CN1164614C - Memory clear protein and its application - Google Patents

Abstract

The present invention relates to a mammalian memory erasing protein gene and its coded product, and its use in promoting memory and forgetting. In addition, the present invention also relates to an antagonist containing the memory erasing protein and its coding gene, as well as pharmaceutical compositions and health products containing the above-mentioned substances.

Description

Memory clear protein and its application

The invention relates to the fields of bioengineering and medicine. More specifically, the present invention relates to mammalian hippyragranin gene and its encoded product, and its use in promoting memory and forgetting. In addition, the present invention also relates to an antagonist containing the memory clearing protein gene and its protein, as well as pharmaceutical compositions and health products containing the above substances.

Learning and memory are basic functions of the brain. In higher animals, damage to the hippocampus, a brain structure in the medial temporal lobe, prevents new memories from being formed. Synapses in the hippocampus are highly plastic. Hippocampal synapses can be induced to produce and maintain long-term potentiation (LTP). After the gene encoding any protein related to the induction or maintenance of LTP is knocked out (knockout), the hippocampus no longer has LTP. phenomena, and animals cannot form memories. Therefore, hippocampus is an important structure of learning and memory, and LTP is a synaptic mechanism of learning and memory, which has been accepted by neuroscientists.

Memory and forgetting are twin brothers. Memory ability is very important, but forgetting also has its rationality and necessity. Without forgetting, people would be overwhelmed by all kinds of remembered information, making it impossible to effectively deal with real life. In addition, although the capacity of the brain is extremely large, it may still not be large enough to store the information received by a person in a lifetime without forgetting. Also, in some cases, such as for certain painful memories, there needs to be a way to forget them. However, there is currently no effective way to promote memory forgetting.

In conclusion, so far little is known about the various proteins involved in or affecting memory. Therefore, there is an urgent need in this field to discover new proteins related to memory and to develop drugs that promote memory or forgetting.

Therefore, the object of the present invention is to provide a protein related to memory and forgetting-hippyragranin and its coding sequence, and its use in improving forgetting.

Another object of the present invention is to provide an antagonist of amnestic protein and its use in inhibiting amnesia.

Another object of the present invention is to provide pharmaceutical compositions and health products containing amnestic proteins, encoded proteins or antagonists.

In the first aspect of the present invention, an isolated amnestic protein polypeptide is provided, which comprises: a polypeptide having an amino acid sequence of SEQ ID NO: 2, or a conservative variant polypeptide thereof, or an active fragment thereof, or an active derivative thereof . Preferably, the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.

In a second aspect of the present invention there is provided an isolated polynucleotide comprising a nucleotide sequence having at least 70% identity to a nucleotide sequence selected from the group consisting of:

(a) a polynucleotide encoding the aforementioned amnestic protein polypeptide;

(b) A polynucleotide complementary to polynucleotide (a).

Preferably, the polynucleotide encodes a polypeptide having the amino acid sequence shown in SEQ ID NO:2. More preferably, the sequence of the polynucleotide is selected from the following group:

(a) has the sequence of positions 29-523 in SEQ ID NO: 1;

(b) having the sequence of positions 1-1920 in SEQ ID NO:1.

In the third aspect of the present invention, a vector containing the above polynucleotide and a host cell containing the vector are provided.

In a fourth aspect of the present invention, a method for preparing a polypeptide having amnestic activity is provided, the method comprising:

(a) cultivating the above-mentioned host cells under conditions suitable for expressing the amnestic protein;

(b) isolating a polypeptide having amnestic activity from the culture.

In the fifth aspect of the present invention, an antibody capable of specifically binding to the amnestic protein is provided.

In the sixth aspect of the present invention, a pharmaceutical composition is provided, which contains a safe and effective amount of the above-mentioned amnestic protein polypeptide, or the above-mentioned polynucleotide, and a pharmaceutically acceptable carrier. The present invention also provides a health care product, which contains the above-mentioned amnestic protein polypeptide or the above-mentioned polynucleotide.

In the seventh aspect of the present invention, there is provided a method for screening compounds that promote or inhibit amnesia, comprising the steps of:

(1) Load the cDNA of the amnestic protein gene into an expression vector, transfect a mammalian cell line, and prepare a cell line expressing the amnestic protein:

(2) Add test compound to the cell line culture fluid expressing amnestic protein in step (1), detect the change of amnestic protein expression level; The compound that promotes the increase of amnesic protein expression level is exactly the compound that promotes forgetting, suppresses memory Compounds that increase the expression of scavenging proteins are compounds that inhibit amnesia.

In an eighth aspect of the present invention, a method for judging individual memory is provided, which comprises the steps of:

detecting the sequence of the amnestic gene, transcript and/or protein in the individual and comparing it to the normal sequence of the amnestic gene, transcript and/or protein;

The presence of a sequence difference indicates that the individual has a higher memory than the normal population; or

or measuring the amount of amnestic transcript and/or protein in the individual and comparing it with normal levels of the amount of amnestic transcript and/or protein;

A higher than normal level indicates that the individual has a lower than normal group memory, and a lower than higher normal level indicates that the individual has a higher than normal group memory.

Preferably, the amnestic gene or transcript is detected, and the difference is compared with the normal amnestic nucleotide sequence.

In the ninth aspect of the present invention, a method for promoting amnesia is provided, comprising the step of: administering a safe and effective amount of normal amnestic protein to a subject who needs to forget. In addition, a method for inhibiting forgetting is also provided, comprising the step of: administering a safe and effective amount of an antagonist of amnestic protein to a subject in need of inhibiting forgetting. A typical antagonist is the antisense fragment of the amnestic protein gene (including the coding region and the non-coding region), and its length ranges from 10-1920bp or longer.

In the tenth aspect of the present invention, a kit for detecting the memory level of an individual is provided, which includes primers for specifically amplifying the amnesic gene or transcript, or an antibody specifically binding to the amnesic protein.

In the attached picture,

Figures 1A and 1B are the cDNA sequence and protein sequence of rat amnestic protein, respectively.

Figure 2 is the expression profile of rat amnestic protein mRNA. Lanes 1-8 and M are respectively: 1. hippocampus; 2. heart; 3. small intestine; 4. kidney; 5. liver; 6. lung; 7. skeletal muscle; 8. spleen; and M. molecular weight standard.

Figure 3 shows the expression pattern of the amnestic gene in adult rats.

Figure 4 shows that local injection of amnestic protein gene antisense sequence nucleic acid in the LV region of rats can significantly improve the performance of rats in the behavioral task of the water maze. Wherein, Fig. 4A has shown the time that rat finds platform required in training process, each group (every group of 12) the average value (mean ± SEM) of 8 times in two days, Fig. 4B has shown rats find in each training stage The average number of successful platforms (mean ± SEM), Figure 4C is a top-view roadmap of different mice finding the platform, and the "○" in the figure indicates the location of the hidden platform. ^* , p<0.05.

Figure 5 shows that local injection of amnestic gene antisense sequence nucleic acid in the CA1 region of rats can significantly improve the performance of rats in the behavioral task of the water maze. Wherein, Fig. 5 A has shown the time that rat finds platform required in training process, each group (every group of 12) the average value (mean ± SEM) of 8 times in two days, Fig. 5 B has shown rats find in each training stage The mean (mean ± SEM) of the number of platform successes. ^* , p<0.05.

Figure 6 shows that after local injection of antisense nucleic acid gene of amnestic protein, the long-term memory of rats is significantly enhanced. Among them, Figures 6A and 6B respectively show the memory of scene fear after local injection of amnestic protein antisense nucleic acid in CA1 and LV regions.

After extensive and in-depth research, the inventor not only cloned the amnestic protein cDNA sequence of rats, but also used different animal behavior models to study the function of the amnestic protein gene in the process of learning and memory. Surprisingly, amnestics were found to be involved in memory forgetting.

The present inventors obtained the differentially expressed amnestic protein in denervated hippocampus and normal hippocampus by screening the sub-library of lowly expressed genes in rats by using differential expression screening technology. The complete sequence determination included the clone of the amnestic protein gene, and it was found that it contained the full length cDNA of 1920bp, including the start codon and PolyA. The putative amnestic protein comprises a total of 165 amino acid residues (Figures 1A and 1B). After homology comparison, it is known that the gene is a brand new gene.

The expression pattern of the memory clear protein gene in rats was studied. RT-PCR results showed that in addition to expression in the hippocampus, the amnestic protein gene was expressed in many other tissues, such as the small intestine, heart, kidney, spleen, and lung; no amnesia was detected in the liver and muscle Expression of protein genes. The expression of amnesic protein gene in various tissues is not the same, its expression is the highest in the small intestine, the expression is the weakest in the lung, and the expression in the hippocampus, heart, kidney and spleen is relatively close (Figure 2). Using the in situ hybridization technique of brain slices, the specific distribution of the memory erasing protein gene in the adult rat brain was studied in detail. The results showed that the memory erasing protein gene mRNA was mainly expressed in the hippocampus, and there was a very weak expression in the granule cells of the cerebral cortex. No expression of this gene was detected in other regions of the brain. In the hippocampus, the region of mRNA expression of the amnestic protein gene was restricted to neurons in the CA1, CA3, and DG neuronal cell layers (Fig. 3).

Using rats as experimental animals, the function of the memory erasing protein gene was tested through behavioral experimental models such as water maze (Water Maze) and scene fear (Fear-conditioning to Context). Targeted injection of amnestic gene antisense nucleic acid into hippocampal CA1 and LV regions, it was found that rat amnestic gene antisense nucleic acid can significantly promote the learning and memory ability of rats (Fig. 4, 5, and 6).

The structure of mammalian amnestic proteins and the specificity of tissue distribution suggest that amnestic proteins have the same function in different mammals, that is, to promote forgetting. Therefore, human amnesiac is also related to human forgetting, and the drugs and diagnosis and treatment techniques designed according to the amnesiac gene and its expression products can be used to diagnose and treat amnesia in humans, and can improve memory and motor coordination.

As used herein, the terms "amnestic protein" and "amnestic protein polypeptide" are used interchangeably to refer to a protein that is expressed specifically in the hippocampus of mammals and has an amino acid sequence similar to that of human, rat or mouse amnestic protein Polypeptides with more than 70%, preferably more than 80%, more preferably 90%, and most preferably more than 95% homology. The amnestic proteins in different mammals can be obtained through routine experiments based on the sequence information disclosed in the present invention. In addition, active fragments, conserved polypeptides, or active derivatives of amnestic proteins can be used in the present invention.

As used herein, "isolated" means that the material is separated from its original environment (if the material is native, the original environment is the natural environment). For example, polynucleotides and polypeptides in the natural state in living cells are not isolated and purified, but the same polynucleotides or polypeptides are isolated and purified if they are separated from other substances that exist together in the natural state .

As used herein, "isolated amnestic protein or polypeptide" refers to an amnestic protein polypeptide that is substantially free of other proteins, lipids, carbohydrates, or other substances with which it is naturally associated. Those skilled in the art can purify amnestic proteins using standard protein purification techniques. Substantially pure polypeptides yield a single major band on non-reducing polyacrylamide gels.

The polypeptide of the present invention can be a recombinant polypeptide, a natural polypeptide, a synthetic polypeptide, preferably a recombinant polypeptide. Polypeptides of the present invention may be naturally purified, or chemically synthesized, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insect and mammalian cells). Depending on the host used in the recombinant production protocol, the polypeptides of the invention may be glycosylated, or may be non-glycosylated. Polypeptides of the invention may or may not include an initial methionine residue.

The invention also includes fragments, derivatives and analogs of amnestic proteins. As used herein, the terms "fragment", "derivative" and "analogue" refer to a polypeptide that substantially retains the same biological function or activity of the native amnestic protein of the present invention. The polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues It may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a mature polypeptide in combination with another compound (such as a compound that extends the half-life of the polypeptide, e.g. polyethylene glycol), or (iv) an additional amino acid sequence fused to the polypeptide sequence (such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or with Formation of fusion proteins of antigen IgG fragments). Such fragments, derivatives and analogs are within the purview of those skilled in the art in light of the teachings herein.

In the present invention, the term "amnestic polypeptide" refers to a polypeptide having the sequence of SEQ ID NO. 2 having amnestic activity. The term also includes variants of the sequence of SEQ ID NO. 2 that have the same function as amnestics. These variations include (but are not limited to): deletions and insertions of several (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acids and/or substitution, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminal and/or N-terminal. For example, in the art, substitutions with amino acids with similar or similar properties generally do not change the function of the protein. As another example, adding one or several amino acids at the C-terminus and/or N-terminus usually does not change the function of the protein. The term also includes active fragments and active derivatives of amnestic proteins.

Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, encoded by DNA that can hybridize with memory erasing protein DNA under high or low stringency conditions Proteins, and polypeptides or proteins obtained using antiserum against amnestic polypeptides. The invention also provides other polypeptides, such as fusion proteins comprising amnestic polypeptides or fragments thereof. In addition to substantially full-length polypeptides, the present invention also includes soluble fragments of amnestic polypeptides. Typically, the fragment has at least about 10 contiguous amino acids, usually at least about 30 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 80 contiguous amino acids of the amnestic polypeptide sequence. About 100 consecutive amino acids.

The invention also provides analogs of amnestic proteins or polypeptides. The difference between these analogues and natural amnestic polypeptides may be the difference in amino acid sequence, or the difference in the modified form which does not affect the sequence, or both. These polypeptides include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, but also by site-directed mutagenesis or other techniques known in molecular biology. Analogs also include analogs with residues other than natural L-amino acids (eg, D-amino acids), and analogs with non-naturally occurring or synthetic amino acids (eg, β, γ-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

Modified (usually without altering primary structure) forms include: chemically derivatized forms of polypeptides such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from polypeptides that are modified by glycosylation during synthesis and processing of the polypeptide or during further processing steps. Such modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylation enzyme. Modified forms also include sequences with phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that have been modified to increase their resistance to proteolysis or to optimize solubility.

In the present invention, "amnesic protein conservative variant polypeptide" refers to amino acid sequence of SEQ ID NO: 2 at most 10, preferably at most 8, more preferably at most 5, most preferably at most Three amino acids are replaced by amino acids with similar or similar properties to form a polypeptide. These conservative variant polypeptides are preferably produced by amino acid substitutions according to Table 1.

Table 1 initial residue representative replacement preferred substitution Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala His(H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Leu

A polynucleotide of the invention may be in the form of DNA or RNA. Forms of DNA include cDNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be either the coding strand or the non-coding strand. The coding region sequence encoding the mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant. As used herein, "degenerate variant" in the present invention refers to a nucleic acid sequence that encodes a protein with SEQ ID NO: 2, but differs from the sequence of the coding region shown in SEQ ID NO: 1.

A polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: a coding sequence encoding only the mature polypeptide; a coding sequence for the mature polypeptide and various additional coding sequences; a coding sequence for the mature polypeptide (and optional additional coding sequences) and non-coding sequence.

The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may also include additional coding and/or non-coding sequences.

The present invention also relates to variants of the above-mentioned polynucleotides, which encode polypeptides or polypeptide fragments, analogs and derivatives having the same amino acid sequence as the present invention. Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is an alternative form of a polynucleotide which may be a substitution, deletion or insertion of one or more nucleotides without substantially altering the function of the polypeptide it encodes .

The present invention also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The invention particularly relates to polynucleotides which are hybridizable under stringent conditions to the polynucleotides of the invention. In the present invention, "stringent conditions" refers to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with There are denaturing agents, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, etc.; or (3) only if the identity between the two sequences is at least 90%, more Preferably, hybridization occurs above 95%.

The present invention also relates to nucleic acid fragments that hybridize to the above-mentioned sequences. As used herein, a "nucleic acid fragment" is at least 15 nucleotides in length, preferably at least 30 nucleotides in length, more preferably at least 50 nucleotides in length, most preferably at least 100 nucleotides in length. Nucleic acid fragments can be used in nucleic acid amplification techniques (eg, PCR) to identify and/or isolate polynucleotides encoding amnestic proteins. And the antisense nucleic acid fragment can be used as an antagonist of amnestic protein to inhibit forgetting and promote memory.

The full-length sequence of amnestic protein nucleotides or its fragments can usually be obtained by PCR amplification, recombination or artificial synthesis. For the PCR amplification method, primers can be designed according to the nucleotide sequences of known amnestic proteins, mouse amnestic proteins or rat amnestic proteins disclosed in the present invention, especially the open reading frame sequence, and commercially available A cDNA library or a cDNA library prepared by a conventional method known to those skilled in the art is used as a template to amplify to obtain related sequences. When the sequence is long, it is often necessary to carry out two or more PCR amplifications, and then splice together the amplified fragments in the correct order.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. Usually, it is cloned into a vector, then transformed into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods.

In addition, related sequences can also be synthesized by artificial synthesis, especially when the fragment length is relatively short. Often, fragments with very long sequences are obtained by synthesizing multiple small fragments and then ligating them.

At present, the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

The present invention also relates to vectors containing the polynucleotides of the present invention, host cells produced by genetic engineering using the vectors or amnestic protein coding sequences of the present invention, and methods for producing the polypeptides of the present invention through recombinant techniques.

The polynucleotide sequences of the present invention can be used to express or produce recombinant amnestic polypeptides by conventional recombinant DNA techniques (Science, 1984; 224:1431). Generally speaking, there are the following steps:

(1). Transform or transduce a suitable host cell with the polynucleotide (or variant) encoding the amnestic protein polypeptide of the present invention, or with a recombinant expression vector containing the polynucleotide;

(2). Host cells cultured in a suitable medium;

(3). Isolate and purify protein from culture medium or cells.

Specifically, the amnesiac protein of the present invention can be expressed by introducing the corresponding coding sequence into the host cell (directly or by introducing a vector containing the amnesiac protein coding sequence), and culturing the transformed host cell under appropriate conditions to express the memory Clear the protein, and then isolate and purify the amnestic protein.

The obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. The medium used in the culture can be selected from various conventional media according to the host cells used. The culture is carried out under conditions suitable for the growth of the host cells. After the host cells have grown to an appropriate cell density, the selected promoter is induced by an appropriate method (such as temperature shift or chemical induction), and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method can be expressed inside the cell, or on the cell membrane, or secreted outside the cell. The recombinant protein can be isolated and purified by various separation methods by taking advantage of its physical, chemical and other properties, if desired. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, osmotic disruption, supertreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

Specifically, the amnestic protein or polypeptide of the present invention has multiple uses. These uses include (but are not limited to): promoting amnesia, and for screening antibodies, polypeptides or other ligands that promote amnestic protein function. Screening a polypeptide library with expressed recombinant amnestics can be used to find therapeutically valuable polypeptide molecules that can inhibit or stimulate the function of amnestics.

On the other hand, the present invention also includes polyclonal antibodies and monoclonal antibodies, especially monoclonal antibodies, specific for amnestic protein DNA or polypeptides encoded by its fragments. Here, "specificity" means that the antibody is capable of binding to the amnestic gene product or fragment. Preferably, it refers to those antibodies that can bind to amnestic protein gene products or fragments but do not recognize and bind to other irrelevant antigenic molecules. Antibodies of the present invention include those molecules capable of binding to and inhibiting amnestic proteins, as well as those antibodies that do not affect the function of amnestic proteins.

The invention includes not only intact monoclonal or polyclonal antibodies, but also immunologically active antibody fragments, such as Fab' or (Fab) ₂ fragments; antibody heavy chains; antibody light chains; or chimeric antibodies, such as with murine antibodies Antibodies that bind specificity but retain the antibody portion from humans.

Antibodies of the present invention can be prepared by various techniques known to those skilled in the art. For example, purified amnestic protein gene products, or antigenic fragments thereof, can be administered to animals to induce polyclonal antibody production. Similarly, cells expressing amnestic proteins or antigenic fragments thereof can be used to immunize animals to produce antibodies. The monoclonal antibodies of the present invention can be produced using hybridoma technology. Various types of antibodies of the present invention can be obtained by using fragments or functional regions of the amnestic protein gene product through conventional immunization techniques. These fragments or functional regions can be prepared using recombinant methods or synthesized using a polypeptide synthesizer. Antibodies that bind the unmodified form of the amnestic gene product can be produced by immunizing animals with the gene product produced in prokaryotic cells (e.g., E. protein or polypeptide), which can be obtained by immunizing an animal with a gene product produced in a eukaryotic cell (eg, yeast or insect cell).

Antibodies against amnestic protein can be used in immunohistochemical techniques to detect amnestic protein in biopsy specimens.

For the production of polyclonal antibodies, animals such as rabbits and sheep can be immunized with amnestic proteins or polypeptides. Various adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant and the like.

Using the protein of the present invention, substances that interact with the memory erasure protein, such as inhibitors, agonists or antagonists, can be screened out through various conventional screening methods. At the time of screening, the amnestic protein can be added to a bioanalytical assay to determine whether the compound is an antagonist by determining its effect on the interaction between the amnestic protein and its receptor. Alternatively, a test compound can be administered to experimental animals together with amnestics, and changes in the animal's ability to remember or forget as compared to controls indicate that the compound is an agonist or antagonist of amnestics.

In addition, the cDNA of the amnesic protein gene can also be loaded into an expression vector, transfected into a mammalian cell line, and a cell line with high expression of amnesic protein can be prepared: and the amnesic protein in the cell line can be used as the target site to screen for Drugs that activate or inhibit memory proteins. A test compound is added to the culture medium of the cell line expressing the amnesic protein to detect the change of the expression level of the amnesic protein. A compound that promotes the expression of amnestics is a compound that promotes forgetting, while a compound that inhibits the expression of amnestics can be used as a compound that inhibits forgetting and promotes memory.

When the protein of the present invention and its antibody, inhibitor, agonist, antagonist, etc. are administered (administered) therapeutically, various effects can be provided. Generally, these materials can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is usually about 5-8, preferably about 6-8, although the pH value can be changed according to the Depending on the nature of the substance formulated and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intramuscular, intravenous, subcutaneous, oral, or topical administration.

Normal amnestic peptides can be used directly in the treatment of diseases, for example, to promote forgetting in the treatment of trauma and the wish to forget unpleasant memories. When using the antagonist of the amnestic protein gene or protein (such as antisense nucleic acid fragment) of the present invention, memory can be promoted and forgetting can be inhibited.

The present invention also provides a pharmaceutical composition, which contains a safe and effective amount of the amnestic protein of the present invention, its encoding nucleic acid, and/or antisense nucleic acid, and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to: saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of injection, for example, by conventional methods using physiological saline or aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods. Pharmaceutical compositions such as injections, solutions, tablets and capsules are preferably manufactured under sterile conditions. The active ingredient is administered in a therapeutically effective amount, for example about 0.01 microgram/kg body weight to about 5 mg/kg body weight per day. In addition, the polypeptides of the invention can also be used with other therapeutic agents.

When using the pharmaceutical composition, a safe and effective amount of amnestic protein or its antagonist, agonist is administered to the mammal, wherein the safe and effective amount is usually at least about 0.01 μg/kg body weight, and in most cases no more than about 10 mg/kg body weight, preferably the dosage is about 0.01 microgram/kg body weight to about 100 microgram/kg body weight. Of course, factors such as the route of administration and the health status of the patient should also be considered for the specific dosage, which are within the skill of skilled physicians.

Polynucleotides of amnestic proteins are also useful for various therapeutic purposes. Gene therapy technology can be used to treat inability to forget due to non-expression of amnestic protein or abnormal/inactive expression of amnestic protein, or memory loss caused by excessive expression or activity of amnestic protein. The method for constructing a recombinant viral vector carrying the amnestic protein gene can be found in existing literature (Sambrook, et al.). In addition, the recombinant amnesiac gene can be packaged into liposomes and then transferred into cells.

The methods for introducing polynucleotides into tissues or cells include: directly injecting polynucleotides into tissues in the body; or first introducing polynucleotides into cells in vitro through vectors (such as viruses, phages, or plasmids, etc.) , and then transplant the cells into the body, etc.

The invention also relates to a diagnostic test method for quantitative and localized detection of amnestic protein levels. These assays are well known in the art and include FISH assays and radioimmunoassays. The level of amnesic protein detected in the test can be used to explain the importance of amnesic protein in various diseases and to diagnose diseases in which amnesic protein acts.

A method for detecting the presence of amnestic protein in a sample is to use an antibody specific for amnesic protein for detection, which includes: contacting the sample with an antibody specific for amnesic protein; observing whether an antibody complex is formed, an antibody complex is formed It means that there is amnestic protein in the sample.

The polynucleotide of amnesic protein can be used for diagnosis and treatment of diseases related to amnesic protein. In terms of diagnosis, the polynucleotide of amnesic protein can be used to detect the expression of amnesic protein or the abnormal expression of amnesic protein in a disease state. For example, the amnestic protein DNA sequence can be used for hybridization of biopsy specimens to determine the abnormal expression of amnestic protein. Hybridization techniques include Southern blotting, Northern blotting, in situ hybridization, and the like. These technical methods are all open and mature technologies, and relevant kits are available from commercial sources. Part or all of the polynucleotides of the present invention can be immobilized as probes on microarrays or DNA chips (also known as "gene chips") for analysis of differential expression of genes in tissues and gene diagnosis. Transcripts of the amnesic gene can also be detected by RNA-polymerase chain reaction (RT-PCR) in vitro amplification using amnesic-specific primers.

Detection of mutations in the amnesin gene can also be used to diagnose amnestic-related disorders. The mutated form of amnesic protein includes point mutation, translocation, deletion, recombination and any other abnormality compared with the normal wild-type amnesic protein DNA sequence. Mutations can be detected using established techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression, so Northern blotting and Western blotting can be used to indirectly determine whether a gene has a mutation.

The amnestic protein and its antagonist of the present invention can not only be used for treatment, but also can be used for promoting or inhibiting the improvement of the forgetting ability of normal individuals. Therefore, the present invention also provides a health product, which contains mammalian amnestic protein or its coding sequence, or its antagonist (such as antisense nucleic acid). The health product of the present invention can be prepared by mixing mammalian amnestic protein or its coding sequence, or its antagonist (such as antisense nucleic acid) with a suitable diluent, food, etc. by conventional methods in the field of health care products . Preferred nutraceuticals are in the form of tablets, granules, orally.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods not indicating specific conditions in the following examples are usually according to conventional conditions such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's suggested conditions.

Example

Example 1

Acquisition of Rat Amnestic Protein cDNA Sequence

Female SD rats weighing about 250 g were cut off the penetrating fibers of the bilateral entorhinal cortex, anesthetized by intraperitoneal injection of pentobarbital sodium one month later, executed by decapitation, separated the hippocampus, removed the hippocampus feet and lower support, weighed, and put into liquid nitrogen. Store at -70°C, coded as (D). The control group was female rats of the same age, which were obtained in the same operation and numbered (N).

Denervated hippocampus (D) and normal hippocampus (N) mRNA were extracted by conventional methods. 2ug mRNA was reverse-transcribed into single-stranded DNA, and then the single-stranded DNA of denervated hippocampus (D) and normal hippocampus (N) were used as templates respectively, and probes were labeled with α- ³² P-dATP. Two probes were used to screen the low-abundance expression gene sub-library of rats constructed by conventional methods, and differentially expressed clones were selected for sequencing. Sequencing results showed that the product contained the full-length cDNA of the rat amnestic protein gene. The length of the cDNA is 1920bp, and the specific sequence is shown in SEQ ID NO:1. Wherein, the open reading frame (Open Reading Frame, ORF) is 29-523, and the encoded rat amnestic protein contains 165 amino acids (SEQ ID NO: 2).

The homologous comparison showed that the homology between memory erasure protein and other proteins was very low, and it was a new protein.

Example 2

Tissue Distribution of Memnestic Protein

On the basis of the known cDNA sequence, a pair of primers were designed, and the primer sequences were as follows: 5' primer 5'-cca gtt ctc tga acc act ga-3' (SEQ ID NO: 5) and 3' primer 5'-ctt ata tcc att ggt cct gc-3' (SEQ ID NO: 6), by RT-PCR method, to detect the distribution of amnestic protein in various tissues.

Total RNA was extracted from various tissues of rats, and then 2 μg of RNA was reverse transcribed into single-stranded cDNA (GIBCO, SuoerscriptII Kit). PCR was performed using 100ng RNA reverse transcription product as a template. The PCR conditions are as follows: annealing temperature 56° C., reaction cycle number 35 times, each cycle of denaturation for 1 minute, annealing for 1 minute, and extension for 1 minute. The fragment amplified by PCR was 299bp in length.

RT-PCR results showed that, in addition to the expression of rat amnesia in the hippocampus, the amnesia gene was expressed in many other tissues, such as the small intestine, heart, kidney, spleen and lung; there was no expression in the liver and muscle. Expression of the amnestic protein gene can be detected. The expression of amnesic protein gene in various tissues is not the same, its expression is the highest in the small intestine, the expression is the weakest in the lung, and the expression in the hippocampus, heart, kidney and spleen is relatively close (Figure 2).

Example 3

Distribution of memory clear protein in the brain

In situ hybridization: According to conventional methods, the animal brain tissue samples were fixed with paraformaldehyde and then made into paraffin-embedded sections. (Digoxin)-labeled probes were hybridized, and then the distribution of the target gene in the brain was detected by an alkaline phosphatase-linked anti-digoxigenin antibody.

The results of in situ hybridization showed that the gene of memory erasing protein was mainly expressed in the hippocampus, and there was a very weak expression in the granule cells of the cerebral cortex, and the expression of the gene could not be detected in other regions of the brain. In the hippocampus, the region of mRNA expression of the amnestic protein gene was restricted to neurons in the CA1, CA3, and DG neuronal cell layers (Fig. 3).

Example 4

Morris water maze experiment

1. Experimental equipment and environment:

Water maze: a black cylinder with a diameter of 150cm and a height of 54cm;

Platform: about 10cm in diameter, submerged in the water about 2cm, fixed in the center of a certain quadrant;

Camera and corresponding software: provided by San Diego Instrument, USA; the camera lens is suspended above the center of the water maze, and can be connected with the microcomputer through the camera system, and the microcomputer will record the swimming distance and trajectory of the rats in the water maze and record the time.

Environment: water depth, 0.34m; temperature 25±1°C; dye harmless to rats was added to the water, so that rats could not see the platform hidden under the water surface. The indoor temperature was controlled at around 27±1°C. Obvious marks were made on the surrounding walls so that the rats could recognize the direction according to the marks. During the experiment, all objects in the room were placed in a fixed position to avoid disturbance to the rats.

2. Experimental animals and nucleic acid sequences:

Male SD rats with a body weight of 200g-250g. Divided into three groups, 12 rats in the amnestic protein antisense nucleic acid sequence group; 12 rats in the random nucleic acid sequence group; and blank control group (12 rats).

Amnestic protein antisense nucleic acid sequence:

The amnesic protein antisense sequence nucleic acid can specifically block the expression of the amnesic protein (ie N4) mRNA. The molecular weight is 6056.0, containing 20 bases in total, wherein A=4, G=3, C=8, T=4, 5=0 (mixed base=0) base sequence is as follows:

5'>CCT CTG AGG CAA CTG CAC TC<3' (SEQ ID NO: 3)

The control sequence is a random sequence nucleic acid with the same base number and molecular weight as the antisense sequence nucleic acid, but it cannot block the expression of any gene in the brain. Its base composition is A=2, G=5, C=7, T=6, X=0 (mixed base=0), and the base sequence is as follows:

5'>TAG CTT CGG CTC GCT CGC TA<3' (SEQ ID NO: 4)

3. Surgery:

Catheters were implanted in the CA1 region of the bilateral hippocampus of the rats for administration of behavioral experiments. The rats were trained in the Morris water maze 7-12 days after surgery.

4. Administration:

Dose in CA1 area 6 hours before behavioral training. The concentration of amnestic protein-antisense sequence and random sequence was 0.25 nmol/μl, and the dose was 1.5 μl of LV region or CA1 region on each side.

5. Training method:

Rats were put into randomly from four positions of the water maze facing the cylinder wall in each training (but it was ensured that the rats had an equal chance of starting from each position). Let it search in the water for 60s, if it finds a platform, let it stay on the platform for 30s, then put it back into the cage to rest for 30s, and then proceed to the next training; if it does not find a platform, guide it artificially to the platform and stay for 60s After that, start the next training session. The training was carried out in two steps. After each rat had been trained for 6 consecutive times, it was rested for 1 hour, and then the next step of training was carried out. A total of 12 training times were performed. Record the rat's swimming route, speed and the time needed to find the platform.

6. Test method:

1. Test method of hidden platform:

The test was carried out 48 hours after the training, in order to detect the memory retention ability of the rats. The test was carried out 3 times in total, and each time the rat started from a fixed position and searched in the water for 60 seconds. If the platform is found within 60 seconds, put it back into the cage immediately, and start the next test after resting for 60 seconds; if the platform is not found within the specified 60 seconds, guide it to the platform and stay for 30 seconds, then put it back into the cage to rest 30s, then start the next test, and record the time and route needed for the rat to find the platform.

2. The test method of the exposed platform:

This test is performed after all tests are completed; the platform is out of the water. The purpose was to see if the rat had impairments in visual cognition that affected its ability to find the platform. The test was carried out 3 times in total, each time the rats started from a fixed position, but the position of the exposed platform was changed randomly. For each test, the rats were placed on the platform for 30 seconds to adapt, and then put into the water from a fixed position and searched for 60 seconds. If you find the platform within 60 seconds, put it back into the cage and rest for 60 seconds before starting the next test. If you don’t find the platform, take it out of the water, put it back in the cage and rest for 60 seconds before starting the next test, and record the time it takes to find the platform. time, route and speed.

7. Experimental results:

The results of injecting antisense sequences in the LV region are shown in Figures 4A-4C. The abscissa is the number of days of training, and the ordinate is the time required for the rats to find the platform (the average value of 3 training or testing). After 12 hours of bilateral injection of the antisense nucleic acid sequence of the amnestic protein gene in the LV region of the hippocampus, the spatial learning and memory processes of rats were significantly improved ( FIG. 4A ). Figure 4B shows that there is no significant difference in visual cognition among the three groups of rats. Figure 4C visually shows that rats injected with the antisense nucleic acid sequence of the amnestic protein gene have a shorter route to find the platform than the control group.

The results of injection of antisense sequences in the CA1 region are shown in Figures 5A-5B. The abscissa is the number of days of training, and the ordinate is the time required for the rats to find the platform (the average value of 3 training or testing). After 12 hours of injection of the antisense nucleic acid sequence of the amnestic protein gene in the CA1 region of the bilateral hippocampus, the spatial learning and memory process of the rat was significantly improved ( FIG. 4A ). Figure 4B shows that there is no significant difference in visual cognition among the three groups of rats.

Example 5

Scenario fear (Freezing) experiment

1. Equipment (provided by San Diego Instrument, USA)

Electric shock reaction box: the box wall is made of transparent plexiglass, length: 36cm; width: 23cm; height: 18cm. The bottom consists of 14 stainless steel tubes through which electricity can flow. The diameter of the stainless steel tube is 0.5 cm, and the distance between two stainless steel tubes is 1.2 cm.

Microcomputer and corresponding software: Infrared ray (photobeam) monitors the activity of rats, and the computer records them. Sounds and stimuli are generated by automatically matching the stimuli and sound generators through program control.

2. Experimental animals and medicines

(1) Experimental animals

Balb/c♂ mice (18-22g). Provided by the Experimental Animal Center. Before and after the operation, 3 mice per cage were given sufficient water and feed every day. The daily light time and dark time are natural light.

(2) Experimental drugs: same water maze experiment

3. Experimental steps

1) CA1 bilateral spraying: manual injection. Taking the Bregma point as the reference point, 2.1mm backward, 1.5mm sideways, the spraying depth is 1.5mm. The inner diameter of the injection tube is 0.2 mm, and 2 μl of drug is injected on each side (it takes about 3 minutes to inject, and then the needle is stopped for 2 minutes). The concentration of antisense and scramble is 0.25nmol/μl.

2) LV bilateral spraying and injection: The spraying site takes the Bregma point as the reference point, 1mm backward, 1mm sideways, and the spraying depth is 1.5mm. Others are sprayed with CA1 area.

3) Training After 20 hours of drug administration, start training. Put the mouse gently into the reaction box (pay attention not to put the mouse in a state of stress). After about 1 minute, start to give the sound, and the sound lasts for 10 seconds. When the sound lasted for 8s, the electric shock was started, the current intensity was 0.6mA, the electric shock time was 2s, and the electric shock was given again with the same sound after 30s. After 30s of electric shock, the test was performed for 30s, and the machine automatically recorded the time of immediate freezing. The less than standard is that the animal does not perform any activities other than the necessary breathing.

4) Test: test after 24 hours, 48 hours and 96 hours of training, put the mouse gently into the reaction box, detect for 3 minutes, and record the immobility time. The recording method and test standard are exactly the same as those of the immediate test.

1. Local injection in CA1 area

After local injection in CA1 area, the performance of rats in scene fear test. Immediately, 24 hours and 48 hours after the electric shock, the memory of the rats to the scene fear. Antisense nucleic acid group (n=12), Normal group (n=12), ^* , P<0.05.

The results are shown in FIG. 6A , after local injection of the antisense nucleic acid gene of amnestic protein, the long-term memory of rats was significantly enhanced.

2. Local injection in the LV area

After local injection in the LV area, the performance of the rats in the scene fear test. Immediately, 24 hours and 48 hours after the electric shock, the memory of the rats to the scene fear. Antisense nucleic acid group (n=12), Normal group (n=12), ^* , P<0.05.

The results are shown in FIG. 6B , after local injection of the antisense nucleic acid gene of amnestic protein, the long-term memory of rats was significantly enhanced.

Example 6

Expression of memory clear protein

1. Obtaining the coding sequence of amnestic protein by RT-PCR

A pair of primers were synthesized, wherein the 5' primer was: 5'-atg tgg aag cca aga cca a-3' (SEQ ID NO: 7) and the 3' primer 5'-tta aca ctt g tt ctt gct g-3' ( SEQ ID NO: 8). According to the same method in Example 3, the DNA sequence of the reading frame of the amnestic protein was obtained by RT-PCR, which was verified to be identical to the sequence at positions 29-526 in SEQ ID NO: 1 by sequencing.

2. Cloning and expression of amnestic protein

Design and synthesize a pair of primers, wherein the 5' primer is: 5'-ggg gta cca tgt gga agc caa gaccaa-3' (SEQ ID NO: 9) and the 3' primer 5'-ccc aag ctt aca ctt gtt ctt gct gaa g-3' (SEQ ID NO: 10). Using the RT-PCR product obtained in step (1) as a template, the DNA sequence of the reading frame of the amnestic protein is obtained by PCR. Alternatively, the reading frame DNA sequence of the amnestic protein is obtained directly by RT-PCR.

Then, using the restriction enzyme sites KpnI and HindIII introduced in the primers, the reading frame DNA of the amnestic protein was cloned into the same enzyme site in pQE-30 (QIAexpress Kit, Qiagene) (the specific operation steps are according to the QIAexpress Kit, Qiagene instructions). The PCR reaction conditions are as follows: the annealing temperature is 56° C., the number of reaction cycles is 30 times, and each cycle is denatured for 1 minute, annealed for 1 minute, and extended for 1 minute.

After obtaining the prokaryotic expression vector pQE-30 containing the amnestic protein reading frame DNA, it was transformed into M15 Escherichia coli by using an electrotransformer. M15 Escherichia coli with amnestic protein was cultured in LB, and under the induction of IPTG with a final concentration of 1mM, it expressed amnestic protein with 6-histidine tag. Utilizing the characteristic of binding 6-histidine to Ni ions, the expressed amnestic protein with 6-histidine tag was purified from total protein by Ni-NTA chromatography column. The molecular weight is about 18kDa.

Example 7

Preparation of Anti-Amnesic Antibody

10-100 μg of the amnestic protein obtained in Example 6 was mixed with FCA adjuvant and injected into the rabbit's ear vein for primary immunization. Two weeks later, the obtained amnestic protein and FIA adjuvant were mixed and injected into the rabbit's ear vein for a second immunization (to induce IgG production). A third immunization is given one month later to maximize antibody production. Two months after immunization, polyclonal anti-memory protein was purified from the sera of immunized rabbits.

Sequence Listing

<110>Shanghai Institute of Biochemistry, Chinese Academy of Sciences

  Shanghai Institute of Physiology, Chinese Academy of Sciences

  Shanghai Bioengineering Research Center, Chinese Academy of Sciences

Fudan University

<120>menesic protein and its application

<130>012976

<160>10

<170>PatentIn version 3.0

<210>1

<211>1920

<212>DNA

<213> Rat

<220>

<221> CDS

<222>(29)..(523)

<400>1

cagcatctct accttccttc cctgctag atg tgg aag cca aga cca agg aga 52

                                                                      , 

1 5

aac caa ttg aat cct gcc ctg aag ttg ctt tat gtt ctg gat ttg gag 100

Asn Gln Leu Asn Pro Ala Leu Lys Leu Leu Tyr Val Leu Asp Leu Glu

10 15 20

agc atg ggg caa gac aaa gta ggt gtt ttg cct gtg tat atg tct gcg 148

Ser Met Gly Gln Asp Lys Val Gly Val Leu Pro Val Tyr Met Ser Ala

25 30 35 40

cac cac ttg agt ggt tgg tgc tgg cgg aga cca aaa gag gcc atg gag 196

His His Leu Ser Gly Trp Cys Trp Arg Arg Pro Lys Glu Ala Met Glu

45 50 55

cca gaa gag agc atg gat ccc cta gaa ctg gaa tta cag acg gtt gtg 244

Pro Glu Glu Ser Met Asp Pro Leu Glu Leu Glu Leu Gln Thr Val Val

60 65 70

gtt tgc caa gtg ggt gct ggg aat gca acc caa gcc ctt tgg aaa aat 292

Val Cys Gln Val Gly Ala Gly Asn Ala Thr Gln Ala Leu Trp Lys Asn

75 80 85

aac cag ttc tct gaa cca ctg agc aat ctc tcc agc ccc tct tta ttt 340

Asn Gln Phe Ser Glu Pro Leu Ser Asn Leu Ser Ser Pro Ser Leu Phe

90 95 100

tat aat tac atg tct gtg tct gtg tct gtg tct gtg tct gct tgt ggg 388

Tyr Asn Tyr Met Ser Val Ser Val Ser Val Ser Val Ser Ala Cys Gly

105 110 115 120

tat gtg cag atg agt gca gtt gcc tca gag gtc aga aaa ggg cac tgg 436

Tyr Val Gln Met Ser Ala Val Ala Ser Glu Val Arg Lys Gly His Trp

125 130 135

atc ctc tgg tgc tgg tgt tac acg cag ttg tca gcc cct gcc atg ggt 484

Ile Leu Trp Cys Trp Cys Tyr Thr Gln Leu Ser Ala Pro Ala Met Gly

140 145 150

gct agg aag caa act cag atc ttc agc aag aac aag tgt taaccgctgg 533

Ala Arg Lys Gln Thr Gln Ile Phe Ser Lys Asn Lys Cys

155 160 165

gccaactctt cagcctctcc tagacatttt taatagaatt gcaggaccaa tggatataag 593

tatcattccc catggttat actttactga tgggttctaa tgagaacaat attgttaatc 653

ttcttctttg tagtgagaat atttcttact ctttttatgt ctgtgtatgt gttttgagac 713

agggtctctc tatgaatcct tgctgttccg gagtttacta tgcaggttag ccttaaggtc 773

acagaactac tcctgcctca gcttcccaag tgctgaaatt aaaggcatac accaccatac 833

ctggctttaa tcttcctttt tttgaattct acaatagaaa cacaagggca agcctctatg 893

gagacattct ttatcatagt aggtagcttg cttgtttctt ttatccttcc ttccttcctt 953

ccttccttcc tttttccttt ctttgttttg gtttcattcg ttcattcaac acagggtttc 1013

tctgtatagt cttggctgtc ctggagtttg ctctgtagac ctgtcttggt ctcccaagtt 1073

tctttgagac aggctcttct gtagacttgg ctatctcaag cctgttacat agttgaagaa 1133

gacattgaac ttctaatcct cctgcctcta cctcccaggt actgaagtca gaggttgttt 1193

cactgtatct tgattttata gttttttttt aattgaaata attatagatt gactttcagt 1253

ttaagaaatg ataaccaaaa tcggggggta gtggttcatg catgtcatct tagtactcaa 1313

aaggctgaaa caaagaattg tctaagtttg agatcagttt ggcctacaga gtaagtttca 1373

ggtcagctgg gggtaaagga aaacccagcc atcacaaaaa caaatccaaa caagacaggc 1433

tgagtgtggt ggcgcatctt tttataatca cagcagtcag gattctgtga gcttgaggcc 1493

agcctggtgt acagagttct agctgtcata gctaaagagt gagctgaggc tggggaatag 1553

ttcattgagt tgatgtgaat gccacatgaa cacgacagca tgagttgttc accgttctcc 1613

acagatgaat ttgtagcccg cgtcctaggg gttgggtgga aacgggtggc tcccagagtt 1673

gctgagtagc cgttctcagc agtcgctgag aattgttgat tctttggctc aaagagagat 1733

cctgtgtaaa gaaatcaggt gaagagtgat acccatcaat ctttaggatc taccgtggtg 1793

cataatcaca tatgtatttt cacagacaaa tacagacaca caaaaacaag aggcccctga 1853

atggtattac tttgcaaaac tgtaataaaa atatatctaa atttcaaaaa aaaaaaaaaa 1913

aaaaaaa 1920

<210>2

<211>165

<212>PRT

<213> Rat

<400>2

Met Trp Lys Pro Arg Pro Arg Arg Asn Gln Leu Asn Pro Ala Leu Lys

1 5 10 15

Leu Leu Tyr Val Leu Asp Leu Glu Ser Met Gly Gln Asp Lys Val Gly

20 25 30

Val Leu Pro Val Tyr Met Ser Ala His His Leu Ser Gly Trp Cys Trp

35 40 45

Arg Arg Pro Lys Glu Ala Met Glu Pro Glu Glu Ser Met Asp Pro Leu

50 55 60

Glu Leu Glu Leu Gln Thr Val Val Val Cys Gln Val Gly Ala Gly Asn

65 70 75 80

Ala Thr Gln Ala Leu Trp Lys Asn Asn Gln Phe Ser Glu Pro Leu Ser

85 90 95

Asn Leu Ser Ser Pro Ser Leu Phe Tyr Asn Tyr Met Ser Val Ser Val

100 105 110

Ser Val Ser Val Ser Ala Cys Gly Tyr Val Gln Met Ser Ala Val Ala

115 120 125

Ser Glu Val Arg Lys Gly His Trp Ile Leu Trp Cys Trp Cys Tyr Thr

130 135 140

Gln Leu Ser Ala Pro Ala Met Gly Ala Arg Lys Gln Thr Gln Ile Phe

145 150 155 160

Ser Lys Asn Lys Cys

165

<210>3

<211>20

<212>DNA

<213> Synthetic oligonucleotides

<220>

<221>misc_feature

<223>The antisense sequence of sequence 400-419 in SEQ ID NO:1

<400>3

cctctgaggc aactgcactc 20

<210>4

<211>20

<212>DNA

<213> Synthetic oligonucleotides

<400>4

tagcttcggc tcgctcgcta 20

<210>5

<211>20

<212>DNA

<213> Synthetic oligonucleotides

<400>5

ccagttctct gaaccactga 20

<210>6

<211>20

<212>DNA

<213> Synthetic oligonucleotides

<400>6

cttatatcca ttggtcctgc 20

<210>7

<211>19

<212>DNA

<213> Synthetic oligonucleotides

<400>7

atgtggaagc caagaccaa 19

<210>8

<211>19

<212>DNA

<213> Synthetic oligonucleotides

<400>8

ttaacacttg ttcttgctg 19

<210>9

<211>27

<212>DNA

<213> Synthetic oligonucleotides

<400>9

ggggtaccat gtggaagcca agaccaa 27

<210>10

<211>28

<212>DNA

<213> Synthetic oligonucleotides

<400>10

cccaagctta cacttgttct tgctgaag 28

Claims (12)

1. An isolated amnestic protein polypeptide, characterized in that the polypeptide is selected from the group consisting of: (a) a polypeptide having the amino acid sequence of SEQ ID NO: 2; (b) A polypeptide derived from (a) formed by substituting, deleting or adding 1-10 amino acid residues to the amino acid sequence of SEQ ID NO: 2, and having the function of erasing memory. 2. The polypeptide according to claim 1, wherein the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO:2. 3. An isolated polynucleotide, characterized in that it comprises a nucleotide sequence selected from the group consisting of: (a) polynucleotide encoding polypeptide as claimed in claim 1; (b) A polynucleotide complementary to polynucleotide (a). 4. The polynucleotide of claim 3, wherein the polynucleotide encodes a polypeptide having an amino acid sequence shown in SEQID NO:2. 5. The polynucleotide of claim 3, wherein the sequence of the polynucleotide is selected from the group consisting of: (a) has the sequence of positions 29-523 in SEQ ID NO: 1; (b) having the sequence of positions 1-1920 in SEQ ID NO:1. 6. A vector comprising the polynucleotide according to claim 3. 7. A genetically engineered host cell, characterized in that it contains the vector according to claim 6. 8. A method for preparing a polypeptide having amnestic protein activity, characterized in that the method comprises: (a) cultivating the host cell according to claim 7 under conditions suitable for expressing the amnestic protein; (b) isolating a polypeptide having amnestic activity from the culture. 9. An antibody capable of specifically binding to the amnestic protein of claim 1. 10. A pharmaceutical composition, characterized in that it contains a safe and effective amount of the amnestic polypeptide according to claim 1, or the polynucleotide according to claim 3, and a pharmaceutically acceptable carrier. 11. A health care product, characterized in that it contains the amnestic protein polypeptide of claim 1 or the polynucleotide of claim 3. 12. A method for screening compounds that promote or inhibit forgetting, characterized in that it comprises the steps of: (1) Load the cDNA of the amnestic protein gene into an expression vector, transfect a mammalian cell line, and prepare a cell line expressing the amnestic protein: (2) Add test compound to the cell line culture fluid expressing amnestic protein in step (1), detect the change of amnestic protein expression level; The compound that promotes the increase of amnesic protein expression level is exactly the compound that promotes forgetting, suppresses memory Compounds that increase the expression of scavenging proteins are compounds that inhibit amnesia.

Images