CN102504016B - Amyloid fibrillar oligomer conformational epitope polypeptide and application thereof - Google Patents
Amyloid fibrillar oligomer conformational epitope polypeptide and application thereof Download PDFInfo
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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
Technical field
The present invention relates to biological technical field, relate in particular to a kind of amyloid fibrillar oligomer conformational epitope polypeptide and application thereof.
Background technology
In recent years, the various diseases that the gathering of amyloid or polypeptide self causes has caused increasing harm to the mankind's health.The nontoxicity of some protein own or toxicity are very little, they there is the oligomer (oligomer) of toxic action or fibrous material (fibril) and cause a series of diseases but can be gathered into self, as the alzheimer's disease (being commonly called as senile dementia) being caused by Beta-amyloid (A-Beta) (Alzheimer ' s disease, AD), the Parkinson's disease that caused by alpha-synuclein (Parkinson ' s disease, PD), more than at least ten of the humans and animals including mad cow disease being caused by prion protein (Prion protein (PrP)) is planted encephalopathic, the at least 9 kinds of heredity nerve degenerative diseases that comprise Huntington Chorea (Huntington ' s disease) that caused by the polypeptide that contains poly glumine (PolyQ), by islet amyloid polypeptide (islet amyloid polypeptide, IAPP, amylin) disease that the type ii diabetes causing and N,O-Diacetylmuramidase (lysozyme) the gathering deposition being caused by long-time dialysis cause etc.Wherein, be AD and PD and type ii diabetes to human health risk maximum.Medical statistics shows, 5～6% suffer from AD, and sickness rate rises year by year in China and American-European countries in over-65s the elderly.This disease has been listed in and has caused dead the fourth-largest disease, is only second to heart trouble, cancer and apoplexy.1% the over-65s the elderly of separately having an appointment suffers from PD.And the number of suffering from type ii diabetes can reach the more than 5% of total population.These diseases have caused increasing harm to the mankind's health, and the basic reason (or partly cause) of its morbidity is the gathering of amyloid or polypeptide self.
Research shows, 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.First form the spherical oligomer by the solubility of electronics or the about 3-10nm of the observable size of atomic force microscope, some oligomer can be further combined with becoming bending pliable and tough filament (protofibril), and then form the smooth surface that diameter is 6-10nm or be the fiber of spiral.The protein of non-homology finally can form the protein polymer with analog structure.The fiber being formed by various amyloid aggregations such as A-Beta, alpha-synuclein and amylin all contains " cross-Beta-sheet " structure, the skeleton that wherein Beta lamella forms is vertical with the fiber longitudinal axis, and hydrogen bond net in skeleton is parallel with the longitudinal axis.Polypeptide is arranged as parallel Beta chain in Beta lamella, and in Beta lamella, amino acid has accurate position.Even the oligomer of different sources or fiber also have similar specific structure, oligomer or fiber-specific identification antibody can be combined with the oligomer and the fiber that are formed by the amyloid protein monomers of different sources respectively, and are not combined with their monomer.This shows that oligomer or fiber can form the common oligomer or the distinctive epitope of fiber that are independent of outside its amino acid primary sequence by protein polypeptide skeleton.Research discovery, amyloid can form a few class oligomer, comprising fibering oligomer (fibrillar oligomer, FO), front fibering oligomer (profibrillar oligomer, PFO) and other type oligomer.FO has formed to amyloid fiber has similar space structure, and the FO that the amyloid with different aminoacids primary sequence forms also has the space structure of this class, the antibody (OC) of FO specific combination forming with A-beta, can be combined with A-beta fiber, can also be combined with FO and the fiber of the formation such as alpha-synuclein, IAPP and PolyQ, but not be combined with the monomer of each amyloid.What past people thought amyloid disease always is that the insoluble fiber being become by protein aggregation causes.A large amount of research in recent years shows, the crucial paathogenic factor of many amyloid diseases is water miscible oligomers of small volume.And the pathogenic effects of FO will obviously be better than PFO.The disease being caused by amyloid oligomer exists similar mechanism, and cell membrane damage, oxidative stress, mitochondrial function imbalance, necrocytosis, signal transmission are abnormal etc.But the detailed mechanism that people affect the normal physiological activity of cell to oligomer it be unclear that.How are the topological framework of oligomer and epitope thereof, and these are all the problems that people urgently inquire into and solve how could effectively to suppress its cytotoxicity etc.
Thereby application initiatively and the gathering of passive immunological technique treatment AD inhibition A-Beta, and the removing of accelerating A-Beta is one of focus of AD research field.2000-2002 U.S. scientific research personnel applies the vaccine that A-Beta makes and has carried out 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.Meanwhile, apply anti-A-Beta antibody the passive immunotherapy of laboratory animal has also been received to good curative effect.But, unfortunately, in phase ii clinical trial, although most patient still shows good result, there is 6% patient to show meningitic symptom and pathological change, even there is death in some patients.Therefore, the experimental study of A-Beta vaccine and antibody is stopped immediately.However, can find out that from above-mentioned research A-Beta vaccine should affirm the result for the treatment of of AD.Research shows that the side effect being caused by A-Beta vaccine may be the autoimmune response by the T cell mediated of this vaccine-induced body generation, and the generation of this side effect is caused by Th1 rather than the immune response of Th2 type.In order to overcome the side effect of A-Beta vaccine, in the time of vaccine design, should strengthen bringing out B cell response and the immunoreactive generation of Th2 type, and avoid bringing out the immune response of Th1 type.Vaccine based on A-Beta oligomer epitope peptide design can easily be taken into account above-mentioned characteristic, also can inducing machine body form the antibody of anti-A-Beta monomer.A rear characteristic can thoroughly be eliminated the possibility having side effects between A-Beta monomer in vaccine and body, and can not affect the normal physiological function of A-beta.Therefore, will there is potential using value according to the peptide vaccine of A-Beta oligomer epitope development, and obtain the prerequisite that A-Beta oligomer epitope is this peptide vaccine design and development.So, in scientific research and clinical study and application, all need to obtain amyloid protein oligomer conformation type epitope polypeptide.
Summary of the invention
An object of the present invention is to provide a kind of amyloid fibrillar oligomer conformational epitope polypeptide.
Polypeptide provided by the invention is following 1) or 2):
1) polypeptide being formed by the aminoacid sequence shown in sequence in sequence table 1;
2) by aminoacid sequence shown in sequence in sequence table 1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and with the polypeptide being derived by sequence 1 with identical function.
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 being formed by the aminoacid sequence shown in sequence in sequence table 2;
B, the polypeptide being formed by the aminoacid sequence shown in sequence in sequence table 3;
C, the polypeptide being formed by the aminoacid sequence shown in sequence in sequence table 4;
D, the polypeptide being formed by the aminoacid sequence shown in sequence in sequence table 5.
The antibody that aforementioned polypeptides obtains as antigen is the scope of protection of the invention.
Another object of the present invention is to provide a kind of amyloid aggregation inhibitor.
Inhibitor provided by the invention, its activeconstituents is above-mentioned antibody; Described amyloid is specially A β 42 (amyloid beta), PrP (PrPC), amylin (amylin) or α-synuclein (α synapse nucleoprotein).
The 3rd object of the present invention is to provide a kind of cytotoxicity inhibitor of amyloid.
Inhibitor provided by the invention, its activeconstituents is above-mentioned antibody; Described cell is specially SH-SY5Y cell, and described amyloid is specially A β 42, PrP, amylin or α-synuclein.
The 4th object of the present invention is to provide a kind of diseases induced product of amyloid aggregation that prevents and/or treats.
Product provided by the invention, its activeconstituents is following 1) or 2): 1) above-mentioned polypeptide; 2) above-mentioned antibody; Described product is specially medicine or vaccine; The diseases induced alzheimer's disease that is specially of described amyloid aggregation.
Above-mentioned antibody is also the scope of protection of the invention in the application that suppresses amyloid aggregation and/or prepare in amyloid aggregation inhibitor; In this application, described amyloid is specially A β 42, PrP, amylin or α-synuclein.
Above-mentioned antibody is also the scope of protection of the invention in the application that suppresses the cytotoxicity of amyloid and/or prepare in the cytotoxicity inhibitor of amyloid; In this application, described cell is specially SH-SY5Y cell, and described amyloid is specially A β 42, PrP, amylin or α-synuclein.
The application that aforementioned polypeptides or above-mentioned antibody prevent and/or treat in the diseases induced product of amyloid aggregation in preparation is also the scope of protection of the invention; Described product is specially medicine or vaccine; The diseases induced alzheimer's disease that is specially of described amyloid aggregation.
The effect that aforementioned polypeptides prevents and/or treats the diseases induced product of amyloid aggregation in preparation is embodied in the quantity that improves spatial memory capacity, reduces senile plaque in brain and/or reduces the application in A β content; In this application, described A β is specially A β 42 or A β 40.
Of the present inventionly experiment showed, that polypeptide provided by the invention can be combined with antibody OC in testing in vitro, proved that this polypeptide is one of corresponding epitope of polyclonal antibody OC; This polypeptide has good antigenic characteristic, can produce antibody by effective stimulus body; This polypeptide is common epitope on various amyloid protein 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 identifying amyloid protein oligomer by effective stimulus body, and this antibody can suppress gathering and the cytotoxicity of amyloid.
Based on vaccine of the present invention may not can inducing machine body form the antibody of anti-A-Beta monomer, can eliminate the possibility having side effects between A-Beta monomer in vaccine and body, and can not affect the normal physiological function of A-beta.
3. polypeptide of the present invention has good immunogenicity, can effective stimulus body produces antibody, thereby can be used as good immunogen in the treatment of amyloid disease or prevention with PEOS aspect the development of vaccine.
Accompanying drawing explanation
Fig. 1 is that the ELISA that PEOS is combined with OC measures
Fig. 2 is that the ELISA of being combined with OC through the PEOS of displacement, disappearance, insertion amino acid or change amino-acid sequence measures
Fig. 3 is that the antibody in mice serum is combined with PEOS after PEOS immunity
Fig. 4 is that the antibody in the rabbit anteserum after PEOS immunity is combined with PEOS
Fig. 5 is that PEOS antibody is combined with oligomer and the fiber of various amyloid protein
Fig. 6 is the gathering of ThT fluorometric assay PEOS antibody suppression amyloid
Fig. 7 is the cytotoxicity of PEOS antibody suppression amyloid
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
The experimental technique using in following embodiment if no special instructions, is ordinary method.
Material, reagent etc. used in following embodiment, 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 is made into 1 liter, adjusting pH is 7.2).
In following embodiment, all experimental datas of PEOS are all by least 3 independently experiment acquisitions, and experimental data is expressed as mean number plus-minus standard deviation.The statistical study of data is that application One-way ANOVA software carries out.The Two-way ANOVA that is application two factor repeating datas for the Treatment Analysis of the data memory of water maze carries out.
Amyloid in embodiment is A β 42 (amyloid beta), PrP (PrPC), amylin (amylin) or α-synuclein (α synapse nucleoprotein) below.
The preparation of embodiment 1, polypeptide PEOS and derivative and antibody
One, the acquisition of polypeptide PEOS
The present invention's application phage display technology is screening substrate with the polyclonal antibody OC (catalog number is AB2286 for Millipore, USA) of specific recognition fibering oligomer and fiber, by 1 × 10
8kind of annular seven peptides have carried out four-wheel elutriation, application Phage-ELISA therefrom filtered out can with the phage clone of the obvious combination of OC, obtain by 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, can synthetic, (preparation method is referring to Weng C C to synthesize according to a conventional method preparation by the biochemical company limited of Shanghai gill, 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); PEOS purity is for being more than or equal to 95%; PEOS is stored in-20 ℃, avoids multigelation.
The synthetic polypeptide PEOS (C hold connection) of 6 Histidines as label that be connected with simultaneously.
Two, the acquisition of the derivative of PEOS
Aminoacid sequence shown in polypeptide PEOS (sequence 1), through replacing, lack, add amino acid or changing amino-acid sequence, is obtained to the derivative of following PEOS:
Specific as follows:
PEOS-Δ T: PEOS polypeptide (sequence 1) is deleted to a polypeptide that Threonine obtains, and sequence is: Cys-Trp-Thr-His-Gln-Arg-Ser-Cys (sequence 2, CWTHQRSC);
PEOS-W/Y: the tryptophane of PEOS polypeptide (sequence 1) is replaced as to the polypeptide that tyrosine obtains, and sequence is: Cys-Tyr-Thr-Thr-His-Gln-Arg-Ser-Cys (sequence 3, CYTTHQRSC);
PEOS-G: will insert the polypeptide that glycine obtains in 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 to glycine and aspartic acid, sequence is: Cys-His-Gln-Thr-Gly-Ser-Arg-Asp-Cys (sequence 5, CHQTGSRDC).
Synthetic polypeptide PEOS-Δ T, PEOS-W/Y, PEOS-G and the PEOS-WT/GD (C end be connected) of 6 Histidines as label that be connected with simultaneously.
Three, the acquisition of polypeptide PEOS antibody
1, PEOS can bring out body generation antibody
Application shows that the M13 phage of PEOS polypeptide (can express the gene fusion of PEOS polypeptide the M13 phage of PEOS polypeptide (7 peptides expression in disulfide linkage ring are at the N-terminal of pIII to being built on the less important capsid protein of M13 phage (pIII), first halfcystine is next to after L-Ala, after second halfcystine, there is a bit of interval polypeptide, formed by Gly-Gly-Gly-Ser, then be wild-type pIII albumen, M13 phage (New England Biolab Inc, catalog number is E8120SC) immune BALB/c mouse (Beijing Vital River Experimental Animals Technology Co., Ltd., catalog number is BALB/c mouse SPF level) (7/group), 1010, every each immune phage.Immunity in every 2 weeks once, is total to immunity 4 times.The 4th immunity blood sampling in latter 15 days, separation of serum.Application ELISA detects the content of anti-PEOS antibody in serum: by the not tagged PEOS of 100 μ l (because PEOS peptide chain is short, in order to make PEOS be adsorbed on preferably on elisa plate, and do not block the site of itself and antibodies, add two strong-hydrophobicity amino acid at the C of PEOS end, the peptide sequence of coated use is CWTTHQRSCIV) (1 μ g/ hole) coated 96 hole enzyme linked immunological microwell plates, put 37 ℃ of 2h, with 37 ℃ of sealing 2h of 3%BSA (200 μ L/ hole).Wash plate 3 times with PBS, add the serum of 2000 times of dilutions, after 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 100 μ l dilutions, after 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 then every hole adds 50 μ l 1mmol/L sulfuric acid termination reactions, measures OD value (wavelength 450nm) on enzyme-linked immunosorbent assay instrument.
As shown in Figure 3, 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 to result; All, than the height after the M13 phage immunity that does not have polypeptide to show, can find out, 7 mouse of immune PEOS can produce the antibody of anti-PEOS, show that PEOS has good antigenic characteristic, can produce antibody by effective stimulus body.
2, the preparation of polypeptide PEOS antibody
By the PEOS (the C end of PEOS connects KLH) that connects KLH (keyhole limpet hemocyanin), (fundamental immunity is added equal-volume Freund Freund's complete adjuvant (Sigma to immune new zealand white rabbit for the first time, F-5881), later booster immunization adds equal-volume freund 's incomplete adjuvant (Sigma, F5506)).Once, each antigen consumption is 200 μ g/ in immunity in every 2 weeks.Immunity 4 times altogether.After last immune 15 days, gather rabbit anteserum, and the ELISA method of application experiment 1 evaluation serum antibody titer (with the coated elisa plate of PEOS-KLH), negative control is not immune collection serum; Result as shown in Figure 4.Rabbit anteserum after 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 PEOS has good antigenic characteristic again, can produce anti-PEOS antibody by effective stimulus body.
With the sepharose that is connected with PEOS polypeptide, rabbit anteserum is carried out to affinity chromatography, adopt the sepharose (5ml) (being prepared by general epoxidation method of attachment by gill (Shanghai) biochemical corp) of coupling PEOS, elutriant is glycine buffer (the 0.2M glycine solution of pH3.0, with hydrochloric acid adjust pH to 3.0), flow velocity is 0.7ml/min, 3 column volumes of wash-out, merge and preserve 7-13ml elutriant, being 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, the combination of PEOS antibody and each amyloid fibering oligomer and fiber
By the A β 42 (U.S. American Peptide Co. buying, 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) be handled as follows: be dissolved to 1mg/ml with hexafluoroisopropanol (HFIP), room temperature (25 ℃) ultrasonication 10min, divide and install in epidorf pipe, HFIP volatilizees in vacuum environment, in-20 ℃ of preservations.Place 20min in room temperature with front A β 42, amylin, the α-synuclein that HFIP was processed, add dimethyl sulfoxide (DMSO) (DMSO), making each protein concentration is 5mg/ml, is then diluted to desired concn with the PBS damping fluid of 0.02M pH7.4.PrP is directly prepared into desired concn with 0.02M pH 7.4 PBS.Now, each amyloid is free state.
The preparation of A β 42 fibering oligomer is that monomer A β 42 obtained above 0.3mg is dissolved in 150 μ l HFIP, and room temperature is placed 20min.Then be diluted with water to 80 μ M, sample, with the centrifugal 15min of 14,000 × G, is got supernatant and is blown and steam HFIP with nitrogen, and sample is put the micro-rod of room temperature and stirred 24h (rotating speed 500RPM).
The preparation of A β 42 fibers is diluted to 10 μ M by A β 42 monomers that HFIP processed, DMSO dissolves with the PBS damping fluid of 0.02M pH7.4, hatches 24h for 37 ℃, gets precipitation with the centrifugal 15min of 14,000 × G.
The preparation of amylin fibering oligomer is diluted to 20 μ M by the amylin monomer that HFIP processed, DMSO dissolves 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 37 ℃ of above-mentioned amylin solution are hatched to 5h, gets precipitation with the centrifugal 15min of 14,000 × G.
The preparation of the fibering oligomer of α-synuclein is diluted to 40 μ M by α-synuclein monomer that HFIP processed, DMSO dissolves 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 is that 37 ℃ of α-synuclein solution are hatched to 4d, gets precipitation 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 is that 37 ℃ of PrP solution are hatched to 5d, gets precipitation with the centrifugal 15min of 14,000 × G.
After above-mentioned sample preparation is complete, measure protein concentration with the BCA protein detection kit (U.S. Merck, production number is 71285-3) of commonly using.And the exactness of observing the accumulation shape confirmation sample of amyloid in sample with transmission scanning electron microscope.
By the monomer of above-mentioned 100 μ l (monomer), fibering oligomer (oligomer) and fiber (fibril) form (1 μ g/ hole) coated 96 hole enzyme linked immunological microwell plates respectively, the negative contrast of BSA.Using the antibody of the above-mentioned anti-PEOS of purifying being obtained by embodiment 1 as primary antibodie, with goat-anti rabbit LgG (Beijing Bo Aosen Bioisystech Co., Ltd of HRP mark, catalog number is bse-0295G) as two anti-, and carry out experimental implementation by above-mentioned ELISA method.
As shown in Figure 5, the OD value after anti-PEOS antibody is combined with monomer, the BSA of A β, PrP, amylin, α-synuclein is respectively respectively 0.11,0.13,0.08,0.06,0.07 to result;
OD value after anti-PEOS antibody is combined with fibering oligomer, the BSA of A β, PrP, amylin, α-synuclein is respectively respectively 0.72,0.66,0.51,0.38,0.085;
OD value after anti-PEOS antibody is combined with fiber, the BSA of A β, PrP, amylin, α-synuclein is respectively respectively 0.58,0.52,0.41,0.53,0.066;
Can find out, anti-PEOS antibody can be combined with fibering oligomer and the fiber of A β, PrP, amylin, α-synuclein respectively, shows that PEOS is epitope common on various amyloid protein fibering oligomer and fiber.
The antibody of the oligomer of this specific combination amyloid can be used for experimental study, and treatment and clinical diagnosis to amyloid disease also has potential using value.
2, PEOS antibody in vitro suppresses the gathering of amyloid
1) prepare monomer, method is with above-mentioned 1, and A β 42, the amylin, the α-synuclein monomer PBS that obtain dilute, and obtain A β 42, PrP, amylin, α-synuclein solution monomer;
2) antibody of the above-mentioned anti-PEOS of purifying being obtained by embodiment 1 is diluted with the PBS damping fluid of 0.02M pH7.4, then join in A β 42, PrP, amylin, 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 is respectively 1: 1,10: 1,1: 1 and 2: 1.With the protein solution of antibody that do not add the anti-PEOS of purifying as contrast.A β 42, PrP, amylin, α-synuclein sample are 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 to 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.After mixing, in multi-functional microplate reader, measure the fluorescence intensity of ThT with the emission wavelength of 450nm excitation wavelength and 482nm.The fluorescence intensity of each sample is deducted to the background fluorescence of ThT itself, and significant difference situation between analytic sample.Add the albumen fluorescence intensity reducing after PEOS antibody to be the inhibiting rate of antibody to protein aggregation divided by the albumen fluorescence intensity that does not add antibody.
Result 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, the cytotoxicity of PEOS antibody suppression amyloid
With containing the substratum (MEM) of 10% foetal calf serum by SH-SY5Y cell (Chinese Academy of Medical Sciences's tumour cell storehouse, production number is SH-SY5Y) be made into 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 the sample (mixture of the antibody of amyloid A β 42, PrP, amylin, α-synuclein and the above-mentioned anti-PEOS of purifying being obtained by embodiment 1, amyloid A β 42, PrP, amylin, α-synuclein oligomer and PBS contrast separately), the final concentration of A β 42, PrP, amylin, α-synuclein albumen is 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, every hole adds MTT solution (5mg/mL) 10 μ L, hatch 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, fully dissolve crystallisate.On multi-functional enzyme-linked immunosorbent assay instrument, measure each hole absorbance value with 570nm wavelength.Using the absorbancy of sample divided by the absorbancy of cell that does not add sample as the activity index of cell, and significant difference situation between analytic sample (contrasts and compares with each amyloid, *, P < 0.05; #, P < 0.01).
As shown in Figure 7, the Cell relative activity of amyloid A β 42, PrP, amylin, α-synuclein is respectively 58.8%, 46%, 59%, 53% to result separately;
The Cell relative activity of the mixture of amyloid A β 42, PrP, amylin, α-synuclein and PEOS antibody is respectively 88%, 82%, 78%, 70%;
Illustrate, PEOS antibody can improve cytoactive, thereby shows that PEOS antibody can obviously suppress the toxicity to SH-SY5Y cell of A β 42, PrP, amylin, α-synuclein.
Two, the application of PEOS polypeptide
1, polypeptide PEOS and derivative thereof are combined with polyclonal antibody OC
1), PEOS is combined with polyclonal antibody OC
By coated 100 μ l polyclonal antibody OC (0.2 and 1 μ g/ hole) 96 hole enzyme linked immunological microwell plates (using the hole of coated OC not as negative control), put 37 ℃ of 2h, with 37 ℃ of sealing 2h of 3%BSA, 200 μ L/ holes.Wash plate 3 times with PBS.Every hole adds 100 μ l with histidine-tagged PEOS, 0.5 μ g/ hole), room temperature (25 ℃) is hatched after 1h, with the PBS washing that contains 0.1%Tween-20 3 times.After 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 100 μ l dilutions, after 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 then every hole adds 50 μ l 1mmol/L sulfuric acid termination reactions, measures OD value (wavelength 450nm) on enzyme-linked immunosorbent assay instrument.
As shown in Figure 1, the OD value of 1 μ gOC, 0.2 μ gOC, negative control is respectively 0.72,0.28,0.051 to result; Can find out, compared with negative control, 1 μ gOC and 0.2 μ gOC can be identified by PEOS, also can say, PEOS can be by OC specific recognition.PEOS is the polypeptide obtaining as screening substrate using OC, it with the combination of OC confirmed it be one of corresponding epitope of polyclonal antibody OC (compared with the negative control of not coated PEOS, *, P < 0.05, #, P < 0.01).
2), the derivative of PEOS is combined with polyclonal antibody OC
In order to detect the combination situation of improved polypeptide and OC, these polypeptide are competed and are combined with OC (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 of the not transformation with histidine-tagged, then detect the combination situation of the latter and OC with ELISA.
Specific as follows: 1 μ g is added in the hole that is coated with OC with the mixture 100 μ l of 5 μ g PEOS-Δ T, PEOS-W/Y, PEOS-G, PEOS-WT/GD, PEOS respectively with the PEOS of label, 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, take PEOS-His tag as contrast, combination with ELISA detection with PEOS and the OC of label, measure OD value, method is with above-mentioned 1.With the negative contrast of BSA.
The results are shown in Figure 2, wherein, the column diagram group in left side is the combination of each group of polypeptide and OC, and the column diagram group on right side is the combination of each group of polypeptide and negative control (BSA),
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 combination 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) combination is respectively 0.076,0.044,0.031,0.035,0.051,0.048;
Can find out, certain amino acid of PEOS polypeptide through displacement, disappearance, in polypeptide, increase amino acid or change and still can be combined with the PEOS effective competition with histidine-tagged of unmodified transformation OC after amino-acid sequence, cause with the PEOS of label and the combination of OC and obviously reduce.
2, PEOS polypeptide improves the effect embodiment of AD disease
1) PEOS polypeptide improves the memory capability of AD transgenic mice
Experimental technique:
(1) the M13 phage of the displaying PEOS polypeptide that application is obtained by embodiment 1 and the immune 8 month female AD transgenic mices of M13 phage (in contrast) that do not have polypeptide to show (turn APP and PS1 gene (APPswe/PS1dE9) mouse purchased from Jackson Lab, Bar Harbor, ME, the U.S., catalog number is: 004462, after breeding through identifying that the mouse that obtains containing APP and PS1 gene is for turning the AD female mice of APP and PS1 gene (APPswe/PS1dE9)), obtain two groups of mouse: AD+PEOS and AD con.; (turn APP and PS1 gene (APPswe/PS1dE9) mouse purchased from Jackson Lab using 8 wild-type mices as WT group, 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) be contrast.
Three groups altogether, 8 every group; Every each immune phage 10
10individual.Immunity in every 2 weeks once, is total to immunity 4 times.The 4th immunity starts water maze laboratory for latter 15 days.
(2) memory training: be placed on 25 ℃ of room temperatures, the environmental adaptation of humidity 46% 3 days before Mice water maze training.All study of behaviour detect the mode that all adopts randomized, double-blind in test.Before training, remove platform, at tank, central authorities put into gently, allow 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.Before training, allow for the first time mouse stand in the upper 15s of platform (10 centimetres of platform diameter), allow it remember the locus of pond (1.1 meters of diameters) middle platform.Platform is placed on the 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 to pool wall, put into gently water, be allowed to condition at 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 (purchased from medicine institute of Chinese medical courses in general institute) records its track and from entering water to the time of climbing up platform, i.e. latent period during this time.If mouse is found platform in 90s, be allowed to condition at and on platform, stop 10s.If mouse can not find platform in 90s, after 90s, guide its upper mounting plate by experimenter, and be allowed to condition at and on platform, stop 10s.Continuously training 5 days, trains 2 every day, between twice, is spaced apart 3-4h.Then interval 24h, removes platform, allows mouse in water, find platform 90s.With the result of video recording and the training in 5 days of software system record and exploration experiment, and process experimental result with the amount of repetition number two-factor analysis of variance (two-way ANOVA, repeated measures).
As shown in Figure 8, along with the growth of training time, wild group of mouse finds shorten gradually the latent period of platform to experimental result, reaches equilibrium state after 4 days.The AD mouse (AD+PEOS) of injection PEOS finds the latent period of platform in the time training by the 3rd, 4,5 days, with contrast AD mouse (AD con.) and compare and there is significant difference (compared with AD control mice group, #, P < 0.05), the results are shown in Figure 8A.Removing the AD mouse (AD+PEOS) of injecting PEOS after platform finds and is also significantly less than AD control group (AD con.) (Fig. 8 B) latent period of platform.The AD mouse (AD+PEOS) of injection PEOS is significantly higher than AD control group (AD con.) (Fig. 8 C) through the number of times of platform position, and the time at AD mouse place in target quadrant after injection PEOS is significantly higher than AD control mice (Fig. 8 D).These results show that AD mouse spatial memory capacity after immune PEOS is significantly better than the AD control group of not injecting PEOS.
2), PEOS polypeptide reduces the quantity of senile plaque in AD Transgenic Mice Brain
The impact of application ThS fluorescent dye measuring PEOS on senile plaque in AD Transgenic Mice Brain:
(1) by above-mentioned 1 AD+PEOS group, AD con. group, WT group mouse heart perfusion, get cerebral tissue, application organizes refrigerating fulid and liquid nitrogen to carry out freezing treatment, and is 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) application 1mg/ml ThS (with 70% ethanol preparation) contaminates section 10min, then with 70% alcohol flushing 3 times.
(3) application fluorescent microscope gathers image (Olympus BX60).
Detected result is shown in 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, the senile plaque quantity in AD (AD con.) mouse brain cortex (A) and the hippocampus (C) of injection PBS is obviously more than (D) senile plaque quantity in injecting immune polypeptide PEOS AD mouse (AD+PEOS group) cortex (B) and 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).
Result as 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 in the 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, the ratio that the mouse senile plaque area of injecting immune polypeptide PEOS accounts for section area obviously reduces (compared with injection PBS group, *, P < 0.01).
3), PEOS polypeptide reduces A β 40 and A β 42 levels in AD Transgenic Mice Brain
The cerebral tissue of above-mentioned 1 AD+PEOS group, AD con. group mouse is containing homogenate in the PBS of proteinase inhibitor (pH7.2).Then with the centrifugal 30min of 15000rpm, collect supernatant.Precipitation adds the Guanidinium hydrochloride (with the preparation of tris-HCl damping fluid, pH8.0) of 5M, and then centrifugal treating, collects supernatant.The A β 40 and A β 42 levels that are dissolved in and be insoluble to PBS are measured according to ELISA method by test kit (catalog number is respectively for Invotrigen, USA: KHB3441 and KHB3482).
The A β amount (ng or mg) that A β level contains according to every Borneo camphor tissue represents, measures OD value and A β typical curve with ELISA method.(standard substance are to be equipped with in 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 OD value, and y is the quality of A β 40 or A β 42.)
The results are shown in Figure 11-13, 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.
In Figure 11, in the AD transgenic mice (AD+PEOS) of injection PEOS polypeptide, AD transgenic mice (AD con.) brain of injection PBS, the content of solubility A β 40 is respectively 16.4ng/g brain albumen and 119.2ng/g brain albumen),
In Figure 12, inject the content of solubility A β 42 in AD transgenic mice (AD con.) brain of AD transgenic mice (AD+PEOS), injection PBS of PEOS polypeptide and be respectively 0.82ng/g brain albumen and 2.35ng/g brain albumen,
In Figure 13, 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;
Result shows, in AD injected in mice immunity PEOS hindbrain, PBS solubility A β 40 (Figure 11) obviously reduce with A β 42 levels (Figure 13) (compared with injection PBS group with A β 42 (Figure 12) level and insoluble A β 40, *, P < 0.05; #, P < 0.01).
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles 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.
Claims (8)
1. a peptide species, the polypeptide being formed by the aminoacid sequence shown in sequence in sequence table 1.
2. the antibody obtaining as antigen using polypeptide described in claim 1.
3. an amyloid aggregation inhibitor, its activeconstituents is antibody claimed in claim 2; Described amyloid is A β 42, PrP, amylin or α-synuclein.
4. a cytotoxicity inhibitor for amyloid, its activeconstituents is antibody claimed in claim 2; Described cell is SH-SY5Y cell, and described amyloid is A β 42, PrP, amylin or α-synuclein.
5. prevent and/or treat the diseases induced product of amyloid aggregation, its activeconstituents is following 1) or 2): 1) polypeptide claimed in claim 1; 2) antibody claimed in claim 2; Described product is medicine or vaccine; Described amyloid aggregation is diseases induced is alzheimer's disease.
6. described in claim 2, antibody suppresses amyloid aggregation and/or prepares the application in the aggregation inhibitor of amyloid in preparation; Described amyloid is A β 42, PrP, amylin or α-synuclein.
Described in claim 2 antibody suppressing the cytotoxicity of amyloid and/or prepare the application in the cytotoxicity inhibitor of amyloid; Described cell is SH-SY5Y cell, and described amyloid is A β 42, PrP, amylin or α-synuclein.
8. polypeptide claimed in claim 1 or antibody claimed in claim 2 prevent and/or treat the application in the diseases induced product of amyloid aggregation in preparation; Described product is medicine or vaccine; Described amyloid aggregation is diseases induced is alzheimer's disease.
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