CN102516360B - Polypeptide capable of inhibiting beta-secretase digestion effect and application thereof - Google Patents

Abstract

The invention discloses a polypeptide capable of inhibiting a beta-secretase digestion effect, and application thereof. The polypeptide provided by the invention is characterized in that 1, the polypeptide has an amino acid sequence shown in the sequence 1 of the sequence table; or 2, the polypeptide has an amino acid sequence which is derived from the amino acid sequence shown in the sequence 1 through replacement and/or deletion and/or addition of one or more amino acid residues, and has functions the same as functions of the polypeptide having the amino acid sequence shown in the sequence 1. A result of an experiment shows that the polypeptide has a capability of simultaneous combination with a beta-secretase digestion site and A beta42 of an amyloid precursor protein (APP), has effects of inhibiting a beta-secretase digestion effect and A beta42 production, can inhibit A beta42 cytotoxicity, and can protect SH-SY5Y neurobiastoma cells from A beta42 cytotoxicity influences.

Description

A kind ofly suppress polypeptide and the application thereof that the beta-secretase enzyme is cut effect

Technical field

The present invention relates to biological technical field, relate in particular to a kind of polypeptide and application thereof that the beta-secretase enzyme is cut effect that suppress.

Background technology

Alzheimer's disease Alzheimer ' s Disease, hereinafter to be referred as AD, be commonly called as senile dementia, assemble to form by amyloid-beta monomer molecule (β-Amyloid 40/42 (A β 40/42 is hereinafter to be referred as A β)) the elderly that the aggregation of tool toxic action causes mainly to form senile plaque as the nerve degenerative diseases of feature take hypomnesis and brain.Medical statistics shows, in China and American-European countries more than 60 years old the elderly have 5～6% to suffer from the A Zihaimo disease.This disease has been listed in and has caused dead the fourth-largest disease, is only second to heart trouble, cancer and apoplexy.The existing population approximately 100,000,000 more than 60 years old of China is 5% estimation according to population person in middle and old age dementia morbidity more than 60 years old, and may there be patients with Alzheimer disease approximately 5,000,000 in the whole nation.This disease brings huge burden for family and society.The researchist estimates, if prophylactico-therapeutic measures can not get improving, the former senile dementia that is in the 4th of Death causes will become 21 century senior health and fitness's No.1 killer, will have 1/3rd over-65s old man and suffer from senile dementia.At present, the clinical treatment of AD is just used some neurotrophies and the anti-inflammatory type medicine carries out symptomatic treatment, still the medicine without any specific treatment AD emerges.

Based on the pathogenesis of A β, at present to the research of AD treatment preparation mainly from three aspects: reduce by suppressing enzymatic reaction the removing that A β produced, suppressed the cohesion of A β and accelerates A β.A β mainly is comprised of 39-42 amino-acid residue, and wherein A β 42 gatherings are very fast, cytotoxicity is larger, is main AD paathogenic factor.It comes from the precursor protein that molecular weight is 110-135KD (Amyloid Precursor Protein, APP).APP produces the aminoterminal of A β through the effect of beta-secretase, the gamma secretase effect produces the carboxyl terminal of A β in after birth.Under normal circumstances, before the beta-secretase effect, the Lys16-Leu17 place of α Secretases in the middle of the A β is cracked into APP APPs α and is embedded in carboxyl fragment C83 shorter on film.APPs α has growth regulating and neurotrophic effect, can extend neuronic survival, stablizes and strengthen the structure of cynapse, strengthens neuronic function etc.In the situation that body is abnormal, β, gamma secretase increased activity, or the reduction of α secretase activity will produce the A β of more amount, and cause the generation of AD.So, reduce the effect of cutting of β or gamma secretase enzyme or reinforcing alpha Secretases enzyme and cut one of focus that the research of effect becomes AD research field in the recent decade.At present, people have had than quantum jump the understanding research of molecular biological characteristic, the mechanism of action and the moiety etc. of this three fermentoid.The multienzyme complex that gamma secretase is comprised of a plurality of components, decapacitation cracking APP produces outside A β, also participates in the generation that other more than 30 kinds have the I type membranin of important physiological function.Therefore, the generation that the activity that suppresses merely this fermentoid reduces A β will affect the normal function of other many associated protein.Similarly, validity and the side-effect problem of the inhibitor of inhibition beta-secretase also never are resolved.People not yet find so far and can be applicable to clinical inhibition β or the gamma secretase enzyme is cut the active drug of effect.

Studies show that, beta-secretase restriction enzyme site amino acid conservative property is very strong.The people such as Arbel in 2005 as target spot, prepare the specific antibody of anti-this restriction enzyme site with beta-secretase restriction enzyme site in the APP peptide chain.This antibody can with the effective combination of APP, stop the enzyme of beta-secretase to cut effect, thereby reduce the generation of cell A β.This shows the combination of beta-secretase restriction enzyme site in Cucumber and APP peptide chain, can check the enzymatic action of beta-secretase, and this will become one of effective way that reduces A β generation.Yet antibody molecule is larger, be difficult for by hemato encephalic barrier, and antibody itself has antigenicity preferably, easily causes the side effects such as transformation reactions.

Summary of the invention

An object of the present invention is to provide a kind of polypeptide and application thereof that the beta-secretase enzyme is cut effect that suppress.

Polypeptide provided by the invention is following 1) or 2):

1) polypeptide that is formed by the aminoacid sequence shown in sequence in sequence table 1;

2) with the aminoacid sequence shown in sequence in sequence table 1 through replacement and/or disappearance and/or the interpolation of one or several amino-acid residue and have identical function by the derivative polypeptide of sequence 1.

In above-mentioned albumen, the replacement of one or several amino-acid residue and/or disappearance and/or interpolation refer to replacement and/or disappearance and/or the interpolation of no more than ten amino-acid residues.

In aforementioned polypeptides, 2) polypeptide shown in is any one in following a, b, c:

A, the polypeptide that is formed by the aminoacid sequence shown in sequence in sequence table 2;

B, the polypeptide that is formed by the aminoacid sequence shown in sequence in sequence table 3;

C, the polypeptide that is formed by the aminoacid sequence shown in sequence in sequence table 4.

Another object of the present invention is to provide the Cytotoxic preparation of a kind of inhibition A β.

Inhibitor provided by the invention, its activeconstituents are above-mentioned polypeptide; Described cell is specially the SH-SY5Y cell, and described A β is specially A β 42.

The 3rd purpose of the present invention is to provide the preparation that a kind of A of inhibition β produces.

Inhibitor provided by the invention, its activeconstituents are above-mentioned polypeptide, and described A β is specially A β 40 or A β 42.

The 4th purpose of the present invention is to provide a kind of product for the treatment of alzheimer's disease for preparing.

Product provided by the invention, its activeconstituents are above-mentioned polypeptide; Described product is specially medicine.

The application of aforementioned polypeptides in suppressing A β cytotoxicity and/or the Cytotoxic preparation of preparation inhibition A β is also the scope of protection of the invention, and described A β is specially A β 42; Described inhibition A β 42 cytotoxicities are embodied in the activity that improves cell; Described cell is specially the SH-SY5Y cell.

The application of aforementioned polypeptides in the preparation of the generation that suppresses A β and/or preparation inhibition A β generation is also the scope of protection of the invention; Described A β is specially A β 40 or A β 42.

Above-mentioned inhibition A β produces the enzymolysis that also can be embodied in the upper β of vitro inhibition APP site.

The application of above-mentioned polypeptide in the product of preparation treatment alzheimer's disease is also the scope of protection of the invention; Described product is specially medicine.

The effect of above-mentioned polypeptide in the product of preparation treatment alzheimer's disease is embodied in as follows: improve spatial memory capacity, reduce the quantity of senile plaque and/or reduce A β content; Described A β is specially A β 40 or A β 42

Of the present invention experiment showed, the invention provides a peptide species, is called YS1, has the polypeptide that suppresses the effect of cutting of beta-secretase enzyme and suppress A β cytotoxic effect, this polypeptide have with APP on the beta-secretase restriction enzyme site and the ability of A β 42 combinations simultaneously; This polypeptide can suppress the cytotoxicity of A β 42, and protection SH-SY5Y neuroblastoma cell is avoided the impact of A β 42 toxicity.The animal memory experiment is carried out in the brain side room that this polypeptide is expelled to AD transgenic mice (APPswe/PS1dE9 double transgenic), result shows, this polypeptide can obviously improve the spatial memory capacity of mouse, reduces the quantity of senile plaque in brain and the output of A β 40 and A β 42.This polypeptide molecular weight is little, easily passes hemato encephalic barrier, and it is relatively poor to compare the antigenicities such as other high molecular weight protein such as antibody, and side effect is less, is being with a wide range of applications aspect the prevention of alzheimer's disease and treatment.

Description of drawings

Fig. 1 is that beta-secretase restriction enzyme site and the A β ELISA of being combined of YS1 on APP measures

Fig. 2 for through displacement, lack or insert beta-secretase restriction enzyme site and the A β ELISA mensuration of being combined of amino acid whose YS1 with APP on

Fig. 3 is the cytotoxicity that YS1 suppresses A β 42

Fig. 4 is that the enzyme in β site on Western-blot detection YS1 inhibition APP is cut effect

Fig. 5 is to protein band amount of densities fractional analysis result in Fig. 4

Fig. 6 is the extracellular generation that YS1 suppresses A β 40

Fig. 7 is the extracellular generation that YS1 suppresses A β 42

Fig. 8 is the water maze laboratory of the AD transgenic mice of injection YS1 polypeptide

Fig. 9 is the variation of the AD transgenic mice brain senile plaque of injection YS1 polypeptide

Figure 10 is the variation of the AD transgenic mice brain senile plaque area of injection YS1 polypeptide

Figure 11 is solubility A β 40 levels in the AD Transgenic Mice Brain of injection YS1 polypeptide

Figure 12 is solubility A β 42 levels in the AD Transgenic Mice Brain of injection YS1 polypeptide

Figure 13 is insoluble A β 40 and 42 levels in the AD Transgenic Mice Brain of injection YS1 polypeptide

Embodiment

The experimental technique that uses in following embodiment is ordinary method if no special instructions.

In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.

It is that (formula is: 5.84g NaCl, 4.72g Na for 7.2 damping fluid that PBS damping fluid in following embodiment is pH value ₂HPO ₄, 2.64g NaH ₂PO ₄.2H ₂O, water are made into 1 liter, and transferring pH is 7.2);

In following embodiment, all experimental datas are all independently tested by at least 3 times except the water maze memory experiment and are obtained, and experimental data is expressed as mean number plus-minus standard deviation.The statistical study of data is to use One-way ANOVA software to carry out, and uses the Two-way ANOVA of repeating data for the comparative analysis of many groups repetitive memory power determination data.

The acquisition of embodiment 1, Pro-Gln-Val-Gly-His-Leu and derivative thereof

One, the acquisition of Pro-Gln-Val-Gly-His-Leu

According to the nosogenesis of alzheimer's disease AD, the basic method for the treatment of AD should be generation, the gathering that suppresses A β that reduces amyloid beta (A β) or the removing of accelerating A β.Use phage display technology, to contain the upper beta-secretase restriction enzyme site of APP and A beta portion amino terminal sequence as the screening substrate, with 1 * 10 ⁸Plant seven peptides and carried out the four-wheel elutriation, use Phage-ELISA therefrom filtered out many can be simultaneously with APP on the beta-secretase restriction enzyme site and the polypeptide of A β 42 obvious combinations, binding characteristic by comparing beta-secretase restriction enzyme site on many peptide chains and APP and A β 42 and on A beta peptide aggregation and Cytotoxic impact, finally the combination selection by the peptide interchain goes out Pro-Gln-Val-Gly-His-Leu.

The aminoacid sequence of YS1 is: and Pro-Gln-Val-Gly-His-Leu (sequence 1, PQVGHL).

Pro-Gln-Val-Gly-His-Leu is one or six peptides, by Shanghai gill biochemical company limited according to a conventional method synthetic preparation (preparation method is referring to Weng C C, Peter D W.Fmoc Solid Phase Peptide Synthesis:A Practical Approach.Oxford:University Press, 2000.1-8; Atherton E, Sheppard R C.Solid Phase Peptide Synthesis:A Practical Approach.Oxford:IRL Press, 1989), obtaining YS1 polypeptide purity is more than or equal to 95%, YS1 is stored in-20 ℃, avoids multigelation.

Synthesize simultaneously and be connected with 6 Histidines as the Pro-Gln-Val-Gly-His-Leu of label.

Two, the acquisition of Pro-Gln-Val-Gly-His-Leu derivative

The Pro-Gln-Val-Gly-His-Leu derivative for the amino acid of YS1 through displacement, lack or insert the polypeptide that other amino acid obtains, specific as follows:

The polypeptide of YS1-Δ H for aforementioned polypeptides YS1 (sequence 1) disappearance Histidine is obtained, sequence be Pro-Gln-Val-Gly-Leu (sequence 2, PQVGL);

YS1-A inserts the polypeptide that L-Ala obtains in will aforementioned polypeptides YS1 (sequence 1), sequence be Pro-Gln-Val-Gly-Ala-His-Leu (sequence 3, PQVGAHL).

YS1-HL/PRT is for to be replaced as with the Histidine in aforementioned polypeptides YS1 (sequence 1) and leucine the polypeptide that proline(Pro), arginine and Threonine obtain, sequence be Pro-Gln-Val-Gly-Pro-Rrg-Thr (sequence 4, PQVGPRT); But the aforementioned polypeptides derivative is synthetic all.

Syntheticly simultaneously be connected with 6 Histidines as the Pro-Gln-Val-Gly-His-Leu of label-Δ H, YS1-A and YS1-HL/PRT.

The application of embodiment 2, Pro-Gln-Val-Gly-His-Leu

One, the specific binding of the beta-secretase restriction enzyme site on Pro-Gln-Val-Gly-His-Leu and derivative thereof and APP and A β 42

1, the specific binding of the beta-secretase restriction enzyme site on Pro-Gln-Val-Gly-His-Leu and APP and A β 42

Respectively with the beta-secretase restriction enzyme site polypeptide (Glu-Val-Lys-Met-Asp-Glu-Phe) on the APP of PBS damping fluid preparation (Shanghai gill polypeptide company is synthetic), A β 42 (American Peptide Co., catalog number is 62-0-80B) and negative control polypeptide (Lys-Thr-Asp-Met-Lys-His-Met-Ala-Gly-Ala-Ala-Ala-Ala-Gly-Ala-Val-Val-Gly-Gly-Leu-Gly) with the coated 96 hole Aminated ELISA Plates in 1 μ g/ hole, 4 ℃ are spent the night.With the blank site of not being combined with polypeptide on BSA sealase target, then add to be connected with 6 Histidines as the YS1 that is obtained by embodiment 1 of label (1 μ g/ hole), room temperature (25 ℃) effect 2h.After washing, add can with the antibody (Beijing Bo Aosen biotech company, catalog number is bs-0899R) of the coupling HRP of histidine-tagged combination, room temperature effect 1h.After washing, add substrate TMB, develop the color after 20 minutes, measure the absorbancy at 450nm place with multi-functional microplate reader.

Triplicate under identical condition the results are shown in Figure data in 1, figure and is the mean value of three times, and error bar is SD,

β site polypeptide is that YS1 is combined with beta-secretase restriction enzyme site polypeptide, and absorbancy is 0.2;

A β 42 is YS1 and A β 42 combinations, and absorbancy is 0.17;

The contrast polypeptide is that YS1 is combined with the negative control polypeptide, and absorbancy is 0.07;

Can find out, compare with the negative control polypeptide, *, P＜0.01, show YS1 can with APP on beta-secretase restriction enzyme site and A β 42 combinations simultaneously.

2, Pro-Gln-Val-Gly-His-Leu derivative and A β 42 specific combination

To be connected histidine-tagged with YS1-HL/PRT by Pro-Gln-Val-Gly-His-Leu derivative YS1-Δ H, the YS1-A that embodiment 1 obtains, then detect specific binding with beta-secretase restriction enzyme site (β site polypeptide), negative control polypeptide (negative control) and A β 42 according to above-mentioned 1 test method, the every hole of each polypeptide adds 1 μ g/ hole, measures the OD value with the ELISA method.

The results are shown in shown in Figure 2ly, YS1-Δ H, YS1-A and the YS1-HL/PRT OD450nm after β site polypeptide is combined respectively are respectively 0.18,0.18,0.16;

YS1-Δ H, YS1-A and YS1-HL/PRT respectively with A β 42 in conjunction with after OD450nm be respectively 0.16,0.14,0.15;

YS1-Δ H, YS1-A and the YS1-HL/PRTY OD450nm after the contrast polypeptide is combined respectively are respectively 0.04,0.03,0.04;

Show certain amino acid of YS1 through displacement, disappearance or still keep increase amino acid in polypeptide after with APP on the beta-secretase restriction enzyme site and the ability (comparing P＜0.01 with negative control) of A β 42 while combinations.

Two, the cytotoxicity of YS1 vitro inhibition A β 42

With the substratum (MEM that contains 10% foetal calf serum, Invotrigen) with SH-SY5Y cell (Chinese Academy of Medical Sciences's tumour cell storehouse, production number is SH-SY5Y) be made into the individual cells suspension, be inoculated into 96 porocyte culture plates with 10000, every hole cell, every pore volume 100 μ L.Cell is in 37 ℃ of cultivations 24 hours, incubator CO ₂Concentration is 5%.Every hole adds following sample:

A β 42+5 μ M is 1 μ M by the final concentration of YS1:A β 42 albumen that embodiment 1 obtains, and the final concentration of YS1 is 5 μ M;

A β 42+20 μ M is 1 μ M by the final concentration of YS1:A β 42 albumen that embodiment 1 obtains, and the final concentration of YS1 is 20 μ M;

The final concentration of A β 42:A β 42 albumen is 1 μ M;

Take PBS as contrast.

Above-mentioned cell continues to cultivate after 48 hours, every hole adds MTT solution (Sigma, catalog number is that M5655, solvent are PBS, 5mg/mL) 10 μ L, hatched 3 hours for 37 ℃, stop cultivating, every hole adds 100 μ L dissolution of crystals liquid (10%SDS and 5% isopropylcarbinol are dissolved in 0.01M HCL), 37 ℃ of overnight incubation are fully dissolved crystallisate.Measure each hole absorbance value with the 570nm wavelength on multi-functional enzyme-linked immunosorbent assay instrument.After deducting background, with the absorbancy of sample divided by the absorbancy of the cell that does not the add sample activity index as cell, and the significant difference situation between analytic sample.

Result is (*, P＜0.05 as shown in Figure 3; #, P＜0.01),

The Cell relative activity of A β 42+5 μ M YS1 is 75.2%;

The Cell relative activity of A β 42+20 μ M YS1 is 86.0%

The Cell relative activity of A β 42 is 62.5%;

Compare with independent A β 42 samples, YS1 adds the activity that can significantly improve cell, shows that namely YS1 can significantly suppress the cytotoxicity of A β 42.

Three, the enzymolysis in the upper β of YS1 vitro inhibition APP site and the generation of A β

In application A β 40 and A β 42 test kits (catalog number is respectively for Invotrigen, USA: KHB3441 and KHB3482) mensuration cell culture fluid, the amount of A β 40 and A β 42, carry out experiment test according to shop instruction.Be summarized as follows:

(7PA2 is by the present of medical college of Harvard University, Podlisny with the Chinese hamster ovary cell of transfection APP/Val717Phe gene, M.B., Selkoe, D.J. etc. (1995) J.Biol.Chem.270,9564-9570, the public can obtain from Tsing-Hua University.) cultivate (substratum is for containing 10% foetal calf serum and G418 (500 μ g/ml) DMEM) on 6 orifice plates, when being 90% to abundance, change serum free medium, the YS1 that the YS1 that to add PBS (YS1 that 0 μ M is obtained by embodiment 1), final concentration be 50 μ M is obtained by embodiment 1 and 200 μ M are obtained by embodiment 1, continue to cultivate after 15 hours, collecting cell and nutrient solution, respectively APP, C83 and C99 in cell being carried out Western-blot detects, A β 40 in nutrient solution and A β 42 levels are carried out the ELISA detection, specific as follows:

1) Western-blot measures APP, C83 and C99 level in the 7PA2 cell

7PA2 cell and cell lysis buffer solution (contain 150mM NaCl, 50mM Tris, 0.5%w/vNa-deoxycholate, 1%v/v Nonidet P-40,0.1%SDS and proteinase inhibitor, pH 7.4) are mixed, hatch 10min on ice.Cell pyrolysis liquid is mixed with the sample buffer of equivalent, boiled after mixing 1 minute.Join in 16%Tris-SDS gel sample hole, carry out electrophoresis.Concentrated glue 60V, separation gel 80V makes tetrabromophenol sulfonphthalein to sheet glass lower rim 1cm place.The gel that will contain sample downcuts, with sample with 80V wet method transferase 12 7min to 0.45 μ m pvdf membrane (Millipore, Billerica, MA, USA).After shifting end, pvdf membrane is rinsed a little, with 37 ℃ of sealing 2h of 8% skim-milk; PBST washes film 1 time, adds to contain the antibody A 8717 (how anti-the rabbit source is, can be combined with APP, C83, C99, Sigma-Aldrich Corp.St.Louis, MO, USA) of the TBST dilution of 3% skim-milk, room temperature effect 1 hour.Flushing membrane, add the goat anti-rabbit antibody (Beijing longitude and latitude is won thing scientific and technological development company limited of Hang Seng, and catalog number is B006,1: 5000) that connects horseradish peroxidase (HRP) to act on 1 hour again.Transferring to protein band applied chemistry luminous detection box on film (IL, USA, catalog number is 32209 for Pierce, Rockford) detects.Exposure 3min, development 30s.

The results are shown in Figure 4 and shown in Figure 5, Fig. 4 is qualitative analysis, Fig. 5 be in Fig. 4 band with respect to β Actin muscle (Sigma-aldrich, catalog number is A5316-.2ML) optical density(OD), Figure 4 and 5 show, (APP can form the C99 fragment after β site hydrolysis, form the C83 fragment from α site hydrolysis in the hydrolysis that can obviously suppress the upper β of APP site that adds of YS1.C99 reduces explanation from the reaction reduction of β site hydrolysis APP, and C83 rising explanation raises from the reaction of α site hydrolysis APP), cause the minimizing of C99 enzymolysis generation.Correspondingly, the hydrolysis in the upper α of APP site is promoted, and what cause that the C83 enzymolysis produces increases.The amount to APP that adds of YS1 has no significant effect.

2) ELISA measures A β 40/A β 42 levels in the 7PA2 cell culture fluid

(1) A β is detected elisa plate in box with 200 μ l 2% skim-milks sealings, put 37 ℃ 2 hours.

(2) add each cell culture fluid of 100 μ l (1 μ g/ml), put 37 ℃ 1 hour.

(3) get rid of sample solution, to contain 0.05% Tween-20PBS washing microwell plate 3 times.

(4) add respectively the mouse source monoclonal antibody (carrying in test kit) of 100 μ l (1: 2000 dilution) specific combination A β 40 or A β 42, put 37 ℃ 1 hour.

(5) wash same step (3).

(6) add the sheep anti mouse two of 100 μ l (1: 1000 dilution) HRP mark anti-, put 37 ℃ 1 hour.

(7) wash same step (3).

(8) add 100 μ l TMB colour developings, with the sulfuric acid color development stopping reaction of 1N.

(9) use the absorbance value that enzyme mark detector is measured the 450nm place.

(A β 40 standard substance are that test kit carries to result, and the function of the typical curve of A β 40 is y=625x-14.8, and x is the OD value, and y is the quality of A β 40 as shown in Figure 6.), can find out, add A β 40 content in the YS1 cell of 0 μ M YS1,50 μ M YS1,200 μ M to be respectively 100%, 91%, 76% (with A β 40 amounts that do not add YS1 as 100%);

(A β 42 standard substance are that test kit carries to result, and the function of the typical curve of A β 42 is y=636.62x-7.15, and x is the OD value, and y is the quality of A β 42 as shown in Figure 7.), add A β 42 content in the cell of 0 μ M YS1,50 μ M YS1,200 μ M YS1 to be respectively 100%, 78%, 60% (with A β 42 amounts that do not add YS1 as 100%);

Can find out, add after the YS1 polypeptide level of the A β 40 (seeing Fig. 6) in cell culture fluid and A β 42 (seeing Fig. 7) all obviously to reduce and (compare *, P＜0.05 with adding the PBS contrast; #, P＜0.01).

Four, YS1 improves the effect embodiment of AD disease

1, improve the memory capability of AD transgenic mice

Experimental technique:

1) brain side room injection: the AD female mice that turns APP and PS1 gene that 8 monthly ages are large (turns APP and PS1 gene (APPswe/PS1dE9) mouse available from Jackson Lab, Bar Harbor, ME, the U.S., catalog number is: 004462, obtain containing the mouse of APP and PS1 gene for turning the AD mouse of APP and PS1 gene (APPswe/PS1dE9) through identifying after breeding) be divided at random and inject Pro-Gln-Val-Gly-His-Leu (AD+YS1) and PBS (AD) group, 8 every group.Simultaneously take 8 ages identical brood wild females mouse (as non-AD type, (turn APP and PS1 gene (APPswe/PS1dE9) mouse available from Jackson Lab, Bar Harbor, ME, the U.S., catalog number is: 004462, after breeding through identifying that the mouse that obtains not containing APP and PS1 gene is wild-type mice; WT) as contrast.Totally three groups.

Before intracerebral injection, 12h can't help water to the mouse fasting.At first with mouse anesthesia, anaesthesia dosage is: 25g mouse peritoneal injection 0.1mL concentration is 10% Chloral Hydrate.Mouse is fixed on stereotaxic instrument, and reference standard collection of illustrative plates (Academic Publish) carries out side room, the three-dimensional location of brain drug administration by injection.Mouse intracerebroventricular positional parameter is: 1.8mm after anterior fontanelle, center line is other to be opened ± 1.8mm, 2.5mm under dura mater.With sterilized water dissolving YS1 to 1mg/ml, injection speed 0.2 μ l/min, injected dose is 5 μ L.The 5min that injected that let the acupuncture needle remain at a certain point.Every injection in 7 days once, inject altogether 4 times.In last injection rear 5 days, mouse is carried out the water maze memory capability detect.

2) memory training: be placed on 25 ℃ of room temperatures, the environmental adaptation of humidity 46% 3 days before the Mice water maze training.All study of behaviour detect the mode that all adopts randomized, double-blind in test.Before training, remove platform, central authorities put into gently at tank, allow its 60s that freely moves about.Measure every group of mouse swimming quadrant preference, select the wall of opposite tank, as this mouse initial release position.Allow for the first time mouse stand in the upper 15s of platform (10 centimetres of platform diameter) before the training, allow its remember the locus of platform in lower storage reservoir (1.1 meters of diameters).Platform is placed in the middle part of the second quadrant, and the upper surface of platform is apart from 1.5 centimetres of the waters surface.Chi Shuizhong adds milk powder, to increase the visual contrast of animal, is beneficial to image recording.Mouse is faced pool wall, put into gently water, be allowed to condition at the tank went swimming.The mouse 2s that stands on platform just stops timing, thinks and appears on the stage, and the training time is at every turn the longest is 90s.Application software (available from medicine institute of Chinese medical courses in general institutes) records its track and from entry to the time of climbing up platform, i.e. latent period during this time.Be allowed to condition at if mouse is found platform in 90s and stop 10s on platform.If mouse can not find platform in 90s, guide its upper mounting plate by the experimenter after 90s, and be allowed to condition at and stop 10s on platform.Continuously training is 5 days, trains every day 2 times, is spaced apart 3-4h between twice.Record experimental result with video recording and software system, and process experimental result with the amount of repetition number two-factor analysis of variance (two-way ANOVA, repeated measures).

Experimental result as shown in Figure 8, along with the growth of training time, wild group of mouse (WT) finds shorten gradually the latent period of platform, namely reaches equilibrium state after 4 days.The latent period that the AD mouse (AD+YS1) of injection YS1 is found platform is in training during by the 4th day and 5 days the time, compare with the AD mouse (AD) of injection PBS contrast and have significant difference and (compare with injection PBS AD mouse group, *, P＜0.05), show that the AD mouse spatial memory capacity of injection after YS1 is significantly better than the control group of injection PBS.

2, YS1 reduces the quantity of senile plaque in the AD Transgenic Mice Brain

The impact of application ThS fluorescent dye measuring YS1 on senile plaque in the AD Transgenic Mice Brain:

1) with above-mentioned 1 three groups of AD+YS1, AD that obtain, WT group mouse heart perfusion, get cerebral tissue, use and organize refrigerating fulid and liquid nitrogen to carry out freezing treatment.And be stored in-80 ℃.Used time cuts into slices with freezing-microtome, and slice thickness 16 μ m get the observation of dyeing every 9 sections.

2) use 1mg/ml ThS (with 70% ethanol preparation) and contaminate section 10min, then with 70% alcohol flushing 3 times.

3) use fluorescent microscope and gather image (Olympus BX60, excitation wavelength 488nm, 4 * object lens).

Detected result as shown in Figure 9, a is AD contrast rat cerebral cortex; B is the AD rat cerebral cortex of injection YS1 polypeptide; C is AD contrast mouse hippocampus; D is the AD mouse hippocampus of injection YS1 polypeptide.Can find out, the AD mouse brain cortex (a) of injection PBS and the senile plaque quantity in hippocampus (d) are obviously more than (e) senile plaque quantity (c, f are respectively cortex and the hippocampus of wild-type mice) in injection Pro-Gln-Val-Gly-His-Leu AD mouse brain cortex (b) and hippocampus.

Application software system (visiopharm) is measured the senile plaque area of injection polypeptide (AD+YS1, n=8) and control mice (AD, n=8) hippocampus and cortex (every each location detection 10-12 of animal opens section).

Results of statistical analysis is injected the hippocampus of polypeptide group (AD+YS1) and the senile plaque area proportion (visual field person in middle and old age's spot area accounts for the ratio in the whole visual field) of cortex and is respectively 3.3% and 7.85% as shown in figure 10; The hippocampus of injection PBS group (AD) and the senile plaque area proportion of cortex are respectively 10.9% and 13.2%;

Show, the ratio that the mouse senile plaque area of injection Pro-Gln-Val-Gly-His-Leu accounts for the section area obviously reduces (comparing *, P＜0.01 with injection PBS group).

3, YS1 reduces A β 40 and A β 42 levels in the AD Transgenic Mice Brain

With the cerebral tissue of above-mentioned 1 three groups of AD+YS1, AD that obtain, WT group mouse contain the PBS of proteinase inhibitor (pH7.2, proteinase inhibitor, Calbiochem company, catalog number is: homogenate 539131,100 times of dilutions).Then with the centrifugal 30min of 15000rpm, collect supernatant.Precipitation adds the Guanidinium hydrochloride of 5M (with the preparation of tris-HCl damping fluid, pH8.0).Then centrifugal treating, collect supernatant.The A β 40 and A β 42 levels that are dissolved in and are insoluble to PBS are measured according to the method shown in above-mentioned three by test kit (catalog number is respectively for Invotrigen, USA: KHB3441 and KHB3482).(A β 40 and A β 42 are that test kit carries for standard substance.The function of the typical curve of A β 40 and A β 42 is respectively y=625x-14.8; Y=636.62x-7.15.X is the OD value, and y is the quality of A β 40 or A β 42) A β amount (ng or mg) expression that A β level contains according to every Borneo camphor tissue, the results are shown in Figure 11-13,

In Figure 11, in AD transgenic mice (AD) brain of the AD transgenic mice (AD+YS1) of injection YS1 polypeptide, injection PBS polypeptide, the content of solubility A β 40 is respectively 30.8ng/g brain albumen and (compares with injection PBS group with 119.1ng/g brain albumen, *, P＜0.01).

In Figure 12 in AD transgenic mice (AD) brain of the AD transgenic mice (AD+YS1) of injection YS1 polypeptide, injection PBS polypeptide the content of solubility A β 42 be respectively 1.03ng/g brain albumen and (compare with injection PBS group with 2.35ng/g brain albumen, *, P＜0.05).

For being respectively 6.1mg/g brain albumen, insoluble A β 40 content in AD transgenic mice (AD) brain of the AD transgenic mice (AD+YS1) of injection YS1 polypeptide, injection PBS polypeptide (compare with injection PBS group with 9.5mg/g brain albumen in Figure 13, *, P＜0.05), insoluble A β 42 content are respectively 7.0mg/g brain albumen and (compare with injection PBS group with 11.2mg/g brain albumen, *, P＜0.05).

Result shows PBS solubility A β 40 (Figure 11) and A β 42 (Figure 12) level and insoluble A β 40 and all obviously reductions of A β 42 levels (Figure 13) in AD injected in mice YS1 hindbrain.

The above is only the preferred embodiment of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.

Studies show that, YS1 produces at the external A β that can obviously reduce, and can suppress the cytotoxicity of A β.In the body that application AD transgenic animal carry out, experimental result shows, YS1 can obviously improve the spatial memory capacity of mouse, reduces the quantity of senile plaque in brain and the generation of A β 40 and A β 42.The YS1 molecular weight is very little, easily pass hemato encephalic barrier performance curative effect, and antigenicity is relatively poor, and side effect is less, is a polypeptide that has AD treatment potentiality.

Claims (7)

1. a peptide species, the polypeptide that is formed by the aminoacid sequence shown in sequence in sequence table 1.

2. one kind is suppressed the Cytotoxic preparation of A β, and its activeconstituents is polypeptide claimed in claim 1; Described cell is specially the SH-SY5Y cell, and described A β is concrete A β 42.

3. one kind is suppressed the preparation that A β produces, and its activeconstituents is polypeptide claimed in claim 1, and described A β is specially A β 40 or A β 42.

4. one kind prepares the product for the treatment of alzheimer's disease, and its activeconstituents is polypeptide claimed in claim 1; Described product is specially medicine.

5. the application of the described polypeptide of claim 1 in preparation inhibition A β 42 Cytotoxic preparations; Described A β is concrete A β 42; Described cell is specially the SH-SY5Y cell.

6. the application of the described polypeptide of claim 1 in the preparation that preparation inhibition A β produces; Described A β is specially A β 40 or A β 42.

7. the application of polypeptide claimed in claim 1 in the product of preparation treatment alzheimer's disease; Described product is specially medicine.

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