CN114369146B

CN114369146B - Acremonium Amuc-2172 protein and preparation method and application thereof

Info

Publication number: CN114369146B
Application number: CN202210041825.2A
Authority: CN
Inventors: 张明明; 蒋怡
Original assignee: Renji Hospital Shanghai Jiaotong University School of Medicine
Current assignee: Renji Hospital Shanghai Jiaotong University School of Medicine
Priority date: 2022-01-14
Filing date: 2022-01-14
Publication date: 2023-05-23
Anticipated expiration: 2042-01-14
Also published as: CN114369146A

Abstract

The invention provides an Amuc_2172 protein of Acremonium, which contains an amino acid sequence shown as SEQ ID NO.1 or SEQ ID NO. 5. The invention also provides a preparation method of the Amuc_2172 protein of the Acremonium. The invention also provides a pharmaceutical composition comprising a therapeutically effective amount of an Amuc_2172 protein of Acremonium. The invention also provides an application of the Amuc_2172 protein of the Acremonium in preparing a medicament for inhibiting Th17 cell differentiation or intestinal inflammation or intestinal cancer. The invention discovers that an active ingredient Amuc_2172 protein of the Ackermansia can inhibit Th17 cells, treat and relieve intestinal inflammation and intestinal tumor occurrence and development, and can be used for treating various autoimmune diseases related to Th17, including but not limited to Crohn disease, ulcerative colitis, multiple sclerosis, rheumatoid arthritis and systemic lupus erythematosus.

Description

Acremonium Amuc-2172 protein and preparation method and application thereof

Technical Field

The invention belongs to the field of biological medicine, and relates to an Amuc_2172 protein of Acremonium, in particular to an Amuc_2172 protein of Acremonium, a preparation method and application thereof.

Background

Intestinal diseases are worldwide health problems, and unhealthy diet and mental stress cause global intestinal inflammation and gradual deterioration of epidemiology of tumors, which adversely affect physical, psychological and domestic and national economic production of patients. Ulcerative colitis (Ulcerative Colitis, UC) and Crohn's Disease (CD) belong to inflammatory bowel Disease (Inflammatory Bowel Disease, IBD), and UC mainly involves colorectal, a chronic Disease characterized by repeated bloody purulent stool, which is currently incurable, and once diagnosis is confirmed for life, brings great physical and psychological burden to patients and their families. Repeated inflammation of the UC intestinal tract promotes colorectal cancer (UC related Colorectal Cancer, UC-CRC), the cancer transformation rate of patients with the UC disease course of more than 10 years is 2%, the cancer transformation rate of patients with the disease course of more than 20 years is 8%, and the cancer transformation rate of patients with the disease course of more than 30 years is 18%; although UC-CRC is only 1-2% of all colorectal cancers (Colorectal Cancer, CRC), it is 15% of the deaths in UC patients. Colorectal cancer is a common malignancy of the digestive tract, 180 Mo Xin cases are all around the world in 2018 only; in recent years, the incidence rate and the death rate of colorectal cancer in China are continuously increased due to the change of dietary structures of the Chinese people, and the colorectal cancer is a serious health problem endangering the national life. The etiology of intestinal inflammation and cancer is not clear, but it is currently believed that intestinal flora disorders promote intestinal inflammation and inflammatory cancer transformation. Intestinal flora disorders significantly promote increased differentiation of Th17 cells (T helper 17 cells), and previous studies have demonstrated that Th17 cells and associated pathways promote a variety of inflammatory and tumor progression. Th17 cells are a type of proinflammatory cell subgroup which is characterized by expressing retinoic acid related orphan receptor gamma t (retinoic acid receptor-related orphan receptor gamma t, ROR-gamma t), are highly expressed in intestinal mucosa and peripheral blood of UC patients, and can play a role in promoting intestinal inflammation and intestinal tumor by secreting Interleukin 17 (IL-17 ). In addition to the intestinal diseases Crohn's disease and ulcerative colitis, th17 cell-related autoimmune inflammatory diseases include multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, and the like. In addition to intestinal tumors, th17 cell-related tumors also include lung cancer, gastric cancer, liver cancer, ovarian cancer and the like.

The human intestinal tract contains more than 1000 different kinds of bacteria, and the total number is about 10 ¹⁴ And each. Intestinal dysregulation is associated with many diseases including intestinal inflammation and tumors, and supplementation of the intestinal tract with absent probiotics or probiotic components can significantly inhibit Th17 cell numbers while significantly alleviating intestinal lesions: previous studies found that clostridium intestinal tract praecox (Faecalibacterium prausnitzii, f.prausiitzi), bacteroides fragilis (Bacteroides fragilis, b.fragilis), bifidobacterium longum (Bifidobacterium longum, b.longum) were significantly reduced, and supplementation of these bacteria and bacterial products reduced Th17 cell levels while alleviating intestinal inflammation in UC model mice. Probiotics of Acremodelling Alkermana muciniphila in patients with enteritis and intestinal cancer (Akkermansia muciniphila, A. Muciniphila) (hereinafter referred to as "ackermanni") decreases, while pathogenic bacteria increases, thereby affecting the occurrence and development of intestinal diseases. Acremonium is a gram-negative, strictly anaerobic flora, colonizing the colon, accounting for about 1-3% of the total flora in the intestine. Most studies consider that ackermannia is a probiotic bacterium having effects of correcting abnormal glucose tolerance, anti-tumor, etc., and further identify that the active ingredient of ackermannia for lowering blood glucose and anti-tumor is a membrane protein (amuc_1100) (hereinafter referred to as "amuc_1100"). The database shows the protein information of amuc_1100 as:>tr|b2ur41|b2ur41_akkm Uncharacterized protein OS = Akkermansia muciniphila, the source strain is: ATCC BAA-835/DSM 22959/JCM 33894/BCRC 81048/CCUG 64013/CIP 107961/Muc.

The amino acid sequence is:

MSNWITDNKPAAMVAGVGLLLFLGLSATGYIVNSKRSELDKKISIAAKEIKSANAAEITPSRSSNEELEKELNRYAKAVGSLETAYKPFLASSALVPTTPTAFQNELKTFRDSLISSCKKKNILITDTSSWLGFQVYSTQAPSVQAASTLGFELKAINSLVNKLAECGLSKFIKVYRPQLPIETPANNPEESDEADQAPWTPMPLEIAFQGDRESVLKAMNAITGMQDYLFTVNSIRIRNERMMPPPIANPAAAKPAAAQPATGAASLTPADEAAAPAAPAIQQVIKPYMGKEQVFVQVSLNLVHFNQPKAQEPSED (database website "https:// www.uniprot.org/uniprot/B2UR 41") (SEQ ID NO. 4).

Another Chinese invention application with the application number cn201380070847.0 and the publication number cn104918626a discloses the use of Acremonium for treating metabolic disorders.

The invention is different from the prior research and discovery and application purposes of the invention: although increasing the levels of akaman bacteria in the intestinal tract of a model animal or directly using recombinant amyc_1100 protein can improve metabolic disorders, studies have found that supplementation with akaman bacteria significantly aggravates intestinal inflammation, and in addition, there is no report of the inhibition of intestinal inflammation by amyc_1100. The invention adopts a brand new Amuc_2172 protein (the used strain is ATCC BAA-835), the database shows that the protein information of Amuc_2172 is: > tr|B2UPV1|B2UPV1_AKM8GCN 5-related N-acetyltransferase OS = Akkermansia muciniphila, and the source strain is: ATCC BAA-835/DSM 22959/JCM 33894/BCRC 81048/CCUG 64013/CIP 107961/Muc.

The amino acid sequence is:

MPTLRQAAKDDITLIHELACQAFPATYRDLLSREQIDFMMDWMYSPANLEKQMEEGHVYFIASHEGKDCGYLSVQPEGPGVFHLQKIYVLPGFQGLHIGSFLFRHAISYIRSIHPEPCLMRLNVNRDNTRAVEFYQRMGMRTLERGDFHIGHGYYMTDYIMGLDIA (database website "https:// www.uniprot.org/uniprot/B2UPV 1") (SEQ ID NO. 1).

The invention is different from the administration route which is found and invented in the prior research: the prior Acremonium preparations are mostly living or inactivated thalli and composite preparations thereof, and the single administration dosage is gram-grade and can only be orally administered. The purified Amuc_2172 protein can be used for single administration of microgram level, can be orally or injected, has small toxicity and good tolerance, and is easy to clinically and commercially popularize.

Disclosure of Invention

Aiming at the technical problems in the prior art, the invention provides an Amuc_2172 protein of the Acremonium, a preparation method and application thereof, and the Amuc_2172 protein of the Acremonium, the preparation method and the application thereof aim to solve the technical problems of poor treatment effect on intestinal inflammation and cancers in the prior art.

The invention provides an Amuc_2172 protein of Acremonium, which contains an amino acid sequence shown as SEQ ID NO.1 or SEQ ID NO. 5.

The invention also provides a preparation method of the Amuc_2172 protein of the Acremonium, which comprises the following steps:

1) Cloning Amuc_2172 gene of Acremonium, and the gene sequence is shown as SEQ ID NO. 2.

2) And (3) connecting a target gene to a pET-30a prokaryotic plasmid with a His tag, respectively converting the plasmids into tool cells E.coli BL21 after sequence verification, further amplifying and culturing, and purifying a recombinant target protein by using a nickel column after the harvested bacteria are cracked and centrifuged and detecting the protein concentration by using a BSA colorimetric method to obtain the recombinant protein.

The invention also provides a pharmaceutical composition, which contains an effective dose of Amuc_2172 protein of Acremonium, and the protein contains an amino acid sequence shown as SEQ ID NO.1 or SEQ ID NO. 5.

Further, the pharmaceutical composition contains pharmaceutically acceptable auxiliary materials.

The invention also provides an application of the Amuc_2172 protein of the Acremonium in preparing a medicament for inhibiting Th17 cell differentiation or intestinal inflammation or intestinal cancer, wherein the Amuc_2172 protein of the Acremonium contains an amino acid sequence shown as SEQ ID NO.1 or SEQ ID NO. 5.

The invention also provides application of the Amuc_2172 protein of the Acremonium in preparing a medicament for autoimmune diseases, wherein the Amuc_2172 protein of the Acremonium contains an amino acid sequence shown as SEQ ID NO.1 or SEQ ID NO. 5.

Further, the autoimmune diseases include, but are not limited to, crohn's disease, ulcerative colitis, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus.

Most of researches at present consider that the Acremonium is a probiotics and has the effects of correcting abnormal glucose tolerance, resisting tumors and the like, but the Acremonium can aggravate intestinal inflammation, so that the Acremonium simultaneously contains active ingredients and pathogenic ingredients. The traditional invention is mainly completed by purifying, culturing and preparing active Acremonium and a drug derivative thereof, and has mixed drug components and low medicinal value. The invention obtains the purified active ingredients of the Acremonium by completely different processes, has simple preparation process and is easy for commercialization preparation.

The previous research has identified that the active ingredient of the ackermannia anti-tumor is a membrane protein Amuc_1100, and further research has found that the recombinant protein Amuc_1100 promotes the anti-tumor activity of intestinal CD8 toxic cells and cannot inhibit Th17 related intestinal inflammation and intestinal tumor. The invention uses Amuc_1100 protein as a control, and discovers the Amuc_2172 protein which is an active ingredient of novel ackermannia for the first time, and discovers that only Amuc_2172 protein can inhibit Th17 cells and relieve intestinal lesions at the same time, and Amuc_1100 protein cannot inhibit Th17 cells.

The invention discovers a novel effective component of the ackermanni for relieving intestinal inflammation and tumor, and constructs a method for obtaining high-purity active component, which can be widely applied to treatment of various inflammation and tumor related to Th 17.

The invention comprises recombinant proteins cloned from Amuc_2172 genes of Acremonium, and analogues and derivatives of Amuc_2172 proteins. The present invention encompasses the efficacy of the Amuc_2172 protein in inhibiting Th17 cells, in treating and alleviating inflammation in the gut and the development of intestinal neoplasia, and in treating a variety of Th 17-related autoimmune diseases.

Compared with the prior art, the invention has obvious technical progress.

1. Innovative, the invention discovers that Amuc_2172 protein, an active ingredient of Ackermansia, can inhibit Th17 cells, treat and relieve intestinal inflammation and intestinal tumor occurrence and development, and can be used for treating various autoimmune diseases related to Th17, including but not limited to Crohn's disease, ulcerative colitis, multiple sclerosis, rheumatoid arthritis and systemic lupus erythematosus.

2. The invention obtains the recombinant Amuc_2172 protein pure product by cloning the Amuc_2172 gene of the Ackermans in vitro and utilizing a prokaryotic cell system to amplify and purify the target protein, and has the advantages of simple preparation process, low cost, high drug effect and easy commercialization preparation and popularization. Overcomes the defects of mixed medicinal components, low medicinal value and the like caused by the process of mainly purifying, culturing and preparing active bacteria of the Acremonium and medicinal derivatives thereof in the prior invention.

3. The Amuc_2172 protein of the present invention has high safety, is effective for oral administration or intraperitoneal injection administration, and has no toxic or side effect. The Amuc_2172 protein has the concentration of microgram level, is favorable for the use and popularization of the pharmaceutical preparation, and avoids the defects of single administration route and huge dosage of a large amount of pure Acremonium bacteria preparation.

Drawings

Fig. 1: the flow chart is constructed by recombinant Amuc_2172 protein and Amuc_1100 protein of Acremonium.

Fig. 2: recombinant Amuc_2172 protein characterization and validation of Acremonium.

Fig. 3: and (5) the characteristics and verification of the recombinant Amuc_1100 protein of the Acremonium.

Fig. 4: inhibition of Th17 cells by recombinant Amuc_2172 and Amuc_1100 proteins from Acremonium.

Fig. 5: abdominal injection and tail vein injection of recombinant Amuc_2172 protein of Ackermansia on weight of mice.

Fig. 6: abkemosla recombinant Amuc_2172 protein was injected intraperitoneally to induce colitis and Th17 cell inhibition in mouse DSS.

Fig. 7: inhibition of intestinal tumor by recombinant Amuc_2172 protein of Alkermansia gastrolavage.

Fig. 8: the recombinant Amuc_2172 protein of Acremonium was shown to inhibit Th17 cell levels and symptoms of multiple sclerosis.

Detailed Description

Example 1

Purifying Acremonium protein: cloning of the Amuc_2172 Gene (see https:// www.ncbi.nlm.nih.gov/nuccore/CP001071.1from=2650234 & to= 2650734) (gene sequence: atgccgactc tccgccaagc cgccaaggac gacatcacgc tgatccatga actggcctgc caggcttttc cggccacata ccgggacctc ctttccagag agcagataga tttcatgatg gactggatgt attcccccgc caacctggaa aaacagatgg aggaggggca cgtgtacttc atcgcctccc atgagggaaa ggactgcggc tacctgtccg tccagccgga aggccccggc gtcttccatt tgcagaaaat ctacgttctg cccggcttcc agggcctgca tataggaagc tttctgttcc gccacgccat cagctacatc agaagcatcc acccggaacc gtgcctgatg cgcctgaacg tgaaccgcga taacacccgg gcggtggaat tctaccagcg catgggcatg cggaccctgg aacgcgggga tttccacatc ggccacggtt actacatgac ggactacatc atgggactgg atatagcctg a) (shown in SEQ ID NO. 2) and the Amuc_1100 Gene (see https:// www.ncbi.nlm.nih.gov/nuccore/CP001071.1from=1314411 & to= 1315364) (gene sequence: ttaatcttca gacggttcct gagccttggg ctggttgaag tggaccagat tcagggagac ctggacaaag acctgctcct tgcccatgta aggcttgatg acttgctgga tggccggggc tgcaggtgca gccgcctcat ccgccggagt cagggaagcc gcacccgtgg cgggttgggc cgcggcaggt ttggcggctg ccggattggc gatgggaggg ggcatcatcc gttcgttgcg gatacggatg gagttgaccg tgaacagata gtcctgcatg ccggttatgg cgttcatggc tttcaataca ctttcccggt cgccctggaa ggctatttcc agaggcatgg gagtccatgg ggcctggtcg gcttcgtccg attcttccgg attgttcgcc ggggtttcaa tggggagctg ggggcggtac accttgatga atttggacag gccgcattcc gccagtttgt tgaccaggct gttgatggct ttcaattcaa aacccagcgt ggaggccgcc tgaacagagg gagcctgggt gctgtaaacc tggaaaccga gccaggagga tgtgtccgtt atgagaatgt tctttttctt gcaggaggag atcagggaat ccctgaatgt tttcagttca ttctggaatg ccgtgggcgt ggtggggacc agcgcggagg aggcaaggaa gggcttgtag gccgtttcca ggctgcccac ggccttggca tagcggttca gttctttttc cagctcttcg ttggatgatc ggctcggagt gatttccgca gcattggcgg acttgatttc cttggcggcg atgctgattt ttttgtccag ttcactgcgt ttggaattga cgatgtaccc tgtcgcggat aaccccagga ataagagaag tcccacgccc gcgaccatgg cggcgggctt gttgtctgta atccaattgc tcat) (shown in SEQ ID NO. 3), ligation of the gene of interest into His-tagged pET-30a prokaryotic plasmid, sequence verification, transformation of the plasmid into tool cells E.coli BL21 and further amplification culture, lysis of the harvested bacteria, centrifugation followed by purification of the recombinant protein of interest using a nickel column, and detection of protein concentration using BSA colorimetric method, successfully obtaining recombinant protein (FIG. 1. Nickel column chromatography, fig. 2 and 3).

The invention is different from the prior research and discovery and the preparation process of the invention: the traditional invention is mainly completed by purifying, culturing and preparing the active bacteria of the Acremonium and the drug derivative thereof, the drug ingredients are mixed, the abundance of the active ingredients is low, and the pro-inflammatory ingredients of the Acremonium are mixed, so that the medicinal value is low. The invention constructs prokaryotic expression plasmid in vitro by cloning Ackermansia genes, converts prokaryotic bacteria and amplifies the prokaryotic bacteria, and then obtains single high-purity active ingredients by using protein nickel column purification.

The recombinant Amuc_2172 protein has 174 amino acids in length, a molecular weight of 20359.8 and an equipotential of 6.87, and the total amino acid sequence is as follows: MHHHHHHHHP TLRQAAKDDI TLIHELACQA FPATYRDLLS REQIDFMMDW MYSPANLEKQ MEEGHVYFIA SHEGKDCGYL SVQPEGPGVF HLQKIYVLPG FQGLHIGSFL FRHAISYIRS IHPEPCLMRL NVNRDNTRAV EFYQRMGMRT LERGDFHIGH GYYMTDYIMG LDIA (SEQ ID NO. 5).

The invention is different from the active ingredients found and invented by the prior research: the previous research has identified that the active component of the ackermanni anti-tumor is a membrane protein Amuc_1100, and further research has found that the recombinant protein Amuc_1100 promotes the anti-tumor activity of intestinal CD8 toxic cells. The present invention also purifies recombinant Amuc_1100 protein: the recombinant Amuc_1100 protein has 295 amino acids in length, a molecular weight of 32228.8, an equipotential of 7.33 and the total amino acid sequence: MHHHHHHHHV NSKRSELDKK ISIAAKEIKS ANAAEITPSR SSNEELEKEL NRYAKAVGSL ETAYKPFLAS SALVPTTPTA FQNELKTFRD SLISSCKKKN ILITDTSSWL GFQVYSTQAP SVQAASTLGF ELKAINSLVN KLAECGLSKF IKVYRPQLPI ETPANNPEES DEADQAPWTP MPLEIAFQGD RESVLKAMNA ITGMQDYLFT VNSIRIRNER MMPPPIANPA AAKPAAAQPA TGAASLTPAD EAAAPAAPAI QQVIKPYMGK EQVFVQVSLN LVHFNQPKAQ EPSED (SEQ ID NO. 6).

Example 2 evaluation of Amuc_2172 effect on Th 17:

wild type C57BL/6J (8-10 weeks old) mice were sacrificed by pulling the neck and spleens were removed under aseptic conditions. In a beaker containing 10ml of physiological saline at 4 ℃, the cells are sheared and crushed by ophthalmology, and the mixture is gently ground through a 200-mesh nylon net and then sieved to collect cell suspension. The suspension was centrifuged at 600 Xg for 5min. The supernatant was discarded and the pellet cell volume was observed at 10: adding the erythrocyte lysate into the mixture according to the volume ratio of 1, uniformly mixing, standing for 2min, immediately adding 10ml of physiological saline at 4 ℃, centrifuging, and carrying out 600 Xg for 5min. The supernatant was discarded, and the cells were repeatedly washed with physiological saline at 4℃for 2 times. Cell counting was performed before the last centrifugation, and cells were resuspended in RPMI-1640 complete medium (RPMI-1640 containing 10% fetal bovine serum by volume percent with 100U/ml penicillin and streptomycin each) to a cell concentration of 1X 10 ⁷ /ml. Spleen cell suspension cells were seeded in 24-well plates for culture at 1 ml/well. Adding cytokine inducing differentiation of Th17 cells, 3ng/ml TGF-beta (100-21, peproTech, chicago, IL, USA) (240-B-002, R)&D Systems,Minneapolis,MN,USA),40ng/ml IL-6(200-06,PeproTech),30ng/ml IL-23(200-23,PeproTech),20ng/ml Tumor Necrosis Factor(TNF)-α(300-01A,PeproTech)and 10ng/ml IL-1β(200-01B,PeproTech)。

The experimental sub-Control group (Ctrl, control group), the recombinant amyc_2172 protein group of example 1 (amyc_2172), the recombinant amyc_1100 protein group (amyc_1100), the final recombinant protein intervention concentration was 5 μg/ml, and each group was repeated 3 times (n=3). After 4 days of in vitro differentiation of Th17 cells with recombinant protein of Ackermans, CD 3-labeled fluorescent antibody was sorted, CD 8-and IL-17-labeled antibody, CD 3-positive CD 8-negative IL-17-positive cells were defined as Th17 cells, and flow cytometry examined Th17 cell levels, and Amuc_2172 protein was found to inhibit Th17 cell differentiation, whereas Amuc_1100 protein did not inhibit Th17 cells (FIG. 4).

Example 3amuc_2172 safety assessment of different modes of administration:

wild type C57BL/6J (8-16 weeks old) mice were taken, up to 5 mice per cage were housed, and the mice were labeled and the reference body weight was weighed. Thirty mice were randomly divided into three groups, and were tail-injected with normal saline in Control animals (Ctrl, control group), 150 μg/kg body weight of recombinant Amuc_2172 protein of example 1 in the intraperitoneal injection group, and 150 μg/kg body weight of Amuc_2172 protein in the tail-injected group. Animals were injected three times a week starting on day 0, and animals were observed daily for general conditions, water intake, body weight, and stool characteristics.

The percent weight loss per day of the animal was calculated as follows: [ (body weight-reference body weight)/reference body weight ] X100. At day 10 of feeding, the experiment was ended and animals were sacrificed. Evaluating the survival and body weight of mice, it was found that there was no significant change in the administration of the Amuc_2172 protein relative to the control group, indicating that the Amuc_2172 protein was not significantly toxic (FIG. 5);

example 4 effect of amuc_2172 on intestinal lesions during enteritis:

wild-type C57BL/6J (8-16 weeks old) mice, sterile water and Dextran Sodium Sulfate (DSS) (molecular weight 35000-50000Da;MP Biomedicals) were taken. At most, no more than 5 mice are raised in each cage. Mice were labeled and reference body weights were weighed. Preparing 3-5% (w/v) DSS aqueous solution, preparing DSS with sterile water, filtering with filter membrane, and changing DSS aqueous solution every 1-2 days. Normal sterile water was used in Control group (Ctrl) animals. The animals were observed daily for general conditions, water intake, body weight, stool characteristics, and presence or absence of hematochezia.

The percent weight loss per day of the animal was calculated as follows: [ (body weight-reference body weight)/reference body weight ] X100. On day 20 of feeding, animals were sacrificed, intestinal tissues were dissected and pathological sections were prepared and HE stained. Using example 1 recombinant amuc_2172 protein 150 μg/kg body weight to administer an intraperitoneal injection intervention to mice, three injections per week, assessing the effect of different treatments on intestinal inflammation in model animals, flow cytometry detecting Th17 cell levels, assessing mouse body weight and intestinal length, it was found that amuc_2172 protein could inhibit Th17 cell differentiation, and alleviate intestinal inflammation (fig. 6);

example 5 effect of amuc_2172 on intestinal tumors during inflammatory cancer transformation:

wild-type C57BL/6J (8-16 weeks old) mice, sterile water and Dextran Sodium Sulfate (DSS) (molecular weight 35000-50000Da;MP Biomedicals) were taken. At most, no more than 5 mice are raised in each cage. Mice were labeled and reference body weights were weighed. The DNA alkylation product of the action of the chemical carcinogen 1, 2-dimethylhydrazine Azoxymethane (AOM) in combination with DSS established a colon cancer induction model. AOM (10 mg/kg) was injected intraperitoneally once, then 3-5% (w/v) aqueous DSS was formulated, DSS was formulated with sterile water and filtered with filter membranes, and the aqueous DSS solution was changed every 1-2 days as prepared.

Normal sterile water was used in Control group (Ctrl) animals and DSS aqueous solution was used in experimental groups. The animals were observed daily for general conditions, water intake, body weight, stool characteristics, and presence or absence of hematochezia. The percent weight loss per day of the animal was calculated as follows: [ (body weight-reference body weight)/reference body weight ] X100. After feeding for 5-7d, normal drinking water is used to replace DSS water solution for 7d. This is a cycle period, typically 3-5 cycles. On the last day of the last cycle, animals were sacrificed, intestinal tissues were dissected and pathological sections were prepared and HE stained. Using 150. Mu.g/kg body weight of recombinant Amuc_2172 protein from example 1, mice were lavaged three times a week to evaluate the effect of different treatments on the conversion of inflammatory cancer in the intestine of model animals, and the intestinal mucosal tumor status was evaluated, and Amuc_2172 protein was found to inhibit the intestinal tumor (FIG. 7).

Example 6 influence of amuc_2172 on disease score during multiple sclerosis:

wild type C57BL/6J (8-16 weeks old) mice were randomly grouped into at least 10 mice per group, and at most 5 mice per cage were bred. Mice were labeled and reference body weights were weighed. The mice were induced to develop multiple sclerosis following subcutaneous injections with a chemical agent Myelin Oligodendrocyte Glycoprotein (MOG 35-55) polypeptide, freund's adjuvant and pertussis toxin. Mice were given an intraperitoneal injection intervention using 150 μg/kg body weight of recombinant Amuc_2172 protein of example 1, three injections per week, and control groups were injected with saline. Mice motor ability was recorded and mice were scored for multiple sclerosis.

After the end of the experiment, animals were sacrificed, spleen tissues were dissected, cells were isolated, and Th17 cell levels were detected. As a result, amuc_2172 protein was found to inhibit Th17 cell levels and symptoms of multiple sclerosis (FIG. 8).

Sequence listing

<110> Shanghai transportation university medical college affiliated ren Ji hospital

<120> Acremonium Amuc_2172 protein, preparation method and use thereof

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 166

<212> PRT

<213> Ackermans mucin-philic protein (Akkermansia muciniphila)

<400> 1

Met Pro Thr Leu Arg Gln Ala Ala Lys Asp Asp Ile Thr Leu Ile His

1 5 10 15

Glu Leu Ala Cys Gln Ala Phe Pro Ala Thr Tyr Arg Asp Leu Leu Ser

20 25 30

Arg Glu Gln Ile Asp Phe Met Met Asp Trp Met Tyr Ser Pro Ala Asn

35 40 45

Leu Glu Lys Gln Met Glu Glu Gly His Val Tyr Phe Ile Ala Ser His

50 55 60

Glu Gly Lys Asp Cys Gly Tyr Leu Ser Val Gln Pro Glu Gly Pro Gly

65 70 75 80

Val Phe His Leu Gln Lys Ile Tyr Val Leu Pro Gly Phe Gln Gly Leu

85 90 95

His Ile Gly Ser Phe Leu Phe Arg His Ala Ile Ser Tyr Ile Arg Ser

100 105 110

Ile His Pro Glu Pro Cys Leu Met Arg Leu Asn Val Asn Arg Asp Asn

115 120 125

Thr Arg Ala Val Glu Phe Tyr Gln Arg Met Gly Met Arg Thr Leu Glu

130 135 140

Arg Gly Asp Phe His Ile Gly His Gly Tyr Tyr Met Thr Asp Tyr Ile

145 150 155 160

Met Gly Leu Asp Ile Ala

165

<210> 2

<211> 501

<212> DNA

<213> Ackermans mucin-philic protein (Akkermansia muciniphila)

<400> 2

atgccgactc tccgccaagc cgccaaggac gacatcacgc tgatccatga actggcctgc 60

caggcttttc cggccacata ccgggacctc ctttccagag agcagataga tttcatgatg 120

gactggatgt attcccccgc caacctggaa aaacagatgg aggaggggca cgtgtacttc 180

atcgcctccc atgagggaaa ggactgcggc tacctgtccg tccagccgga aggccccggc 240

gtcttccatt tgcagaaaat ctacgttctg cccggcttcc agggcctgca tataggaagc 300

tttctgttcc gccacgccat cagctacatc agaagcatcc acccggaacc gtgcctgatg 360

cgcctgaacg tgaaccgcga taacacccgg gcggtggaat tctaccagcg catgggcatg 420

cggaccctgg aacgcgggga tttccacatc ggccacggtt actacatgac ggactacatc 480

atgggactgg atatagcctg a 501

<210> 3

<211> 954

<212> DNA

<213> Ackermans mucin-philic protein (Akkermansia muciniphila)

<400> 3

ttaatcttca gacggttcct gagccttggg ctggttgaag tggaccagat tcagggagac 60

ctggacaaag acctgctcct tgcccatgta aggcttgatg acttgctgga tggccggggc 120

tgcaggtgca gccgcctcat ccgccggagt cagggaagcc gcacccgtgg cgggttgggc 180

cgcggcaggt ttggcggctg ccggattggc gatgggaggg ggcatcatcc gttcgttgcg 240

gatacggatg gagttgaccg tgaacagata gtcctgcatg ccggttatgg cgttcatggc 300

tttcaataca ctttcccggt cgccctggaa ggctatttcc agaggcatgg gagtccatgg 360

ggcctggtcg gcttcgtccg attcttccgg attgttcgcc ggggtttcaa tggggagctg 420

ggggcggtac accttgatga atttggacag gccgcattcc gccagtttgt tgaccaggct 480

gttgatggct ttcaattcaa aacccagcgt ggaggccgcc tgaacagagg gagcctgggt 540

gctgtaaacc tggaaaccga gccaggagga tgtgtccgtt atgagaatgt tctttttctt 600

gcaggaggag atcagggaat ccctgaatgt tttcagttca ttctggaatg ccgtgggcgt 660

ggtggggacc agcgcggagg aggcaaggaa gggcttgtag gccgtttcca ggctgcccac 720

ggccttggca tagcggttca gttctttttc cagctcttcg ttggatgatc ggctcggagt 780

gatttccgca gcattggcgg acttgatttc cttggcggcg atgctgattt ttttgtccag 840

ttcactgcgt ttggaattga cgatgtaccc tgtcgcggat aaccccagga ataagagaag 900

tcccacgccc gcgaccatgg cggcgggctt gttgtctgta atccaattgc tcat 954

<210> 4

<211> 317

<212> PRT

<213> Ackermans mucin-philic protein (Akkermansia muciniphila)

<400> 4

Met Ser Asn Trp Ile Thr Asp Asn Lys Pro Ala Ala Met Val Ala Gly

1 5 10 15

Val Gly Leu Leu Leu Phe Leu Gly Leu Ser Ala Thr Gly Tyr Ile Val

20 25 30

Asn Ser Lys Arg Ser Glu Leu Asp Lys Lys Ile Ser Ile Ala Ala Lys

35 40 45

Glu Ile Lys Ser Ala Asn Ala Ala Glu Ile Thr Pro Ser Arg Ser Ser

50 55 60

Asn Glu Glu Leu Glu Lys Glu Leu Asn Arg Tyr Ala Lys Ala Val Gly

65 70 75 80

Ser Leu Glu Thr Ala Tyr Lys Pro Phe Leu Ala Ser Ser Ala Leu Val

85 90 95

Pro Thr Thr Pro Thr Ala Phe Gln Asn Glu Leu Lys Thr Phe Arg Asp

100 105 110

Ser Leu Ile Ser Ser Cys Lys Lys Lys Asn Ile Leu Ile Thr Asp Thr

115 120 125

Ser Ser Trp Leu Gly Phe Gln Val Tyr Ser Thr Gln Ala Pro Ser Val

130 135 140

Gln Ala Ala Ser Thr Leu Gly Phe Glu Leu Lys Ala Ile Asn Ser Leu

145 150 155 160

Val Asn Lys Leu Ala Glu Cys Gly Leu Ser Lys Phe Ile Lys Val Tyr

165 170 175

Arg Pro Gln Leu Pro Ile Glu Thr Pro Ala Asn Asn Pro Glu Glu Ser

180 185 190

Asp Glu Ala Asp Gln Ala Pro Trp Thr Pro Met Pro Leu Glu Ile Ala

195 200 205

Phe Gln Gly Asp Arg Glu Ser Val Leu Lys Ala Met Asn Ala Ile Thr

210 215 220

Gly Met Gln Asp Tyr Leu Phe Thr Val Asn Ser Ile Arg Ile Arg Asn

225 230 235 240

Glu Arg Met Met Pro Pro Pro Ile Ala Asn Pro Ala Ala Ala Lys Pro

245 250 255

Ala Ala Ala Gln Pro Ala Thr Gly Ala Ala Ser Leu Thr Pro Ala Asp

260 265 270

Glu Ala Ala Ala Pro Ala Ala Pro Ala Ile Gln Gln Val Ile Lys Pro

275 280 285

Tyr Met Gly Lys Glu Gln Val Phe Val Gln Val Ser Leu Asn Leu Val

290 295 300

His Phe Asn Gln Pro Lys Ala Gln Glu Pro Ser Glu Asp

305 310 315

<210> 5

<211> 174

<212> PRT

<213> Ackermans mucin-philic protein (Akkermansia muciniphila)

<400> 5

Met His His His His His His His His Pro Thr Leu Arg Gln Ala Ala

1 5 10 15

Lys Asp Asp Ile Thr Leu Ile His Glu Leu Ala Cys Gln Ala Phe Pro

20 25 30

Ala Thr Tyr Arg Asp Leu Leu Ser Arg Glu Gln Ile Asp Phe Met Met

35 40 45

Asp Trp Met Tyr Ser Pro Ala Asn Leu Glu Lys Gln Met Glu Glu Gly

50 55 60

His Val Tyr Phe Ile Ala Ser His Glu Gly Lys Asp Cys Gly Tyr Leu

65 70 75 80

Ser Val Gln Pro Glu Gly Pro Gly Val Phe His Leu Gln Lys Ile Tyr

85 90 95

Val Leu Pro Gly Phe Gln Gly Leu His Ile Gly Ser Phe Leu Phe Arg

100 105 110

His Ala Ile Ser Tyr Ile Arg Ser Ile His Pro Glu Pro Cys Leu Met

115 120 125

Arg Leu Asn Val Asn Arg Asp Asn Thr Arg Ala Val Glu Phe Tyr Gln

130 135 140

Arg Met Gly Met Arg Thr Leu Glu Arg Gly Asp Phe His Ile Gly His

145 150 155 160

Gly Tyr Tyr Met Thr Asp Tyr Ile Met Gly Leu Asp Ile Ala

165 170

<210> 6

<211> 295

<212> PRT

<213> Ackermans mucin-philic protein (Akkermansia muciniphila)

<400> 6

Met His His His His His His His His Val Asn Ser Lys Arg Ser Glu

1 5 10 15

Leu Asp Lys Lys Ile Ser Ile Ala Ala Lys Glu Ile Lys Ser Ala Asn

20 25 30

Ala Ala Glu Ile Thr Pro Ser Arg Ser Ser Asn Glu Glu Leu Glu Lys

35 40 45

Glu Leu Asn Arg Tyr Ala Lys Ala Val Gly Ser Leu Glu Thr Ala Tyr

50 55 60

Lys Pro Phe Leu Ala Ser Ser Ala Leu Val Pro Thr Thr Pro Thr Ala

65 70 75 80

Phe Gln Asn Glu Leu Lys Thr Phe Arg Asp Ser Leu Ile Ser Ser Cys

85 90 95

Lys Lys Lys Asn Ile Leu Ile Thr Asp Thr Ser Ser Trp Leu Gly Phe

100 105 110

Gln Val Tyr Ser Thr Gln Ala Pro Ser Val Gln Ala Ala Ser Thr Leu

115 120 125

Gly Phe Glu Leu Lys Ala Ile Asn Ser Leu Val Asn Lys Leu Ala Glu

130 135 140

Cys Gly Leu Ser Lys Phe Ile Lys Val Tyr Arg Pro Gln Leu Pro Ile

145 150 155 160

Glu Thr Pro Ala Asn Asn Pro Glu Glu Ser Asp Glu Ala Asp Gln Ala

165 170 175

Pro Trp Thr Pro Met Pro Leu Glu Ile Ala Phe Gln Gly Asp Arg Glu

180 185 190

Ser Val Leu Lys Ala Met Asn Ala Ile Thr Gly Met Gln Asp Tyr Leu

195 200 205

Phe Thr Val Asn Ser Ile Arg Ile Arg Asn Glu Arg Met Met Pro Pro

210 215 220

Pro Ile Ala Asn Pro Ala Ala Ala Lys Pro Ala Ala Ala Gln Pro Ala

225 230 235 240

Thr Gly Ala Ala Ser Leu Thr Pro Ala Asp Glu Ala Ala Ala Pro Ala

245 250 255

Ala Pro Ala Ile Gln Gln Val Ile Lys Pro Tyr Met Gly Lys Glu Gln

260 265 270

Val Phe Val Gln Val Ser Leu Asn Leu Val His Phe Asn Gln Pro Lys

275 280 285

Ala Gln Glu Pro Ser Glu Asp

290 295

Claims

1. Use of an akkermansia amuc_2172 protein in the manufacture of a medicament for inhibiting Th17 cell differentiation or intestinal inflammation or intestinal cancer or multiple sclerosis, wherein the amino acid sequence of the akkermansia amuc_2172 protein is shown as SEQ ID No.1 or SEQ ID No. 5.