CN102504016A - Amyloid fibrillar oligomer conformational epitope polypeptide and application thereof - Google Patents

Abstract

The invention discloses an amyloid fibrillar oligomer conformational epitope polypeptide and application of the amyloid fibrillar oligomer conformational epitope polypeptide. The polypeptide provided by the invention is a protein (1) or (2) as follows: the polypeptide (1) has an amino acid sequence shown in SEQ ID NO.1 in the sequence table, and the polypeptide (2) is derived from the polypeptide (1) by replacement and/or deletion and/or insertion of one or more amino acid residues in the amino acid sequence of SEQ ID NO.1 in the sequence table and has the same function as the polypeptide (1). The studies show that the polypeptide and antibodies thereof have the following benefits: the polypeptide can effectively stimulate production of a specific antibody against amyloid fibrillar oligomer in vivo, and the antibody can inhibit aggregation and cytotoxicity of amyloid, improve space memory in experimental animals, and reduce content of senile plaques and/or content of amyloid-beta (A-beta) in the brain.

Description

Amyloid fibering oligomer conformation type antigen epitope polypeptide and application thereof

Technical field

The present invention relates to biological technical field, relate in particular to a kind of amyloid fibering oligomer conformation type antigen epitope polypeptide and application thereof.

Background technology

In recent years, the multiple disease that causes of the gathering of amyloid or polypeptide self has caused increasing harm to human beings'health.Nontoxicity of some protein own or toxicity are very little; But they self can be gathered into oligomer (oligomer) with toxic action or fibrous material (fibril) and cause a series of diseases; As the alzheimer's disease (being commonly called as senile dementia) that causes by Beta-amyloid (A-Beta) (Alzheimer ' s disease; AD); The Parkinson's disease that cause by alpha-synuclein (Parkinson ' s disease, PD), at least ten surplus kinds of encephalopathics of the humans and animals that comprises mad cow disease that causes by prion protein (Prion protein (PrP)); At least 9 kinds of heredity nerve degenerative diseases that comprise Huntington Chorea (Huntington ' s disease) that cause by the polypeptide that contains poly glumine (PolyQ); (islet amyloid polypeptide, IAPP, type ii diabetes that amylin) causes and the N,O-Diacetylmuramidase (lysozyme) that is caused by long-time dialysis assemble the disease that deposition causes etc. by islet amyloid polypeptide.Wherein, to human health risk maximum be AD and PD and type ii diabetes.Medical statistics shows, 5～6% suffer from AD among the over-65s the elderly in China and the American-European countries, and sickness rate rises year by year.This disease has been classified as causes dead the fourth-largest disease, is only second to heart trouble, cancer and apoplexy.Other have an appointment 1% over-65s the elderly suffers from PD.And the number of suffering from type ii diabetes can reach more than 5% of total population.These diseases have caused increasing harm to human beings'health, and the basic reason of its morbidity (or partly cause) is the gathering of amyloid or polypeptide self.

Research shows that the gathering of different proteins starts from protein (or polypeptide) monomer of false folding or sex change at first, and the formation of monomer polypeptide interchain hydrogen bond has caused the polymerization of protein molecular.At first form spherical oligomer by the solubility of electronics or the about 3-10nm of the observable size of AFM; Some oligomer can further be combined into crooked flexible silk (protofibril), and then the formation diameter is the smooth surface of 6-10nm or fiber in the shape of a spiral.The protein of non-homology finally can form the protein polymer with analog structure.The fiber that is formed by various amyloid aggregations such as A-Beta, alpha-synuclein and amylin all contains " cross-Beta-sheet " structure; Wherein the skeleton of Beta lamella formation is vertical with the fiber longitudinal axis, and the hydrogen bond net in the skeleton is parallel with the longitudinal axis.Polypeptide is arranged as parallel Beta chain in the Beta lamella, in the Beta lamella, amino acid all has accurate position.Even the oligomer of different sources or fiber also have similar specific structure, oligomer or fiber-specific identification antibody can be respectively with amyloid monomer by different sources forms oligomer and fiber combine, and do not combine with their monomer.This shows that oligomer or fiber can form common oligomer or the distinctive epitope of fiber that is independent of outside its amino acid primary sequence by the protein polypeptide skeleton.Discover that amyloid can form several types of oligomer, comprising the fibering oligomer (fibrillar oligomer, FO), preceding fibering oligomer (profibrillar oligomer, PFO) and other type oligomer.FO has formed with the amyloid fiber has similar space structure; And the formed FO of the amyloid with different aminoacids primary sequence also has the space structure of this type; The antibody (OC) of the FO specific combination that promptly forms with A-beta; Can combine with the A-beta fiber, can also combine with the FO and the fiber of formation such as alpha-synuclein, IAPP and PolyQ, but do not combine with the monomer of each amyloid.What the past people thought the amyloid disease always is to be caused by the insoluble fiber that protein aggregation becomes.A large amount of in recent years researchs show that the crucial paathogenic factor of many amyloid diseases is the less water-soluble oligomeric things of volume.And the pathogenic effects of FO will obviously be better than PFO.The disease that is caused by the amyloid oligomer exists similar mechanism, and promptly cell membrane damage, oxidative stress, mitochondrial function imbalance, necrocytosis, signal transmission are unusual etc.But people it be unclear that the detailed mechanism that oligomer influences the normal physiological activity of cell.How are the topological framework of oligomer and epitope thereof, and these all are that people demand the problem inquiring into and solve urgently how could to suppress its cytotoxicity etc. effectively.

Thereby use the gathering that active and passive immunological technique treatment AD suppress A-Beta, and the removing of quickening A-Beta is one of focus of AD research field.2000-2002 U.S. scientific research personnel uses the vaccine that A-Beta processes and has carried out an animal experiment and a clinical trial phase.Experimental result is encouraging, and laboratory animal and patient's clinical symptom obviously alleviates, and hypomnesis is checked, and pathological change is also significantly gone down.Simultaneously, use anti-A-Beta antibody the passive immunotherapy of laboratory animal has also been received better curative effect.Yet, unfortunately in phase ii clinical trial,, have 6% patient table to reveal meningitic symptom and pathological change, the patient who has even take place dead though most patient still shows good result.Therefore, the experimental study to A-Beta vaccine and antibody stops immediately.However, can find out that from above-mentioned research the A-Beta vaccine should affirm the result of treatment of AD.Research shows that the spinoff that is caused by the A-Beta vaccine possibly be the autoimmune response by the T cell mediated of this vaccine-induced body generation, and the generation of this spinoff is caused by Th1 rather than the immunoreation of Th2 type.In order to overcome the spinoff of A-Beta vaccine, when vaccine design, should strengthen bringing out B cell response and the immunoreactive generation of Th2 type, and avoid bringing out the immunoreation of Th1 type.Vaccine based on the design of A-Beta oligomer epitope peptide can easily be taken into account above-mentioned characteristic, also can not induce body to form the monomeric antibody of anti-A-Beta.Back one characteristic can thoroughly be eliminated the possibility that has side effects between the A-Beta monomer in vaccine and the body, and can not influence the normal physiological function of A-beta.Therefore, the peptide vaccine of developing according to A-Beta oligomer epitope will have the potential using value, and obtain the prerequisite that A-Beta oligomer epitope is this peptide vaccine design and development.So, all need obtain amyloid oligomer conformation type antigen epitope polypeptide in scientific research and clinical study with in using.

Summary of the invention

An object of the present invention is to provide a kind of amyloid fibering oligomer conformation type antigen epitope polypeptide.

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

1) polypeptide of forming by the aminoacid sequence shown in the sequence in the sequence table 1;

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

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

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

A, the polypeptide of forming by the aminoacid sequence shown in the sequence in the sequence table 2;

B, the polypeptide of forming by the aminoacid sequence shown in the sequence in the sequence table 3;

C, the polypeptide of forming by the aminoacid sequence shown in the sequence in the sequence table 4;

D, the polypeptide of forming by the aminoacid sequence shown in the sequence in the sequence table 5.

The scope that the antibody that aforementioned polypeptides obtains as antigen is protected for the present invention.

Another object of the present invention provides a kind of amyloid aggregation suppressor factor.

Suppressor factor provided by the invention, its activeconstituents are above-mentioned antibody; Said amyloid is specially A β 42 (amyloid beta), PrP (PrPC), amylin (pancreas opsonin) or α-synuclein (α synapse nucleoprotein).

The 3rd purpose of the present invention provides a kind of cytotoxicity suppressor factor of amyloid.

Suppressor factor provided by the invention, its activeconstituents are above-mentioned antibody; Said cell is specially the SH-SY5Y cell, and said amyloid is specially A β 42, PrP, amylin or α-synuclein.

The 4th purpose of the present invention provides a kind of diseases induced product of amyloid aggregation that prevents and/or treats.

Product provided by the invention, its activeconstituents are following 1) or 2): 1) above-mentioned polypeptide; 2) above-mentioned antibody; Said product is specially medicine or vaccine; The diseases induced alzheimer's disease that is specially of said amyloid aggregation.

The application of above-mentioned antibody in suppressing amyloid aggregation and/or preparation amyloid aggregation suppressor factor also is the scope that the present invention protects; In this was used, said amyloid was specially A β 42, PrP, amylin or α-synuclein.

The application of above-mentioned antibody in the cytotoxicity suppressor factor of cytotoxicity that suppresses amyloid and/or preparation amyloid also is the scope that the present invention protects; In this was used, said cell was specially the SH-SY5Y cell, and said amyloid is specially A β 42, PrP, amylin or α-synuclein.

Aforementioned polypeptides or above-mentioned antibody also are the scopes that the present invention protects in the application that preparation prevents and/or treats in the diseases induced product of amyloid aggregation; Said product is specially medicine or vaccine; The diseases induced alzheimer's disease that is specially of said amyloid aggregation.

Aforementioned polypeptides is embodied in the quantity that improves the spatial memory ability, reduces senile plaque in the brain and/or reduces the application in the A β content in the effect that preparation prevents and/or treats the diseases induced product of amyloid aggregation; In this was used, said A β was specially A β 42 or A β 40.

Of the present inventionly experiment showed, that polypeptide provided by the invention can combine with antibody OC in vitro tests, proved that this polypeptide is one of pairing epitope of polyclonal antibody OC; This polypeptide has good antigenic characteristic, but the effective stimulus body produces antibody; This polypeptide is a common epitope on the multiple amyloid fibering oligomer; The antibody of this polypeptide can obviously suppress amyloid A β 42, PrP, amylin, α-synuclein, lysozyme gathering; This polypeptide antibody can obviously suppress the cytotoxicity of A β 42, PrP, amylin, α-synuclein, lysozyme.

Polypeptide of the present invention and antibody specific thereof have following beneficial effect:

1. polypeptide of the present invention can produce the specific antibody of discerning the amyloid oligomer by the effective stimulus body, and this antibody can suppress the gathering and the cytotoxicity of amyloid.

2. maybe not can induce body to form the monomeric antibody of anti-A-Beta based on vaccine of the present invention, can eliminate the possibility that has side effects between the A-Beta monomer in vaccine and the body, and can not influence the normal physiological function of A-beta.

3. polypeptide of the present invention has better immunogenicity, but the effective stimulus body produces antibody, thereby can be used as good immunogen in amyloid treatment of diseases or prevention PEOS aspect the development of vaccine.

Description of drawings

Fig. 1 is that PEOS and OC bonded ELISA measure

Fig. 2 measures through the PEOS and the OC bonded ELISA of displacement, disappearance, insertion amino acid or change amino-acid sequence

Fig. 3 is that the antibody in the mice serum combines with PEOS after the PEOS immunity

Fig. 4 is that the antibody in the rabbit anteserum after the PEOS immunity combines with PEOS

Fig. 5 is that PEOS antibody combines with the oligomer and the fiber of multiple amyloid

Fig. 6 is the gathering that ThT fluorometric assay PEOS antibody suppresses amyloid

Fig. 7 suppresses the cytotoxicity of amyloid for PEOS antibody

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

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

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

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

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

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

Embodiment

Employed experimental technique is ordinary method like no specified otherwise among the following embodiment.

Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources among the following embodiment.

It is that (prescription is: 5.84g NaCl, 4.72g Na for 7.2 damping fluid that PBS damping fluid among the 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).

All experimental datas of PEOS are all through at least 3 independently experiment acquisitions among the following embodiment, 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.Treatment Analysis for the data memory of water maze is that the Two-way ANOVA that uses two factor repeating datas carries out.

Amyloid among the following embodiment is A β 42 (amyloid beta), PrP (PrPC), amylin (pancreas opsonin) or α-synuclein (α synapse nucleoprotein).

The preparation of embodiment 1, polypeptide PEOS and verivate and antibody

One, the acquisition of polypeptide PEOS

The present invention uses phage display technology, with the polyclonal antibody OC (catalog number is AB2286 for Millipore, USA) of specific recognition fibering oligomer and fiber for the screening substrate, with 1 * 10 ⁸Kind of annular seven peptides have carried out the four-wheel elutriation, use phage E LISA therefrom filtered out can with the obvious bonded phage clone of OC, obtain through amino acid sequencing.Polypeptide PEOS, its aminoacid sequence is: Cys-Trp-Thr-Thr-His-Gln-Arg-Ser-Cys (sequence 1, CWTTHQRSC)

The synthesis preparation method of PEOS is:

PEOS is a cyclic peptide; 2 halfcystines that add two ends are 9 peptides; But synthetic; (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 by the synthetic preparation of ordinary method by the biochemical ltd of Shanghai gill; Atherton E, Sheppard R C.Solid Phase Peptide Synthesis:A Practical Approach.Oxford:IRL Press, 1989); PEOS purity is more than or equal to 95%; PEOS is stored in-20 ℃, avoids multigelation.

The synthetic simultaneously polypeptide PEOS (C hold connection) of 6 Histidines that be connected with as label.

Two, the acquisition of the verivate of PEOS

Aminoacid sequence shown in the polypeptide PEOS (sequence 1) through replacing, lack, add amino acid or changing amino-acid sequence, is obtained the verivate of following PEOS:

Specific as follows:

PEOS-Δ T: with polypeptide that Threonine obtains of PEOS polypeptide (sequence 1) deletion, sequence is: and Cys-Trp-Thr-His-Gln-Arg-Ser-Cys (sequence 2, CWTHQRSC);

PEOS-W/Y: the tryptophane of PEOS polypeptide (sequence 1) is replaced as the polypeptide that tyrosine obtains, and sequence is: and Cys-Tyr-Thr-Thr-His-Gln-Arg-Ser-Cys (sequence 3, CYTTHQRSC);

PEOS-G: with inserting the polypeptide that glycocoll obtains in the PEOS polypeptide (sequence 1), sequence is: Cys-Trp-Thr-Thr-His-Gln-Gly-Arg-Ser-Cys (sequence 4, CWTTHQGRSC),

PEOS-WT/GD: PEOS polypeptide (sequence 1) change aminoacid sequence while replaced color propylhomoserin and Threonine are respectively glycocoll and aspartic acid, and sequence is: and Cys-His-Gln-Thr-Gly-Ser-Arg-Asp-Cys (sequence 5, CHQTGSRDC).

Synthetic simultaneously polypeptide PEOS-Δ T, PEOS-W/Y, PEOS-G and the PEOS-WT/GD (the C end is connected) that is connected with 6 Histidines as label.

Three, the acquisition of polypeptide PEOS antibody

1, PEOS can bring out body generation antibody

Use to show that ((disulfide linkage intra-annular 7 peptides are expressed in the N-terminal of pIII can the gene fusion of PEOS polypeptide to be built into the M13 phage of expression PEOS polypeptide to the less important capsid protein of M13 phage (pIII) for the M13 phage of PEOS polypeptide; After first halfcystine is next to L-Ala; A bit of interval polypeptide is arranged behind second halfcystine; Form by Gly-Gly-Gly-Ser; Be wild-type pIII albumen then, the immune BALB/c mouse of M13 phage (New England Biolab Inc, catalog number is E8120SC) (Beijing Vital River Experimental Animals Technology Co., Ltd.; Catalog number is a BALB/c mouse SPF level) (7/group), 1010 in every each immune phage.Per 2 all immunity once are total to immunity 4 times.The 4th immunity blood sampling in back 15 days, separation of serum.Use the content that ELISA detects anti-PEOS antibody in the serum: with the not tagged PEOS of 100 μ l (because PEOS peptide chain weak point; In order to make PEOS be adsorbed on the elisa plate preferably; And do not block the site of itself and antibodies, add two strong-hydrophobicity amino acid, then encapsulate the peptide sequence that uses and be CWTTHQRSCIV at the C of PEOS end) (1 μ g/ hole) encapsulate 96 hole enzyme linked immunological microwell plates; Put 37 ℃ of 2h, with 37 ℃ of sealings of 3%BSA 2h (200 μ L/ hole).Wash plate 3 times with PBS, add the serum of 2000 times of dilutions, behind the incubated at room 1h, with the PBS washing that contains 0.1%Tween-20 3 times.Every hole adds the sheep anti mouse ELIAS secondary antibody (1: 3000) after the 100 μ l dilution, behind the incubated at room 1h, with the PBS washing that contains 0.1%Tween-20 3 times.Every hole adds substrate solution TMB 100 μ L, places 37 ℃ of 20min, and every then hole adds 50 μ l 1mmol/L sulfuric acid termination reactions, on enzyme-linked immunosorbent assay instrument, measures OD value (wavelength 450nm).

The result is as shown in Figure 3, and the OD value of 7 mouse of immune PEOS (wavelength 450nm) is respectively 0.45,0.52,0.58,0.61,0.66,0.82,0.76; All, can find out that 7 mouse of immune PEOS can both produce the antibody of anti-PEOS, show that PEOS has good antigenic characteristic, but the effective stimulus body produces antibody than the height after the M13 phage immunity that does not have polypeptide to show.

2, the preparation of polypeptide PEOS antibody

(fundamental immunity is added equal-volume and is added Fu Shi Freund's complete adjuvant (Sigma for the first time will to connect the immune new zealand white rabbit of PEOS (C of PEOS end connects KLH) of KLH (keyhole limpet hemocyanin); F-5881); Later booster immunization adds equal-volume freund 's incomplete adjuvant (Sigma, F5506)).Per 2 all immunity once, each antigen consumption be 200 μ g/ only.Immunity is 4 times altogether.After last immune 15 days, gather rabbit anteserum, and the ELISA method of application experiment 1 evaluation serum antibody titer (encapsulating elisa plate with PEOS-KLH), negative control is not immune collection serum; The result is as shown in Figure 4.Rabbit anteserum after the immunity is after 1000,4000,16000,64000 times of dilutions, and the OD value that ELISA measures is respectively 1.42,1.1,0.74,0.32, and negative control sera is 0.146.This result shows that once more PEOS has good antigenic characteristic, but the effective stimulus body produces anti-PEOS antibody.

Sepharose to be connected with the PEOS polypeptide carries out affinity chromatography to rabbit anteserum; Adopt the sepharose (5ml) (being prepared by general epoxidation method of attachment by gill (Shanghai) biochemical corp) of coupling PEOS, elutriant is the glycine buffer (the 0.2M glycine solution is with hydrochloric acid adjust pH to 3.0) of pH3.0; Flow velocity is 0.7ml/min; 3 column volumes of wash-out merge and preserve the 7-13ml elutriant, are the antibody of the anti-PEOS of purifying.

The application of embodiment 2, polypeptide PEOS and antibody thereof

One, the application of PEOS antibody

1, PEOS antibody and each amyloid fibering oligomer and fiber combines

With the A β 42 (U.S. American Peptide Co. that buys; Catalog number is 62-0-80B), amylin (U.S. American Peptide Co.; Catalog number is 74-5-14B), α-synuclein (U.S. rpeptide, catalog number S-1001-2), PrP (U.S. American Peptide Co., catalog number is 62-0-07B) handle as follows: (HFIP) is dissolved to 1mg/ml with hexafluoroisopropanol; Room temperature (25 ℃) ultrasonication 10min; Divide to install in the epidorf pipe, the HFIP that volatilizees in the vacuum environment is in-20 ℃ of preservations.Place 20min with preceding A β 42, amylin, the α-synuclein that HFIP was handled in room temperature, add DMSO 99.8MIN. (DMSO), making each protein concentration is 5mg/ml, and the PBS damping fluid with 0.02M pH7.4 is diluted to desired concn then.PrP then directly is prepared into desired concn with 0.02M pH 7.4 PBS.At this moment, each amyloid is a free state.

The preparation of A β 42 fibering oligomer is that the above-mentioned monomer A β that obtains 42 of 0.3mg is dissolved among the 150 μ l HFIP, and room temperature is placed 20min.Be diluted with water to 80 μ M then, sample is got supernatant and is blown steaming HFIP with nitrogen with the centrifugal 15min of 14,000 * G, and sample is put room temperature and stirred 24h (rotating speed 500RPM) with little rod.

The preparation of A β 42 fibers with HFIP handled, DMSO dissolved A β 42 monomers are diluted to 10 μ M with the PBS damping fluid of 0.02M pH7.4, hatch 24h for 37 ℃, get deposition with the centrifugal 15min of 14,000 * G.

The preparation of amylin fibering oligomer with HFIP handled, DMSO dissolved amylin monomer is diluted to 20 μ M with the PBS damping fluid of 0.02M pH7.4, hatches 2h for 37 ℃, gets supernatant with the centrifugal 15min of 14,000 * G.

The preparation of amylin fiber is that above-mentioned amylin solution is hatched 5h for 37 ℃, gets deposition with the centrifugal 15min of 14,000 * G.

The preparation of the fibering oligomer of α-synuclein with HFIP handled, DMSO dissolved α-synuclein monomer is diluted to 40 μ M with the PBS damping fluid of 0.02M pH7.4, hatches 2d for 37 ℃, gets supernatant with the centrifugal 15min of 14,000 * G.

The preparation of α-synuclein fiber then is that α-synuclein solution is hatched 4d for 37 ℃, gets deposition with the centrifugal 15min of 14,000 * G.

The preparation of the fibering oligomer of PrP is that PrP solution is diluted to 200 μ M with the PBS damping fluid of 0.02M pH7.4, hatches 2d for 37 ℃, gets supernatant with the centrifugal 15min of 14,000 * G.

The preparation of PrP fiber then is that PrP solution is hatched 5d for 37 ℃, gets deposition with the centrifugal 15min of 14,000 * G.

After above-mentioned specimen preparation is intact, measure protein concentration with BCA protein detection kit (U.S. Merck, production number are 71285-3) commonly used.And confirm the exactness of sample with the accumulation shape of amyloid in the transmission scanning electron microscope observation sample.

Monomer (monomer), fibering oligomer (oligomer) and fiber (fibril) form (1 μ g/ hole) of above-mentioned 100 μ l are encapsulated 96 hole enzyme linked immunological microwell plates, the negative contrast of BSA respectively.Antibody with the above-mentioned anti-PEOS of purifying that is obtained by embodiment 1 is anti-as one, and is anti-as two with the goat-anti rabbit LgG (Beijing Bo Aosen Bioisystech Co., Ltd, catalog number is bse-0295G) of HRP mark, and by the operation that experimentizes of above-mentioned ELISA method.

The result is as shown in Figure 5, and anti-PEOS antibody is respectively 0.11,0.13,0.08,0.06,0.07 with OD value after monomer, the BSA of A β, PrP, amylin, α-synuclein combine respectively;

Anti-PEOS antibody is respectively 0.72,0.66,0.51,0.38,0.085 with OD value after fibering oligomer, the BSA of A β, PrP, amylin, α-synuclein combine respectively;

Anti-PEOS antibody is respectively 0.58,0.52,0.41,0.53,0.066 with OD value after fiber, the BSA of A β, PrP, amylin, α-synuclein combine respectively;

Can find out that anti-PEOS antibody can combine with fibering oligomer and the fiber of A β, PrP, amylin, α-synuclein respectively, show that PEOS is a common epitope on multiple amyloid fibering oligomer and the fiber.

The antibody of the oligomer of this specific combination amyloid can be used for experimental study, and amyloid treatment of diseases and clinical diagnosis are also had the potential using value.

2, the gathering of PEOS antibody vitro inhibition amyloid

1) preparation monomer, method is with above-mentioned 1, and the A β 42 that obtains, amylin, α-synuclein monomer dilute with PBS, obtain A β 42, PrP, amylin, α-synuclein solution monomer;

2) with the PBS damping fluid dilution of the antibody of the above-mentioned anti-PEOS of purifying that obtains by embodiment 1 with 0.02M pH7.4; Join then among A β 42, PrP, amylin, the α-synuclein solution (the PBS damping fluid of pH7.4), make the final concentration of each albumin A β 42, PrP, amylin, α-synuclein be respectively 10,200,20,40 μ M.The mol ratio of the antibody of each albumen and the anti-PEOS of purifying was respectively 1: 1,10: 1,1: 1 and 2: 1.With the protein solution of the antibody that do not add the anti-PEOS of purifying as contrast.A β 42, PrP, amylin, α-synuclein sample were placed respectively 1 day, 5 days, 5 hours and 4 days in 37 ℃.

3) it is 5 μ M that the phosphate buffered saline buffer that thioflavine (ThT) is dissolved in pH6.5,50mM makes its concentration.The sample 20 μ l that get after hatching join in the black enzyme plate that contains 180 μ l ThT solution.On multi-functional ELIASA, measure the fluorescence intensity of ThT behind the mixing with the emission wavelength of 450nm excitation wavelength and 482nm.The fluorescence intensity of each sample is deducted the background fluorescence of ThT itself and the significant difference situation between the analytic sample.Add the albumen fluorescence intensity that reduces behind the PEOS antibody and be the inhibiting rate of antibody protein aggregation divided by the albumen fluorescence intensity that does not add antibody.

The result is as shown in Figure 6; Can find out; The antibody of the anti-PEOS of purifying is respectively 93%, 96%, 65%, 35% to the gathering inhibiting rate of A β 42, amylin, α-synuclein, PrP, shows that the antibody of anti-PEOS can obviously suppress the gathering of each amyloid.

3, PEOS antibody suppresses the cytotoxicity of amyloid

Substratum (MEM) with containing 10% foetal calf serum is made into the individual cells suspension with SH-SY5Y cell (Chinese Academy of Medical Sciences tumour cell storehouse, production number are SH-SY5Y), with 10000 cell inoculations in every hole to 96 porocyte culture plates, every pore volume 100 μ L.Cell was cultivated incubator CO 24 hours in 37 ℃ ₂Concentration is 5%.Every hole adds the sample (mixture of the antibody of amyloid A β 42, PrP, amylin, α-synuclein and the above-mentioned anti-PEOS of purifying that is obtained by embodiment 1; Amyloid A β 42, PrP, amylin, α-synuclein oligomer and PBS contrast separately); A β 42, PrP, amylin, the proteic final concentration of α-synuclein are respectively 2,200,5,2 μ M, and the final concentration of PEOS antibody is 1,20,2.5,1 μ M.Cell continued to cultivate after 48 hours, and every hole adds MTT solution (5mg/mL) 10 μ L, hatches 3 hours for 37 ℃; Stop cultivating; Every hole adds 100 μ L dissolution of crystals liquid (10%SDS and 5% isopropylcarbinol are dissolved among the 0.01M HCL), and 37 ℃ of incubated overnight are fully dissolved crystallisate.On multi-functional enzyme-linked immunosorbent assay instrument, measure each hole absorbance value with the 570nm wavelength.With the absorbancy of sample divided by the absorbancy of the cell that does not add sample as the cell activity index, and the significant difference situation between the analytic sample (is compared *, P＜0.05 with each amyloid contrast; #, P＜0.01).

The result is as shown in Figure 7, and the cell relative reactivity of amyloid A β 42, PrP, amylin, α-synuclein is respectively 58.8%, 46%, 59%, 53% separately;

The cell relative reactivity of the mixture of amyloid A β 42, PrP, amylin, α-synuclein and PEOS antibody is respectively 88%, 82%, 78%, 70%;

Explain that PEOS antibody can improve cytoactive, thereby show that PEOS antibody can obviously suppress the toxicity to the SH-SY5Y cell of A β 42, PrP, amylin, α-synuclein.

Two, the application of PEOS polypeptide

1, polypeptide PEOS and verivate thereof combine with polyclonal antibody OC

1), PEOS combines with polyclonal antibody OC

100 μ l polyclonal antibody OC (0.2 and 1 μ g/ hole) are encapsulated 96 hole enzyme linked immunological microwell plates (with the hole that do not encapsulate OC as negative control), put 37 ℃ of 2h, with 37 ℃ of sealings of 3%BSA 2h, 200 μ L/ holes.Wash plate 3 times with PBS.Every hole adds 100 μ l and has histidine-tagged PEOS, 0.5 μ g/ hole), after room temperature (25 ℃) is hatched 1h, with the PBS washing that contains 0.1%Tween-20 3 times.Behind the incubated at room 1h, with the PBS washing that contains 0.1%Tween-20 3 times.Every hole adds the anti-histidine-tagged antibody of the HRP mark after the 100 μ l dilution, behind the incubated at room 1h, with the PBS washing that contains 0.1%Tween-20 3 times.Every hole adds substrate solution TMB 100 μ L, places 37 ℃ of 20min, and every then hole adds 50 μ l 1mmol/L sulfuric acid termination reactions, on enzyme-linked immunosorbent assay instrument, measures OD value (wavelength 450nm).

The result is as shown in Figure 1, and the OD value of 1 μ gOC, 0.2 μ gOC, negative control is respectively 0.72,0.28,0.051; Can find out, compare with negative control that 1 μ gOC and 0.2 μ gOC can be discerned by PEOS, also we can say, PEOS can be by the OC specific recognition.PEOS is the polypeptide that obtains as the screening substrate with OC, and it has confirmed that with the combination of OC it is one of pairing epitope of polyclonal antibody OC (comparing *, P＜0.05, #, P＜0.01 with the negative control that does not encapsulate PEOS).

2), the verivate of PEOS combines with polyclonal antibody OC

In order to detect the situation that combines of improved polypeptide and OC; These polypeptide are combined to competition (PEOS-His tag+PEOS-Δ T, PEOS-His tag+PEOS-W/Y, PEOS-His tag+PEOS-G, PEOS-His tag+PEOS-WT/GD, PEOS-His tag, PEOS-His tag+PEOS) with the PEOS that has histidine-tagged not transformation with OC, then with the combine situation of the ELISA detection latter with OC.

Specific as follows: the PEOS that 1 μ g is had a label adds with the mixture 100 μ l of 5 μ g PEOS-Δ T, PEOS-W/Y, PEOS-G, PEOS-WT/GD, PEOS respectively and is coated with in the hole of OC; Obtain PEOS-His tag+PEOS-Δ T, PEOS-His tag+PEOS-W/Y, PEOS-His tag+PEOS-G, PEOS-His tag+PEOS-WT/GD, PEOS-His tag+PEOS; With PEOS-His tag is contrast; Have the PEOS of label and combining of OC with the ELISA detection; Measure the OD value, method is with above-mentioned 1.With the negative contrast of BSA.

The result sees Fig. 2, and wherein, the column diagram group in left side combines for each group polypeptide and OC's, and the column diagram group on right side is organized combining of polypeptide and negative control (BSA) for each,

OD value after PEOS-His tag+PEOS-Δ T, PEOS-His tag+PEOS-W/Y, PEOS-His tag+PEOS-G, PEOS-His tag+PEOS-WT/GD, PEOS-His tag+PEOS, PEOS-His tag and OC combine is respectively 0.31,0.22,0.33,0.42,0.23,0.75;

OD value after PEOS-His tag+PEOS-Δ T, PEOS-His tag+PEOS-W/Y, PEOS-His tag+PEOS-G, PEOS-His tag+PEOS-WT/GD, PEOS-His tag+PEOS, PEOS-His tag and negative control (BSA) combine is respectively 0.076,0.044,0.031,0.035,0.051,0.048;

Can find out; Certain amino acid of PEOS polypeptide still can combine OC with having of unmodified transformation of histidine-tagged PEOS effective competition through displacement, disappearance, after in polypeptide, increasing amino acid or changing amino-acid sequence, cause the PEOS that has label and OC combine obviously reduce.

2, the PEOS polypeptide improves the sick effect embodiment of AD

1) the PEOS polypeptide improves the memory capability of AD transgenic mice

Experimental technique:

(1) the immune 8 month female AD transgenic mices of M13 phage (as contrast) of using the M13 phage of the displaying PEOS polypeptide that is obtained by embodiment 1 and not having polypeptide to show (change APP and PS1 gene (APPswe/PS1dE9) mouse available from Jackson Lab; Bar Harbor; ME; The U.S.; Catalog number is: 004462, obtain containing the AD female mice of the mouse of APP and PS1 gene through identifying after the breeding for commentaries on classics APP and PS1 gene (APPswe/PS1dE9)), obtain two groups of mouse: AD+PEOS and AD con.; (change APP and PS1 gene (APPswe/PS1dE9) mouse with 8 wild-type mices as the WT group available from Jackson Lab; Bar Harbor, ME, the U.S.; Catalog number is: 004462, after the breeding through identifying that the mouse that is not contained APP and PS1 gene is a wild-type mice; WT) be contrast.

Three groups altogether, 8 every group; Every each immune phage 10 ¹⁰Individual.Per 2 all immunity once are total to immunity 4 times.The 4th immunity beginning in back 15 days water maze laboratory.

(2) memory training: be placed on 25 ℃ of room temperatures, the environmental adaptation of humidity 46% 3 days before the training of mouse water maze.All study of behaviour detect the mode that all adopts randomized, double-blind in the test.Before the training, remove platform, central authorities put into gently at tank, let its 60s that freely moves about.Measure every group of mouse swimming quadrant preference, select the wall of preference opposite tank, as this mouse initial release position.Let mouse stand in platform (10 centimetres of platform diameter) for the first time before the training and go up 15s, let it remember the locus of platform in the lower storage reservoir (1.1 meters of diameters).Platform is placed on second quadrant middle part, 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 water gently, be allowed to condition in the tank and swim.The mouse 2s that on platform, stands just stops timing, thinks and appears on the stage, and the training time is the longest at every turn to be 90s.During this time application software (available from medicine institute of Chinese medical courses in general institute) write down its track and from entry to the time of climbing up platform, i.e. latent period.Be allowed to condition at if mouse is found platform in 90s and stop 10s on the platform.If mouse can not find platform in 90s, then behind 90s, guide its upper mounting plate, and be allowed to condition at and stop 10s on the platform by the experimenter.Continuously training is 5 days, trains every day 2 times, is spaced apart 3-4h between twice.Interval 24h removes platform then, lets mouse in water, seek platform 90s.With video recording and training in 5 days of software system record and exploration result of experiment, and with the amount of repetition number two-factor analysis of variance (two-way ANOVA, repeated measures) processing experimental result.

Experimental result is as shown in Figure 8, and along with the growth of training time, wild group of mouse finds shorten gradually the latent period of platform, promptly reaches equilibrium state after 4 days.Compare and have significant difference (comparing #, P＜0.05 with AD control mice group) with contrast AD mouse (AD con.) latent period that the AD mouse (AD+PEOS) of injection PEOS is found platform when training the 3rd, 4,5 day, and the result sees Fig. 8 A.Removing behind the platform AD mouse (AD+PEOS) of injection PEOS finds and also is significantly less than AD control group (AD con.) (Fig. 8 B) latent period of platform.The number of times that the AD mouse (AD+PEOS) of injection PEOS passes the platform position is significantly higher than AD control group (AD con.) (Fig. 8 C), and the time that the AD mouse behind the injection PEOS belongs in the target quadrant is significantly higher than AD control mice (Fig. 8 D).These results show that the AD mouse spatial memory ability behind the immune PEOS obviously is better than the AD control group of not injecting PEOS.

2), the PEOS polypeptide reduces the quantity of senile plaque in the AD Transgenic Mice Brain

Use the influence of ThS fluorescent dye measuring PEOS to senile plaque in the AD Transgenic Mice Brain:

(1) with above-mentioned 1 AD+PEOS group, AD con. group, WT group mouse heart perfusion, get cerebral tissue, application organizes refrigerating fulid and liquid nitrogen carry out freezing treatment, and are stored in-80 ℃.Time spent cuts into slices with freezing-microtome, and slice thickness 16 μ m whenever get the observation of dyeing at a distance from 9 sections.

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

(3) use fluorescent microscope and gather image (Olympus BX60).

Detected result is seen Fig. 9, and wherein A is AD con. group mouse brain cortex; B is the mouse brain cortex of AD+PEOS group; C is AD con. group mouse hippocampus; D is the mouse hippocampus of AD+PEOS group; Can find out that AD (AD con.) the mouse brain cortex (A) of injection PBS and the senile plaque quantity in the hippocampus (C) are obviously more than (D) senile plaque quantity in injecting immune polypeptide PEOS AD mouse (AD+PEOS group) cortex (B) and the hippocampus.

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

The result is shown in figure 10, can find out, the senile plaque area proportion (visual field person in middle and old age's spot area accounts for the ratio of whole visual field) of AD con. group mouse brain cortex is 11.2%;

The senile plaque area proportion of the mouse brain cortex of AD+PEOS group is 3.7%;

The senile plaque area proportion of AD con. group mouse hippocampus is 13.5%;

The senile plaque area proportion of the mouse hippocampus of AD+PEOS group is 4.9%;

Statistical analysis shows that the ratio that the mouse senile plaque area of injecting immune polypeptide PEOS accounts for the section area obviously reduces (comparing *, P＜0.01 with injection PBS group).

3), the PEOS polypeptide reduces A β 40 and A β 42 levels in the AD Transgenic Mice Brain

Cerebral tissue homogenate in containing the PBS of proteinase inhibitor (pH7.2) of above-mentioned 1 AD+PEOS group, AD con. group mouse.With the centrifugal 30min of 15000rpm, collect supernatant then.(with the preparation of tris-HCl damping fluid, pH8.0), centrifugal treating is collected supernatant to the Guanidinium hydrochloride of deposition adding 5M then.The A β 40 that is dissolved in and is insoluble to PBS is measured according to the ELISA method by test kit (catalog number is respectively for Invotrigen, USA: KHB3441 and KHB3482) with A β 42 levels.

A β level is measured OD value and A β typical curve according to A β amount (ng or the mg) expression that every Borneo camphor tissue contains with the ELISA method.(standard substance are to be equipped with in the test kit, Invotrigen, and USA, catalog number is respectively: KHB3441 and KHB3482, 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.)

The result sees Figure 11-13, and wherein Figure 11 is solubility A β 40 levels in the AD Transgenic Mice Brain of injection PEOS polypeptide; Figure 12 is solubility A β 42 levels in the AD Transgenic Mice Brain of injection PEOS polypeptide; Figure 13 is insoluble A β 40 and 42 levels in the AD Transgenic Mice Brain of injection PEOS polypeptide.

Among Figure 11, the content of solubility A β 40 is respectively 16.4ng/g brain albumen and 119.2ng/g brain albumen in the AD transgenic mice (AD+PEOS) of injection PEOS polypeptide, AD transgenic mice (AD con.) brain of injection PBS),

Among Figure 12 in AD transgenic mice (AD con.) brain of the AD transgenic mice (AD+PEOS) of injection PEOS polypeptide, injection PBS the content of solubility A β 42 be respectively 0.82ng/g brain albumen and 2.35ng/g brain albumen,

For insoluble A β 40 content in AD transgenic mice (AD con.) brain of the AD transgenic mice (AD+PEOS) of injection PEOS polypeptide, injection PBS are respectively 3.2mg/g brain albumen and 8.9mg/g brain albumen, insoluble A β 42 content are respectively 3.48mg/g brain albumen and 9.46mg/g brain albumen among Figure 13;

The result shows that PBS solubility A β 40 (Figure 11) (compare *, P＜0.05 with injection PBS group with A β 42 (Figure 12) level and insoluble A β 40 with obviously reductions of A β 42 levels (Figure 13) in the AD injected in mice immunity PEOS hindbrain; #, P＜0.01).

The above only is a preferred implementation 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 improvement and retouching, these improvement and retouching also should be regarded as protection scope of the present invention.

Claims (9)

1. a peptide species is following 1) or 2):

1) polypeptide of forming by the aminoacid sequence shown in the sequence in the sequence table 1;

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

2. polypeptide according to claim 1 is characterized in that:

2) polypeptide shown in is any one among following a, b, the c:

A, the polypeptide of forming by the aminoacid sequence shown in the sequence in the sequence table 2;

B, the polypeptide of forming by the aminoacid sequence shown in the sequence in the sequence table 3;

C, the polypeptide of forming by the aminoacid sequence shown in the sequence in the sequence table 4;

D, the polypeptide of forming by the aminoacid sequence shown in the sequence in the sequence table 5.

3. the antibody that obtains as antigen with polypeptide shown in claim 1 or 2.

4. amyloid aggregation suppressor factor, its activeconstituents is the described antibody of claim 3; Said amyloid is specially A β 42, PrP, amylin or α-synuclein.

5. the cytotoxicity suppressor factor of an amyloid, its activeconstituents is the described antibody of claim 3; Said cell is specially the SH-SY5Y cell, and said amyloid is specially A β 42, PrP, amylin or α-synuclein.

6. one kind prevents and/or treats the diseases induced product of amyloid aggregation, and its activeconstituents is following 1) or 2): 1) claim 1 or 2 described polypeptide; 2) the described antibody of claim 3; Said product is specially medicine or vaccine; The diseases induced alzheimer's disease that is specially of said amyloid aggregation.

7. the application of the said antibody of claim 3 in the aggregation inhibitor that suppresses amyloid aggregation and/or preparation amyloid; Said amyloid is specially A β 42, PrP, amylin or α-synuclein.

8. the application of the said antibody of claim 3 in the cytotoxicity suppressor factor of cytotoxicity that suppresses amyloid and/or preparation amyloid; Said cell is specially the SH-SY5Y cell, and said amyloid is specially A β 42, PrP, amylin or α-synuclein.

9. claim 1 or 2 described polypeptide or the described antibody of claim 3 prevent and/or treat the application in the diseases induced product of amyloid aggregation in preparation; Said product is specially medicine or vaccine; The diseases induced alzheimer's disease that is specially of said amyloid aggregation.

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