 8 Wherein, the methods of AB-PAS and immunohistochemical staining are consistent with those in Example 4;

[0127] The immunofluorescence staining method is as follows:

[0128] (1) Take the frozen tissue sections out of the -80°C freezer and place them at room temperature for 30 min to 1 h.

[0129] (2) Precool acetone on ice for 30 min. Place tissue sections in precooled acetone and fix at 4°C for 10 min.

[0130] (3) Wash with PBS three times, 3 min each time.

[0131] (4) Precool 3% H2O2 (methanol) on ice for 30 min and place the tissue sections in it for 10 min to remove endogenous peroxidase.

[0132] (5) Wash with PBS three times, 3 min each time.

[0133] (6) Blocking: Add 5% goat serum to the sections and block for 1 h at room temperature.

[0134] (7) Primary antibody: Use 5% goat serum with MUC2 antibody (1:500, Proteintech) and incubate overnight at 4°C.

[0135] (8) Remove the slices from the 4°C refrigerator and allow to warm to room temperature for 1 hour.

[0136] (9) Wash with PBS five times, 5 min each time.

[0137] (10) Secondary antibody: Use fluorescently labeled 488 goat anti-rabbit secondary antibody, prepared with 5% goat serum, incubate at room temperature for 1 h in the dark.

[0138] (11) Wash with PBS five times, 5 min each time.

[0139] (12) Nuclear staining: DAPI (1:1000, in PBS) staining for 10 min.

[0140] (13) Wash with PBS three times, 5 min each time.

[0141] (14) Seal the sections with quench-resistant sealing media and store at 4°C for two weeks.

[0142] (15) Use confocal microscopy to observe and take pictures.

[0143] according to Figure 8 It can be seen that the number of colon goblet cells in mice that received EcN (BAD-904) gavage for one week increased significantly, and the secretion of intestinal MUC2 increased compared with the blank control group and the blank plasmid control group.

[0144] Example 7

[0145] according to Fig. 9 Process A in the middle verifies the preventive effect of engineered bacteria EcN (BAD-904) on DSS enteritis in mice. The specific steps are as follows:

[0146] (1) After 3 days of adaptive feeding, male 6- to 8-week-old C57BL / 6J mice were randomly divided into a blank control group (Control), an experimental group (EcN (BAD-904)), and a blank plasmid control group (EcN (BAD-vector)), with 8 mice in each group; each male mouse in each treatment group was gavaged with 500 μL AVMN once a day to eliminate intestinal flora;

[0147] (2) Five days after intragastric administration of AVMN, each group was treated as follows:

[0148] Experimental group: Each male mouse was gavaged with 200 μL of 25×10 8 CFU / mL of Akkermansia muciniphila TMU bacterial solution (solvent is PBS) of the engineered bacteria EcN (BAD-904) of Example 5, i.e., 5×10 8 CFU engineered bacteria EcN (BAD-904) / male mouse;

[0149] Blank control group: 200 μL PBS solution was gavaged every day.

[0150] Blank plasmid control group: Each male mouse was gavaged with 200 μL 25×10 8 CFU / mL Comparative Example 1 The bacterial solution of the engineered bacteria EcN (BAD-vector) (the solvent is PBS), that is, 5×10 8 CFU engineered bacteria EcN (BAD-vector) / male mouse;

[0151] The frequency of gavage for each treatment group was once every two days. Five days after the first gavage, DSS was added to the drinking water of male mice in each treatment group to a final concentration of 2.5 g / 100 mL to induce acute colitis, and LPS (lipopolysaccharide) was gavaged at a dose of 8 mg / kg mouse / d to aggravate enteritis for 6 days. While DSS and LPS induced acute colitis, the experimental group and the blank plasmid control group were gavaged with the corresponding strains, and the blank control group was gavaged with PBS; 2 wt.% arabinose was added to the drinking water of C57BL / 6J male mice in each treatment group to induce Amuc_0904 expression, and the drinking water was replaced every 2 to 3 days.

[0152] (3) During the intragastric administration, the weight and feces of the male mice were recorded daily according to the method of Example 3, and the weight change percentage and disease activity index were calculated. The results are as follows: Fig. 9 As shown in B and C.

[0153] (4) After 6 days of DSS treatment, the male mice in each treatment group were euthanized and the complete colon was removed to measure the length. Fig. 9 As shown in D; tissue sectioning, HE staining and histological scoring were performed in the manner of Example 3, as shown in Fig. 9 As shown in E. AB-PAS and immunohistochemical staining were performed in the manner of Example 4. Fig. 9 As shown in F and G.

[0154] according to Fig. 9 It can be seen that oral administration of EcN (BAD-904) effectively inhibited colitis in mice. The colon tissue morphology of the EcN (BAD-904) treatment group was more complete, with less inflammatory cell infiltration, while the number of goblet cells increased significantly and MUC2 expression increased. These results indicate that EcN (BAD-904) can effectively prevent the occurrence of enteritis.

[0155] Example 8

[0156] according to Fig.10 Process A in the middle verifies the therapeutic effect of engineered bacteria EcN (BAD-904) on DSS enteritis in mice. The specific steps are as follows:

[0157] (1) After 3 days of adaptive feeding, male 6- to 8-week-old C57BL / 6J mice were randomly divided into a blank control group (Control), an experimental group (EcN (BAD-904)), and a blank plasmid control group (EcN (BAD-vector)), with 8 mice in each group; DSS was added to the drinking water of male mice in each treatment group to a final concentration of 2.5 g / 100 mL to induce acute colitis;

[0158] (2) After 2 days of DSS pretreatment, mice in each treatment group were treated as follows:

[0159] Experimental group: Each male mouse was gavaged with 200 μL of 25×10 8 CFU / mL of Akkermansia muciniphila TMU bacterial solution (solvent is PBS) of the engineered bacteria EcN (BAD-904) of Example 5, i.e., 5×10 8 CFU engineered bacteria EcN (BAD-904) / male mouse;

[0160] Blank control group: 200 μL PBS solution was gavaged every day.

[0161] Blank plasmid control group: Each male mouse was gavaged with 200 μL 25×10 8 CFU / mL Comparative Example 1 The bacterial solution of the engineered bacteria EcN (BAD-vector) (the solvent is PBS), that is, 5×10 8 CFU engineered bacteria EcN (BAD-vector) / male mouse;

[0162] The frequency of gavage in each treatment group was once every two days, and 2 wt.% arabinose was added to the drinking water of C57BL / 6J male mice in each treatment group to induce the expression of Amuc_0904, and the drinking water was replaced every 2 to 3 days.

[0163] (3) During the intragastric administration, the weight and feces of the male mice were recorded daily according to the method of Example 3, and the weight change percentage and disease activity index were calculated. The results are as follows: Fig.10 As shown in B and C.

[0164] (4) After 7 days of DSS treatment, the male mice in each treatment group were euthanized and the complete colon was removed to measure the length. Fig.10 As shown in D; tissue sectioning, HE staining and histological scoring were performed in the manner of Example 3, as shown in Fig.10As shown in E. AB-PAS and immunohistochemical staining were performed in the manner of Example 4. Fig.10 As shown in F and G.

[0165] according to Fig.10 It can be seen that the mice in the EcN (BAD-904) treatment group had less weight loss and DAI score, the longest colon length, less damage to colon tissue, more complete morphological structure, and significantly increased goblet cell number and MUC2 production. These results indicate that EcN (BAD-904) has a certain therapeutic effect on enteritis.

[0166] Based on the above content, it can be seen that the Akkermansia muciniphila TMU provided by the present invention, or its metabolite outer membrane protein Amuc_0904, can effectively promote the number of intestinal goblet cells and mucin secretion, improve the intestinal mucosal barrier, maintain intestinal homeostasis, and relieve enteritis in mice, providing a promising method for the clinical treatment of IBD.

[0167] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of Akkermansia muciniphila TMU, with a deposit number of CGMCC No.30820.

2. The outer membrane protein Amuc_0904 produced by the metabolism of Akkermansia muciniphila TMU according to claim 1, It is characterized in that The amino acid sequence of the outer membrane protein Amuc_0904 is shown in SEQ ID NO.

1.

3. A gene encoding the outer membrane protein Amuc_0904 according to claim 2, It is characterized in that The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

4. A bacterial agent, It is characterized in that The bacterial agent includes the Akkermansia muciniphila TMU according to claim 1.

5. The bacterial agent according to claim 4, It is characterized in that The viable bacterial count of Akkermansia muciniphila TMU in the bacterial agent is 5×10 8 ~25×10 8 CFU / mL.

6. A recombinant protein, It is characterized in that The recombinant protein comprises the Escherichia coli outer membrane protein OmpX and the outer membrane protein Amuc_0904 according to claim 2; The outer membrane protein Amuc_0904 is inserted between 76aa and 77aa of the extracellular region of the Escherichia coli outer membrane protein OmpX.

7. A gene encoding the recombinant protein according to claim 6.

8. A reloaded expression vector, It is characterized in that The reloaded expression vector comprises a basic vector and the gene according to claim 7 inserted into the basic vector.

9. An engineered bacterium, It is characterized in that The engineered bacteria comprises the heavy-loaded expression vector described in claim 8.

10. Use of Akkermansia muciniphila TMU and / or its metabolites according to claim 1, or the outer membrane protein Amuc_0904 according to claim 2, or the gene according to claim 3 or 7, or the bacterial agent according to claim 4 or 5, or the recombinant protein according to claim 6, or the overloaded expression vector according to claim 8, or the engineered bacteria according to claim 9 in one or more of the following: 1) Preparation of drugs for preventing and / or treating inflammatory bowel disease; 2) preparing drugs that promote the increase of intestinal goblet cell number and / or mucin secretion; 3) Prepare drugs for protecting the intestinal mucosal barrier.

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