CN1273614C - Synaptic knob association membrane protein 25 and its use - Google Patents

Abstract

The invention relates to mammalian synaptosome-associated membrane protein 25 (SNAP-25) gene and its coded product, and its use in promoting memory and the like. In addition, the present invention also relates to an antagonist containing SNAP-25 and its coding gene, as well as pharmaceutical compositions and health products containing the above substances.

Description

Synaptic knob association membrane protein 25 and application thereof

Invention field

The present invention relates to biotechnology and medical field.More specifically, the present invention relates to mammiferous synaptic knob association membrane protein 25 (synaptosomal-associated membrane protein-25 abbreviates " SNAP-25 " as) gene and coded product thereof, and in aspect purposes such as promotion memories.In addition, the invention still further relates to and contain SNAP-25 gene and proteic antagonist thereof, and the pharmaceutical composition and the healthcare products that contain above-mentioned substance.

Technical field

Learning and memory is the basic function of brain.Higher animal, hippocampus is a brain structure of Medial Temporal Lobe, and after it damage, new memory can't form.The cynapse of hippocampus has very strong plasticity-.The hippocampus cynapse can be induced to produce and keep long time-histories enhancing (long-term potentiation, LTP), after inducing with LTP or keeping relevant any one the proteinic encoding gene disallowable (knockout) of link, hippocampus just no longer includes the LTP phenomenon, and animal also just can not form memory.Therefore, hippocampus is as the important structure of learning and memory, and LTP is accepted by numerous neuroscientists as the cynapse mechanism of learning and memory.

The SNAP-25 gene belongs to a gene family very conservative in evolution, and mostly to tell behavior relevant with the extracellular for its member.There is the SNAP-25 sequence of 7 kinds of species to be disclosed, comprises people, rat, mouse, Xenopus laevis, zebra fish, nematode and fruit bat.Different neurones and neuroendocrine cell are expressed two kinds of different shearing product [Oyler GA. of this gene in the different steps of growing, Polli JW., Wilson MC., Billingsley ML.Developmental expression of the 25-kDa synaptosomal-associated protein (SNAP-25) in rat brain.Proc.Natl.Acad.Sci.USA 1991,88 (12): 5247-51]: SNAP-25a and SNAP-25b, and and the some other gene of its homologous.SNAP-25 albumen is positioned on cytoplasmic cytoplasmic membrane and secretory vesicle film.It and other the two kinds albumen relevant with exocytosis and synaptic vesicle, be syntaxin (syntaxin) and synaptobrevin, stable ternary complex of common formation, play an important role at exocytosis and neurotransmitter dispose procedure [Hodel A.SNAP-25.Int.J Biochem.CellBiol.1998, (10): 1069-73].

Prove with experiment in vitro in the body that in the late period that spinous process extends, SANP-25 albumen is expressed at growing tip, suppress it with antisense nucleic acid and express the extension that just can suppress spinous process.This shows SNAP-25 [the Osen-Sand A that plays an important role in the plasticity-of axon growth and nerve ending, Catsicas M, Staple JK, Jones KA, AyalaG, Knowles J, Grenningloh G, Catsicas S.Inhibition of axonal growth bySNAP-25 antisense oligonucleotides in vitro and in vivo.Nature 1993,364 (6436): 445-8].In cerebral hippocampal, cause LTP and be considered to closely related with the plasticity-of cynapse.People such as RobertsLA [Roberts LA, Morris BJ, O ' Shaughnessy CT.Involvement of two isoformsof SNAP-25 in the expression of long-term potentiation in the rathippocampus.Neuroreport 1998,9 (1): 33-6] experimental result shows, the high frequency stimulation tooth returns granulosa cell and causes LTP after 2 hours, and the mRNA of SNAP-25 expression amount in the rat hippocampus of isolated culture significantly increases.

Yet, still there be not the SNAP-25 albumen evidence directly related up to now with mammiferous learning and memory, whether remember by participation in learning to SNAP-25 for people, and play which kind of effect and do not remove in learning and memory.

In view of also knowing little about it for the range protein that participates in or influence is remembered at present.Therefore, this area presses for finds new and memory proteins associated matter, and exploitation promotes the medicine of memory.

Summary of the invention

Purpose of the present invention just provides a kind of and memory proteins associated-SNAP-25 albumen, and the purposes aspect diagnosis, improvement or treatment hypomnesis and amnesia.

Another object of the present invention provides and contains the proteic pharmaceutical composition of SNAP-25, healthcare products.

In a first aspect of the present invention, a kind of method that the amnesia susceptibility of individuality is diagnosed is provided, it comprises step:

Detect this individual SNAP-25 gene, transcript and/or albumen, and compare with normal SNAP-25 gene, transcript and/or albumen,

The possibility that there are differences with regard to showing this individuality trouble amnesia is higher than normal population.

Preferably, detected is gene or the transcript of people SNAP-25, and with normal people SNAP-25 nucleotide sequence comparing difference.

In a second aspect of the present invention, a kind of method for the treatment of amnesia is provided, comprise step: the normal SNAP-25 albumen of using safe and effective amount to the patient of the described treatment of needs.

In a third aspect of the present invention, a kind of method of remembering of promoting is provided, comprise step: promote the object of remembering to use the SNAP-25 of safe and effective amount for needs.

In a fourth aspect of the present invention, a kind of healthcare products are provided, it contains mammiferous SNAP-25 albumen or its active fragments.

In a fifth aspect of the present invention, a kind of pharmaceutical composition is provided, it contains the mammiferous SNAP-25 albumen and the pharmaceutically acceptable carrier of safe and effective amount.Preferably, described SNAP-25 albumen is selected from down the SNAP-25 albumen of treated animal: people, rat and mouse.

In a sixth aspect of the present invention, a kind of test kit that detects amnesia susceptibility or individual memory level is provided, it comprises the primer of specific amplification SNAP-25 gene or transcript, perhaps with SNAP-25 protein-specific bonded antibody.

In a seventh aspect of the present invention, a kind of method of screening the medicine of treatment amnesia is provided, it comprises step:

(1) cDNA of SNAP-25 gene is packed into expression vector, the transfection mammalian cell strain, the proteic cell strain of SNAP-25 is expressed in preparation;

(2) add test compounds in the proteic cell strain nutrient solution of the expression SNAP-25 in step (1), detect the variation of SNAP-25 expressing quantity; The compound that promotes the SNAP-25 expressing quantity to increase is exactly the drug candidate of treatment amnesia.

In a eighth aspect of the present invention, a kind of isolating SNAP-25 albumen is provided, it comprises the aminoacid sequence shown in the SEQ ID NO:2.

In a ninth aspect of the present invention, a kind of isolating polynucleotide are provided, it comprises a nucleotide sequence, and this nucleotide sequence is shown at least 70% homogeny with a kind of nucleotides sequence that is selected from down group:

(a) polynucleotide of the above-mentioned SNAP-25 polypeptide of coding;

(b) with polynucleotide (a) complementary polynucleotide.

Preferably, this polynucleotide encoding has the polypeptide of aminoacid sequence shown in the SEQ ID NO:2.More preferably, the sequence of these polynucleotide is selected from down group:

(a) has the sequence of 200-817 position among the SEQ ID NO:1;

(b) has the sequence of 1-2040 position among the SEQ ID NO:1.

In a tenth aspect of the present invention, a kind of carrier that contains above-mentioned polynucleotide is provided, contain the host cell of described carrier.Preparation method with the active polypeptide of SNAP-25 also is provided, and the method comprising the steps of:

(a) under the condition that is fit to expression SNAP-25, cultivate above-mentioned host cell;

(b) from culture, isolate and have the active polypeptide of SNAP-25.

Description of drawings

Fig. 1 has shown at the CA1 district of rat local injection SNAP-25 gene antisense sequencing nucleic acid, can obviously suppress the performance (*, p＜0.05) of rat in water maze behavior task, comprising:

Figure 1A: the front and rear part be respectively in the training process and after 48 hours in the test process rat find the needed time of platform, per three times mean value (mean ± SEM).Antisense nucleic acid group rat finds the platform required time significantly to be longer than other two groups.

Figure 1B: in the test of exposing platform, rat is found the needed time of platform, and the eyesight between each group does not show difference.

Fig. 1 C: the swimming rate of rat in the water maze training process.That show is the average swimming rate (mean ± SEM), each group between do not show difference of rat in former and later two stages of training process (1-6 time and 7-12 time).

Fig. 1 D: in the training process and after 48 hours in the test process rat in per three training, find the number of times of platform, wherein train with 7-9 time and 10-12 time and in test process difference the most remarkable.

Fig. 1 E: each organizes rat typical swimming track in test.The antisense nucleic acid group is that the distance that finds platform to swim significantly is longer than other two groups.The position that platform is hidden in " zero " expression among the figure.

Fig. 2 significantly strengthens the long-term memory of rat after having shown the antisense nucleic acid gene of local injection SNAP-25.Comprising:

Fig. 2 A: after the electric shock, rat respectively instant, after 24 hours and 48 hours to scene Horrible Memory situation.

Fig. 2 B: rat is to the reaction of electric shock in training process.When 70 seconds and 100 seconds, give twice electric shock of rat respectively.Each measuring point is represented the situation of rat freezing in 0.5 minute.

Fig. 2 C and 2D: when expression training is tested after 24 hours and 48 hours respectively, the fear reaction that rat showed in each time period, each measuring point is represented the mean value (mean ± SEM) in 0.5 minute.The point on right side is represented the average response in the whole test process.

After Fig. 3 had shown the antisense nucleic acid gene fragment of CA1 district local injection SNAP-25, damage Schaffer side shoot path LTP induced and keeps situation (CA3 district result is similarly), comprising;

Fig. 3 A: the mean value (mean ± SEM) that induces the pEPSP before and after the LTP in four groups of rat CA1 districts.Give high frequency stimulation (200Hz at the arrow place, 3 strings, 20 square waves of every string stimulate, string is 30s at interval), record 30min before tetanic, 3 or 6 hours (only showing 3 hours) are write down in tetanic back, and the rat of injection antisense nucleic acid can not induce LTP, can not keep down for a long time even if perhaps can derive.And other group rats can induce LTP, and can keep down for a long time.

Fig. 3 B: each organizes tetanic preceding, the tetanic back 10min of rat, tetanic back 3 hours typical pEPSP waveform.

Fig. 3 C: the comparison of wave shape before and after the injection antisense sequences nucleic acid.

Fig. 4 A and Fig. 4 B are undergo training the Northern trace figure and the analysis chart of rat hippocampus SNAP-25mRNA expression amount, show that SNAP-25mRNA expresses to have significantly to increase.

Fig. 5 A and 5B are in situ hybridization photo and the analysis chart of SNAP-25, and the result shows the rat hippocampus CA1 that undergoes training, and CA3 and DG district SNAP-25mRNA express to have and increase, and be wherein the most remarkable with the CA3 district.

Fig. 6 A and 6B are SNAP-25 proteic Western trace photo and analysis chart, and the result shows that the rat hippocampus SNAP-25 protein expression of undergoing training has significantly and increases.

Fig. 7 A is people SNAP-25cDNA sequence and protein sequence, is the position of antisense sequences nucleic acid in the square frame wherein; Fig. 7 B is the cDNA homology comparison diagram of people and rat SNAP-25.

Embodiment

The inventor has not only cloned the SNAP-25cDNA sequence of rat, but also has utilized different animal ethology models through extensive and deep research, and the function of SNAP-25 gene in the learning and memory process studied.Be surprised to find that SNAP-25 is relevant with learning and memory.

The inventor utilizes different animal ethology models, and the function of SNAP-25 gene in the learning and memory process studied.At rat hippocampus CA1 and CA3 district pipe laying injection SNAP-25 gene antisense nucleic acid, and establish injecting normal saline and stochastic sequence nucleic acid (Scramble) is control group, observation experiment group and the control group performance difference in the Morris water maze (Morris water maze) of investigating the learning and memory ability and two kinds of study of behaviour detection models of conditionality environment fear (Fearconditioning to context) then, and the gained data are carried out statistical study.The result of two kinds of study of behaviour detection models shows, after the injection SNAP-25 gene antisense nucleic acid, the learning and memory ability of rat has significantly and weakens.Simultaneously, electrophysiological result shows that after the injection SNAP-25 gene antisense nucleic acid, hippocampus LTP can not form or significantly weaken.By the Northern engram analysis, in situ hybridization and Western engram analysis have confirmed that in the study of behaviour detection model, the SNAP-25 expression of gene all is significantly increased in the trained rat hippocampus on mRNA and protein level.

As used herein, term " SNAP-25 " and " SNAP-25 polypeptide " are used interchangeably, finger is specific expressed in mammiferous hippocampus, the SNAP-25 aminoacid sequence of aminoacid sequence and people, rat or mouse has more than 70%, preferably more than 80%, more preferably 90%, the polypeptide of 95% above homology best.SNAP-25 in the different Mammalss can obtain by routine test according to sequence information disclosed by the invention.In addition, the active fragments of SNAP-25, conservative property polypeptide or reactive derivative can be used for the present invention.

According to protein homology relatively, human SNAP-25 albumen and mouse and rat SNAP-25 albumen height homology (homogeny is more than 90%).The specificity of proteic structure of Mammals SNAP-25 and tissue distribution is all pointed out, and SNAP-25 albumen has identical functions in different Mammalss, can improve memory.Therefore human SNAP-25 albumen is same relevant with man memory power, according to the medicine and the diagnoses and treatment technology of people SNAP-25 gene and expression product design thereof, can be used for diagnosing and treating human amnesia, and can improve memory.

SNAP-25 Nucleotide full length sequence or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be according to the nucleotide sequence of known people SNAP-25, mouse SNAP-25 or the disclosed rat SNAP-25 of the present invention, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.When sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.

In case obtained relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.

In addition, also the method for available synthetic is synthesized relevant sequence, especially fragment length more in short-term.Usually, by first synthetic a plurality of small segments, and then connect and to obtain the very long fragment of sequence.

At present, can be fully obtain the dna sequence dna of code book invention albumen (or its fragment, or derivatives thereof) by chemosynthesis.This dna sequence dna can be introduced in various existing dna moleculars as known in the art (or as carrier) and the cell then.In addition, also can will suddenly change and introduce in the protein sequence of the present invention by chemosynthesis.

The present invention also relates to comprise the carrier of polynucleotide of the present invention, and the host cell that produces through genetically engineered with carrier of the present invention or SNAP-25 encoding sequence, and the method that produces polypeptide of the present invention through recombinant technology.

Recombinant DNA technology (Science, 1984 by routine; 224:1431), can utilize polymerized nucleoside acid sequence of the present invention to can be used to express or produce the SNAP-25 polypeptide of reorganization.In general following steps are arranged:

(1). with the polynucleotide (or varient) of coding of the present invention SNAP-25 polypeptide, or with the recombinant expression vector that contains these polynucleotide proper host cell that transforms or transduce;

(2). the host cell of in suitable medium, cultivating;

(3). separation, protein purification from substratum or cell.

Particularly, SNAP-25 of the present invention can be by introducing corresponding coding sequence host cell (directly introducing or contain by introducing the carrier of SNAP-25 encoding sequence), and under appropriate condition, cultivate transformed host cells to express SNAP-25, separate then and be purified into SNAP-25.

The extracellular can be expressed or be secreted into to recombinant polypeptide in the above methods in cell or on cytolemma.If desired, can utilize its physics, the separating by various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation handles, with protein precipitant handle (salt analysis method), centrifugal, the broken bacterium of infiltration, superly handle, the combination of super centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods.

SNAP-25 polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthetic polypeptide, preferred recombinant polypeptide.Polypeptide of the present invention can be the product of natural purifying, or the product of chemosynthesis, or uses recombinant technology to produce from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell).The host used according to the recombinant production scheme, polypeptide of the present invention can be glycosylated, maybe can be nonglycosylated.Polypeptide of the present invention also can comprise or not comprise initial methionine residues.

In the present invention, term " SNAP-25 polypeptide " refers to have the active SEQ ID of SNAP-25 NO.2 polypeptide of sequence.This term also comprises having and the variant form SNAP-25 identical function, SEQ ID NO.2 sequence.These variant forms comprise (but being not limited to): several (are generally 1-50, preferably 1-30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or replacement, and add one or several at C-terminal and/or N-terminal and (be generally in 20, preferably being in 10, more preferably is in 5) amino acid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.The present invention also comprises active fragments, derivative and the analogue of SNAP-25.

The variant form of this polypeptide comprises: homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, under high or low tight degree condition can with the coded albumen of the DNA of SNAP-25DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-SNAP-25 polypeptide to obtain.The present invention also provides other polypeptide, as comprises SNAP-25 polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of SNAP-25 polypeptide.Usually, this fragment have the SNAP-25 peptide sequence at least about 10 continuous amino acids, usually at least about 30 continuous amino acids, preferably at least about 50 continuous amino acids, more preferably at least about 80 continuous amino acids, best at least about 100 continuous amino acids.

Invention also provides the analogue of SNAP-25 or polypeptide.The difference of these analogues and natural SNAP-25 polypeptide can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps haves both at the same time.These polypeptide comprise natural or the inductive genetic variant.The induce variation body can obtain by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, also can pass through site-directed mutagenesis method or the biological technology of other known moleculars.Analogue also comprises having the analogue that is different from the amino acid whose residue of natural L-(as D-amino acid), and has non-natural analogue that exist or synthetic amino acid (as β, gamma-amino acid).Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide that exemplifies.

Particularly, SNAP-25 albumen of the present invention or polypeptide are of use in many ways.These purposes include, but is not limited to: the direct disease (as hypomnesis) due to the low or forfeiture and be used to screen antibody, polypeptide or other part that promotes the SNAP-25 protein function as pharmacological agent SNAP-25 protein function.The peptide molecule that can stimulate people SNAP-25 protein function that can be used for seeking therapeutic value with the reorganization SNAP-25 protein screening peptide library of expressing.

On the other hand, the present invention also comprises SNAP-25DNA or the polypeptide of its fragment coding has specific polyclonal antibody and monoclonal antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into SNAP-25 gene product or fragment.Preferably, refer to that those can combine with SNAP-25 gene product or fragment but nonrecognition and be incorporated into the antibody of other irrelevant antigen molecule.Among the present invention antibody comprise those can in conjunction with and suppress the proteic molecule of SNAP-25, comprise that also those do not influence the antibody of SNAP-25 protein function.

The present invention not only comprises complete mono-clonal or polyclonal antibody, but also comprises having immunocompetent antibody fragment, as Fab ' or (Fab) ₂Fragment; Heavy chain of antibody; Light chain of antibody; Or chimeric antibody, as have the murine antibody binding specificity but still keep antibody from people's antibody moiety.

Antibody of the present invention can be prepared by the known various technology of those skilled in that art.For example, the SNAP-25 gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing SNAP-25 albumen or its has antigenic segmental cell and can be used to immune animal and produce antibody.Monoclonal antibody of the present invention can utilize hybridoma technology to prepare that (see people such as Kohler, Nature 256; 495,1975; People such as Kohler, Eur.J.Immunol.6:511,1976; People such as Kohler, Eur.J.Immunol.6:292,1976; People such as Hammerling, In Monoclonal Antibodiesand T Cell Hybridomas, Elsevier, N.Y., 1981).Each antibody-like of the present invention can utilize for example fragment or the functional zone of people, rat or mouse SNAP-25 gene product, obtains by the routine immunization technology.These fragments or functional zone can utilize recombinant methods or utilize Peptide synthesizer synthetic.Can come immune animal and produce with the gene product of producing in the prokaryotic cell prokaryocyte (for example E.Coli) with the unmodified form bonded antibody of SNAP-25 gene product; With posttranslational modification form bonded antibody (as the albumen or the polypeptide of glycosylation or phosphorylation), can come immune animal and obtain with the gene product that produces in the eukaryotic cell (for example yeast or insect cell).

The proteic antibody of anti-SNAP-25 can be used in the immunohistochemistry technology, detects the SNAP-25 albumen in the biopsy specimen.

Available SNAP-25 albumen of the production of polyclonal antibody or polypeptide immune animal, as rabbit, sheep etc.Multiple adjuvant can be used for the enhancing immunity reaction, includes but not limited to freund's adjuvant etc.

Utilize albumen of the present invention,, can filter out with SNAP-25 albumen interactional material takes place, as inhibitor, agonist or antagonist etc. by various conventional screening methods.When screening, SNAP-25 albumen can be added in the bioanalysis mensuration, determine by the interaction of measuring between compounds affect SNAP-25 albumen and its acceptor whether compound is antagonist.In addition, also test compounds can be applied to laboratory animal with SNAP-25 albumen, exist the variation of animal memory just to show compared with the control, this compound is SNAP-25 agonist or an antagonist certainly.

In addition, the expression vector of also cDNA of SNAP-25 gene can being packed into, the transfection mammalian cell strain, preparation high expression level SNAP-25 proteic cell strain: and be target site with the SNAP-25 albumen in this cell strain, screening has SNAP-25 albumen and activates or inhibiting medicine.And in the proteic cell strain nutrient solution of described expression SNAP-25, add test compounds, detect the variation of SNAP-25 expressing quantity.The compound that promotes the SNAP-25 protein expression is exactly the drug candidate of treatment amnesia, and suppresses to promote the compound of SNAP-25 protein expression can be used as people are forgotten not wish the medicine of the memory wanted.

Albumen of the present invention and antibody thereof, inhibitor, agonist, antagonist etc. when using (administration) in treatment, can provide different effects.Usually, can these materials are formulated in nontoxic, inert and the pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5-8 usually, and preferably pH is about 6-8, although the pH value can be with being changed to some extent by preparation Substance Properties and illness to be treated.The pharmaceutical composition for preparing can carry out administration by conventional route, comprising (but being not limited to): intramuscular, intravenously, subcutaneous, oral or topical.

Normal SNAP-25 polypeptide can be directly used in disease treatment, for example, is used for the treatment of amnesia aspect.When using SNAP-25 albumen of the present invention, also can use the medicament of other treatment amnesia simultaneously.

The present invention also provides a kind of pharmaceutical composition, and it contains SNAP-25 albumen of the present invention and the pharmaceutically acceptable carrier or the vehicle of safe and effective amount.This class carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Pharmaceutical preparation should be complementary with administering mode.Pharmaceutical composition of the present invention can be made into the injection form, for example is prepared by ordinary method with the physiological saline or the aqueous solution that contains glucose and other assistant agents.Pharmaceutical composition such as tablet and capsule can be prepared by ordinary method.Pharmaceutical composition such as injection, solution, tablet and capsule should be made under aseptic condition.The dosage of activeconstituents is the treatment significant quantity, for example every day about 0.1 microgram/kg body weight-Yue 5 mg/kg body weight.In addition, polypeptide of the present invention also can use with the other treatment agent.

When making pharmaceutical composition, be that the SNAP-25 albumen of safe and effective amount or its antagonist, agonist are applied to Mammals, wherein this safe and effective amount is usually at least about 0.1 microgram/kg body weight, and in most of the cases be no more than about 10 mg/kg body weight, preferably this dosage is about 0.1 microgram/kg body weight-Yue 100 micrograms/kg body weight.Certainly, concrete dosage also should be considered factors such as route of administration, patient health situation, and these all are within the skilled practitioners skill.

The proteic polynucleotide of SNAP-25 also can be used for multiple therapeutic purpose.Gene therapy technology can be used for treating because cell proliferation, growth or the metabolic disturbance due to the proteic expression of SNAP-25 of the proteic nothing expression of SNAP-25 or unusual/non-activity.The method that structure carries the recombinant viral vector of SNAP-25 gene is found in existing document (Sambrook, et al.).Recombinant human SNAP-25 gene can be packaged in the liposome in addition, and then is transferred in the cell.

Polynucleotide imports tissue or intracellular method comprises: directly be injected into polynucleotide in the in-vivo tissue; Or external by carrier (as virus, phage or plasmid etc.) earlier with the polynucleotide transfered cell in, again cell is transplanted in the body etc.

The invention still further relates to the diagnostic testing process of quantitative and detection and localization SNAP-25 protein level.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.The SNAP-25 protein level that is detected in the test can be with laying down a definition the importance of SNAP-25 albumen in various diseases and be used to the disease of diagnosing SNAP-25 albumen to work.

Whether having the proteic method of SNAP-25 in a kind of test sample is to utilize the proteic specific antibody of SNAP-25 to detect, and it comprises: sample is contacted with the SNAP-25 protein specific antibody; Observe whether form antibody complex, formed antibody complex and just represented to exist in the sample SNAP-25 albumen.

The proteic polynucleotide of SNAP-25 can be used for the diagnosis and the treatment of SNAP-25 protein related diseases.Aspect diagnosis, the proteic polynucleotide of SNAP-25 can be used for detecting the proteic expression of SNAP-25 SNAP-25 abnormal exprssion whether or under morbid state.Can be used for the hybridization of biopsy specimen to judge the proteic abnormal expression of SNAP-25 as the SNAP-25DNA sequence.Hybridization technique comprises the Southern blotting, Northern blotting, in situ hybridization etc.These technological methods all are disclosed mature technologies, and relevant test kit all can obtain from commercial channels.Part or all of polynucleotide of the present invention can be used as probe stationary on microarray (microarray) or DNA chip (being called " gene chip " again), is used for analyzing the differential expression analysis and the gene diagnosis of tissue gene.Carry out RNA-polymerase chain reaction (RT-PCR) amplification in vitro with the special primer of SNAP-25 albumen and also can detect SNAP-25 gene transcription product.

The sudden change that detects the SNAP-25 gene also can be used for the disease of diagnosing SNAP-25 albumen relevant.The form of SNAP-25 protein mutation comprises that the point mutation compared with normal wild type SNAP-25DNA sequence, transposition, disappearance, reorganization and other are any unusual etc.Available existing technology such as Southern blotting, dna sequence analysis, PCR and in situ hybridization detect sudden change.In addition, sudden change might influence proteic expression, therefore can judge indirectly that with Northern blotting, Western blotting gene has or not sudden change.

SNAP-25 albumen of the present invention not only can be used for treating hypomnesis or defective object, also can be used for improving the memory of normal individual.Therefore, the present invention also provides a kind of healthcare products, and it contains mammiferous SNAP-25 albumen or its active fragments.Healthcare products of the present invention, method that can be by the field of health care products routine prepares by mammiferous SNAP-25 albumen or its active fragments and suitable diluent, food etc. are mixed.Preferred healthcare products are tablet, granule, oral preparation form.

Polynucleotide of the present invention can be dna form or rna form.Dna form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or double-stranded.DNA can be coding strand or noncoding strand.The coding region sequence of encoding mature polypeptide can be identical with the coding region sequence shown in the SEQ ID N0:1 or the varient of degeneracy.As used herein, " varient of degeneracy " is meant that in the present invention coding has the protein of SEQ ID NO:2, but with the differentiated nucleotide sequence of coding region sequence shown in the SEQ ID NO:1.

The polynucleotide of the mature polypeptide of coding SEQ ID NO:2 comprise: the encoding sequence of an encoding mature polypeptide; The encoding sequence of mature polypeptide and various additional code sequence; Encoding sequence of mature polypeptide (with optional additional code sequence) and non-coding sequence.

The invention still further relates to nucleic acid fragment with above-mentioned sequence hybridization.As used herein, the length of " nucleic acid fragment " contains 15 Nucleotide at least, better is at least 30 Nucleotide, is more preferably at least 50 Nucleotide, preferably more than at least 100 Nucleotide.Nucleic acid fragment can be used for the amplification technique (as PCR) of nucleic acid to determine and/or to separate the polynucleotide of coding SNAP-25.And the nucleic acid fragment of antisense can be used as the antagonist of SNAP-25, is used for suppressing memory.

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:Cold SpringHarbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment 1

The Morris water maze laboratory

One, experimental installation and environment:

Water maze: be a dark circles cylinder, diameter is 150cm, highly is 54cm;

Platform: diameter is about 10cm, does not have the about 2cm of upstream face, is fixed on the central authorities of some quadrants;

Video camera and corresponding software: by San Diego Instrument, USA provides; Camera lens hangs on the Centromedian top of water maze, can be connected with microcomputer by camera system, by microcomputer with under swimming distance, track and the time record of rat in water maze.

Environment: the depth of water, 0.34m; 25 ± 1 ℃ of temperature; Add the dyestuff harmless in the water, rat be can't see be hidden in undersurface platform rat.Room temp is controlled at about 27 ± 1 ℃.Rat makes tangible mark on the surroundings wall, so that can be recognized direction according to mark.The putting position of indoor all objects is fixed in the experimentation, in order to avoid rat is produced interference.

Two, laboratory animal and nucleotide sequence:

Male SD rat, body weight are 200g-250g.Be divided into three groups, 12 of SNAP-25 anti sense nucleotide sequence groups; 12 of random nucleic acid sequence set; 12 of blank groups.

The SNAP-25 anti sense nucleotide sequence:

SNAP-25 antisense sequences nucleic acid is the nucleic acid of can specific blockage SNAP-25mRNA expressing.Molecular weight is 6056.0, contains 20 bases altogether, A=3 wherein, and G=4, C=6, T=7,5=0 (mixing base=0) base sequence is as follows:

5’-CCA?GCA?TCT?TTG?TTG?CAC?GT-3’(SEQ?ID?NO：3)

This antisense sequences nucleic acid can be specifically and (the annotating: the ten fens homologies of SNAP-25 that are used for people and rat corresponding to 790-809 position among the people SEQ IDNO:1 of SNAP-25cDNA open reading frame stage casing, this antisense sequences nucleic acid is also corresponding to 754-773 position among the rat SNAP-25cDNA (Fig. 7 B center line marks part)) the base sequence combination, blocking-up SNAP-25 is proteinic synthetic.

Antisense sequences nucleic acid is a kind of macromolecule nucleic acid substances, because its chemical property may cause the reaction that some and sequence have nothing to do.Therefore, it is very important contrast to be set.Stochastic sequence nucleic acid (Scramble) is a kind of crt gene commonly used, and its base quantity and molecular weight are identical with antisense sequences nucleic acid, but can not block any expression of gene in the brain.Its based composition is A=2, G=5, and C=3, T=6,5=0,6=0,7=0,8=0 (mixing base=0), base sequence is as follows:

5’＞tag?ctt?cgg?ctc?gct?cgc?ta＜3’(SEQ?ID?NO：4)

The medicine preparation: according to the content of medicine, accurately add physiological saline, making concentration is 1nmol/ μ l.

Three, operation;

At rat bilateral hippocampus CA1 district's heeling-in guiding tube, be used for the behavioral experiment administration, the training of Morris water maze is carried out in rat operation back 7-12 days.

Four, administration:

(1) CA3 bilateral pipe laying

Preceding every the rat of operation enters the laboratory and conforms a week.(concentration is 50mg/ml with vetanarcol; Dosage is 40mg/kg; Intraperitoneal administration) after the anesthesia, the animal head is fixed on (by Narishige, Japan produces) on the stereotaxic instrument, cuts off skin of head, expose skull, determine the pipe laying site.As with reference to point, 3.3mm then, is a fundamental point with the centre joint backward with the Bregma point in the site, the other to the left and right 2.6mm that opens ^[57]With the ophthalmology brill skull in this site is opened a duck eye, note being sure not to injure pallium.On skull, bore three duck eyes (arranging with equilateral triangle as much as possible) again, screw on screw, with fixing dentistry cement around two pipe laying sites.With two length is 8mm, and internal diameter is that the guiding tube of 0.4mm inserts respectively in two holes, and the degree of depth is 2.5mm, buries dentistry cement.To guide the mouth of pipe to seal then, to protect from infection with stopper.After treating that rat revives, send Animal House back to and raise.After animal recovers a week, training.

(2) CA1 bilateral pipe laying

The same CA3 of method.The site is: with the Bregma point is reference point, 3.3mm backward, and left and right sides 1.8mm, pipe laying depth are 1.5mm.

(3) injection

Perform the operation after 7 days injection.The medicine-pouring pipe internal diameter is 0.2mm, and the injection degree of depth in CA1 district is 2.8mm, and the injection degree of depth in CA3 district is 4.0mm.Every side injection 1.5 μ l (needed inject approximately in 3 minutes, stopped pin then 2 minutes).The concentration of antisense nucleic acid and stochastic sequence nucleic acid is 1nmol/ μ l.

Five, training method:

After the administration 6 hours, begin training.Training is divided into two stages, and each stage training 6 times is trained 12 times altogether.Each training 60s, the minor tick time is 60s.The timed interval between two training stages is one hour.Rat is put into from the intersection of 4 quadrants respectively, the position of at every turn putting at random, but the fixed order that all rats are put into.When rat is put into,, put into gently, avoid and to immerse in the water with rat head towards the labyrinth wall.During training,, after stopping 30s on the platform, take off, steam again rest 30s then, the training of beginning next round if rat is found platform in 60s; If can not find platform in the 60s, it is guided on the platform, take off behind the 30s, behind the slow-witted 30s, the beginning next round is trained in cage.Write down swimming route, the speed of rat and find the needed time of platform.

Six, testing method:

1. hide the testing method of platform:

Train and tested in back 48 hours, purpose is in order to detect the memory hold facility of rat.Test is carried out altogether 3 times, and each rat is to search 60s in water from a fixed position equally.If in 60s, find platform; Just it is put back to cage immediately, behind the rest 60s, beginning is test next time; If do not find platform in 60 second time in regulation, just it is directed on the platform behind the stop 30s, put back to cage rest 30s, begin then to test next time, write down rat equally and find needed time of platform and route.

2. the testing method of exposing platform:

This test is after all end of test (EOT); Platform surfaced carry out.Purpose is in order to observe rat whether the obstacle of visual cognition aspect to be arranged, thereby influences the ability that it seeks platform.Test is carried out altogether 3 times, and each rat is all from a fixed position, but the position of exposing platform but is random fluctuation.Each test is placed on rat earlier and adapts to 30s on the platform, puts into water from the fixed position then, searches 60s.If find platform in 60s, after putting back to cage rest 60s immediately, beginning is test next time, if do not find platform, just it is taken out from water, put back to beginning test next time behind the cage rest 60s, write down it and find required time of platform and route, speed.

Seven, experimental result:

Inject the result of antisense sequences shown in Figure 1A-4E in the CA1 district.X-coordinate is the training fate, and ordinate zou is that rat is found the needed time of platform (being the mean value of 3 training or detection).Behind the anti sense nucleotide sequence of CA1 district injection SNAP-25 gene, significantly improve space learning and the memory process (Figure 1A) of rat.Figure 1B shows the visual cognition aspect there was no significant difference of three groups of rats.Fig. 1 C shows the visual cognition aspect there was no significant difference of three groups of rats.Fig. 1 D shown in the training process and after 48 hours in the test process rat in per three training, find the number of times of platform, wherein train with 7-9 time and 10-12 time and in test process difference the most remarkable.Fig. 1 E has shown that intuitively the rat of the anti sense nucleotide sequence of injection SNAP-25 gene finds the route of platform to be longer than control group.

Embodiment 2

Scene fear (Freezing) experiment

One. equipment (by San Diego Instrument, USA provides)

The electric shock reaction box: tank wall is formed by transparent organic glass, and is long: 36cm; Wide: 23cm; High: 18cm.The bottom is made up of 14 stainless steel tubes, can pass to electric current.The stainless steel tube diameter is 0.5cm, and the spacing between two stainless steel tubes is 1.2cm.

Microcomputer and corresponding software: the active situation of infrared rays (photobeam) monitoring rat, by the microcomputer record.Sound and stimulation are by mating generation automatically by time variable control stimulation and sound generator.

Two. laboratory animal and medicine

(1) laboratory animal: same water maze laboratory

(2) experimental drug: same water maze laboratory

Three. experimental procedure

(1) CA1 or CA3 bilateral pipe laying: same water maze laboratory

(2) injection: same water maze laboratory

(3) training

After the administration 6 hours, begin training.Rat is put into reaction box gently, and (noting not making rat be in stress situation) after about 1 minute, begins to sound as far as possible, and sound continues 10s.When continuing to 9s, sound begins electric shock, strength of current 1mA, and electric shock time 1s gives once same sound-electric shock again behind the 30s.Detect 30s behind the electric shock 30s, write down the time of its motionless immediately (immediate freezing) by machine automatically.Less than standard be animal except essential breathing, do not have other any activity.

(4) test: train test after 24 hours and 48 hours, mouse is put into reaction box gently, detect 3min, the record dead time.Recording method and testing standard are all and instant test identical.

Four, result

After the local injection in CA1 district, the performance situation of rat in the frightened experiment of scene.After the electric shock, rat respectively instant, after 24 hours and 48 hours to scene Horrible Memory situation.Antisense nucleic acid group (n=12), Normal organizes (n=12), *, P＜0.05.

The result behind the antisense nucleic acid gene of local injection SNAP-25, significantly strengthens the long-term memory of rat as shown in Figure 2.After Fig. 2 A is electric shock, rat respectively instant, after 24 hours and 48 hours to scene Horrible Memory situation.The result shows that the number of success of antisense sequences nucleic acid group significantly descends.

Fig. 2 B: rat is to the reaction of electric shock in training process.When 70 seconds and 100 seconds, give twice electric shock of rat respectively.Each measuring point is represented the situation of rat motionless in 0.5 minute (freezing).The result shows, when rat has just been put into the electric shock reaction box, can probe into reaction box, and the motionless time does not seldom have difference between each group.After shocking by electricity, each dead time of organizing rat all shows relative rising gradually, illustrates that respectively organizing rat is not having visual defects in the kinetic force.The average motionless per-cent of rat is as broad as long between each group in 0.5min.

Fig. 2 C and 2D: when expression training is tested after 24 hours and 48 hours respectively, the fear reaction that rat showed in each time period, each measuring point is represented the mean value (mean ± SEM) in 0.5 minute.The point on right side is represented the average response in the whole test process.The result shows, after 24 hours, when again rat being put into the electric shock reaction box, injection antisense nucleic acid rat total dead time in 3min is lower than other three groups slightly, but do not have the significance difference (Figure 10, C).Put into electric shock reaction box 1min just, the motionless degree of antisense nucleic acid group (n=10) rat significantly is lower than blank group (n=15), salt solution group (n=14) and stochastic sequence nucleic acid (n=10) group (P＜0.05).But afterwards in each time period (is unit with 0.5min) respectively to organize the reaction of rat as broad as long.When testing after 48 hours, rat total motionless degree (57.69% ± 4.7%) in 3min of injection antisense nucleic acid group significantly is lower than stochastic sequence nucleic acid group (81.83% ± 3.12%) (P＜0.01), salt solution group (78.31% ± 3.2%) (P＜0.001) and blank group (80.42% ± 3.92%) (P＜0.01).The motionless degree of the rat of antisense nucleic acid group all is starkly lower than other three groups in each time period, wherein with 1min left and right sides difference significantly (P＜0.001) (Figure 10, D).Above presentation of results, after the expression of CA1 district local injection antisense nucleic acid blocking-up SNAP-25, significance influence the long-term memory of rat to the scene fear.

Embodiment 3

Damage Schaffer side shoot path LTP induces and keeps situation

One, plant and instrument

Stereotaxic apparatus: by Narishige, Japan produces.

Electrical stimulator, storage oscilloscope, stimulation isolator and average hardware wired averager: be Japanese NIHON KOHDEN company product.

Two, laboratory animal and medicine: with embodiment 1.

Three, the preparation of concentric electrode

(1) preparation of stimulating electrode:

A. be about 3cm with one earlier, the lacquer of enameled wire one end of diameter 120 μ m is wiped off at this end folder lastblock silver strip (to increase bonding area), and is standby.

B. use No. 4 syringe needles or syringe needle for transfusion device (internal diameter is about 200 μ m), long one section of intercepting 8mm, enameled wire of interpolation (stopping up the mouth of pipe when preventing to weld).An end of getting a silver strip and needle tubing welds together.(note: must weld, and not stop up the mouth of pipe.)

C. a stainless steel terminal stud is welded on the silver strip, notes making it parallel with needle tubing.

D. the enameled wire among another root stainless steel terminal stud and a is forward welded, these two terminal studs are clamped with mosquito forceps, make it parallel, silica gel is melted, be coated between two terminal studs with electric iron.Attention must smoothen, in order to avoid influence experimental implementation.

E. silica gel is drawn a threads, be wrapped in the needle tubing surface, and with electric iron it melted, smear, what make needle tubing is coated with the very thin silica gel of last layer on every side with insulation, and silica gel does not have at needle tubing mouth of pipe place.The tip of enameled wire is cut off, make it to expose slightly the needle tubing tip.

2) preparation of recording electrode:

Be about 3cm with one earlier, the lacquer of enameled wire one end of diameter 120 μ m is wiped off at this end folder lastblock silver strip (to increase bonding area), gets a stainless steel terminal stud and enameled wire and forward welds, and makes it and the enameled wire keeping parallelism.Then, with enameled wire folding 90 degree, be enclosed within the outside of enameled wire with a needle tubing that is about 2mm, with solid shape.Roll over 90 degree downwards then, get No. 8 needle tubings (making guiding tube) that are about 8mm and weld together with silica gel with enameled wire.As above scheme.Enameled wire is cut off, made it most advanced and sophisticated than the long 1mm of needle tubing, medicine-pouring pipe promptly writes down the site and puts 0.5mm apart from injection, the place that medicine can be diffused into than guiding pipe range 0.5mm.

Four, experimental procedure

(1) with Ethylurethanm (urethane carbamate concentration: 250mg/ml; Dosage: 1250mg/kg; Intraperitoneal administration) behind the anesthetized animal, the animal head is fixed, cut off skin of head, expose skull, as with reference to point, determine the record site and stimulate the site with the Bregma point.In the CA1 district, stimulating electrode is 4.9mm backward, and 3.8mm is opened on the side, turns forward 15 °; Recording electrode is 3.4mm backward, and 2.5mm (1,2) is opened on the side.In the CA3 district, stimulating electrode is 4.1mm backward, and 1.8mm is opened on the side, turns forward 15 °; Recording electrode is 3.4mm backward, and 3.0mm is opened on the side.Stimulating electrode is a bipolar electrode, and internal diameter is 0.2mm; Recording electrode is a monopolar electrode, and other near it is a medicine-introducing pipe, and the medicine-introducing pipe internal diameter is 0.4mm.

(2) determining of stimulating electrode and recording electrode position: (Populationexitory postsnaptic potential when pEPSP) maximum appears in amplitude, fixes the position of two electrodes to colony's excitatory postsynaptic potential (EPSP).In the electrode folding process, the variation of the counter-rotating of current potential phase place, preclinical length, current potential size etc. all are to judge whether stimulating electrode and recording electrode arrive the important indicator in CA1 district.

After electrode position is determined, by medicine-introducing pipe injection SNAP-25 antisense nucleic acid (1.0 μ g/ μ l in the CA1 district; At CA3 district 1.0nmol/ μ l), stochastic sequence nucleic acid (1.0 μ g/ μ l in the CA1 district; At CA3 district 1.0nmol/ μ l), physiological saline 1.0 μ l (4min injection) or injectable drug not.

After (3) 6 hours, select suitable stimulus intensity and magnification.Method is the line of writing music of running business into strong one, and selects 2/3rds of saturated stimulus intensity to be the stimulus intensity of suitable test.

(4) 30 minutes normal pEPSP of record give high frequency stimulation then and (are 200HZ in the CA1 district, 3 strings, 20 square-wave pulses of every string, string interval 30s in contrast; In the CA3 district is 100HZ, 3 strings, and 30 square-wave pulses of every string, string is 20s at interval) and carry out tetanic stimulation, induce LTP.Write down pEPSP then; And the gained result carried out statistical study.

Five, statistical procedures

Each is organized experimental data and all adopts mean number (mean) ± standard error (SEM) expression.The significance of difference variance T checks such as the two samples of two tails between each group.With bilateral P＜0.05 is significant difference.P＜0.01 is that difference is very remarkable.

Six, test-results

The result after the injection antisense nucleic acid, even if can not induce preferably LTP or can derive, can not keep down as shown in Figure 3 for a long time, within 2-3 hour, just is reduced to below 130%.And behind injection stochastic sequence nucleic acid and the physiological saline, can induce LTP, and can keep down (more than 6 hours) for a long time.As Figure 12 (A), at tetanic back about 2.5 hours, the slope of antisense nucleic acid group (n=6) EPSP drops to 119% ± 3.19%, and stochastic sequence nucleic acid group (n=8) (140% ± 7.2%) (P＜0.05), salt solution group (n=10) (142% ± 6.2%) (P＜0.05) normal control group (n=16) (157.8% ± 6.6%) have significant difference between (P＜0.001).Therefore, after near the local injection hippocampus CA1 district recording electrode, significance damage LTP inducing and keeping.After the antisense nucleic acid gene fragment of CA1 district local injection SNAP-25, damage Schaffer side shoot path LTP induces and keeps situation (CA3 district result is similarly), comprising:

Fig. 3 A: the mean value (mean ± SEM) that induces the pEPSP before and after the LTP in four groups of rat CA1 districts.Give high frequency stimulation (200Hz at the arrow place, 3 strings, 20 square waves of every string stimulate, string is 30s at interval), record 30min before tetanic, 3 or 6 hours (only showing 3 hours) are write down in tetanic back, and the rat of injection antisense nucleic acid can not induce LTP, can not keep down for a long time even if perhaps can derive.And other group rats can induce LTP, and can keep down for a long time.Fig. 3 B: each organizes tetanic preceding, the tetanic back 10min of rat, tetanic back 3 hours typical pEPSP waveform.Fig. 3 C: the comparison of wave shape before and after the injection antisense sequences nucleic acid.

Embodiment 4

The Northern engram analysis

Extract total RNA of rat cerebral tissue, behind the sex change gel electrophoresis RNA is transferred on the nylon membrane, this film and α- ³²The probe of P-dATP mark is hybridized, and shows results of hybridization by radioautograph at last, learns the expression and distribution situation of goal gene.

Northern blot hybridization result demonstration, the rat hippocampus SNAP-25mRNA expression amount of undergoing training have significantly increases (Fig. 4 A and Fig. 4 B).

Embodiment 5

The in situ hybridization analysis of SNAP-25

According to a conventional method, animal brain's sample is made specimens paraffin embedding slices with the fixing back of Paraformaldehyde 96, processes such as tissue slice is fixing through dewaxing, rehydration, fixing, digestion, back, prehybridization, hybridize with the probe of digoxin (Digoxin) mark, afterwards the distribution of anti digoxin antibody testing goal gene in brain that connects by alkaline phosphatase.

In situ hybridization is the result show, the rat hippocampus CA1 that undergoes training, CA3 and DG district SNAP-25mRNA express to have and increase, wherein with CA3 district the most remarkable (Fig. 5 A and 5B).

Embodiment 6

The Western engram analysis

Collect sample, after PBS washes, add protein lysate (50mmol/L Tri s-HCl, 150mmol/L NaCl, 0.1%SDS, 10mmol/L EDTA, 1% Sodium desoxycholate, 1%TritonX-100,20mmol/L NaF, 0.25mmol/L PMSF, 5 μ g/ml Leupeptin, pH8.2), 4 ℃ 15 minutes, 12, centrifugal 20 minutes of 000rpm.Supernatant liquor is with ethanol sedimentation (containing the 0.013mol/L Potassium ethanoate), and after the dissolving, electrophoresis on 7%-15%SDS-PAGE glue is transferred to albumen on the pvdf membrane then again.Film room temperature effect in confining liquid added one and resists after 2 hours, and 4 ℃ are spent the night.With PBS/TB (PBS, 0.05%Tween, 1%BSA) wash film 3 times after, with two anti-room temperature incubations 1 hour, after PBS/TB washes twice of film, under the room temperature at NBT-BCIP solution (4.5 μ L 75mg/ml NBT, 3.5 μ L 50mg/ml BCIP is melted into 1mL 0.1mol/L NaCl, 0.05mol/L MgCl ₂, 0.1mol/L Tris-HCl, pH9.5) in lucifuge reaction, positive band appears and after, add stop buffer (20mmol/L Tris-HCl, 1mmol/LEDTA) termination reaction.

The Western engram analysis is the result show, the rat hippocampus SNAP-25 protein expression of undergoing training has significantly and increases (Fig. 6 A and 6B).

Embodiment 7

The expression of people SNAP-25

1.RT-PCR obtain the SNAP-25 encoding sequence

Synthetic a pair of primer, wherein 5 ' primer is: 5 '-atg gcc gaa gac gca gac at-3 ' (SEQ ID NO:5) and 3 ' primer 5 '-tta acc act tcc cag cat ct-3 ' (SEQ ID NO:6).To be template,, identical with 200-820 bit sequence among the SEQ ID NO:1 through sequence verification by the proofreading dna sequence of RT-PCR acquisition SNAP-25 with the extractive normal hippocampus of ordinary method (N) mRNA.

2.SNAP-25 cloning and expression

Design and synthesize a pair of primer, wherein 5 ' primer is: 5 '-ggg gta cca tgg ccg aag acg cagaca t-3 ' (SEQ ID NO:7) and 3 ' primer 5 '-ccc aag ctt tta acc act tcc cag cat ct-3 ' (SEQ ID NO:8).The RT-PCR product that obtains with step (1) is a template, obtains the proofreading dna sequence of SNAP-25 by PCR.Perhaps, directly obtain the proofreading dna sequence of SNAP-25 by RT-PCR.

Then, utilize the restriction enzyme site KpnI and the HindIII that introduce in the primer, the proofreading dna of SNAP-25 is cloned into pQE-30 (QIAexpress Kit, Qiagene) (the concrete operations step is pressed QIAexpress Kit, and the Qiagene specification sheets carries out) on the identical restriction enzyme site in.The PCR reaction conditions is as follows: 56 ℃ of annealing temperatures, and several 30 times of reaction cycle, the sex change 1 minute that at every turn circulates was annealed 1 minute, extended 1 minute.

After obtaining to comprise the prokaryotic expression carrier pQE-30 of SNAP-25 proofreading dna, utilize electric conversion instrument, it is transformed in the M15 intestinal bacteria.The M15 intestinal bacteria that have SNAP-25 are cultivated in LB, are that the IPTG of 1mM induces down at final concentration, give expression to the SNAP-25 that contains 6 histidine marks.Utilize the characteristic of 6 Histidines and Ni ionic bond, the SNAP-25 that the contains 6 histidine marks purifying from total protein that will express by the Ni-NTA chromatography column comes out.Molecular weight is about 25kDa.

Embodiment 8

The preparation of anti-SNAP-25 antibody

SNAP-25 protein 10-100 μ the g and the FCA adjuvant that obtain among the embodiment 7 are mixed the back from the injection of rabbit ear vein, carry out initial immunity.After two weeks, SNAP-25 albumen and the FIA adjuvant that obtains mixed the back from the injection of rabbit ear vein, carry out immunity (inducing the generation of IgG) for the second time.Carry out immunity for the third time after one month again, can make the antibody generation reach maximum like this.Immunity is purified into memory cleaning albumen and resists more after two months from the serum of immune rabbit.

All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

Sequence table

＜110〉Shanghai Inst. of Biochemistry, Chinese Academy of Sciences

Shanghai Physiology Inst., Chinese Academy of Sciences

Shanghai Research Center of Biotechnology

Fudan University

＜120〉synaptic knob association membrane protein 25 and application thereof

<130>012977

<160>8

<170>PatentIn?version?3.0

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<211>2040

<212>DNA

<213>Homo?sapiens

<220>

<221>CDS

<222>(200)..(817)

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ggcagagctc?acgttgcatt?gaagacgaaa?cctcggggag?gtcaggcgct?gtctttcctt 60

ccctccctgc?tcggcggctc?caccacagtt?gcaacctgca?gaggcccgga?gaacacaacc 120

ctcccgagaa?gcccaggtcc?agagccaaac?ccgtcactga?ccccccagcc?caggcgccca 180

gccactcccc?accgctacc?atg?gcc?gaa?gac?gca?gac?atg?cgc?aat?gag?ctg 232

Met?Ala?Glu?Asp?Ala?Asp?Met?Arg?Asn?Glu?Leu

1 5 10

gag?gag?atg?cag?cga?agg?gct?gac?cag?ttg?gct?gat?gag?tcg?ctg?gaa 280

Glu?Glu?Met?Gln?Arg?Arg?Ala?Asp?Gln?Leu?Ala?Asp?Glu?Ser?Leu?Glu

15 20 25

agc?acc?cgt?cgt?atg?ctg?caa?ctg?gtt?gaa?gag?agt?aaa?gat?gct?ggt 328

Ser?Thr?Arg?Arg?Met?Leu?Gln?Leu?Val?Glu?Glu?Ser?Lys?Asp?Ala?Gly

30 35 40

atc?agg?act?ttg?gtt?atg?ttg?gat?gaa?caa?gga?gaa?caa?ctg?gaa?cgc 376

Ile?Arg?Thr?Leu?Val?Met?Leu?Asp?Glu?Gln?Gly?Glu?Gln?Leu?Glu?Arg

45 50 55

att?gag?gaa?ggg?atg?gac?caa?atc?aat?aag?gac?atg?aaa?gaa?gca?gaa 424

Ile?Glu?Glu?Gly?Met?Asp?Gln?Ile?Asn?Lys?Asp?Met?Lys?Glu?Ala?Glu

60 65 70 75

aag?aat?ttg?acg?gac?cta?gga?aaa?ttc?tgc?ggg?ctt?tgt?gtg?tgt?ccc 472

Lys?Asn?Leu?Thr?Asp?Leu?Gly?Lys?Phe?Cys?Gly?Leu?Cys?Val?Cys?Pro

80 85 90

tgt?aac?aag?ctt?aaa?tca?agt?gat?gct?tac?aaa?aaa?gcc?tgg?ggc?aat 520

Cys?Asn?Lys?Leu?Lys?Ser?Ser?Asp?Ala?Tyr?Lys?Lys?Ala?Trp?Gly?Asn

95 100 105

aat?cag?gac?gga?gtg?gtg?gcc?agc?cag?cct?gct?cgt?gta?gtg?gac?gaa 568

Asn?Gln?Asp?Gly?Val?Val?Ala?Ser?Gln?Pro?Ala?Arg?Val?Val?Asp?Glu

110 115 120

cgg?gag?cag?atg?gcc?atc?agt?ggc?ggc?ttc?atc?cgc?agg?gta?aca?aat 616

Arg?Glu?Gln?Met?Ala?Ile?Ser?Gly?Gly?Phe?Ile?Arg?Arg?Val?Thr?Asn

125 130 135

gat?gcc?cga?gaa?aat?gaa?atg?gat?gaa?aac?cta?gag?cag?gtg?agc?ggc 664

Asp?Ala?Arg?Glu?Asn?Glu?Met?Asp?Glu?Asn?Leu?Glu?Gln?Val?Ser?Gly

140 145 150 155

atc?atc?ggg?aac?ctc?cgt?cac?atg?gcc?ctg?gat?atg?ggc?aat?gag?atc 712

Ile?Ile?Gly?Asn?Leu?Arg?His?Met?Ala?Leu?Asp?Met?Gly?Asn?Glu?Ile

160 165 170

gat?aca?cag?aat?cgc?cag?atc?gac?agg?atc?atg?gag?aag?gct?gat?tcc 760

Asp?Thr?Gln?Asn?Arg?Gln?Ile?Asp?Arg?Ile?Met?Glu?Lys?Ala?Asp?Ser

175 180 185

aac?aaa?acc?aga?att?gat?gag?gcc?aac?caa?cgt?gca?aca?aag?atg?ctg 808

Asn?Lys?Thr?Arg?Ile?Asp?Glu?Ala?Asn?Gln?Arg?Ala?Thr?Lys?Met?Leu

190 195 200

gga?agt?ggt?taagtgtgcc?cacccgtgtt?ctcctccaaa?tgctgtcggg 857

Gly?Ser?Gly

205

caagatagct?ccttcatgct?tttctcatgg?tattatctag?taggtctgca?cacataacac 917

acatcagtcc?acccccattg?tgaatgttgt?cctgtgtcat?ctgtcagctt?cccaacaata 977

ctttgtgtct?tttgttctct?cttggtctct?ttctttccaa?aggttgtaca?tagtggtcat 1037

ttggtggctc?taactccttg?aggtcttgag?tttcattttt?cattttctct?cctcggtggc 1097

atttgctgaa?taacaacaat?ttaggaatgc?tcaatgtgct?gttgattctt?tcaatccaca 1157

gtattgttct?tgtaaaactg?tgacattcca?cagagttact?gccacggtcc?tttgagtgtc 1217

aggctctgaa?tctctcaaaa?tgtgccgtct?ttggttcctc?atggctgtta?tctgtcttta 1277

tgatttcatg?attagacaat?gtggaattac?ataacaggca?ttgcactaaa?agtgatgtga 1337

tttatgcatt?tatgcatgag?aactaaatag?atttttagat?tcctacttaa?acaaaaactt 1397

tccatgacag?tagcatactg?atgagacaac?acacacacac?acaaaacaac?agcaacaaca 1457

acagaacaac?aacaaagcat?gctcagtatt?gagacactgt?caagattaag?ttataccagc 1517

aaaagtgcag?tagtgtcact?tttttcctgt?caatatatag?agacttctaa?atcataatca 1577

tcctttttta?aaaaaaagaa?ttttaaaaaa?gatggatttg?acacactcac?catttaatca 1637

tttccagcaa?aatatatgtt?tggctgaaat?tatgtcaaat?ggatgtaata?tagggtttgt 1697

ttgctgcttt?tgatggctat?gttttggaga?gagcaatctt?gctgtgaaac?agtgtggatg 1757

taaattttat?aaggctgact?cttactaacc?accatttccc?ctgtggtttg?ttatcagtac 1817

aattctttgt?tgcttaatct?agagctatgc?acaccaaatt?gctgagatgt?ttagtagctg 1877

ataaagaaac?cttttaaaaa?aataatataa?atgaatgaaa?tataaactgt?gagataaata 1937

tcattatagc?atgtaatatt?aaattcctcc?tgtctcctct?gtcagtttgt?gaagtgattg 1997

acattttgta?gctagtttaa?aattattaaa?aattatagac?tcc 2040

<210>2

<211>206

<212>PRT

<213>Homo?sapiens

<400>2

Met?Ala?Glu?Asp?Ala?Asp?Met?Arg?Asn?Glu?Leu?Glu?Glu?Met?Gln?Arg

1 5 10 15

Arg?Ala?Asp?Gln?Leu?Ala?Asp?Glu?Ser?Leu?Glu?Ser?Thr?Arg?Arg?Met

20 25 30

Leu?Gln?Leu?Val?Glu?Glu?Ser?Lys?Asp?Ala?Gly?Ile?Arg?Thr?Leu?Val

35 40 45

Met?Leu?Asp?Glu?Gln?Gly?Glu?Gln?Leu?Glu?Arg?Ile?Glu?Glu?Gly?Met

50 55 60

Asp?Gln?Ile?Asn?Lys?Asp?Met?Lys?Glu?Ala?Glu?Lys?Asn?Leu?Thr?Asp

65 70 75 80

Leu?Gly?Lys?Phe?Cys?Gly?Leu?Cys?Val?Cys?Pro?Cys?Asn?Lys?Leu?Lys

85 90 95

Ser?Ser?Asp?Ala?Tyr?Lys?Lys?Ala?Trp?Gly?Asn?Asn?Gln?Asp?Gly?Val

100 105 110

Val?Ala?Ser?Gln?Pro?Ala?Arg?Val?Val?Asp?Glu?Arg?Glu?Gln?Met?Ala

115 120 125

Ile?Ser?Gly?Gly?Phe?Ile?Arg?Arg?Val?Thr?Asn?Asp?Ala?Arg?Glu?Asn

130 135 140

Glu?Met?Asp?Glu?Asn?Leu?Glu?Gln?Val?Ser?Gly?Ile?Ile?Gly?Asn?Leu

145 150 155 160

Arg?His?Met?Ala?Leu?Asp?Met?Gly?Asn?Glu?Ile?Asp?Thr?Gln?Asn?Arg

165 170 175

Gln?Ile?Asp?Arg?Ile?Met?Glu?Lys?Ala?Asp?Ser?Asn?Lys?Thr?Arg?Ile

180 185 190

Asp?Glu?Ala?Asn?Gln?Arg?Ala?Thr?Lys?Met?Leu?Gly?Ser?Gly

195 200 205

<210>3

<211>20

<212>DNA

＜213〉primer

<220>

<221>misc_feature

＜223〉antisense sequences of 790-809 bit sequence among the SEQ ID N0:1

<400>3

ccagcatctt?tgttgcacgt 20

<210>4

<211>20

<212>DNA

＜213〉primer

<400>4

tagcttcggc?tcgctcgcta 20

<210>5

<211>20

<212>DNA

＜213〉primer

<400>5

atggccgaag?acgcagacat 20

<210>6

<211>20

<212>DNA

＜213〉primer

<400>6

ttaaccactt?cccagcatct 20

<210>7

<211>28

<212>DNA

＜213〉primer

<400>7

ggggtaccat?ggccgaagac?gcagacat 28

<210>8

<211>29

<212>DNA

＜213〉primer

<400>8

cccaagcttt?taaccacttc?ccagcatct 29

Claims (9)

1. a SNAP-25 gene or proteic purposes is characterized in that, are used to prepare the reagent that detects the Mammals memory level.

2. purposes as claimed in claim 1 is characterized in that, described SNAP-25 gene or albumen are people SNAP-25 gene or albumen.

3. a SNAP-25 gene or proteic purposes is characterized in that, are used to prepare the medicine that promotes the Mammals memory level.

4. purposes as claimed in claim 3 is characterized in that, described SNAP-25 gene or albumen are people SNAP-25 gene or albumen.

5. a pharmaceutical composition that promotes Mammals memory is characterized in that, it contains the Mammals SNAP-25 albumen and the pharmaceutically acceptable carrier of safe and effective amount.

6. the pharmaceutical composition of promotion Mammals memory as claimed in claim 5 is characterized in that described SNAP-25 albumen is selected from down the SNAP-25 albumen of treated animal: people, rat and mouse.

7. a test kit that detects mammalian subject memory level is characterized in that, it comprises and SNAP-25 protein-specific bonded antibody.

8. method of screening the medicine that promotes Mammals memory is characterized in that it comprises step:

(1) cDNA of SNAP-25 gene is packed into expression vector, the transfection mammalian cell strain, the proteic cell strain of SNAP-25 is expressed in preparation;

(2) add test compounds in the proteic cell strain nutrient solution of the expression SNAP-25 in step (1), detect the variation of SNAP-25 expressing quantity; The compound that promotes the SNAP-25 expressing quantity to increase is exactly the drug candidate that promotes Mammals memory.

9. method as claimed in claim 8 is characterized in that, described SNAP-25 albumen has the aminoacid sequence shown in the SEQ ID NO:2.

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