阅读说明：本技术 Amuc1100蛋白的用途 (Application of Amuc1100 protein ) 是由 张晓洁 陈体 傅潇雅 杨铖 李云飞扬 文杰 于 2020-05-06 设计创作，主要内容包括：本发明涉及医药技术领域,尤其涉及Amuc1100蛋白的用途。本发明研究表明Amuc1100蛋白或表达Amuc1100蛋白的宿主具有改善情绪记忆的作用,包括促进负性情绪记忆的消退和/或增强正性情绪记忆。能够用于制备成瘾戒断的药物中的应用,或在制备治疗焦虑症、强迫症、自闭症、痴呆、精神分裂症中至少一种的药物中的应用。(The invention relates to the technical field of medicines, in particular to application of an Amuc1100 protein. The research of the invention shows that the Amuc1100 protein or the host expressing the Amuc1100 protein has the effect of improving emotional memory, including promoting the regression of negative emotional memory and/or enhancing positive emotional memory. Can be used for preparing drugs for addiction withdrawal or for preparing drugs for treating at least one of anxiety disorder, obsessive-compulsive disorder, autism, dementia and schizophrenia.)

Application of Amuc1100 protein in preparation of products for improving emotional memory;

the amino acid sequence of the Amuc1100 protein is shown as SEQ ID NO. 1, or a derivative protein which is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown as SEQ ID NO. 1 and has the activity of the Amuc1100 protein.

2. Application of a host expressing the Amuc1100 protein in preparation of a product for improving emotional memory.

3. Use according to claim 3, wherein the host expressing the Amuc1100 protein is a microorganism of the Verrucomicrobia family or a recombinant host expressing the Amuc1100 protein.

4. Use according to claim 3, wherein the microorganism of the Verrucomicrobia family is Akkermansia Muciniphila.

5. The use according to any one of claims 1 to 4, wherein the improvement of emotional memory comprises promoting regression of negative emotional memory and/or enhancing positive emotional memory;

the causes of negative emotional memory include: major illness, catastrophic time, social phobia, spatial location phobia;

positive emotional memory includes positive emotional memory that satisfies psychophysiological needs for health, safety, food, love, sexual pleasure, and the like.

6. A nucleic acid vector comprising a backbone vector and a polynucleotide encoding an Amuc1100 protein.

7. A recombinant host for expressing the Amuc1100 protein, which is characterized in that the host is a microorganism or a cell strain; the amino acid sequence of the Amuc1100 protein is shown as SEQ ID NO. 1, or a derivative protein which is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown as SEQ ID NO. 1 and has the activity of the Amuc1100 protein.

8. A product comprising at least one of the following components:

I) the Amuc1100 protein of the amino acid sequence shown as SEQ ID NO. 1;

II) derivative protein which is substituted, deleted or added with one or more amino acids in the amino acid sequence shown in SEQ ID NO. 1 and has the activity of the Amuc1100 protein;

III), a host expressing the protein of I) or II);

IV), III) fermentation products of said host;

v), III) an extract of said host and/or of said fermentation product;

VI), other substances with biological activity that improve emotional memory.

9. Use of a product according to claim 8 for the manufacture of a medicament for addiction withdrawal or for the manufacture of a medicament for the treatment of at least one of anxiety, obsessive compulsive disorder, autism, dementia and schizophrenia.

10. A method of addiction withdrawal or of treatment of anxiety, obsessive compulsive disorder, autism, dementia, schizophrenia or addiction withdrawal comprising administering a product according to claim 7.

Technical Field

The invention relates to the technical field of medicines, in particular to application of an Amuc1100 protein.

Background

The enhancement of positive emotional memory capacity and negative emotional memory regression capacity is important for the adaptability of the organism, and the damage is a sign of emotional memory disorders such as posttraumatic stress disorder (PTSD), generalized anxiety disorder, depression, social phobia and spatial phobia. Studies suggest that fear responses and depressed mood are relieved when a new positive emotional memory is developed to suppress the initial negative emotional memory. If the subject is re-exposed to the previous conditioned stimulus without the expected negative unconditional stimulus after establishing the conditioned fear response according to pavlov's conditioned reflex principle, the previously established conditioned fear response will gradually disappear after repeated exposure, a process known as the regression of negative emotional memory. Further studies demonstrated that the function encoding negative emotional memory regression developed and was stored in a unique Basal Lateral Amygdala (BLA) neuronal population that antagonized BLA's original neurons encoding negative emotional memory by driving the behavior of reward memory. In BLAs, neurons coding for negative emotional memory regression function and neurons coding for reward memory (food, love, sexual, and psychoactive substances) overlap significantly. Furthermore, there is a reciprocal phenomenon of the function of these two neuronal subpopulations in driving the behavior of treats and negative mood-abolishing behavior.

Many studies in recent years have shown that the intestinal flora can influence the learning and memory process via the "brain-gut axis". Research finds that intestinal flora influences the memory forming process, such as: mice infected with Citrobacter murinus show impaired memory function after acute stress exposure, and early administration of probiotics inhibits this impaired memory function. Intervention of probiotics can improve the spatial learning and memory function of diabetic animals. Regression of fear memory was inhibited in either the sterile animals or the antibiotic-treated mice. Clinical studies report that healthy volunteers can remarkably promote the memory function of healthy volunteers when taking the bifidobacterium 1714 strain.

Akkermansia Muciniphila Akkermansia mucinuphila (a. mucinuphila) is a common anaerobic bacterium in the intestinal tract, with a relative abundance of about 3% to 5%. It was found by genomic sequencing that many genes encoding mucin-degrading enzymes (more than 61, 11% of the total genes) were present in the genomic sequence thereof. Proteomics analysis found that there are a large number of enzymes in human feces that are used by this bacterium to degrade gastrointestinal mucin, such as: glycosidases, sulfatases, neuraminidases, and the like. The Amuc1100 protein is an outer membrane protein of AAK, the presence of a cluster of genes associated with pili formation suggests that it may be involved in host interactions. Studies have demonstrated that Amuc _1100 can signal cells expressing TLR2 in a similar manner to akkermansia, and furthermore, Amuc _1100 exhibits relative thermostability, and a role in signal transduction can likewise be observed. Also, recent studies have found that Amuc _1100 may be involved in regulating metabolism and fighting obesity to alleviate type ii diabetes.

The research shows that the strain is obviously related to the effectiveness of PD-1 in treating cancer, diabetes, type 2 obesity and other metabolic immune diseases, however, no research or report about the association between the strain and positive emotional memory or negative emotional memory exists in the field.

Disclosure of Invention

In view of the above, the technical problem to be solved by the present invention is to provide an application of the Amuc1100 protein to improve emotional memory, i.e., an application of the Amuc1100 protein to a medicine or a health care product for enhancing positive emotional memory and simultaneously promoting regression of negative emotional memory.

The invention provides application of an Amuc1100 protein in preparation of a product for improving emotional memory;

the amino acid sequence of the Amuc1100 protein is shown as SEQ ID NO. 1, or a derivative protein which is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown as SEQ ID NO. 1 and has the activity of the Amuc1100 protein.

In the invention, the Amuc1100 protein with the amino acid sequence shown as SEQ ID NO. 1 is from Akkermansia Cinciniphila (AKK), the AKK is a strict anaerobic enterobacteria separated from feces, belongs to Microbactera verruculosa, and can grow by using intestinal mucoprotein as a unique carbon source and nitrogen source, and the main metabolite is propionic acid. Analysis of microorganisms in human intestinal tracts by sequencing methods revealed that at least 8 of these strains have greater than 95% similarity to a. muciniphila and are known as Akkermansia-like microorganisms (microbiological report [ J ],2017,44, 1458-1463). Ouwerkerk et al isolated and cultured a new strain having 94.4% similarity to the AKK gene sequence from the boa snake in vivo and found that they also had similar properties, and named the microorganism Akkermansia glycerapila (DOI: 10.1099/ijsem.0.001399).

The research of the invention shows that the Amuc1100 protein or the strain expressing the Amuc1100 protein can improve the emotional memory.

In some embodiments, the amino acid sequence of the Amuc1100 protein is shown as SEQ ID No. 1, or a derivative protein with the activity of the Amuc1100 protein obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown as SEQ ID No. 1.

Application of a host expressing the Amuc1100 protein in preparation of a product for improving emotional memory.

In the present invention, the host is a natural host or a recombinant host prepared by genetic engineering means. Wherein the native host is a microorganism which is present in nature and which is capable of expressing the Amuc1100 protein, for example, the host expressing the Amuc1100 protein is a microorganism of the Verrucomicrobia family; in some embodiments, the microorganism of the Verrucomicrobia family is Akkermansia Cinniphilia. It is to be understood that the Akkermansia Muciniphila of the present invention includes not only Akkermansia muccinifila but also microorganisms having more than 90% similarity in nucleic acid sequence, particularly greater than 95% similarity to a.

In other embodiments, the host is a recombinant host expressing the Amuc1100 protein. For example, by means of genetic engineering, a host that does not express the Amuc1100 protein is allowed to express the Amuc1100 protein, or a host that expresses the Amuc1100 protein at a low level is allowed to produce a larger amount of the Amuc1100 protein. The genetic engineering means include, but are not limited to, ZFNs, TALENs, or CRISPR/Cas 9; such hosts include, but are not limited to, bacteria, fungi, plant cells, or animal cells. The bacteria are selected from Escherichia coli, in the examples of the present invention, Escherichia coli BL21(DE3) is used. The mammalian cell is selected from the HEK293 cell line.

In a recombinant host, the amino acid sequence of the Amuc1100 protein is shown in SEQ ID NO. 1, or a derivative protein which is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO. 1 and has the activity of the Amuc1100 protein. The derivative protein which is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO. 1 and has the activity of the Amuc1100 protein can be a sequence of the Amuc1100 after a signal peptide is removed. In some embodiments, the amino acid sequence is set forth in SEQ ID NO 2.

In the construction process of the recombinant host, in order to enable the Amuc1100 protein to be more smoothly expressed, the invention carries out codon optimization aiming at different hosts. Thus, the invention also provides codon-optimized polynucleotides. In the present invention, the polynucleotide sequence expressing the amino acid sequence shown in SEQ ID NO. 2 is shown in SEQ ID NO. 3.

The invention also provides a nucleic acid vector for constructing the recombinant host. The invention provides a nucleic acid vector, which comprises a skeleton vector and a polynucleotide for coding an Amuc1100 protein. In some embodiments, the backbone vector is pET-28a or pcDNA4a (+).

In some embodiments, the gene sequence encoding Amuc1100 after removal of the signal peptide is amplified by PCR, cloned into pET-28a vector by enzymatic ligation, constructing an expression plasmid of Amuc1100 with an N-terminal fused 6 xhis tag, heterologously expressing using escherichia coli engineering bacteria BL21(DE3) strain cultured overnight on an LB + KanR resistant plate, selecting monoclonal antibodies, inoculating in 5ml of LB medium containing 100 μ g/ml KanR, culturing at 37 ℃ and 180rpm for 4 hours, transferring 1:100 to a new medium at mid-exponential phase (0D600 ═ 0.6), culturing at 180rpm for 4 hours, adding IPTG to a final concentration of 0.5-2mM, and inducing at 28 ℃ and 180rpm overnight. The bacterial liquid was centrifuged at 8,000rpm for 5 minutes to precipitate cells, and the cells were stored at-20 ℃.

The invention also provides a recombinant host for expressing the Amuc1100 protein, wherein the host is a microorganism or a cell strain; the amino acid sequence of the Amuc1100 protein is shown as SEQ ID NO. 1, or a derivative protein which is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown as SEQ ID NO. 1 and has the activity of the Amuc1100 protein. In the embodiment of the invention, the host is Escherichia coli. In particular to an engineering bacterium BL21 of escherichia coli (DE 3).

In the embodiment of the invention, thallus is uniformly mixed in 10 times volume of ultrasonic lysis solution, 4ml of PMSF containing lysozyme, DNase/RNase and 1mM of final concentration is added, the mixture is uniformly mixed, the mixture is placed on ice for ultrasonic lysis and fragmentation for 90min until the solution is thoroughly clear, the cell lysis solution is centrifuged at 12,000rpm, 30min and 4 ℃, supernatant is collected and placed on ice to obtain Amuc1100 recombinant protein, the Amuc1100 protein is obtained through Ni-16 NTAHis-Bind resin affinity chromatography purification, and the Amuc1100 protein is subpackaged and stored at-80 ℃ for later use to prepare the proper concentration.

The invention also provides a product comprising at least one of the following components:

I) the Amuc1100 protein of the amino acid sequence shown as SEQ ID NO. 1;

II) derivative protein which is substituted, deleted or added with one or more amino acids in the amino acid sequence shown in SEQ ID NO. 1 and has the activity of the Amuc1100 protein;

III), a host expressing the protein of I) or II);

IV), III) fermentation products of said host;

v), III) an extract of said host and/or of said fermentation product;

VI), other substances with biological activity that improve emotional memory.

The product of the invention is a medicine, food or health product.

The invention also provides application of the product in improving emotional memory.

In the present invention, the improvement of emotional memory includes promoting regression of negative emotional memory and/or enhancing positive emotional memory;

the causes of negative emotional memory include: major illness, catastrophic time, social phobia, spatial location phobia;

positive emotional memory includes positive emotional memory that satisfies psychophysiological needs for health, safety, food, love, sexual pleasure, and the like.

The product of the invention is applied to the preparation of drugs for addiction withdrawal or the preparation of drugs for treating at least one of anxiety disorder, obsessive-compulsive disorder, autism, dementia and schizophrenia.

The medicine provided by the invention comprises the product and pharmaceutically common auxiliary materials.

The pharmaceutically acceptable adjuvants are one or more of fruit powder, edible essence, sweetener, sour agent, bulking agent, lubricant, antiseptic, suspending agent, edible pigment, diluent, emulsifier, disintegrating agent or plasticizer.

The dosage form is tablet, pill, oral liquid, capsule, syrup, drop pill or granule.

In some embodiments provided herein, the capsule is a hard or soft capsule.

In some embodiments provided herein, the tablet is an oral tablet or buccal tablet.

Oral tablets refer to tablets intended for oral administration, most of which act by absorption through the gastrointestinal tract, and some of which act locally in the gastrointestinal tract. In some embodiments provided herein, the oral tablet is a compressed tablet, a dispersible tablet, an effervescent tablet, a chewable tablet, a coated tablet, or a sustained release tablet.

In the present invention, the withdrawal of addiction includes withdrawal of addiction caused by ***e, methamphetamine and opioids.

The invention also provides a method of addiction withdrawal or a method of treatment of anxiety, obsessive compulsive disorder, autism, dementia, schizophrenia by administering a product according to the invention.

It is verified that in the conditioned place preference rat model induced by psychoactive substances (such as methamphetamine), the psychoactive substances do not have a significant influence on the diversity of intestinal flora or the relative abundance of AKK, but compared with the low-sensitivity individuals, the individuals with high reward sensitivity to psychoactive substances and the relative environmental memory capacity of the psychoactive substances have a significant increase in the relative abundance of AKK, and the individuals with higher reward sensitivity to psychoactive substances and the relative environmental memory capacity of the psychoactive substances are positively correlated with each other, so that the psychoactive substances can be applied to withdrawal treatment of the psychoactive substances, such as: making into medicine or health product.

After subjecting the rats treated with broad-spectrum antibiotics to a methamphetamine-induced conditioned place preference test, we found that the antibiotic-treated rats had significantly increased susceptibility to psychoactive substance reward and their associated environmental cues of memory as compared to the control group. Compared with the control group, the diversity and the total abundance of intestinal flora of rats treated by the broad-spectrum antibiotic are obviously reduced, while the relative abundance of AKK is obviously increased.

In the conditioned fear experiment, in the rat group with the obvious increase of the relative abundance of AKK, the process of the regression of the fear memory shows obvious accelerated regression. The difference of spatial learning and memory abilities is evaluated by adopting the water maze, the basic activity or anxiety level is evaluated by an open field experiment, and no obvious difference is found among groups.

Any one of the products of the invention has the functions of promoting the increase of positive emotional memory experience and simultaneously improving emotional memory (enhancing positive emotional memory and simultaneously promoting the regression of negative emotional memory), and can be developed into a microbial preparation or used as an additive for preparing a health-care product.

Situations that elicit positive emotional memory include: there are many situations in which psychophysiological needs are satisfied due to health, safety, food, love, sexual pleasure, etc.

Situations that arise with negative emotional memory include: the symptoms of the disease can be caused by major diseases (such as anxiety, depression, obsessive compulsive disorder, autism, dementia, schizophrenia and the like), disastrous events, addiction to substances (such as but not limited to ***e, methamphetamine, opioids and the like), social phobia, spatial fear, anorexia, paranoia and the like, and the combination of the symptoms.

The research of the invention shows that the Amuc1100 protein or the host expressing the Amuc1100 protein has the effect of improving emotional memory, including promoting the regression of negative emotional memory and/or enhancing positive emotional memory. Can be used for preparing drugs for addiction withdrawal or for preparing drugs for treating at least one of anxiety disorder, obsessive-compulsive disorder, autism, dementia and schizophrenia.

Drawings

FIG. 1 is a graph showing the results of Conditioned Place Preference (CPP) induced by Methamphetamine (MA), wherein the p-values shown in the graph are examined by Student's t, wherein A is a CPP value of rats in a saline solution group and an MA-exposed group, and B is a graph dividing the MA-exposed group into a high CPP (H-CPP) group and a low CPP (L-CPP) group by the CPP value;

FIG. 2 is the difference between the fecal microbial populations of the L-CPP and H-CPP groups after the CPP training, wherein the A diagram is a box diagram of the phylum, family and genus levels with significant difference between the intestinal microbial populations of the L-CPP group and the H-CPP group, p <0.05 and p <0.01, which is in accordance with Mann-Whitney U test, the B diagram is a histogram of LDA Effect Size (LEfSe) analysis, and a Linear Discriminant Analysis (LDA) score indicates the Size and ranking of the influence of each difference category, and a threshold value of 2 is used;

FIG. 3 is the correlation analysis result of CPP value and relative abundance of partial strains after CPP training;

FIG. 4 shows the results of AKK changes from pre-test period to test period in the physiological saline group, L-CPP group and H-CPP group; a is the relative abundance and change condition of AKK in CPP pre-testing and testing stages, and B is the difference value of the relative abundance of AKK in the testing stage compared with that in the pre-testing stage;

FIG. 5 is a result of antibiotic intervention in gut flora; figure a shows that antibiotics can significantly improve CPP values in MA-induced CPP experiments in rats; the graph B shows that intestinal flora diversity can be obviously changed by antibiotic intervention MA addicted rat intestinal flora, the sample alpha diversity result is displayed by a Chao index to reflect community abundance, the graph C shows part of the result of an LEfSe analysis histogram, and LDA score reflects the influence of flora with obvious difference in abundance among groups;

figure 6 effect of antibiotic intervention on gut flora in conditioned fear experiments; graphs a and C show the immobility time and percentage of immobility time during the development of conditional fear in rats, graphs B and D show the immobility time and percentage of immobility time during the resolution of conditional fear in rats, and the p values of graphs C and D were determined by calculating the area under the curve (AUC) for each rat followed by an inter-group Student's t test, < 0.01;

FIG. 7 effect of antibiotic intervention on intestinal flora on water maze experiments; graph a is the escape latency results for the hidden platform test in the water maze test, and graph B is the time of the rat in each quadrant for the exploration test;

FIG. 8 impact of antibiotic intervention on the open field experiment of intestinal flora; graph A is the percentage of the time length of the rat staying in the central area of the open field to the total time length, graph B is the percentage of the distance of the rat in the central area of the open field to the total distance, and graph C is the distance of the rat moving in the whole open field.

Detailed Description

The invention provides application of an Amuc1100 protein. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The test materials adopted by the invention are all common commercial products and can be purchased in the market.

The invention is further illustrated by the following examples:

the following examples of the present invention employ various species information as follows:

the source of akkermansia muciniphila (zheng zhou jinsi wei chemical limited), originally originated from (ATCC, BAA-835), the working seed lot was 5 generations.