CN114621936B - Protein composition and use thereof - Google Patents

Protein composition and use thereof Download PDF

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CN114621936B
CN114621936B CN202210197701.3A CN202210197701A CN114621936B CN 114621936 B CN114621936 B CN 114621936B CN 202210197701 A CN202210197701 A CN 202210197701A CN 114621936 B CN114621936 B CN 114621936B
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CN114621936A (en
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张晓洁
陈体
罗世林
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Second Xiangya Hospital of Central South University
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    • A61K38/443—Oxidoreductases (1) acting on CH-OH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01001—Alcohol dehydrogenase (1.1.1.1)
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    • C12Y102/0101—Acetaldehyde dehydrogenase (acetylating) (1.2.1.10)
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Abstract

The application discloses a protein composition and application thereof, wherein the protein composition comprises a first recombinant protein and a second recombinant protein, the first recombinant protein is used for catalyzing ethanol to be oxidized into acetaldehyde, and the second recombinant protein is used for catalyzing acetaldehyde to be oxidized into acetic acid, so that ethanol metabolism is realized.

Description

Protein composition and use thereof
Technical Field
The application relates to the technical field of medicines, in particular to a protein composition and application thereof.
Background
Wine culture is a special culture form and plays a special role in the traditional culture of countries in the world, especially China. However, as consumption of drinking wine increases, various negative consequences associated with drinking become more and more prominent, becoming a serious public health problem. The world health organization report indicates that the harmful use of alcohol causes 330 thousands of deaths worldwide each year, with about 5.1% of the disease and injury burden worldwide being caused by drinking. Alcohol impairs human health such as gastrointestinal, liver, neurological and psychiatric disorders, cardiovascular disorders, etc. Wherein, the damage action of alcohol on the nervous system such as violent attack behavior, antisocial behavior, and reduced accuracy of vigilance reaction speed caused by cortical removal inhibition, impulse control disorder, and cognitive impairment, etc. brings serious threat to self health and public safety. About 20-30% of alcohol abusers develop alcoholic liver disease (alcoholic liver disease, ALD), including fatty liver, alcoholic hepatitis, cirrhosis, liver fibrosis, and liver cancer. ALD is increasingly one of the leading causes of morbidity and mortality worldwide. Meanwhile, researches prove that some mood problems such as depression have a very complex relation with alcohol use, on one hand, the depression mood is a precursor factor of harmful use of alcohol, and on the other hand, the serious depression caused by long-term use of alcohol further aggravates alcohol abuse. In summary, alcohol use related disorders are a complex etiology and involve multiple organ system dysfunction.
In recent years, intestinal flora has been found to play an important role in bi-directional regulation in alcohol use disorders, ALD, and depression. On the one hand, alcohol and metabolites thereof can cause intestinal microecological unbalance, further cause the damage of intestinal mucosa barrier and the increase of intestinal permeability, and the cellular components of intestinal microorganisms and neurotransmitters, metabolites and the like synthesized by the intestinal microorganisms can enter the blood circulation, thereby affecting the health of other organs of the organism such as brain and liver. For example, endotoxin (LPS) circulates through the portal into the liver, and binding to Toll-like receptor 4 (tlr 4) activates Kupffer cells in the liver, which is believed to be a major factor in ALD pathogenesis. For example, intestinal microecological imbalance activates immune response, and immune factors enter brain via blood circulation, damage neurons, and act on hypothalamus-pituitary-adrenal Axis (HPA Axis) to cause emotional disorder such as depression. On the other hand, in-intestinal probiotics implants such as lactobacillus, bifidobacterium and AKK, which are known as AKK, significantly improve alcohol damage to liver function; such as Lactobacillus helveticus, and Achroman muciniphilum, significantly improve depression-like behavior. These evidence suggest that the intestinal flora plays an important role in a number of disorders related to alcohol use.
However, due to the complex and varied intestinal flora, the mechanism of action in alcohol use related disorders is not clear, and the mechanism by which probiotics play a protective role is also lacking. Therefore, the action mechanism of probiotics on alcohol needs to be determined, so that the damage of alcohol to human bodies is reduced and resisted to the greatest extent.
Content of the application
The present application aims to provide a protein composition and its use, which is capable of metabolizing alcohol before it enters the human body.
In order to achieve the purpose of the application, the application provides the following technical scheme:
in a first aspect, a protein composition comprising a first class of recombinant proteins and a second class of recombinant proteins;
the first recombinant protein comprises a polypeptide shown as SEQ ID NO: 1. SEQ ID NO:2 and SEQ ID NO:3, or one or more of the amino acid sequences shown in SEQ ID NO: 1. SEQ ID NO:2 and SEQ ID NO:3, wherein one or more of the amino acid sequences shown in 3 is substituted, deleted or added with one or more amino acids, respectively, and has the activity of the recombinant protein of the first type;
the second class of recombinant proteins comprises the amino acid sequence as shown in SEQ ID NO: 4. SEQ ID NO:5 and SEQ ID NO:6, or one or more of the amino acid sequences shown in SEQ ID NO: 4. SEQ ID NO:5 and SEQ ID NO:6, one or more of the amino acid sequences shown in fig. 6 is substituted, deleted or added with one or more amino acids, respectively, and has the activity of said second type of recombinant protein.
In the present application, the above-mentioned SEQ ID NO: 1. SEQ ID NO:2 and SEQ ID NO:3 are all from the intestinal mucosa bacteria Achroman muciniphila Akkermansia muciniphila (A).
muciniphila, AKK), is an in vitro recombinant protein, and the sequence of SEQ ID NO: 1. SEQ ID NO:2 and SEQ ID NO:3 each have the function of alcohol dehydrogenase (Alcohol dehydrogenase, ADH) for metabolizing ethanol to acetaldehyde.
Specifically, SEQ ID NO:1 is Amuc 0233 protein, encoded by SEQ ID NO:7, adding a histidine tag to carry a white and thrombin cleavage site, mutating the glycine of the 42 th amino acid into histidine, and mutating the lysine of the 338 th amino acid into cysteine. SEQ ID NO:2 is Amuc-2116, represented by SEQ ID NO:8, adding a histidine tag to carry a white and thrombin cleavage site, mutating the glycine of the 42 th amino acid into histidine, and mutating the lysine of the 362 th amino acid into cysteine. SEQ ID NO:3 is Amuc-1190 protein, encoded by SEQ ID NO:9 is obtained by adding a histidine tag with a white and thrombin cleavage site, mutating the proline at the 46 th amino acid into histidine, mutating the lysine at the 325 th amino acid into cysteine.
Wherein, SEQ ID NO: 7. SEQ ID NO:8 and SEQ ID NO: the 9 proteins are all proteins included in AKK bacteria.
The alcohol dehydrogenase ADH exists in large quantities in human and animal liver, plants and microbial cells as a key enzyme for the metabolism of major short-chain alcohols in organisms, and can metabolize ethanol to acetaldehyde. Likewise, SEQ ID NO: 1. SEQ ID NO:2 and SEQ ID NO:3, and the derivative protein which is respectively substituted, deleted or added with one or more amino acids and has the activity of the first recombinant protein has the function of alcohol dehydrogenase.
And, SEQ ID NO as described above: 4. SEQ ID NO:5 and SEQ ID NO:6 are also from the intestinal mucosa bacteria akkermansia muciniphila Akkermansia muciniphila (a. Muciniphila, AKK), are recombinant proteins in vitro, and the SEQ ID NO: 4. SEQ ID NO:5 and SEQ ID NO:6 have the function of acetaldehyde dehydrogenase for metabolizing acetaldehyde to acetic acid.
Specifically, SEQ ID NO:4 is Amuc-2066 protein, represented by SEQ ID NO:10 is obtained by adding histidine tag with white and thrombin cleavage site and mutating the 1142 nd amino acid glutamic acid into lysine modification. SEQ ID NO:5 is Amuc-0528 protein, represented by SEQ ID NO:11 is obtained by adding histidine tag with white and thrombin cleavage site and mutating 390 th amino acid glutamic acid into lysine modification. SEQ ID NO:6 is Amuc-1265 protein, encoded by SEQ ID NO:12 is obtained by adding a histidine tag with a white and thrombin cleavage site and mutating amino acid threonine at position 332 into lysine for modification.
Wherein, SEQ ID NO: 10. SEQ ID NO:11 and SEQ ID NO: the three proteins are all proteins included in AKK bacteria.
Acetaldehyde dehydrogenase (acetaldehyde dehydrogenase, ALDH) exists in human and animal livers, and is responsible for catalyzing the reaction of oxidizing acetaldehyde into acetic acid after alcohol enters the human body (acetaldehyde is obtained by catalyzing the ethanol under the action of ethanol dehydrogenase), and when alcohol is metabolized in the liver of the human body, acetaldehyde generated under the action of the ethanol dehydrogenase is further converted into harmless acetic acid under the action of the acetaldehyde dehydrogenase as a substrate, and the acetic acid is finally decomposed into carbon dioxide and water to finish alcohol metabolism. Likewise, SEQ ID NO: 4. SEQ ID NO:5 and SEQ ID NO:6, wherein one or more derivative proteins having the activity of the second type of recombinant protein, each of which has been substituted, deleted or added with one or more amino acids, respectively, have acetaldehyde dehydrogenase function.
In a second aspect, there is provided the use of a protein composition for the preparation of a product for metabolising ethanol;
the protein composition comprises a first type of recombinant protein and a second type of recombinant protein;
the first recombinant protein comprises a polypeptide shown as SEQ ID NO: 1. SEQ ID NO:2 and SEQ ID NO:3, or one or more of the amino acid sequences shown in SEQ ID NO: 1. SEQ ID NO:2 and SEQ ID NO:3, wherein one or more of the amino acid sequences shown in 3 is substituted, deleted or added with one or more amino acids, respectively, and has the activity of the recombinant protein of the first type;
the second class of recombinant proteins comprises the amino acid sequence as shown in SEQ ID NO: 4. SEQ ID NO:5 and SEQ ID NO:6, or one or more of the amino acid sequences shown in SEQ ID NO: 4. SEQ ID NO:5 and SEQ ID NO:6, one or more of the amino acid sequences shown in fig. 6 is substituted, deleted or added with one or more amino acids, respectively, and has the activity of said second type of recombinant protein.
Further, the first recombinant protein is used for metabolizing ethanol to acetaldehyde;
the second recombinant protein is used for metabolizing the acetaldehyde obtained into acetic acid.
In a third aspect, there is provided the use of a host expressing a protein composition in the preparation of a product for metabolising ethanol.
Wherein the protein composition comprises a first type of recombinant protein and a second type of recombinant protein;
the first recombinant protein comprises a polypeptide shown as SEQ ID NO: 1. SEQ ID NO:2 and SEQ ID NO:3, or one or more of the amino acid sequences shown in SEQ ID NO: 1. SEQ ID NO:2 and SEQ ID NO:3, wherein one or more of the amino acid sequences shown in 3 is substituted, deleted or added with one or more amino acids, respectively, and has the activity of the recombinant protein of the first type;
the second class of recombinant proteins comprises the amino acid sequence as shown in SEQ ID NO: 4. SEQ ID NO:5 and SEQ ID NO:6, or one or more of the amino acid sequences shown in SEQ ID NO: 4. SEQ ID NO:5 and SEQ ID NO:6, one or more of the amino acid sequences shown in fig. 6 is substituted, deleted or added with one or more amino acids, respectively, and has the activity of said second type of recombinant protein.
Wherein the host is a natural host or a recombinant host prepared by genetic engineering means.
In some embodiments, the native host is a microorganism that exists in nature and is capable of expressing a first type of recombinant protein and a second type of recombinant protein. For example, the host expressing the first and second recombinant proteins is a microorganism of the Verrucomicrobia family. In some embodiments, the microorganism of the Verrucomicrobia family is AKK bacteria. It is to be understood that the mucin-philic Acremonium of the present application includes not only A.muciniti but also microorganisms having more than 90% similarity in nucleic acid sequence, especially microorganisms having more than 95% similarity to A.muciniti, known as Akkermansia-like.
In other embodiments, the host is a recombinant host expressing a first type of recombinant protein and a second type of recombinant protein. The recombinant host is a microorganism or a cell strain; the recombinant host expresses a protein composition comprising a first type of recombinant protein and a second type of recombinant protein. For example, by genetic engineering means, a host that does not express a first type of recombinant protein or a second type of recombinant protein is caused to produce a large amount of the first type of recombinant protein or the second type of recombinant protein; or using genetic engineering means to replace, add and delete single amino acid or multiple amino acid sites, so that the functions of the first type recombinant protein and the second type recombinant protein are more obvious or inactivated. Such genetic engineering means include, but are not limited to ZFNs, TALENs or CRISPR/Cas9. Hosts include, but are not limited to, bacteria, fungi, plant cells, or animal cells. Wherein the bacteria are selected from E.coli, such as E.coli BL21 (DE 3), and the mammalian cells are selected from HEK293 cell lines.
In a fourth aspect, a nucleic acid vector is provided comprising a backbone vector and a polynucleotide encoding the protein composition.
In a preferred embodiment, the backbone vector is Pet-28 or pcDNA4a (+).
In a fifth aspect, there is provided a product comprising at least one of the following components:
a. a protein composition comprising a first class of recombinant proteins and a second class of recombinant proteins;
the first recombinant protein comprises a polypeptide shown as SEQ ID NO: 1. SEQ ID NO:2 and SEQ ID NO:3, or one or more of the amino acid sequences shown in SEQ ID NO: 1. SEQ ID NO:2 and SEQ ID NO:3, wherein one or more of the amino acid sequences shown in 3 is substituted, deleted or added with one or more amino acids, respectively, and has the activity of the recombinant protein of the first type;
the second class of recombinant proteins comprises the amino acid sequence as shown in SEQ ID NO: 4. SEQ ID NO:5 and SEQ ID NO:6, or one or more of the amino acid sequences shown in SEQ ID NO: 4. SEQ ID NO:5 and SEQ ID NO:6, one or more of the amino acid sequences shown in fig. 6 is substituted, deleted or added with one or more amino acids, respectively, and has the activity of said second type of recombinant protein.
b. A host expressing the protein composition of a;
c. b a fermentation product of said host;
d. b said host and/or c an extract of said fermentation product;
e. other substances having biological activity for metabolizing alcohol.
Further, an application of the product in preparing anti-alcohol medicines is provided.
In a sixth aspect, a pharmaceutical composition is provided, comprising a protein composition as active ingredient, and optionally a pharmaceutically acceptable carrier, excipient, adjuvant and/or diluent;
the protein composition comprises a first type of recombinant protein and a second type of recombinant protein;
the first recombinant protein comprises a polypeptide shown as SEQ ID NO: 1. SEQ ID NO:2 and SEQ ID NO:3, or one or more of the amino acid sequences shown in SEQ ID NO: 1. SEQ ID NO:2 and SEQ ID NO:3, wherein one or more of the amino acid sequences shown in 3 is substituted, deleted or added with one or more amino acids, respectively, and has the activity of the recombinant protein of the first type;
the second class of recombinant proteins comprises the amino acid sequence as shown in SEQ ID NO: 4. SEQ ID NO:5 and SEQ ID NO:6, or one or more of the amino acid sequences shown in SEQ ID NO: 4. SEQ ID NO:5 and SEQ ID NO:6, one or more of the amino acid sequences shown in fig. 6 is substituted, deleted or added with one or more amino acids, respectively, and has the activity of said second type of recombinant protein.
Compared with the prior art, the application has the following beneficial effects:
the application provides a protein composition and application thereof, wherein the protein composition comprises a first recombinant protein and a second recombinant protein, wherein the first recombinant protein is used for catalyzing ethanol to be oxidized into acetaldehyde, and the second recombinant protein is used for catalyzing acetaldehyde to be oxidized into acetic acid, so that metabolism of ethanol is realized, and ethanol dehydrogenase and acetaldehyde dehydrogenase in a human body are replaced.
Drawings
FIG. 1 is an analytical graph of the effect of AKK bacteria on alcohol consumption;
FIGS. 2A and 2B are graphs of the analysis of AKK bacteria on improving anxiety patterns in mice with alcohol models;
FIGS. 3A-3D are graphs of the effect of AKK bacterial intervention on mouse serum liver function index in a mouse chronic alcohol lavage model;
FIGS. 4A and 4B are graphs showing the effect of AKK bacteria on serum alcohol concentration and intestinal permeability change;
FIGS. 5A to 5G are graphs showing intestinal permeability analysis of AKK-bacteria-interfered mice;
FIGS. 6A to 6C are graphs showing the concentration of alcohol in serum versus time for the in vivo calibration of AKK bacteria;
FIG. 7 is a graph showing changes in serum alcohol concentration before and after the treatment of the AKK bacteria by gastric lavage.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the present application more apparent, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present application, and it is apparent that the described embodiments are only some embodiments of the present application, not all embodiments of the present application. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.
In the description of the present application, it should be understood that the terms "first," "second," and the like are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or number of technical features indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature. In the description of the present application, unless otherwise indicated, the meaning of "a plurality" is two or more.
Examples
The test materials used in this example are all commercially available and commercially available.
The various bacterial information employed in this example is as follows:
achroman mucin source: zhengzhou fine waffle limited, its original source (ATCC, BAA-835), work seed lot 5.
The present embodiment provides a protein composition comprising a first type of recombinant protein and a second type of recombinant protein. Wherein the first recombinant protein comprises a polypeptide as shown in SEQ ID NO: 1. SEQ ID NO:2 and SEQ ID NO:3, or one or more of the amino acid sequences shown in SEQ ID NO: 1. SEQ ID NO:2 and SEQ ID NO:3, and one or more of the amino acid sequences shown in 3 is/are substituted, deleted or added with one or more amino acids, respectively, and has the activity of the recombinant protein of the first type. The second class of recombinant proteins comprises the amino acid sequence set forth in SEQ ID NO: 4. SEQ ID NO:5 and SEQ ID NO:6, or one or more of the amino acid sequences shown in SEQ ID NO: 4. SEQ ID NO:5 and SEQ ID NO:6, one or more of the amino acid sequences shown in fig. 6 is substituted, deleted or added with one or more amino acids, respectively, and has the activity of said second type of recombinant protein. Wherein, SEQ ID NO: 1-SEQ ID NO:6 consist of SEQ ID NO: 7-SEQ ID NO:12 is obtained by corresponding site modification, and SEQ ID NO: 7-SEQ ID NO: and 12 is protein included by AKK bacteria.
Protein compositions include, but are not limited to, acremodelling of the mucin Acremodelling broth, recombinant protein purified fluids, and the like. The recombinant protein can also be prepared by a conventional technical means through a mode of introducing a gene sequence into engineering bacteria to ferment, separate and purify. A specific method for preparing recombinant protein, comprising the following steps:
the gene sequence of the recombinant protein is amplified by PCR, the sequence is cloned to a pET-28a vector by an enzyme digestion enzyme linkage method, an expression plasmid of the recombinant protein with an N-terminal fused 6 XHis tag is constructed, the escherichia coli engineering bacterium BL21 (DE 3) strain is utilized for heterogenous expression, the strain is cultured on a LB+KanR resistant plate overnight, monoclonal antibodies are selected, transferred to a 5ml LB culture solution containing 100 mug/ml KanR, cultured for 4 hours at 37 ℃ and 180rpm, 1:100 is transferred to a new culture medium at an exponential metaphase (0 D600=0.6), and after the culture is continued for 4 hours at 180rpm, IPTG is added to a final concentration of 0.5-2 mM, and the culture is induced at 28 ℃ and 180rpm overnight. The bacterial liquid was centrifuged at 8,000rpm for 5 minutes to pellet cells, and the cells were stored at-20 ℃.
The thalli are evenly mixed in 10 times volume of ultrasonic lysate, 4ml of PMSF containing lysozyme, DNase/Rnase and final concentration of 1mM is added, evenly mixed, placed on ice for ultrasonic 90min for cracking and breaking until the solution is thoroughly clear, the cell lysate is centrifuged at 12,000rpm, 30min and 4 ℃, the supernatant is collected and placed on ice, the recombinant protein is obtained through Ni-16NTA His-Bind resin affinity chromatography purification, and the recombinant protein is prepared into proper concentration after being stored at-80 ℃ for standby.
The alcohol metabolizing ability of AKK bacteria expressing the above protein composition was examined by taking the following example.
As shown in fig. 1, a model of the alcoholic diet feeding of mice was established, and AKK bacteria were found to be able to affect the daily intake of alcohol by mice.
Specifically, experiments show that AKK bacteria or solvent (glycerogelatin) contrast gastric lavage intervention is started from the 14 th day of modeling of mice fed with an alcoholic diet, the intake of alcohol by mice in an AKK bacteria intervention group is obviously reduced from the fourth day after gastric lavage, and then the autonomous alcohol consumption of the mice is obviously reduced compared with that of a contrast group in the whole alcoholic diet modeling process, so that the AKK bacteria can reduce the damage of alcohol to organisms by reducing the autonomous alcohol consumption of the mice, and further reduce the damage effect of the alcohol to liver tissues of the mice. Thus, AKK bacteria are thought to be able to affect the daily intake of alcohol by mice.
As shown in fig. 2A and 2B, experiments show that AKK bacteria can interfere and improve anxiety and depression behaviors of the mice with the alcohol model.
Specifically, ALD mice are subjected to AKK bacterium gastric lavage intervention, forced Swimming (FST) and open field experiments (OFT) find that AKK bacterium obviously relieves the depression-like behavior of alcohol model mice and improves the anxiety-like behavior of alcohol model mice. This suggests that AKK bacteria may be a therapeutic target for improving alcohol-induced anxiety depression or anxiety-depressive disorder.
As shown in fig. 3A-3B, a chronic alcohol gastric lavage model is established, and experiments find that AKK bacteria have a protective effect on liver injury caused by alcohol.
After 4 weeks of AKK bacterium gastric lavage intervention, alcohol chronic gastric lavage is carried out, the influence of AKK bacterium intervention on the serum liver function index of mice is analyzed, and the liver injury caused by AKK bacterium intervention group is found to be lighter than that caused by pure alcoholic liver disease group, and the ALT and AST rise degree is also reduced. This suggests that AKK bacteria have a protective effect on liver injury caused by alcohol.
As shown in FIGS. 4A-4B, the effect of AKK bacteria on serum alcohol concentration was found to be independent of intestinal permeability changes.
Specifically, the AKK bacteria or a solvent (glycerolsaline) is adopted to feed mice by alcohol diet for contrast gastric lavage intervention, the intestinal permeability of the mice in the AKK bacteria intervention group is not changed significantly, but the alcohol concentration in serum is reduced significantly, which indicates that the influence of the AKK bacteria on the alcohol concentration in serum is irrelevant to the intestinal permeability change, and the AKK bacteria can play an important role in the alcohol metabolism process.
As shown in fig. 5A-5G, it was found experimentally that AKK bacteria have an effect on serum alcohol concentration before alcohol enters the host intestinal wall and is independent of host cell ADH and ALDH activity.
Alcohol chronic gastric lavage was performed for 8 weeks after pretreatment with AKK bacteria or vehicle (glycerolsaline) control for 4 weeks, and the materials were obtained after 30 minutes of alcohol gastric lavage the last day, and alcohol concentration, ADH activity and ALDH activity in blood, intestinal tissues, and liver were measured, respectively. The results suggest that the alcohol concentration in blood, intestinal tissue and liver of mice pretreated with AKK bacteria is significantly reduced, while the ADH activity and ALDH activity in intestinal tissue and liver tissue are not significantly different, indicating that the effect of AKK bacteria on alcohol concentration in serum may be independent of host cell ADH and ALDH activity before alcohol enters the intestinal wall of the host.
As shown in FIGS. 6A to 6C, it was found that AKK bacteria were colonized in vivo for a sufficient period of time to have an effect on the concentration of alcohol in serum.
Further research shows that after long enough time of field planting, AKK bacteria can obviously reduce the alcohol concentration in blood of mice after single alcohol gavage, and after two gavage and even 15 gavage, mucin-philin Ackermans has no obvious influence on the alcohol concentration in serum. It can be seen that AKK bacteria need to colonize the body for a sufficient period of time to have an effect on the concentration of alcohol in the serum.
As shown in FIG. 7, the experiment shows that AKK bacteria have an effect on the concentration of alcohol in serum after a single alcohol gavage of rats after 21 days of the gavage.
Specifically, the rats are subjected to AKK bacteria gastric lavage treatment for 21 days, tail vein blood sampling is carried out before and after single alcohol gastric lavage for 5 minutes, 10 minutes, 30 minutes, 60 minutes and 120 minutes, and the change of alcohol concentration is measured, so that the results indicate that the alcohol concentration in serum of the rats pretreated by the AKK bacteria is maintained at a remarkably reduced level after single alcohol gastric lavage, and the effect of remarkably reducing the alcohol concentration in serum by the field planting of the AKK bacteria is verified again.
Therefore, the AKK bacteria expressing the protein composition provided by the embodiment can finish alcohol metabolism before alcohol enters a human body, wherein the first recombinant protein is used for catalyzing ethanol to be oxidized into acetaldehyde and has the function of alcohol dehydrogenase, and the second recombinant protein is used for catalyzing acetaldehyde to be oxidized into acetic acid and has the function of acetaldehyde dehydrogenase, so that alcohol in the alimentary canal before the alcohol enters the human body is effectively metabolized, and human body damage, emotion problems and related alcohol use barriers caused by the alcohol are reduced.
Further, the present embodiment also provides a product comprising at least one of the following components:
a. a protein composition comprising a first class of recombinant proteins and a second class of recombinant proteins;
the first recombinant protein comprises a polypeptide shown as SEQ ID NO: 1. SEQ ID NO:2 and SEQ ID NO:3, or one or more of the amino acid sequences shown in SEQ ID NO: 1. SEQ ID NO:2 and SEQ ID NO:3, wherein one or more of the amino acid sequences shown in 3 is substituted, deleted or added with one or more amino acids, respectively, and has the activity of the recombinant protein of the first type;
the second class of recombinant proteins comprises the amino acid sequence as shown in SEQ ID NO: 4. SEQ ID NO:5 and SEQ ID NO:6, or one or more of the amino acid sequences shown in SEQ ID NO: 4. SEQ ID NO:5 and SEQ ID NO:6, one or more of the amino acid sequences shown in fig. 6 is substituted, deleted or added with one or more amino acids, respectively, and has the activity of said second type of recombinant protein.
b. A host expressing the protein composition of a;
c. b a fermentation product of said host;
d. b said host and/or c an extract of said fermentation product;
e. other substances having biological activity for metabolizing alcohol.
Further, the embodiment also provides an application of the product in preparation of anti-alcohol drugs.
And, also provided is a pharmaceutical composition comprising the protein composition as an active ingredient, and optionally a pharmaceutically acceptable carrier, excipient, adjuvant and/or diluent.
Correspondingly, the pharmaceutical composition also has alcohol metabolism function.
Thus, the user takes the pharmaceutical composition before drinking to reserve a certain amount of the pharmaceutical composition in the digestive tract and form a protective barrier. When the user drinks and the alcohol reaches the alimentary canal, the pharmaceutical composition can effectively metabolize the alcohol so as to reduce the absorption of the alcohol by the human body, thereby avoiding the physiological and psychological wounds of the user caused by the alcohol.
All the above optional technical solutions can be combined to form an optional embodiment of the present application, and any multiple embodiments can be combined, so as to obtain the requirements of coping with different application scenarios, which are all within the protection scope of the present application, and are not described in detail herein.
It should be noted that the above-mentioned embodiments are only preferred embodiments of the present application, and are not intended to limit the present application, and any modifications, equivalent substitutions, improvements, etc. within the spirit and principle of the present application should be included in the protection scope of the present application.
Sequence listing
<110> Xiangya two hospitals at university of south China
<120> protein composition and use thereof
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Gln Ala Gly Pro Gly Leu Glu Leu Met Asp Val Pro Met Pro Glu Val
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Gly Pro Asn Asp Val Leu Ile Lys Ile His Lys Thr Ala Ile Cys His
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Thr Asp Leu His Ile Trp Asn Trp Asp Lys Trp Ala Gln Gln Thr Ile
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Pro Val Gly Met His Val Gly His Glu Phe Cys Gly Val Ile Glu Ser
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Val Gly Ser Ser Val Thr Glu Tyr Lys Pro Gly Glu Ile Val Ser Gly
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Glu Gly His Ile Val Cys Gly His Cys Arg Ser Cys Arg Ser Gly Gln
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Lys His Leu Cys Pro Asn Thr Lys Gly Val Gly Val Asn Arg Pro Gly
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Cys Phe Ala Glu Tyr Leu Ser Ile Pro Gln Asp Asn Val Val Arg Ile
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His Lys Ser Ile Pro Met Glu Ile Ala Ser Ile Phe Asp Pro Leu Gly
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Asn Ala Val His Thr Ala Leu Ser Trp Asp Leu Val Gly Glu Asp Val
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Leu Ile Thr Gly Ala Gly Val Ile Gly Cys Met Ala Ala Ala Val Cys
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Lys Lys Ala Gly Ala Lys Thr Val Val Ile Thr Asp Ile Asn Asp Phe
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Asp Ile Ile Asp Asn Ser Arg Asn Gly Ala Asn Ile Ser Leu Leu Gly
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Ile Gln Pro Asp Gly Ser Ser Ile Glu Trp Asn Lys Phe Ile Trp Lys
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Gly Leu Lys Met Lys Gly Ile Tyr Gly Arg Glu Ile Phe Glu Thr Trp
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Thr Ala Pro Lys Thr Phe Glu Ile Arg Glu Tyr Pro Ile Pro Ala Ile
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Gly Asp Asp Glu Met Leu Ile Lys Val Glu Ala Cys Gly Val Cys His
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Thr Asp Gly His Glu Tyr Asn Arg Asp Pro Phe Gly Leu Cys Pro Val
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Val Leu Gly His Glu Gly Thr Gly Glu Ile Val Ala Met Gly Arg Asn
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Val Thr Cys Ile Ile Pro Cys Gly Thr Cys Asp Ala Cys Leu Asn Thr
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Pro Ala Arg Thr Asn Leu Cys Glu Asn Val Gly Val Tyr Gly Leu Met
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Pro Asp Asp Asp Val His Leu Asn Gly Tyr Phe Gly Glu Tyr Leu Val
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Ile Arg Lys Gly Ser Thr Phe Phe Asn Val Ser Gly Met Thr Leu Asp
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Gln Arg Ile Leu Val Glu Pro Ala Ala Val Val Val His Ser Leu Glu
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Arg Ala Lys Ser Thr Gly Leu Leu Lys Phe Asn Ser Val Val Leu Val
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Gln Gly Cys Gly Pro Ile Gly Leu Leu Gln Ile Ala Thr Leu Arg Thr
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Leu Gly Ile Glu Thr Ile Ile Ala Val Asp Gly Asn Asp Ser Arg Leu
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Glu Leu Ala Arg Glu Met Gly Ala Ser Arg Thr Tyr Asn Phe Thr Arg
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Tyr Ala Asp Leu Asn Glu Leu Leu Asp Ala Val Lys Lys Asp Asn Gly
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Gly Arg Leu Ala Asp Phe Val Phe Gln Cys Thr Gly Val Gly Lys Ala
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Gly Ala Asn Ala Trp Lys Phe Val Lys Arg Gly Gly Gly Leu Cys Glu
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Val Gly Phe Phe Met Asp Gly Gly Glu Ser Val Ile Asn His His Tyr
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Asp Leu Cys Asn Lys Glu Val Thr Ala Val Gly Ser Trp Val Tyr Ser
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Pro Gln Asp Tyr Pro Thr Thr Phe Asp Phe Leu Lys Arg Ala Tyr Gly
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Ile Gly Leu Pro Leu Thr Lys Leu Ile Ser His Arg Phe Lys Leu Asp
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Glu Cys Ser Met Lys Pro Gln Glu Val Leu Glu Tyr Val Ser Gly Pro
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Ile Pro Val Pro Glu Glu Gly Glu Val Leu Val Arg Met Lys Ala Ala
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Pro Ile Asn His Ala Asp Ile Asn Phe Val Gln Gly Val Tyr Gly Leu
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Lys Pro Val Leu Pro His Ser Arg Ala Gly Leu Glu Gly Cys Gly Val
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Val Gln Glu Ser Arg Ala Ala Gly Phe Arg Glu Gly Asp Glu Val Ile
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Leu Leu Arg Gly Val Gly Ser Trp Ser Glu Tyr Val Ala Val Pro Ser
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Val Asn Val Met Lys Leu Pro Val Lys Val Asp Pro Val Gln Ala Ala
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Met Leu Lys Val Asn Pro Leu Thr Ala Leu Arg Met Leu Glu Gly Phe
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Val Ser Leu Glu Pro Gly Asp Trp Leu Val Gln Asn Ala Ala Asn Ser
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Gly Val Gly Arg Cys Ile Ile Gln Leu Ala Arg Glu Met Gly Val Lys
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Thr Val Asn Phe Val Arg Arg Pro Asp Glu Leu Arg Asp Glu Leu Thr
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Ala Leu Gly Ala Asp Leu Val Val Gly Glu Asp Asp Gly Asp Val Val
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Lys Asn Thr Leu Ala Arg Leu Asp Gly Lys Arg Pro Val Leu Ala Ser
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Asn Ala Val Gly Gly Glu Ser Ala Leu Arg Leu Met Asp Met Leu Ala
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Pro Gly Gly Ser Met Val Thr Tyr Gly Ala Met Ser Arg Lys Ser Ile
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Lys Val Pro Asn Gly Phe Leu Ile Phe Lys Gly Ile Lys Leu Glu Gly
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Leu Trp Val Thr Gln Trp Leu Lys Asn Ala Pro Val Ser Glu Ile Glu
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Ala Ala Tyr Glu Lys Leu Ala Arg Leu Met Ala Asp Gly Arg Leu Lys
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Gln Ala Val Asp Thr Val Tyr Pro Leu Ser Asp Val Arg Lys Ala Val
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Glu Lys Ala Gln Glu Glu Phe Arg Ser Gly Lys Cys Val Leu Ser Met
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Asp Cys Ala
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Leu Val Pro Arg Gly Ser Met Thr Asp Ser Ser Ile Pro Asp Met Met
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Ala Glu Ala Arg Arg Gly Gln Trp Thr Asp Gln Gln Leu Ala Ala Lys
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Ala Val Glu Leu Ala Glu Ser Ile Leu Lys Gln Ser Asn Ala Gly Met
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Arg Gly Lys Glu Lys Arg Gln Ala Gln Gln Met Glu Arg Met Met Asn
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Asp Pro Ala Gly Lys Ala Phe Thr Leu Ala Leu Ala Asp Arg Val Phe
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Arg Pro Ser Ser Pro Val Arg Gly Ala Glu Leu Phe Arg Tyr Leu Leu
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Asp Gly Tyr Gly Val Pro Arg Tyr Leu Ser Ala Ala Asp Arg Phe Ala
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Ile Lys Met Gly Gly Arg Phe Ser Ala Gln Phe Pro Gly Val Val Met
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Pro Val Ile Thr Ser Gln Leu Arg Lys Glu Ser Ser Asn Val Ile Leu
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Pro Ala Glu Asp Gly Lys Leu Arg Pro His Leu Arg Arg Arg Arg Lys
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Gly Gly Ile Arg Met Asn Ile Asn Gln Leu Gly Glu Ala Ile Leu Gly
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Asp Lys Asp Cys Asp Tyr Ile Ser Val Lys Ile Ser Ala Ile Phe Ser
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Gln Ile His Leu Val Ala Phe Glu Glu Thr Val Lys Leu Ile Gln Glu
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Pro Asp Gly Ser Arg Lys Pro Lys Phe Val Asn Leu Asp Met Glu Glu
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Tyr Arg Asp Leu His Leu Thr Ala Glu Ala Phe Lys Arg Thr Leu Met
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Glu Asp Glu Phe Met Gln Leu Glu Ala Gly Ile Val Leu Gln Ala Tyr
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Leu Pro Asp Ser Trp Glu Glu Gln Met Lys Leu Cys Ala Trp Ala Lys
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Glu Arg Val Glu Gln Gly Gly Ala Arg Ile Lys Ile Arg Leu Val Lys
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Gly Ala Asn Leu Ala Met Glu Lys Val Glu Ala Ser Met His Gly Trp
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Ala Gln Ala Pro Tyr Gly Thr Lys Ala Gln Val Asp Ala Asn Tyr Lys
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Arg Met Leu His Tyr Gly Cys Met Pro Asp Asn Ala Arg Tyr Val Gln
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Phe Gly Val Ala Ser His Asn Leu Phe Asp Leu Cys Tyr Ala Met Leu
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Leu Arg Glu Arg Glu Gly Val Arg Asp Gln Val Glu Phe Glu Met Leu
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Glu Gly Met Ala Asn His Gln Ala Arg Val Ile Arg Gln Ala Ala Asp
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Gly Leu Leu Leu Tyr Ala Pro Val Val Leu Lys Glu Asp Phe His Ser
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Ala Ile Ala Tyr Leu Val Arg Arg Leu Asp Glu Asn Thr Ser Glu Glu
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Asn Phe Leu His Asp Leu Phe Gly Met Thr Pro Gly Ser Arg Ser Trp
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Glu Val Gln Lys Lys Arg Phe Leu Lys Ala Cys Gln Glu Lys Asp Glu
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Val Lys Tyr Gly Pro Asn Arg Thr Gln Asn Arg Ala Ala Asp Pro Ile
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Gln Pro Ser His Tyr Arg Asp Ala Phe Ala Asn Glu Arg Asp Thr Asp
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Trp Ser Leu Arg Gln Asn Ala Glu Trp Ile Asn Gly Leu Ile Ala Ala
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Glu Lys Glu Lys Ser Gly Glu Glu Ile Pro Leu Val Ile Asp Gly Glu
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Glu Ile Thr Thr Asn Leu Trp Gly Val Gly Arg Asp Pro Ser Arg His
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Asn Glu Val Ser Tyr Lys Phe Ala Tyr Ala Asp Phe Asp Gln Val Glu
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His Ala Leu Val Thr Ala Asp Arg Val Arg Ser Ser Trp Ala Ser Lys
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Ser Ile Gly Glu Arg Ala Glu Ile Leu His Arg Ala Ala Gln Glu Leu
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Ser Arg Ile Arg Gly Glu Ala Ile Ala Ala Met Val Arg Asp Ala Gly
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Lys Ala Pro Thr Glu Ala Asp Val Glu Val Ser Glu Ala Ile Asp Phe
625 630 635 640
Cys Arg Tyr Tyr Ala Glu Gly Leu Asp Arg Asp Gly Met Asn Asp Gly
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Val Glu Met Ser Pro Leu Gly Ile Ile Cys Val Met Ser Pro Trp Asn
660 665 670
Phe Pro Phe Ala Ile Pro Thr Gly Gly Val Ala Ala Ala Leu Met Ala
675 680 685
Gly Asn Ala Val Val Phe Lys Pro Ser Glu Leu Ala Val Tyr Thr Ala
690 695 700
Trp Gln Ile Val Gln Ala Phe Trp Arg Ala Gly Val Pro Lys Asn Val
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Leu Gln Phe Val Pro Met Pro Arg Asn Glu Ile Ser Cys Lys Phe Leu
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Met Asp Pro Arg Leu Asn Gly Val Ile Met Thr Gly Ser Tyr Arg Thr
740 745 750
Gly Lys Met Leu Arg Glu Leu Arg Pro Asp Leu His Val Leu Ala Glu
755 760 765
Thr Ser Gly Lys Asp Ala Met Ile Ile Thr Ala Thr Ala Asp Pro Asp
770 775 780
Gln Ala Val Lys Asp Leu Val Lys Ser Ala Phe Gly His Ser Gly Gln
785 790 795 800
Lys Cys Ser Ala Ala Ser Val Ala Ile Val Glu Ala Ser Val Tyr Asp
805 810 815
Asn Pro Ala Phe Leu Arg Gln Leu Lys Asp Ala Ala Ala Ser Leu Lys
820 825 830
Val Gly Gly Ser Trp Glu Val Asn Ser Val Val Thr Pro Leu Ile Arg
835 840 845
Glu Pro Glu Gly Asn Leu Leu Arg Ala Leu Thr Gln Leu Glu Pro Gly
850 855 860
Glu Glu Trp Leu Leu Lys Pro Glu Pro Ser Glu Asp Asn Pro Cys Leu
865 870 875 880
Trp Ser Pro Gly Ile Arg Leu Gly Val Lys Pro Gly Ser Trp Phe His
885 890 895
Gln Thr Glu Cys Phe Gly Pro Val Leu Gly Ile Ile Arg Ala Glu Asn
900 905 910
Leu Glu Glu Ala Ile Asp Ile Gln Asn Asp Ser Glu Phe Gly Leu Thr
915 920 925
Gly Gly Leu Gln Ser Leu Asp Glu Arg Glu Ile Ala Leu Trp Lys Thr
930 935 940
Lys Val Gln Val Gly Asn Ala Tyr Ile Asn Arg Val Ile Thr Gly Ala
945 950 955 960
Ile Val Arg Arg Gln Pro Phe Gly Gly Trp Asn His Ser Ser Met Gly
965 970 975
Pro Gly Ala Lys Ala Gly Gly Lys Asn Tyr Leu Thr Met Leu Gly Ser
980 985 990
Trp Glu Glu Lys Ala Leu Pro Gln Lys Leu Arg Thr Pro Gly Glu Arg
995 1000 1005
Ile Ser Gly Leu Val Glu Lys Leu Cys Ser Glu Leu Pro Asp Cys Ala
1010 1015 1020
Lys Arg Ile Arg Ser Ala Ala Gly Ser Gln Ala Lys Trp Trp Met Glu
1025 1030 1035 1040
Glu Phe Gly Val Asp His Asp Pro Ser Arg Val Tyr Gly Glu Asn Asn
1045 1050 1055
Thr Phe Arg Tyr Ile Pro Val Lys Gly Ile Leu Ala Arg Val Glu Asn
1060 1065 1070
Met Ser Asp Asp Asp Val Ala Ile Leu Leu Leu Gly Ala Lys Leu Cys
1075 1080 1085
Gly Val Leu Leu His Leu Ser Ile Gly Thr Ser Arg Pro Trp Ile Gln
1090 1095 1100
Lys Met His Gly Tyr Tyr Ala Ser Leu Thr Val Glu Thr Glu Ala Glu
1105 1110 1115 1120
Leu Ile Gly Arg Met Pro Glu Ala Leu Pro Gly Ile Arg Phe Leu Arg
1125 1130 1135
Gly Thr Asp Ile Ser Glu Thr Leu Ala Asn Ala Ala Arg Ala Arg Asp
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Val Glu Val Leu Asp Arg Pro Val Leu Ala Asn Gly Arg Leu Glu Leu
1155 1160 1165
Leu Gly Tyr Phe Arg Glu Gln Ser Val Ser Glu Thr Val His Arg Tyr
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Gly Asn Leu Ile Pro Pro Pro Gly Ser Phe Lys Thr Asp Ser Val
1185 1190 1195
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<213> Ackermans mucin (Akkermansia muciniphila)
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Leu Val Pro Arg Gly Ser Met Lys Met Glu Asn Pro Leu Glu Pro Ala
1 5 10 15
Gly Arg Ala Ala Leu Ala Ala Ser Arg Glu Met Leu Ala Leu Glu Pro
20 25 30
Glu Glu Lys Ala Gln Ala Leu Arg Leu Met Ala Ala Gly Val Arg Asn
35 40 45
Ala Ala Glu Lys Ile Ile Arg Glu Asn Val Leu Asp Met Glu Glu Ala
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Arg Lys Ser Gly Arg Asn Ser Ala Phe Leu Asp Arg Leu Leu Leu Thr
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Pro Asp Arg Val Glu His Met Ala Cys Gly Ile Glu Gln Val Ala Ala
85 90 95
Leu Pro Asp Pro Val Gly Glu Cys Ile Arg Glu Trp Thr Arg Pro Asn
100 105 110
Gly Leu Arg Ile Arg Gln Val Arg Val Pro Leu Gly Val Ile Gly Phe
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Ile Tyr Glu Ser Arg Gly Thr Val Thr Cys Asp Ala Ala Ala Leu Cys
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Leu Lys Ser Gly Asn Ala Val Ile Leu Arg Gly Gly Ser Glu Ser Leu
145 150 155 160
Arg Thr Asn Arg Val Leu Ala Glu Thr Leu Arg Ser Ala Leu Glu Lys
165 170 175
Ser Ala Val Pro Ala Asp Ala Val Arg Leu Leu Asp Ser Gly Ser Arg
180 185 190
Asp Glu Val Arg Gln Leu Cys Glu Leu Asp Ser Cys Leu Asn Val Ile
195 200 205
Ile Pro Arg Gly Gly Lys Gly Leu Ile Arg Ala Val Thr Glu Phe Ser
210 215 220
Lys Val Pro Val Leu Lys His Leu Asp Gly Ile Cys His Val Tyr Val
225 230 235 240
Asp Ala Ala Ala Asp Leu Glu Met Ala Val Asn Val Leu Asp Asp Ala
245 250 255
Lys Thr Gln Arg Pro Gly Val Cys Asn Ala Ala Glu Thr Leu Leu Val
260 265 270
Asp Ala Ala Ala Ala Glu Arg Phe Leu Pro Met Ala Ala Glu Arg Met
275 280 285
Arg Met Arg Gly Val Glu Cys Arg Val Cys Glu Arg Ser Arg Pro Phe
290 295 300
Phe Gly Gln Glu Ala Val Pro Ala Glu Glu Arg Asp Trp Ser Thr Glu
305 310 315 320
Tyr Glu Asp Leu Ile Leu Ser Val Lys Val Val Asp Gly Val Arg Asp
325 330 335
Ala Val Glu His Ile Asn Arg Tyr Gly Ser His His Ser Asp Ser Ile
340 345 350
Ile Thr Glu Asp Lys Arg Ala Arg Asp Tyr Phe Met Asn Arg Val Asp
355 360 365
Ser Ala Cys Val Tyr His Asn Cys Ser Thr Arg Phe Ser Asp Gly Glu
370 375 380
Glu Phe Gly Phe Gly Ala Glu Ile Gly Ile Gly Thr Asp Lys Phe His
385 390 395 400
Ala Arg Gly Lys Met Ala Leu Arg Glu Leu Thr Ser Tyr Lys Tyr Val
405 410 415
Ile Glu Gly Asn Gly Gln Leu Lys Glu Pro Ser Arg Ile Pro Gly Glu
420 425 430
Ser Arg Ala
435
<210> 6
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Leu Val Pro Arg Gly Ser Met Asn Gln Ala Pro His Val Ala Ile Val
1 5 10 15
Gly Ala Thr Gly Ala Val Gly Val Glu Ile Leu Ser Cys Leu Glu Thr
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Arg Asn Phe Pro Val Gly Ser Leu Lys Leu Leu Ala Ser Ala Arg Ser
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Ala Gly Lys Gln Val Ala Phe Arg Gly Lys Met Leu Thr Val Glu Glu
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Leu Thr Glu Lys Ser Phe Asp Gly Val Asp Ile Ala Leu Phe Ser Ala
65 70 75 80
Gly Gly Gly Ile Ser Leu Lys Phe Ala Pro Ile Ala Ala Ala Ala Gly
85 90 95
Cys Val Val Ile Asp Asn Ser Ser Ala Phe Arg Gln Glu Pro Asp Val
100 105 110
Pro Leu Val Val Pro Glu Ile Asn Pro Glu Ala Ala Phe Asn His Pro
115 120 125
Arg Asn Ile Ile Ala Asn Pro Asn Cys Thr Thr Ile Ile Thr Leu Met
130 135 140
Ala Leu Phe Pro Leu His Gln Arg Phe Gly Leu Lys Thr Val Ile Ala
145 150 155 160
Ser Ser Tyr Gln Ala Val Ser Gly Ser Gly Gln His Gly Ile Ala Glu
165 170 175
Leu Glu Thr Gln Val Arg Ala Val Val Asp Gly His Pro Val Val Lys
180 185 190
Asn Val Tyr Pro His Gln Ile Ala Phe Asn Leu Leu Pro Gln Ile Asp
195 200 205
Ser Phe Thr Glu Asn Gly Tyr Thr Lys Glu Glu Leu Lys Met Leu Asn
210 215 220
Glu Gly Arg Lys Ile Leu Ser Leu Pro Glu Leu Lys Val Thr Cys Thr
225 230 235 240
Cys Val Arg Val Pro Val Tyr Arg Ser His Ser Ile Ser Val Thr Ala
245 250 255
Gln Phe Glu Lys Pro Val Asp Leu Glu Thr Ala Arg Ser Ala Tyr Glu
260 265 270
Gly Lys Pro Gly Val Ala Leu Met Asp Asn Pro Ala Glu Gly Val Trp
275 280 285
Pro Thr Pro Leu Asp Ser Thr Asn Gly Asp Thr Cys Tyr Val Gly Arg
290 295 300
Met Arg Met Asp Met Ala Ile Asp Asn Ala Leu Thr Leu Trp Val Val
305 310 315 320
Gly Asp Gln Val Arg Lys Gly Ala Ala Leu Asn Ala Val Gln Ile Ala
325 330 335
Lys Leu Leu Val Asn Arg
340
<210> 7
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Met Gly Gly Met Lys Ala Leu Val Lys Thr Gln Ala Gly Pro Gly Leu
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Glu Leu Met Asp Val Pro Met Pro Glu Val Gly Pro Asn Asp Val Leu
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Ile Lys Ile His Lys Thr Ala Ile Cys Gly Thr Asp Leu His Ile Trp
35 40 45
Asn Trp Asp Lys Trp Ala Gln Gln Thr Ile Pro Val Gly Met His Val
50 55 60
Gly His Glu Phe Cys Gly Val Ile Glu Ser Val Gly Ser Ser Val Thr
65 70 75 80
Glu Tyr Lys Pro Gly Glu Ile Val Ser Gly Glu Gly His Ile Val Cys
85 90 95
Gly His Cys Arg Ser Cys Arg Ser Gly Gln Lys His Leu Cys Pro Asn
100 105 110
Thr Lys Gly Val Gly Val Asn Arg Pro Gly Cys Phe Ala Glu Tyr Leu
115 120 125
Ser Ile Pro Gln Asp Asn Val Val Arg Ile His Lys Ser Ile Pro Met
130 135 140
Glu Ile Ala Ser Ile Phe Asp Pro Leu Gly Asn Ala Val His Thr Ala
145 150 155 160
Leu Ser Trp Asp Leu Val Gly Glu Asp Val Leu Ile Thr Gly Ala Gly
165 170 175
Val Ile Gly Cys Met Ala Ala Ala Val Cys Lys Lys Ala Gly Ala Lys
180 185 190
Thr Val Val Ile Thr Asp Ile Asn Asp Phe Arg Leu Gly Leu Ala Lys
195 200 205
Thr Leu Gly Ala Asp Arg Thr Val Asn Val Thr Arg Glu Lys Leu Glu
210 215 220
Asp Val Met Lys Glu Leu Glu Met Thr Glu Gly Phe Asp Val Cys Leu
225 230 235 240
Glu Met Ser Gly Ala Pro Ser Cys Leu Lys Asp Ile Ile Asp Asn Ser
245 250 255
Arg Asn Gly Ala Asn Ile Ser Leu Leu Gly Ile Gln Pro Asp Gly Ser
260 265 270
Ser Ile Glu Trp Asn Lys Phe Ile Trp Lys Gly Leu Lys Met Lys Gly
275 280 285
Ile Tyr Gly Arg Glu Ile Phe Glu Thr Trp His Lys Met Asp Ser Met
290 295 300
Ile Arg Ser Gly Leu Asn Val Ala Pro Ile Ile Thr His Arg Leu Pro
305 310 315 320
Tyr Thr Glu Phe Arg Glu Gly Phe Glu Ala Met Asn Ser Gly Lys Ser
325 330 335
Gly Lys Val Val Leu Asp Trp Ile Val
340 345
<210> 8
<211> 373
<212> PRT
<213> Ackermans mucin (Akkermansia muciniphila)
<400> 8
Met Gln Gln Thr Ala Arg Ala Ala Val Leu Thr Ala Pro Lys Thr Phe
1 5 10 15
Glu Ile Arg Glu Tyr Pro Ile Pro Ala Ile Gly Asp Asp Glu Met Leu
20 25 30
Ile Lys Val Glu Ala Cys Gly Val Cys Gly Thr Asp Gly His Glu Tyr
35 40 45
Asn Arg Asp Pro Phe Gly Leu Cys Pro Val Val Leu Gly His Glu Gly
50 55 60
Thr Gly Glu Ile Val Ala Met Gly Arg Asn Ile Thr Lys Asp Thr Ala
65 70 75 80
Gly Asn Pro Val Ala Leu Gly Asp Lys Ile Val Thr Cys Ile Ile Pro
85 90 95
Cys Gly Thr Cys Asp Ala Cys Leu Asn Thr Pro Ala Arg Thr Asn Leu
100 105 110
Cys Glu Asn Val Gly Val Tyr Gly Leu Met Pro Asp Asp Asp Val His
115 120 125
Leu Asn Gly Tyr Phe Gly Glu Tyr Leu Val Ile Arg Lys Gly Ser Thr
130 135 140
Phe Phe Asn Val Ser Gly Met Thr Leu Asp Gln Arg Ile Leu Val Glu
145 150 155 160
Pro Ala Ala Val Val Val His Ser Leu Glu Arg Ala Lys Ser Thr Gly
165 170 175
Leu Leu Lys Phe Asn Ser Val Val Leu Val Gln Gly Cys Gly Pro Ile
180 185 190
Gly Leu Leu Gln Ile Ala Thr Leu Arg Thr Leu Gly Ile Glu Thr Ile
195 200 205
Ile Ala Val Asp Gly Asn Asp Ser Arg Leu Glu Leu Ala Arg Glu Met
210 215 220
Gly Ala Ser Arg Thr Tyr Asn Phe Thr Arg Tyr Ala Asp Leu Asn Glu
225 230 235 240
Leu Leu Asp Ala Val Lys Lys Asp Asn Gly Gly Arg Leu Ala Asp Phe
245 250 255
Val Phe Gln Cys Thr Gly Val Gly Lys Ala Gly Ala Asn Ala Trp Lys
260 265 270
Phe Val Lys Arg Gly Gly Gly Leu Cys Glu Val Gly Phe Phe Met Asp
275 280 285
Gly Gly Glu Ser Val Ile Asn His His Tyr Asp Leu Cys Asn Lys Glu
290 295 300
Val Thr Ala Val Gly Ser Trp Val Tyr Ser Pro Gln Asp Tyr Pro Thr
305 310 315 320
Thr Phe Asp Phe Leu Lys Arg Ala Tyr Gly Ile Gly Leu Pro Leu Thr
325 330 335
Lys Leu Ile Ser His Arg Phe Lys Leu Asp Glu Ile Ala Glu Ala Leu
340 345 350
Glu Thr Asn Val Gln Met Lys Gly Ile Lys Ile Ala Val Ile Cys Asn
355 360 365
Cys Ser Lys Asn Ile
370
<210> 9
<211> 333
<212> PRT
<213> Ackermans mucin (Akkermansia muciniphila)
<400> 9
Met Ser Glu Asn His Tyr Ala Glu Phe Ser Glu Cys Ser Met Lys Pro
1 5 10 15
Gln Glu Val Leu Glu Tyr Val Ser Gly Pro Ile Pro Val Pro Glu Glu
20 25 30
Gly Glu Val Leu Val Arg Met Lys Ala Ala Pro Ile Asn Pro Ala Asp
35 40 45
Ile Asn Phe Val Gln Gly Val Tyr Gly Leu Lys Pro Val Leu Pro His
50 55 60
Ser Arg Ala Gly Leu Glu Gly Cys Gly Val Val Gln Glu Ser Arg Ala
65 70 75 80
Ala Gly Phe Arg Glu Gly Asp Glu Val Ile Leu Leu Arg Gly Val Gly
85 90 95
Ser Trp Ser Glu Tyr Val Ala Val Pro Ser Val Asn Val Met Lys Leu
100 105 110
Pro Val Lys Val Asp Pro Val Gln Ala Ala Met Leu Lys Val Asn Pro
115 120 125
Leu Thr Ala Leu Arg Met Leu Glu Gly Phe Val Ser Leu Glu Pro Gly
130 135 140
Asp Trp Leu Val Gln Asn Ala Ala Asn Ser Gly Val Gly Arg Cys Ile
145 150 155 160
Ile Gln Leu Ala Arg Glu Met Gly Val Lys Thr Val Asn Phe Val Arg
165 170 175
Arg Pro Asp Glu Leu Arg Asp Glu Leu Thr Ala Leu Gly Ala Asp Leu
180 185 190
Val Val Gly Glu Asp Asp Gly Asp Val Val Lys Asn Thr Leu Ala Arg
195 200 205
Leu Asp Gly Lys Arg Pro Val Leu Ala Ser Asn Ala Val Gly Gly Glu
210 215 220
Ser Ala Leu Arg Leu Met Asp Met Leu Ala Pro Gly Gly Ser Met Val
225 230 235 240
Thr Tyr Gly Ala Met Ser Arg Lys Ser Ile Lys Val Pro Asn Gly Phe
245 250 255
Leu Ile Phe Lys Gly Ile Lys Leu Glu Gly Leu Trp Val Thr Gln Trp
260 265 270
Leu Lys Asn Ala Pro Val Ser Glu Ile Glu Ala Ala Tyr Glu Lys Leu
275 280 285
Ala Arg Leu Met Ala Asp Gly Arg Leu Lys Gln Ala Val Asp Thr Val
290 295 300
Tyr Pro Leu Ser Asp Val Arg Lys Ala Val Glu Lys Ala Gln Glu Glu
305 310 315 320
Phe Arg Ser Gly Lys Lys Val Leu Ser Met Asp Cys Ala
325 330
<210> 10
<211> 1193
<212> PRT
<213> Ackermans mucin (Akkermansia muciniphila)
<400> 10
Met Thr Asp Ser Ser Ile Pro Asp Met Met Ala Glu Ala Arg Arg Gly
1 5 10 15
Gln Trp Thr Asp Gln Gln Leu Ala Ala Lys Ala Val Glu Leu Ala Glu
20 25 30
Ser Ile Leu Lys Gln Ser Asn Ala Gly Met Arg Gly Lys Glu Lys Arg
35 40 45
Gln Ala Gln Gln Met Glu Arg Met Met Asn Asp Pro Ala Gly Lys Ala
50 55 60
Phe Thr Leu Ala Leu Ala Asp Arg Val Phe Arg Pro Ser Ser Pro Val
65 70 75 80
Arg Gly Ala Glu Leu Phe Arg Tyr Leu Leu Asp Gly Tyr Gly Val Pro
85 90 95
Arg Tyr Leu Ser Ala Ala Asp Arg Phe Ala Ile Lys Met Gly Gly Arg
100 105 110
Phe Ser Ala Gln Phe Pro Gly Val Val Met Pro Val Ile Thr Ser Gln
115 120 125
Leu Arg Lys Glu Ser Ser Asn Val Ile Leu Pro Ala Glu Asp Gly Lys
130 135 140
Leu Arg Pro His Leu Arg Arg Arg Arg Lys Gly Gly Ile Arg Met Asn
145 150 155 160
Ile Asn Gln Leu Gly Glu Ala Ile Leu Gly Glu Ser Glu Ala His His
165 170 175
Arg Leu Gln Gln Val Val Asp Arg Leu Thr Asp Lys Asp Cys Asp Tyr
180 185 190
Ile Ser Val Lys Ile Ser Ala Ile Phe Ser Gln Ile His Leu Val Ala
195 200 205
Phe Glu Glu Thr Val Lys Leu Ile Gln Glu Arg Leu Arg Ile Leu Tyr
210 215 220
Arg Ala Ala Ile Thr Asn Ala Val Thr Leu Pro Asp Gly Ser Arg Lys
225 230 235 240
Pro Lys Phe Val Asn Leu Asp Met Glu Glu Tyr Arg Asp Leu His Leu
245 250 255
Thr Ala Glu Ala Phe Lys Arg Thr Leu Met Glu Asp Glu Phe Met Gln
260 265 270
Leu Glu Ala Gly Ile Val Leu Gln Ala Tyr Leu Pro Asp Ser Trp Glu
275 280 285
Glu Gln Met Lys Leu Cys Ala Trp Ala Lys Glu Arg Val Glu Gln Gly
290 295 300
Gly Ala Arg Ile Lys Ile Arg Leu Val Lys Gly Ala Asn Leu Ala Met
305 310 315 320
Glu Lys Val Glu Ala Ser Met His Gly Trp Ala Gln Ala Pro Tyr Gly
325 330 335
Thr Lys Ala Gln Val Asp Ala Asn Tyr Lys Arg Met Leu His Tyr Gly
340 345 350
Cys Met Pro Asp Asn Ala Arg Tyr Val Gln Phe Gly Val Ala Ser His
355 360 365
Asn Leu Phe Asp Leu Cys Tyr Ala Met Leu Leu Arg Glu Arg Glu Gly
370 375 380
Val Arg Asp Gln Val Glu Phe Glu Met Leu Glu Gly Met Ala Asn His
385 390 395 400
Gln Ala Arg Val Ile Arg Gln Ala Ala Asp Gly Leu Leu Leu Tyr Ala
405 410 415
Pro Val Val Leu Lys Glu Asp Phe His Ser Ala Ile Ala Tyr Leu Val
420 425 430
Arg Arg Leu Asp Glu Asn Thr Ser Glu Glu Asn Phe Leu His Asp Leu
435 440 445
Phe Gly Met Thr Pro Gly Ser Arg Ser Trp Glu Val Gln Lys Lys Arg
450 455 460
Phe Leu Lys Ala Cys Gln Glu Lys Asp Glu Val Lys Tyr Gly Pro Asn
465 470 475 480
Arg Thr Gln Asn Arg Ala Ala Asp Pro Ile Gln Pro Ser His Tyr Arg
485 490 495
Asp Ala Phe Ala Asn Glu Arg Asp Thr Asp Trp Ser Leu Arg Gln Asn
500 505 510
Ala Glu Trp Ile Asn Gly Leu Ile Ala Ala Glu Lys Glu Lys Ser Gly
515 520 525
Glu Glu Ile Pro Leu Val Ile Asp Gly Glu Glu Ile Thr Thr Asn Leu
530 535 540
Trp Gly Val Gly Arg Asp Pro Ser Arg His Asn Glu Val Ser Tyr Lys
545 550 555 560
Phe Ala Tyr Ala Asp Phe Asp Gln Val Glu His Ala Leu Val Thr Ala
565 570 575
Asp Arg Val Arg Ser Ser Trp Ala Ser Lys Ser Ile Gly Glu Arg Ala
580 585 590
Glu Ile Leu His Arg Ala Ala Gln Glu Leu Ser Arg Ile Arg Gly Glu
595 600 605
Ala Ile Ala Ala Met Val Arg Asp Ala Gly Lys Ala Pro Thr Glu Ala
610 615 620
Asp Val Glu Val Ser Glu Ala Ile Asp Phe Cys Arg Tyr Tyr Ala Glu
625 630 635 640
Gly Leu Asp Arg Asp Gly Met Asn Asp Gly Val Glu Met Ser Pro Leu
645 650 655
Gly Ile Ile Cys Val Met Ser Pro Trp Asn Phe Pro Phe Ala Ile Pro
660 665 670
Thr Gly Gly Val Ala Ala Ala Leu Met Ala Gly Asn Ala Val Val Phe
675 680 685
Lys Pro Ser Glu Leu Ala Val Tyr Thr Ala Trp Gln Ile Val Gln Ala
690 695 700
Phe Trp Arg Ala Gly Val Pro Lys Asn Val Leu Gln Phe Val Pro Met
705 710 715 720
Pro Arg Asn Glu Ile Ser Cys Lys Phe Leu Met Asp Pro Arg Leu Asn
725 730 735
Gly Val Ile Met Thr Gly Ser Tyr Arg Thr Gly Lys Met Leu Arg Glu
740 745 750
Leu Arg Pro Asp Leu His Val Leu Ala Glu Thr Ser Gly Lys Asp Ala
755 760 765
Met Ile Ile Thr Ala Thr Ala Asp Pro Asp Gln Ala Val Lys Asp Leu
770 775 780
Val Lys Ser Ala Phe Gly His Ser Gly Gln Lys Cys Ser Ala Ala Ser
785 790 795 800
Val Ala Ile Val Glu Ala Ser Val Tyr Asp Asn Pro Ala Phe Leu Arg
805 810 815
Gln Leu Lys Asp Ala Ala Ala Ser Leu Lys Val Gly Gly Ser Trp Glu
820 825 830
Val Asn Ser Val Val Thr Pro Leu Ile Arg Glu Pro Glu Gly Asn Leu
835 840 845
Leu Arg Ala Leu Thr Gln Leu Glu Pro Gly Glu Glu Trp Leu Leu Lys
850 855 860
Pro Glu Pro Ser Glu Asp Asn Pro Cys Leu Trp Ser Pro Gly Ile Arg
865 870 875 880
Leu Gly Val Lys Pro Gly Ser Trp Phe His Gln Thr Glu Cys Phe Gly
885 890 895
Pro Val Leu Gly Ile Ile Arg Ala Glu Asn Leu Glu Glu Ala Ile Asp
900 905 910
Ile Gln Asn Asp Ser Glu Phe Gly Leu Thr Gly Gly Leu Gln Ser Leu
915 920 925
Asp Glu Arg Glu Ile Ala Leu Trp Lys Thr Lys Val Gln Val Gly Asn
930 935 940
Ala Tyr Ile Asn Arg Val Ile Thr Gly Ala Ile Val Arg Arg Gln Pro
945 950 955 960
Phe Gly Gly Trp Asn His Ser Ser Met Gly Pro Gly Ala Lys Ala Gly
965 970 975
Gly Glu Asn Tyr Leu Thr Met Leu Gly Ser Trp Glu Glu Lys Ala Leu
980 985 990
Pro Gln Lys Leu Arg Thr Pro Gly Glu Arg Ile Ser Gly Leu Val Glu
995 1000 1005
Lys Leu Cys Ser Glu Leu Pro Asp Cys Ala Lys Arg Ile Arg Ser Ala
1010 1015 1020
Ala Gly Ser Gln Ala Lys Trp Trp Met Glu Glu Phe Gly Val Asp His
1025 1030 1035 1040
Asp Pro Ser Arg Val Tyr Gly Glu Asn Asn Thr Phe Arg Tyr Ile Pro
1045 1050 1055
Val Lys Gly Ile Leu Ala Arg Val Glu Asn Met Ser Asp Asp Asp Val
1060 1065 1070
Ala Ile Leu Leu Leu Gly Ala Lys Leu Cys Gly Val Leu Leu His Leu
1075 1080 1085
Ser Ile Gly Thr Ser Arg Pro Trp Ile Gln Lys Met His Gly Tyr Tyr
1090 1095 1100
Ala Ser Leu Thr Val Glu Thr Glu Ala Glu Leu Ile Gly Arg Met Pro
1105 1110 1115 1120
Glu Ala Leu Pro Gly Ile Arg Phe Leu Arg Gly Thr Asp Ile Ser Glu
1125 1130 1135
Thr Leu Ala Asn Ala Ala Arg Ala Arg Asp Val Glu Val Leu Asp Arg
1140 1145 1150
Pro Val Leu Ala Asn Gly Arg Leu Glu Leu Leu Gly Tyr Phe Arg Glu
1155 1160 1165
Gln Ser Val Ser Glu Thr Val His Arg Tyr Gly Asn Leu Ile Pro Pro
1170 1175 1180
Pro Gly Ser Phe Lys Thr Asp Ser Val
1185 1190
<210> 11
<211> 429
<212> PRT
<213> Ackermans mucin (Akkermansia muciniphila)
<400> 11
Met Lys Met Glu Asn Pro Leu Glu Pro Ala Gly Arg Ala Ala Leu Ala
1 5 10 15
Ala Ser Arg Glu Met Leu Ala Leu Glu Pro Glu Glu Lys Ala Gln Ala
20 25 30
Leu Arg Leu Met Ala Ala Gly Val Arg Asn Ala Ala Glu Lys Ile Ile
35 40 45
Arg Glu Asn Val Leu Asp Met Glu Glu Ala Arg Lys Ser Gly Arg Asn
50 55 60
Ser Ala Phe Leu Asp Arg Leu Leu Leu Thr Pro Asp Arg Val Glu His
65 70 75 80
Met Ala Cys Gly Ile Glu Gln Val Ala Ala Leu Pro Asp Pro Val Gly
85 90 95
Glu Cys Ile Arg Glu Trp Thr Arg Pro Asn Gly Leu Arg Ile Arg Gln
100 105 110
Val Arg Val Pro Leu Gly Val Ile Gly Phe Ile Tyr Glu Ser Arg Gly
115 120 125
Thr Val Thr Cys Asp Ala Ala Ala Leu Cys Leu Lys Ser Gly Asn Ala
130 135 140
Val Ile Leu Arg Gly Gly Ser Glu Ser Leu Arg Thr Asn Arg Val Leu
145 150 155 160
Ala Glu Thr Leu Arg Ser Ala Leu Glu Lys Ser Ala Val Pro Ala Asp
165 170 175
Ala Val Arg Leu Leu Asp Ser Gly Ser Arg Asp Glu Val Arg Gln Leu
180 185 190
Cys Glu Leu Asp Ser Cys Leu Asn Val Ile Ile Pro Arg Gly Gly Lys
195 200 205
Gly Leu Ile Arg Ala Val Thr Glu Phe Ser Lys Val Pro Val Leu Lys
210 215 220
His Leu Asp Gly Ile Cys His Val Tyr Val Asp Ala Ala Ala Asp Leu
225 230 235 240
Glu Met Ala Val Asn Val Leu Asp Asp Ala Lys Thr Gln Arg Pro Gly
245 250 255
Val Cys Asn Ala Ala Glu Thr Leu Leu Val Asp Ala Ala Ala Ala Glu
260 265 270
Arg Phe Leu Pro Met Ala Ala Glu Arg Met Arg Met Arg Gly Val Glu
275 280 285
Cys Arg Val Cys Glu Arg Ser Arg Pro Phe Phe Gly Gln Glu Ala Val
290 295 300
Pro Ala Glu Glu Arg Asp Trp Ser Thr Glu Tyr Glu Asp Leu Ile Leu
305 310 315 320
Ser Val Lys Val Val Asp Gly Val Arg Asp Ala Val Glu His Ile Asn
325 330 335
Arg Tyr Gly Ser His His Ser Asp Ser Ile Ile Thr Glu Asp Lys Arg
340 345 350
Ala Arg Asp Tyr Phe Met Asn Arg Val Asp Ser Ala Cys Val Tyr His
355 360 365
Asn Cys Ser Thr Arg Phe Ser Asp Gly Glu Glu Phe Gly Phe Gly Ala
370 375 380
Glu Ile Gly Ile Gly Thr Asp Lys Phe His Ala Arg Gly Glu Met Ala
385 390 395 400
Leu Arg Glu Leu Thr Ser Tyr Lys Tyr Val Ile Glu Gly Asn Gly Gln
405 410 415
Leu Lys Glu Pro Ser Arg Ile Pro Gly Glu Ser Arg Ala
420 425
<210> 12
<211> 336
<212> PRT
<213> Ackermans mucin (Akkermansia muciniphila)
<400> 12
Met Asn Gln Ala Pro His Val Ala Ile Val Gly Ala Thr Gly Ala Val
1 5 10 15
Gly Val Glu Ile Leu Ser Cys Leu Glu Thr Arg Asn Phe Pro Val Gly
20 25 30
Ser Leu Lys Leu Leu Ala Ser Ala Arg Ser Ala Gly Lys Gln Val Ala
35 40 45
Phe Arg Gly Lys Met Leu Thr Val Glu Glu Leu Thr Glu Lys Ser Phe
50 55 60
Asp Gly Val Asp Ile Ala Leu Phe Ser Ala Gly Gly Gly Ile Ser Leu
65 70 75 80
Lys Phe Ala Pro Ile Ala Ala Ala Ala Gly Cys Val Val Ile Asp Asn
85 90 95
Ser Ser Ala Phe Arg Gln Glu Pro Asp Val Pro Leu Val Val Pro Glu
100 105 110
Ile Asn Pro Glu Ala Ala Phe Asn His Pro Arg Asn Ile Ile Ala Asn
115 120 125
Pro Asn Cys Thr Thr Ile Ile Thr Leu Met Ala Leu Phe Pro Leu His
130 135 140
Gln Arg Phe Gly Leu Lys Thr Val Ile Ala Ser Ser Tyr Gln Ala Val
145 150 155 160
Ser Gly Ser Gly Gln His Gly Ile Ala Glu Leu Glu Thr Gln Val Arg
165 170 175
Ala Val Val Asp Gly His Pro Val Val Lys Asn Val Tyr Pro His Gln
180 185 190
Ile Ala Phe Asn Leu Leu Pro Gln Ile Asp Ser Phe Thr Glu Asn Gly
195 200 205
Tyr Thr Lys Glu Glu Leu Lys Met Leu Asn Glu Gly Arg Lys Ile Leu
210 215 220
Ser Leu Pro Glu Leu Lys Val Thr Cys Thr Cys Val Arg Val Pro Val
225 230 235 240
Tyr Arg Ser His Ser Ile Ser Val Thr Ala Gln Phe Glu Lys Pro Val
245 250 255
Asp Leu Glu Thr Ala Arg Ser Ala Tyr Glu Gly Lys Pro Gly Val Ala
260 265 270
Leu Met Asp Asn Pro Ala Glu Gly Val Trp Pro Thr Pro Leu Asp Ser
275 280 285
Thr Asn Gly Asp Thr Cys Tyr Val Gly Arg Met Arg Met Asp Met Ala
290 295 300
Ile Asp Asn Ala Leu Thr Leu Trp Val Val Gly Asp Gln Val Arg Lys
305 310 315 320
Gly Ala Ala Leu Asn Ala Val Gln Ile Ala Thr Leu Leu Val Asn Arg
325 330 335

Claims (9)

1. A protein composition, wherein the protein composition is composed of a first type of recombinant protein and a second type of recombinant protein;
the first recombinant protein consists of a polypeptide with an amino acid sequence shown as SEQ ID NO: 1. SEQ ID NO:2 and SEQ ID NO:3, a protein composition shown in the formula;
the second kind of recombinant protein consists of a polypeptide with an amino acid sequence shown as SEQ ID NO: 4. SEQ ID NO:5 and SEQ ID NO: 6.
2. Use of a protein composition for the preparation of a product for metabolising ethanol;
the protein composition consists of a first type of recombinant protein and a second type of recombinant protein;
the first recombinant protein consists of a polypeptide with an amino acid sequence shown as SEQ ID NO: 1. SEQ ID NO:2 and SEQ ID NO:3, a protein composition shown in the formula;
the second kind of recombinant protein consists of a polypeptide with an amino acid sequence shown as SEQ ID NO: 4. SEQ ID NO:5 and SEQ ID NO: 6.
3. The use according to claim 2, wherein the first recombinant protein is used to metabolize ethanol to acetaldehyde;
the second recombinant protein is used for metabolizing the acetaldehyde obtained into acetic acid.
4. Use of a host expressing the protein composition of claim 1 in the preparation of a product for metabolizing ethanol.
5. A nucleic acid vector comprising a backbone vector and a polynucleotide encoding the protein composition of claim 1.
6. A recombinant host expressing the protein composition of claim 1, wherein the recombinant host is a microorganism or cell line; the recombinant host expresses the protein composition of claim 1.
7. A product comprising at least one of the following components:
a. a protein composition comprising a first class of recombinant proteins and a second class of recombinant proteins;
the first recombinant protein consists of a polypeptide with an amino acid sequence shown as SEQ ID NO: 1. SEQ ID NO:2 and SEQ ID NO:3, a protein composition shown in the formula;
the second kind of recombinant protein consists of a polypeptide with an amino acid sequence shown as SEQ ID NO: 4. SEQ ID NO:5 and SEQ ID NO: 6;
b. a host expressing the protein composition of a;
c. b a fermentation product of said host;
d. b said host and/or c an extract of said fermentation product;
e. the protein composition containing a and other substances with biological activity for metabolizing alcohol.
8. Use of the product of claim 7 for the preparation of an anti-hangover medicament.
9. A pharmaceutical composition comprising as active ingredient the protein composition of claim 1, and optionally a pharmaceutically acceptable carrier, excipient, adjuvant and/or diluent.
CN202210197701.3A 2022-03-02 2022-03-02 Protein composition and use thereof Active CN114621936B (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1706479A (en) * 2004-06-08 2005-12-14 美国酶医公司 Methods and compositions for accelerating alcohol metabolism

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JP7033655B2 (en) * 2017-09-28 2022-03-10 コバイオラブス，インコーポレーテッド Composition for diagnosis and treatment of alcoholic liver disease using changes in short-chain fatty acid-producing intestinal bacterial community

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Publication number Priority date Publication date Assignee Title
CN1706479A (en) * 2004-06-08 2005-12-14 美国酶医公司 Methods and compositions for accelerating alcohol metabolism

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Alcohol dehydrogenase zinc-binding domain protein [Akkermansia muciniphila ATCC BAA-835];无;GenBank: ACD05015.1;全文 *
Alcohol dehydrogenase zinc-binding domain protein [Akkermansia muciniphila ATCC BAA-835];无;GenBank: ACD05925.1;全文 *
Aldehyde Dehydrogenase [Akkermansia muciniphila ATCC BAA-835];无;GenBank: ACD05876.1;全文 *
aspartate-semialdehyde dehydrogenase [Akkermansia muciniphila ATCC BAA-835];无;GenBank: ACD05090.1;全文 *
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