CN110317814A - Beta-amyloid protein ring-type ribonucleic acid, polypeptide and its application - Google Patents

Beta-amyloid protein ring-type ribonucleic acid, polypeptide and its application Download PDF

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CN110317814A
CN110317814A CN201910656135.6A CN201910656135A CN110317814A CN 110317814 A CN110317814 A CN 110317814A CN 201910656135 A CN201910656135 A CN 201910656135A CN 110317814 A CN110317814 A CN 110317814A
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莫丁丁
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Abstract

The present invention discloses a kind of beta-amyloid protein ring-type ribonucleic acid, polypeptide and its application.Present invention discover that app gene can generate a variety of circular rnas from the code area A β by reverse splicing, it is named as beta-amyloid protein ring-type ribonucleic acid circA β.By means of newly-established circular rna research method, identify a variety of polypeptides generated by circA β, and such polypeptide can be further processed to form A β, and then beta-amyloid protein patch is formed in primary neuronal culture object, embody the key point of AD neuropathology.CircA β and its albumen of translation represent the novel targets in AD treatment.

Description

Beta-amyloid protein ring-type ribonucleic acid, polypeptide and its application
Technical field
The present invention relates to the fields beta-amyloid protein (A β), more particularly to beta-amyloid protein ring-type ribonucleic acid CircA β, the polypeptide being generated by it and the purposes in preventing, treating or diagnose in Alzheimer disease.
Background technique
Alzheimer disease (AD) is one of most common dementia, Zhan great Yue 70%.AD is a kind of memory capability decline It is the neurodegenerative disease of notable feature with cognition dysfunction.As disease relevant to aging, Alzheimer disease is most Big known risk factor is to increase at the age.The cause of disease of Alzheimer disease is the product of insoluble amyloid-beta peptide (A β) Tired and Protein tau Hyperphosphorylationof.It has been proved that APP (beta-amyloid protein precursor protein) and presenilin genes (participate in P egg Plain boiled water solution processes APP albumen) in mutation accelerate the accumulation of A beta polypeptides.The polymerization of A β leads to toxicity oligomer, assembles again At insoluble beta-amyloid protein patch (A β plaque block), this process eventually leads to Protein tau Hyperphosphorylationof.Then it is in mind Neurofibrillary tangles are formed in member and cause complicated downstream reaction, eventually lead to neuronal death.It is this due to gene Mutation leads to nervus retrogression pathology, referred to as familial Alzheimer disease.Various mutation are in familial Alzheimer disease In effect be studied it is clear；However, this familial Alzheimer disease account for all cases less than 1-5%.Most often The Alzheimer disease form seen is " sporadic ".Such Alzheimer disease is for 65 years old or more patient Common, and potential heredity or molecule reason are still unknown.Although familial and sporadic Alzheimer disease form Between there are potential hereditary difference, but the shared weight of various Pathological Physiology related fields that two kinds of disease subtypes develop in disease Folded, i.e., especially A β accumulation gradually increases caused neurotrosis.However it finds in clinical studies, most A Erci There is no the mutation of APP and related gene by the silent sick patient in sea.Meanwhile normal APP albumen is overexpressed in rat brain, and Significant A β plaque block cannot be generated.These are research shows that the A β generated by APP proteolysis is not the source most important A β.Cause This most key and pathogenic A β is still huge unknown from He Erlai.The source for finding real A β is diagnosis, prevents and treats The key of Alzheimer disease.
Summary of the invention
After extensive studies it inventors have found that Alzheimer disease key gene APP can synthesize in vivo it is related to A β Circular rna (being named as beta-amyloid protein ring-type ribonucleic acid, circA β), and be translated and be processed into Alzheimer Sick key Lethal Factor A beta polypeptides, and A β plaque block is formed outside primary neural cell in a short time, show the A of circA β coding β has important Pathogenic potential.Since the transcription and translation of circA β do not need the mutation of app gene, it is encoded and A β with neurotoxicity can admirably explain why normal population is as aging can also generate Alzheimer disease.At least It is based in part on this discovery and completes the present invention.
The present invention does not only disclose the completely new and A β constructive ways the most main in normal human, and additionally provides A kind of new mechanism about onset of Alzheimer disease, while also being mentioned to be directed to the diagnosis of the disease from now on, preventing and treating A completely new target spot is supplied.
Detailed description of the invention
The identification of circA β and its overexpression in HEK293 cell in Fig. 1 human brain.Wherein: Figure 1A: 5% natural poly- third Acrylamide gel electrophoresis circA β RT-PCR product has anti-in the exons 17 of people's app gene in human brain sample To primer (A β-VF2, A β-VR2)；Use two people's cerebral RNA samples.Figure 1B: the positioning of circA β-a, b, c, d in app gene； 42 sequence of A β is used as reference by location.Has_circ_0007556 is named as circA β-a in our current research.Amyloid protein-β (A β) sequence is located in exon16 and 17；A β-cF and A β-cR are used to expand circA β-a by qRT-PCR.Fig. 1 C:circA β-a is overexpressed construct.CircA β-a expression and overexpression in Fig. 1 D:RT-PCR identifier brain sample (frontal lobe and hippocampus) The HEK293 cell of circA β-a；Control, pCircRNA-DMo empty carrier transfect HEK293；BE-CircA β-a, pCircRNA- BE-A β-a transfects HEK293；DMo-circA β-a, pCircRNA-DMo-A β-a is transfected into HEK293；Another group of few nucleosides Acid is shown in table 1 the RT-PCR verifying of circA β-a.What circA β-a was expressed in Fig. 1 E:HEK293 cell quantifies；Control, Empty carrier (pCircRNA-DMo)；BE-circAβ-a,pCircRNA-BE-Aβ-a,DMo-circAβ-a,pCircRNA-DMo-A β-a.All statistics T inspections are compared with control sample, * * * *, P≤0.0001, n=4.In Fig. 1 F:HEK293 cell The Northern engram analysis of circA β-a expression；The linear homologous ORF mRNA of ORF-mRNA, circA β-a.For RNase R processing digests 15 μ g total serum IgEs 1 hour with 10 unit RNase R at 37 DEG C；Expression does not digest；+ indicate digestion； OligoA β-NB-R1 (CCCACCATGAGTCCAATGATTGCACCTTTGTTTGAACCCACATCTTCTGCAAAGAA CACC) quilt For northern trace；The ethidium bromide staining of Ago-Gel is used as loading control.Fig. 1 G: primer A β-VR2 and A is used The agarose gel electrophoresis of the RT-PCR product (503bp) of β-VF2.Fig. 1 H and Fig. 1 I: sequencing and comparison RT-PCR product, it was demonstrated that CircA β-a is exons 14,15,16 and 17 that is identical and including the not app gene of introne (data are not shown)； The join domain of reverse splicing of the sequencing-of circA β from pCircRNA-BE-A β-a and pCircRNA-DMo-A β-a.
Fig. 2 circA β-a in HEK293 cell translates into A beta related peptides.Wherein: the open reading of Fig. 2A: circA β-a Frame (ORF) is indicated with outer circle；Black gray expandable area, a kind of unique peptide area of protein of circA β-a translation；Grey arrow, translation Initiation codon；Grey rectangle, terminator codon；Interior black arrow shows the beginning of circA β-a.Fig. 2 B:HEK293 cell The Western blotting of middle A beta related peptides；Control, empty carrier (pCircRNA-DMo)；BE-A β-a, pCircRNA-BE-A β-a； DMo-A β-a, pCircRNA-DMoA β-a；The peptide detected is shown on right side: protein derived from A β 175, circA β-a-；β- Actin is used as loading control.Fig. 2 C:A β 175 is horizontal to be quantified；All statistics T are carried out to control sample to examine；*, P≤ 0.05；*, P≤0.01；n≥3.The peptide sequence of Fig. 2 D:A β 175；Black small letter, the peptide sequence and wild type APP albumen of A β 175 It is identical；α indicates alpha-secretase enzyme；β indicates beta-secretase；γ indicates gamma-secretase；Protease site is indicated by an arrow；42 sequence of A β It is classified as small letter band underscore；Light color capitalization indicates unique polypeptide sequence of A β 175；Light color capitalization underscore indicates IP-MS detection The unique polypeptide arrived.Fig. 2 E: the mass spectrum of unique polypeptide exists only in the cyclic annular translation of circA β-a；Exempted from by anti-amyloid beta antibodies The method of epidemic disease precipitate A β 175 prepares the polypeptide；Left side ordinate is relative intensity, and right side ordinate is absolute intensity, and level is vertical Coordinate is m/z.
Fig. 3 circA β-a, which is overexpressed, to be generated A beta polypeptides and A β plaque block is caused to be formed.Wherein: Fig. 3 A: crossing table in circA β-a Up to the IP-WB of the A beta polypeptides in the conditioned medium of cell；The HKE293 CMC model of circA β-a over-express vector will be transfected Base anti-amyloid beta antibodies (6E10,4G8；Mouse antibodies) immunoprecipitation；Control, pCircRNA-DMo；BE-A β-a, pCircRNA- BE-Aβ-a；DMo-A β-a, pCircRNA-DMo-A β-a；A β antibody (D54D2, rabbit antibody) is used for Western blotting；β-flesh Filamentous actin is used as loading control.The A β 42 that 5ng is synthesized in vitro is used as the A β control in Western blotting.The quantization of Fig. 3 B:A；With Control sample is compared, and is carried out all statistics T and is examined；*, P≤0.05；* *, P≤0.001；N=3.Fig. 3 C and Fig. 3 D:circA β- A, which is overexpressed in mouse primary neuron culture, generates A β plaque block；PCircRNA-DMo is used as empty vector control.DMo-A β-a, pCircRNA-DMo-Aβ-a；GFP is shown with brilliant white；A β (6E10) is shown as light grey；Bracket and white arrow indicate A β plaque Block position；DAPI (nuclear targeting) appears dimmed.It is worth noting that, using the starting of identical quantity in each transfection Neuron；The neuron for the different densities observed in image between Fig. 3 C and Fig. 3 D may be caused by the toxicity of A β peptide.
Fig. 4: the alternative route that A β is generated in Alzheimer disease.In top, circA β-a and A beta polypeptides (light gray) The exon sequence for including is compared with overall length app gene.In left side, the linear APP mRNA transcribed from app gene undergoes classical Then montage is translated into overall length APP albumen.The proteolysis processing of APP albumen generates A beta polypeptides (A β 40, A β 42, light gray Color), pathogenic effects are played in AD pathology.On right side, circA β-a is synthesized by the reverse splicing of app gene.It is open Reading frame (ORF) is grey, and A β sequence is light gray, and initiation codon is light grey arrows, and terminator codon is that black is rectangular Shape.The translation of circA β-a generates A beta related peptides (A β 175), and further processing forms A β.
The sequence alignment of Fig. 5 circA β-a-DP, circA β-b-DP, circA β-c-DP and APP695.Solid line indicates A β Sequence；Dotted line indicates the sequence distinct zones (being different from APP) of circA β expression albumen, is named as circA β-DP-SP.
The expression identification of circA β-a derived peptide of the Fig. 6 in human brain.IP-MS in 175 protein sequence of Fig. 6 A:A β Antigenic site and the polypeptide detected；Polypeptide with underscore, the antigen for antibody producing；With the more of black underscore Peptide, the polypeptide detected by IP-MS；Polypeptide _ 1, the polypeptide detected are shown in C；The polypeptide detected in polypeptide _ 2, D.Figure 6B: the Western blot analysis of A β 175 in human brain sample；Control, the transfection of HEK293 cell empty carrier；CircA β-a, HEK293 cell is transfected with pCircRNA-DMo-A β-a；α, β and gamma-secretase is added in the complete cutting of secretase in order to prevent Inhibitor is to allow to cut A β 175 in HEK293 cell partial cut；Human brain sample 1-6 is for this measurement；Band 1,2,3,4 comes The A β 175 for the different length that autocrine digestion is cut processes product.Fig. 6 C and Fig. 6 D: pass through the mass spectrum of the IP-MS polypeptide detected.
Antisense oligonucleotides (the anti-circA β-of Fig. 7 directed against amyloid-beta albumen ring-type ribonucleic acid-a (circA β-a) A-ASO the level of intracellular circA β-a) is reduced.The design diagram of Fig. 7 A:anti-circA β-a-ASO.Fig. 7 B: in ASO Beta-amyloid protein ring-type ribonucleic acid qRT-PCR result under processing；BE-A β indicates to cross table using pCircRNA-BE carrier Up to the plasmid of circA β-a；DMo-A β indicates the plasmid that circA β-a is overexpressed using pCircRNA-DMo carrier；Scr.ASO table Show that negative control tests ASO.Fig. 7 C: the qRT-PCR result of APP mRNA under ASO processing.
The expression system of Fig. 8 circA β and its cDNA.
The external synthesis schematic diagram of Fig. 9 circA β.
Specific embodiment
The existing various exemplary embodiment that the present invention will be described in detail, the detailed description are not considered as to limit of the invention System, and it is understood as the more detailed description to certain aspects of the invention, characteristic and embodiment.
It should be understood that it is to describe special embodiment that heretofore described term, which is only, it is not intended to limit this hair It is bright.In addition, for the numberical range in the present invention, it is thus understood that specifically disclose the range upper and lower bound and they it Between each median.Median and any other statement value in any statement value or stated ranges or in the range Lesser range is also included in the present invention each of between interior median.These small range of upper and lower bounds can be independent Ground includes or excludes in range.
Unless otherwise stated, all technical and scientific terms used herein has the routine in field of the present invention The normally understood identical meanings of technical staff.Although the present invention only describes preferred method and material, of the invention Implement or also can be used and similar or equivalent any method and material described herein in testing.The institute mentioned in this specification There is document to be incorporated by reference into, to disclosure and description method relevant to the document and/or material.It is incorporated to any When document conflicts, it is subject to the content of this specification.Unless otherwise stated, " % " is the percentage based on weight.
In the present invention, term " cyclic annular ribonucleic acid " refers to the ribonucleic acid in contrast with linear nucleic acid, not have 3 ' ends and/or 5 ' ends, end to end ribonucleic acid.The nucleic acid that cyclic annular ribonucleic acid of the invention is typically separate；Or synthesis Nucleic acid, including chemically synthesized cyclic annular ribonucleic acid, the cyclic annular ribonucleic acid also obtained including the use of biosynthesis.
In the present invention, term " polypeptide " refers to the multiple amino acid being connected to each other by peptide bond.Amino acid herein It can be naturally occurring 20 kinds of amino acid or modified amino acid.Polypeptide can be modified by any natural method, such as Post translational processing, or modified by chemical modification technology known in the art.Modification can occur any in polypeptide Place, including peptide backbone, amino acid side chain and amino terminal or carboxyl terminal.It should be noted that in specific polypeptide, it can To carry out the modification of same type in multiple sites, a plurality of types of modifications can also be carried out.These modifications include but is not limited to Acetylation, acylation, ADP- ribosylation, amidation, crosslinking cyclisation, disulfide bond, demethylation, covalent cross-linking, cysteine Change, pyroglutamic acid, formylated, gamma-carboxylation, glycosylation, GPI anchoring, hydroxylating, iodate, methylation, myristoylation, oxidation, Proteolysis and phosphorylation.
In the present invention, term " specific binding " refers to that for other albumen, albumen of the invention preferentially selects Property combining target albumen.In certain embodiments, " specific binding " refers to albumen of the invention peptide special for circA β Or the affinity of its segment is greater than other protein.For example, the equilibrium dissociation constant measured by surface plasmon resonance (KD) value is less than 10-7M, preferably smaller than 10-8M, more preferably less than 10-9M。
In the present invention, term " separation " refer to the substances such as nucleic acid, albumen or polypeptide leave its primal environment (for example, if Be it is naturally occurring, then leave its natural surroundings).For example, being present in the intracorporal naturally occurring nucleic acid of animal living or albumen Or polypeptide be not separation, but isolated from some or all of natural surroundings coexisting substances identical nucleic acid, albumen or Polypeptide is separation.It should be noted that such nucleic acid of a part as carrier；And/or as the combination artificially obtained Such nucleic acid, and/or albumen or polypeptide of a part of object are still separation, and reason is these carriers or composition is not A part of natural surroundings.
In the present invention, term " purifying " is an opposite definition, is not required for Economical Purification." purifying of the invention " it include purifying at least an order of magnitude from the mixture, natural products or other environment manually obtained, preferably purify two A or three orders of magnitude have more preferably purified four or five orders of magnitude.
In the present invention, term " host cell ", also referred to as recombinant host cell refer to and lead recombinant expression carrier Enter cell therein.Host cell not only includes specific subject cell, but also the offspring including this cell.Because certain repair Decorations can occur in the subsequent generation due to mutation or environment influence, so actually such offspring may be with parental cell not It is identical, but be included in the range of term used in the present invention " host cell ".Host cell of the invention includes Such as transfectoma, such as Chinese hamster ovary celI, NS/0 cell and lymphocyte.
In the present invention, term " subject " includes people or non-human animal." non-human animal " includes all vertebrates, such as Mammal and nonmammalian, such as non-human primates, sheep, dog, ox, chicken, amphibian animal, reptiles, rat, mouse, pig Deng.
In the present invention, term " biological sample " refers to tissue or ingredient from subject.Preferably, biological sample is originated from Subject with related disease.Biological sample includes body fluid, tissue fluid or the cell or cell mass isolated from subject. Wherein the example of body fluid includes but is not limited to blood, serum, blood plasma, saliva, urine, ascites, cyst fluid etc..The example of tissue fluid includes The tissue sample of homogenate, such as the tissue sample obtained by biopsy.The cell or cell isolated from subject The type of group is not particularly limited, preferably body cell or body cell mass.Biological sample of the invention can be in examples detailed above One or more mixtures.Preferably, biological sample of the invention comes from brain tissue, such as cerebrospinal fluid.
In the present invention, term " substantially homologous " refers to the phase between object sequence (including base sequence or amino acid sequence) It is 50% or more, such as 60% or more, 80% or more or 90% or more, preferably 95% or more, more preferable 97% or more with degree, Further preferred 99%.
In the present invention, term " stringent condition " or " stringent hybridization condition " will be with its targets including being related to probe under this condition Sequence hybridizes so that detectable degree is greater than the condition of other sequences (for example, relative to background at least 2 times).Stringent condition is sequence It is that Leie relies and different by varying environment.By the stringency and/or wash conditions of control hybridization, can recognize can be with primer Or probe is up to 100% complementary target sequence (same to source detection).Optionally, adjustable stringent condition allows certain mistakes in sequence With the similitude (heterologous detection) to detect lower degree.
Stringent condition of the invention is wherein in pH 7.0 to 8.3 and temperature (the short probe, such as 10 to 50 that is at least about 30 A nucleotide) and at least about 60 DEG C (long probe is greater than 50 nucleotide) under salinity be less than about 1.5M Na ion, allusion quotation Type about 0.01 those of to 1.0M Na ion concentration (or other salt).Stringent condition can also add destabilizing agent such as formamide Or Denhardt ' s solution is realized.Exemplary low stringency condition includes 30 to 35% formamides, 1M at 37 DEG C The buffer solution of NaCl, 1%SDS (lauryl sodium sulfate) hybridizes and 1 at 50 to 55 DEG C × to 2 × SSC (20 × SSC =3.0MNaCl/0.3M trisodium citrate) in washing.Exemplary mild stringent condition includes 40 to 45% formyls at 37 DEG C Amine, 1M NaCl, hybridize in 1%SDS and the 0.5X at 55 to 60 DEG C is washed into 1 × SSC.Exemplary high stringency conditions packet 50% formamide at 37 DEG C, 1M NaCl are included, hybridizes in 1%SDS and is washed in 60 to 65 DEG C of 0.1 × SSC.
For the washing after hybridization, key factor is the ionic strength and temperature of final cleaning solution.It is miscellaneous for DNA-DNA It hands over, TmCan following formula estimation: Tm=81.5 DEG C of+16.6 (log M)+0.41 (%GC) -0.61 (%form) -500/L；Wherein M is single The molar concentration of valence cation, %GC are the percentage of guanine and cytidylic acid in DNA, and %form is that formamide exists Percentage and L in hybridization solution are the length of hybrid in base-pair.TmWherein for (under determining ionic strength and pH) The temperature that 50% complementary target sequence hybridizes with the probe of Optimum Matching.TmMispairing relative to every 1% reduces about 1 DEG C；Therefore, Adjustable Tm, hybridization and/or wash conditions to hybridize with the sequence of desired identity.For example, if searching > 90% identity Sequence, Tm10 DEG C can be reduced.In general, stringent condition selection is under determining ionic strength and pH than distinguished sequence and its complementation Heat fusion joint (the T of bodym) about 5 DEG C low.However, very strict condition can be than heat fusion joint (Tm) it is 1,2,3 or 4 DEG C low at for hybridizing And/or washing；Mild stringent condition can be than heat fusion joint (Tm) it is 6,7,8,9 or 10 DEG C low at for hybridizing and/or wash；It is low tight Glazing bar part can be at lower than heat fusion joint (Tm) 11,12,13,14,15 or 20 DEG C for hybridizing and/or washing.Use the equation, miscellaneous It hands over and washing forms and desired Tm, ordinarily skilled artisan will understand that the variation of the stringency of hybridization and/or cleaning solution is solid It is described.If it is desire to extent of mismatch generate be lower than 45 DEG C (aqueous solutions) or 32 DEG C (formamide solution) Tm, then preferred to increase Add SSC concentration in order to use higher temperature.Unless otherwise indicated, in this application, high stringency is defined as at 65 DEG C 4 × SSC, 5 × Denhardt ' s (5g ficoll, 5g polyvinylpyrrolidone, 5g bovine serum albumin(BSA) in 500ml water), Hybridize in the salmon sperm dna and 25mM phosphate sodium that 0.1mg/ml boils and in 0.1 × SSC, the 0.1%SDS at 65 DEG C Washing.
[beta-amyloid protein ring-type ribonucleic acid]
The first aspect of the present invention provides separation or synthesis beta-amyloid protein ring-type ribonucleic acid, also simple herein Referred to as " cyclic annular ribonucleic acid of the invention " or " circA β ".By separating or synthesizing the cyclic annular ribose core preferably purified Acid.Cyclic annular ribonucleic acid of the invention includes the alkali of at least one exon of amyloid precusor protein (APP) gene of cross-film Basic sequence or part thereof sequence, or with these substantial sequence homologies and from the sequence of same species.Ring nucleus of the invention Ribosomal ribonucleic acid may include whole base sequences of an exon in 18 exons or the segment of an exon, i.e. portion Divide base sequence, or with these substantial sequence homologies and from the sequence of same species.Cyclic annular ribonucleic acid of the invention is also It may include whole base sequences of more than two exons in 18 exons or the segment of part of exon, i.e. part The number of base sequence of exon.Preferably, A β 40 or A β 42 can be expressed or be generated to cyclic annular ribonucleic acid of the invention, or Its segment or cyclic annular ribonucleic acid of the invention include the base sequence of coding A β 40 or A β 42 or their segment.
In certain embodiments, cyclic annular ribonucleic acid of the invention includes the amyloid precusor protein selected from cross-film (APP) base sequence of the exon of at least one of gene Exon 14, exons 15, exon16 and exons 17 Or part thereof sequence, or with these substantial sequence homologies and derive from same species sequence." partial sequence " herein refers to Including at least the base sequence of coding A β 40 or A β 42.
In certain embodiments, cyclic annular ribonucleic acid of the invention includes encoded exon 14, exons 15, exon 16 and exons 17 base sequence or part thereof sequence, or with these substantial sequence homologies and derive from the sequences of same species Column.Preferably, cyclic annular ribonucleic acid of the invention is circA β-a, and sequence is as shown in SEQ ID No.1.
In certain embodiments, cyclic annular ribonucleic acid of the invention includes encoded exon 15, exon16 and outer aobvious The base sequence of son 17 or part thereof sequence, or with these substantial sequence homologies and from the sequence of same species.Preferably, Cyclic annular ribonucleic acid of the invention is circA β-b, and sequence is as shown in SEQ ID No.2.
In certain embodiments, cyclic annular ribonucleic acid of the invention includes the base of encoded exon 16 and exons 17 Sequence or part thereof sequence, or with these substantial sequence homologies and from the sequence of same species.Preferably, ring of the invention Shape ribonucleic acid is circA β-c, and sequence is as shown in SEQ ID No.3.
In certain embodiments, cyclic annular ribonucleic acid of the invention includes base sequence or its portion of encoded exon 17 Sub-sequence, or with these substantial sequence homologies and from the sequence of same species.Preferably, cyclic annular ribonucleic acid of the invention For circA β-d, sequence is as shown in SEQ ID No.4.
In certain embodiments, cyclic annular ribonucleic acid of the invention be circA β-e, circA β-f, circA β-g, circAβ-h、circAβ-i、circAβ-j、circAβ-k、circAβ-l、circAβ-m、circAβ-n、circAβ-o、circA β-p or circA β-q, they are respectively the combined base sequence for encoding different exons or different exons or part thereof sequence Column.
Information about circA β-a to circA β-q is referring to table 1.
The information of table 1- Exemplary circular ribonucleic acid
In certain embodiments, cyclic annular ribonucleic acid of the invention includes in sequence shown in SEQ ID No.1-17 At least one of；Or with these substantial sequence homologies and from the sequence of same species.Although SEQ ID No.1-17 is with line The mode of property sequence shows the sequence of ribonucleic acid, it should be understood that cyclic annular ribonucleic acid of the invention actually with Annular form exists.SEQ ID No.1-17 is only the composition purpose for illustrating sequence.
Cyclic annular ribonucleic acid of the invention can be prepared by known methods to obtain.In illustrative preparation method comprising It include the circA β exon DNA fragmentation of T7 RNA polymerase for example, by PCR or plasmid synthesis, it is then poly- by T7 RNA The linear circA β exon RNA of synthase transcriptional；Linear RNA is connected by T4 RNA ligase again cricoid circAβRNA.The example of circA β includes but is not limited to circA β-a, circA β-b, circA β-c, circA β-d, circA β- e、circAβ-f、circAβ-g、circAβ-h、circAβ-i、circAβ-j、circAβ-k、circAβ-l、circAβ-m、 CircA β-n, circA β-o, circA β-p or circA β-q.
[carrier]
The second aspect of the present invention is provided as that circA β can be expressed or generates the carrier of its cDNA.
In certain embodiments, carrier of the invention includes circular rna expression plasmid, and the example includes but is not limited to PCircRNA-BE-A β, pCircRNA-DMo-A β, pCMV-circA β-ORF, pCMV-circA β-SP and pCMV-circA β- (SP)n.Information about these plasmids is as shown in table 2 below.
Table 2- circular rna expression plasmid or the information for expressing its cDNA plasmid
Number Plasmid designations Expression product Remarks
1 pCircRNA-BE-Aβ circAβ CircA β includes circA β-a-q shown in table 1
2 pCircRNA-Dmo-Aβ circAβ CircA β includes circA β-a-q shown in table 1
3 pCMV-circAβ-ORF ORF cDNA
4 pCMV-circAβ-SP The special peptide of circA β SP is special peptide comprising SEQ ID No.18-23
5 pCMV-circAβ-(SP)n Express the n special peptide of circA β N indicates that n times repeat, and is 1 or more natural number
[cell]
The third aspect of the present invention provides a kind of cell, wherein being overexpressed circA β or its cDNA in the cell.It is preferred that Ground, the cell are Alzheimer disease cell model.
Cell of the invention can be prepared by methods known in the art.In illustrative preparation method comprising will The step of enough promoting circA β expression or expressing the vector introduction host cell of its cDNA.In cell of the invention, circA β It can also be expressed with transient expression with stability.
[the special peptide of separation or synthesis circA β]
The fourth aspect of the present invention, providing the special peptide of separation or synthesis circA β, (present invention is sometimes referred to as " unique Polypeptide " or " unique peptide "), for it is being generated by cyclic annular ribonucleic acid coding of the invention and in natural A PP albumen it is (or wild Type) in the polypeptide that is not present.That is, the special peptide of circA β cannot correspond to any continuous amino acid sequence segment of APP.
In certain embodiments, the special peptide of circA β of the invention includes to be selected from sequence shown in SEQ ID No.18-23 Column, or with these substantial sequence homologies and from the sequence of same species.
The exemplary special peptide of circA β of table 3-
SEQ ID No. Sequence information
18 MSCFRKSKTIQMTSWPT
19 LSLLMPALLPTED
20 GVVEVLG
21 MIYSLSPFDSCAVTQ
22 WVDKYQDGGDL
23 WMQNSDMTQDMKFIIKNWCSLQKMWVQTKVQSLDSW WAVLS
Note: " GVVE " in SEQ ID No.20 is derived from APP, only for embody the purpose of specificity by its with it is true special Sequence " VLG " group be combined into the sequence.
[separation or synthesis A beta related peptides]
The fifth aspect of the present invention provides separation or synthesis A beta related peptides, for by cyclic annular ribose core of the invention The polypeptide that acid generates or coding obtains.A beta related peptides of the invention preferably comprise basic sequence and particular sequence.
Basic sequence of the invention refers to the sequence being made of multiple continuous amino acid identical with APP or its segment. Wherein APP refers to the albumen being made of 18 exons, and segment refers to one of any or part of it in 18 exons. In A beta related peptides of the invention, the quantity of basic sequence is not limited, and can be one, is also possible to multiple.
In certain embodiments, basic sequence of the invention is from APP exons 14, exons 15, exon16 With the exon or its segment of at least one of exons 17.In an exemplary embodiment, basic sequence of the invention includes A The amino acid sequence or its segment of β 40 or A β 42.In other exemplary implementation scheme, basic sequence of the invention does not include The amino acid sequence or its segment of A β 40 or A β 42.
Particular sequence of the invention is identical as the sequence of the special peptide of circA β, is turning over for cyclic annular ribonucleic acid of the invention The sequence being made of multiple continuous amino acids generated in the process is translated or expresses, it is special in the polypeptide obtained for cyclic annular ribonucleic acid Some amino acid sequences cannot correspond to the amino acid sequence for the APP being currently known.
In certain embodiments, particular sequence of the invention be selected from SEQ ID No.18-23 shown in sequence, or and this A little substantial sequence homologies and the sequence for deriving from same species.
In certain embodiments, the structure of A beta related peptides of the invention is basic sequence-particular sequence, particular sequence- Basic sequence, the-the second basic sequence of the first basic sequence-particular sequence or the basic sequence of the second basic sequence-particular sequence-the first Column.Wherein, the first basic sequence and the second basic sequence are corresponding to discrete two segments of APP."-" refers to chemistry herein Key, especially peptide bond.
In certain embodiments, A beta related peptides of the invention include 42 sequence of A β, particular sequence and are located between the two Catenation sequence.The example of catenation sequence includes but is not limited to sequence as TVIVITLVMLKKKQYTSIHHGVVE.
In certain embodiments, A beta related peptides of the invention include the sequence shown in the SEQ ID No.24-40, or With these substantial sequence homologies and from the sequence of same species.
The exemplary A beta related peptides of table 4-
Note: the letter of small letter is A beta related peptides, and what is underlined is unique polypeptide (the unexistent sequence of APP albumen)
[antisense oligonucleotides]
The sixth aspect of the present invention, provides antisense oligonucleotides, and antisense oligonucleotides of the invention refers to comprising being complementary to Target sequence and the oligonucleotides that can hybridize under strict conditions with target sequence.
The example of antisense oligonucleotides of the invention includes naturally occurring nucleic acid molecules, and by more stable with another kind Group replacement phosphate or ribose moieties in the resulting derivative of hydroxyl.The specific reality of this antisense oligonucleotides acid derivative Example includes with the hydroxyl of the substitution phosphates such as sulphur substitution phosphate, methyl acid phosphate group or ribose moieties by Alkoxy such as first The derivative of oxygroup, allyloxy etc. or the substitutions such as amino, fluorine atom.It is preferred that methylation modification, thiophosphoric acid fat modification (phosphorothioate, PS), morpholine modify (morpholino), peptide nucleic acid (peptide nucleic acid), 2'- The chemical modification objects such as O- methylation (2'-O- methyl), 2'-O- (2- methoxyethyl), lock nucleic acid (LNA).Antisense in the present invention Oligonucleotides preferably has sugared (preferably pentose) structure in its structure, because being conducive in this way across cell membrane, nuclear membrane etc. Structure.Antisense oligonucleotides in the present invention can be DNA type or RNA type, but to the angle for keeping higher stability after administration For degree, DNA is preferred.
Target sequence of the invention generally refers in cyclic annular ribonucleic acid by the sequence of multiple base compositions of arbitrary continuation. Preferably, target sequence is the base sequence for encoding the special peptide of circA β.It is highly preferred that target sequence is coding SEQ ID The base sequence of at least one of sequence polypeptide shown in No.18-23.
In certain embodiments, antisense oligonucleotides of the invention includes to be selected from sequence shown in SEQ ID No.41-57 Column, or include the sequence with these substantial sequence homologies.
Table 5- exemplary antisense oligonucleotides
[the inhibition ribonucleic acid for targeting cyclic annular ribonucleic acid]
The seventh aspect of the present invention provides the inhibition ribose for targeting cyclic annular ribonucleic acid of the invention or part thereof sequence Nucleic acid.The example of inhibition ribonucleic acid of the invention includes targeting siRNA, miRNA or sgRNA of cyclic annular ribonucleic acid (to answer For CRISPR/Cas gene editing system).Preferably, inhibition ribonucleic acid of the invention includes antisense widow's core of the invention Thuja acid.It is highly preferred that inhibition ribonucleic acid of the invention includes the sequence shown in the SEQ ID No.41-57, or comprising With the sequence of these substantial sequence homologies.
[the special peptide-binding proteins of circA β]
The eighth aspect of the present invention provides the special peptide-binding proteins of circA β, can be special with circA β of the invention Peptide or its fragments specific combine.
In certain embodiments, the special peptide-binding proteins of circA β of the invention are the special peptide antibody of circA β or it is repaired Jewelry or its conjugate, using the special peptide of circA β or its segment as epitope.Antibody of the invention includes how anti-, single Anti-, chimeric antibody, nano antibody, humanized antibody or complete human antibodies.Antibody of the invention can be single-chain antibody.This The hybridoma for generating monoclonal antibody of the present invention can also be provided in invention.
In the present invention, the modifier of antibody includes the conjugate of chemical modification object and antibody and other materials.Wherein, change The example for learning modifier includes but is not limited to acetylation, acylation, ADP- ribosylation, amidation, the crosslinking cyclisation, two sulphur of antibody Key, demethylation, covalent cross-linking, cysteine, pyroglutamic acid, formylated, gamma-carboxylation, glycosylation, GPI anchoring, hydroxyl Base, iodate, methylation, myristoylation, oxidation, proteolysis and phosphorylation etc..Wherein, the example of conjugate includes but unlimited In the conjugate with nano-macromolecule material, magnetic bead etc..
In certain embodiments, the special peptide-binding proteins of circA β of the invention are antibody derivatives, and the example includes But it is not limited to the supporting structure of one or more complementary determining regions (CDRs) antibody-containing, containing one or more varistructures Supporting structure, the antibody fragment and variant with the special peptide specific binding ability of circA β in domain (heavy chain or light chain).
Antibody of the invention can be prepared by known method.In an exemplary embodiment, the preparation side of antibody Method includes the steps that wherein immunizing antigen is special peptide of circA β or derivatives thereof using the immune animal of immunizing antigen.circAβ The example of special peptide derivant includes but is not limited to the conjugate of the special peptide of circA β or A beta related peptides and carrier protein.The present invention Carrier protein example but be not limited to seralbumin (BSA), chicken ovalbumin (OVA) and keyhole limpet hemocyanin (KLH).? In certain embodiments, immunizing antigen of the invention, which has, is selected from sequence shown in SEQ ID No.58-63.
Table 6- exemplary immunization antigen
SEQ ID No. Sequence information
58 CFRKSKTIQMTSWPT-KLH
59 LSLLMPALLPTED-KLH
60 GVVEVLG-KLH
61 MIYSLSPFDSCAVTQ-KLH
62 WVDKYQDGGDL-KLH
63 WMQNSDMTQDMKFIIKNWCSLQKMWVQTKVQSLDSW WAVLS-KLH
Antibody of the invention can be generated by known method.In an exemplary embodiment, antibody of the invention is more grams Grand antibody, preparation method include using at least one of SEQ ID No.58-63 as immunizing antigen be immunized animal (for example, New Zealand White Rabbit, mouse or rat, alpaca), to obtain polyclonal antibody.
In other exemplary implementation scheme, antibody of the invention is polyclonal antibody, and preparation method includes will The special peptide of circA β and keyhole limpet hemocyanin (KLH) are coupled, and KLH-circA β-DP-SP is made and is used as immunizing antigen, with after purification Antigen-immunized animal (for example, New Zealand White Rabbit, mouse or rat, alpaca), collect blood serum, therefrom isolate and purify out and resist CircA β-DP-SP polyclonal antibody.
[pharmaceutical composition of prevention or treatment Alzheimer disease]
The ninth aspect of the present invention provides the pharmaceutical composition for preventing or treating Alzheimer disease.Of the invention Pharmaceutical composition includes cyclic annular ribonuclease inhibitors of the invention and/or the special inhibitor peptides of circA β and/or A beta related peptides Inhibitor.Optionally, pharmacological-acceptable carrier is further included.
Cyclic annular ribonuclease inhibitors of the invention include that can reduce, reduce or block cyclic annular ribonucleic acid of the invention The substance of generation；It reduces, reduce or blocks by the substance of ring-type ribonucleic acid expression, translation corresponding polypeptide；With this hair of degrading The substance of bright cyclic annular ribonucleic acid.Cyclic annular ribonuclease inhibitors not only include macromolecular compound, such as polypeptide；Further include Small molecule compound.
In certain embodiments, cyclic annular ribonuclease inhibitors of the invention include antisense oligonucleotides of the present invention Acid.Preferably, cyclic annular ribonuclease inhibitors of the invention include and have to be selected from sequence shown in SEQ ID No.41-57, or Oligonucleotides comprising the sequence with these substantial sequence homologies.In certain embodiments, cyclic annular ribonucleic acid of the invention Inhibitor includes inhibition ring-type ribonucleic acid of the invention.
The special inhibitor peptides of circA β of the invention include that can combine and (preferably specifically bind) circA β of the invention The substance of special peptide or its segment；Reduce, reduce or block the substance that the special peptide of circA β is generated by precursor peptide；It reduces, reduce Or block the active substance of the special peptide of circA β；With the substance for degrading or decomposing the special peptide of circA β.Substance of this kind can be Macromolecular compound, such as polypeptide；It is also possible to small molecule compound.
In certain embodiments, the special inhibitor peptides of circA β of the invention include circA β of the present invention special Peptide-binding proteins.Preferably, the special inhibitor peptides of circA β of the invention include the special peptide antibody of circA β.
Pharmacological-acceptable carrier of the invention be in field it is well known, those of ordinary skill in the art can determine it Meet clinical criteria.Pharmacological-acceptable carrier includes diluent and excipient.
Pharmaceutical composition of the invention also may include other compositions to modify, maintain or keep the property of composition, such as PH, Morie osmolarity, viscosity, transparency, color, isotonicity, smell, aseptic, stability, dispersion or rate of release, Absorption or Penetration Signature.The example of suitable other compositions includes but are not limited to amino acid (such as glycine, glutamic acid, asparagus fern Propylhomoserin, arginine or lysine)；Antibacterial agent, antioxidant (such as ascorbic acid, sodium sulfite or sodium hydrogensulfite), buffer It is (such as borate buffer solution, kind carbonate buffer solution, Tris-HCl, citrate buffer, phosphate buffer, other organic Acid buffer), bulking agent (such as mannitol or glycine), chelating agent (such as EDTA), complexing agent (such as caffeine, polyvinyl pyrrole Alkanone, β-cyclodextrin or hydroxypropyl-β-cyclodextrin), filler, monosaccharide, disaccharides and other carbohydrate (such as grapes Sugar, mannose or dextrin), albumen (such as seralbumin, gelatin or immunoglobulin), colorant, flavoring agent or dilution Agent, emulsifier, hydrophilic polymer (such as polyvinylpyrrolidone), low molecular weight polypeptide, at salt counter ion, preservative (such as chlorobenzene Methane ammonium, benzoic acid, salicylic acid, thimerosal, phenethyl ethyl alcohol, methyl hydroxybenzoate, Nipasol, chlorhexidine, mountain Pears acid or hydrogen peroxide), solvent (such as glycerol, propylene glycol or polyethylene glycol), sugar alcohol (such as mannitol or sorbierite), suspending agent, table Face activating agent or wetting agent increase steady agent (sucrose or sorbierite), are tonicity reinforcing agent (such as alkali halide), transport agent, dilute Release agent, excipient and/or medicinal adjuvant etc..
In certain embodiments, pharmaceutical composition of the invention is vaccine, and it includes can express circA of the invention The plasmid of the special peptide of β or circA beta related peptides or their segment.Preferably, plasmid herein includes and can generate or express It the gene of the special peptide of circA β of the invention or circA beta related peptides or their segment and is operatively connected with the gene Operating element.
Those skilled in the art can determine optimal drug according to such as purpose administration route, types of transportation and required dosage Composition.Such composition can influence the physics shape body of specific binding agent, stability, internal rate of release, clear in vivo Except rate.
Primary medium or carrier in pharmaceutical composition of the invention can for natural water at or it is non-aqueous.Such as it closes Suitable medium or carrier can be water, physiological saline or artificial CSF for injection, and can supplement as it is other Common drug administration by injection material in composition.Carrier is in another example be neutral buffered saline or seralbumin-salt mixed liquor.It is other The example of pharmaceutical composition includes Tirs buffer (pH about 7.0-8.5) or acetate buffer solution (pH about 4.0-5.5), can also be into one Step includes sorbierite or its suitable substitute.
It in one embodiment of the invention, can be by composition and optionally freeze-drying when preparation storage pharmaceutical composition Block or the excipient of aqueous solution form mix.Then suitable excipient such as sucrose can be used, by this bonding agent product system At lyophilizate.
May be selected to give pharmaceutical composition by injection system, or pass through respiratory tract or intestinal canal administration, for example, take orally to Medicine or rectally.The preparation method of this pharmaceutically acceptable composition as known to those skilled in the art.
When considering to pass through drug administration by injection, pharmaceutical composition of the invention can be the aqueous solution shape of no heat source and injectable Formula, wherein containing required inhibitor and accommodating its pharmaceutical acceptable carrier.Specially suitable injection carrier is nothing Bacterium distilled water, inhibitor of the invention is made into sterile no solion wherein, and properly saves.Adoptable another kind side Formula is that required molecule and a kind of reagent will be used in combination, such as the microsphere of injectable, biodegradable particle, polymer (polylactic acid, polyglycolic acid), pearl or liposome, these reagents make that required product controls or sustained release, system Preparation is administered by depot injection after.
It is suitble to the pharmaceutical composition of drug administration by injection to may be made as aqueous solution dosage form, the buffer that can preferably hold in physiological condition In, such as Hanks solution, ringer solution or normal saline buffer solution.Water for injection suspension may include improving suspension viscosity Substance, such as sodium carboxymethylcellulose, sorbierite and dextran.In addition, active mixture suspension may be made as suitable oil Property suspensions.Suitable lipophilic solvent or carrier include fatty such as sesame oil, Acrawax such as ethyl oleate, sweet Oily three acid esters, liposome.Suspension optionally comprising suitable stabilizer or can be improved the deliquescent substance of compound, to allow Carry out highly concentrated solution preparation.
The dosage form that can be administered orally can be made in pharmaceutical composition by the present invention.In one embodiment of the invention In, the inhibitor being administered in this way can be made together with (such as tablet or capsule) in solid dosage form common carrier Agent can not also be prepared together with these carriers.For example, can be by capsule designs at when bioavailability is maximum and system is degraded in advance When minimum, the active part of composition is discharged in gastrointestinal tract.Other reagents can be used also to promote the absorption of inhibitor.? Diluent, flavoring agent, low melt wax, vegetable oil, lubricant, suspending agent, tablet disintegrant and adhesive can be used.
In the present invention, it is possible to use the pharmaceutically acceptable carrier of suitable oral administered dosage form well known in the art, by medicine Oral delivery form is made in compositions.These carriers can make pharmaceutical composition form the form absorbed by patient.Such as piece Agent, pill, dragee, capsule, solution, gel, syrup, suspension.
Those skilled in the art also know the pharmaceutical composition of other forms, including continue inhibitor molecules or by controlled release The dosage form put.Known a variety of other lasting or controlled release mode of movement the formulation methods of those skilled in the art, such as lipid Body carrier, bio-erodible microparticles or porous bead.
In the present invention, the pharmaceutical composition for vivo medicine-feeding must be sterile.This can be come by using sterilised membrane filter filtering It realizes.When composition is lyophilized form, this degerming method can carry out (after re-dissolving) before or after freeze-drying.For The composition of injection can be saved under lyophilized form or be saved in the solution.In addition the container for containing composition for injection is usual With aseptic valve, such as use parenteral solutions packet or the plug that can be pierced through by hypodermic needle via one. Once drug combination preparation is completed, can be by it with the shape of solution, suspension, gel, emulsion, solid, dehydration or freeze-dried powder Formula is in sterile vials.The storing mode of these preparations can be form i.e., or the shape to need weight molten before use Formula (as being lyophilized).
In one embodiment of the invention, using packaging kit box to obtain single dose administration unit.It is complete It can contain in packing box there are two types of container, the first contains desiccation protein, contains watery preparation second.Present invention also contemplates that using This kind of packaging kit box contains single-chamber or multi-cavity pre-filled syringe (such as fluid injector and lyosol syringe).
[method of the composition of prevention or treatment Alzheimer disease]
The tenth aspect of the present invention provides the method for preventing or treating Alzheimer disease comprising to have this need The subject that wants apply prevention or therapeutically effective amount cyclic annular ribonuclease inhibitors and/or the special inhibitor peptides of circA β and/ Or A beta related peptides inhibitor or pharmaceutical composition of the invention.
Prevention or treatment effective dose of the invention is depended on such as prevention or Curing circumstance and target.Those skilled in the art It will be understood that suitable therapeutic dose level therefore will height it is different, partly depend on delivery of molecules, binding agent molecule to be used The physique (body kind, body surface area or organ mass) and condition of instruction, administration route and patient (be good for overall by the age Health situation).Therefore clinician, which can be changed dosage and adjust administration route, has reached optimum treatment effect.According to above-mentioned factor, Exemplary dosage ranges can be about 0.1mg/kg-100mg/kg.In other embodiments, dosage range is about 0.1mg/kg- 100mg/kg, perhaps about 1mg/kg-100mg/kg or about 5mg/kg-100mg/kg.
It should be noted that the correlative factor of the subject that exact dose will treat as needed determines.Adjust dosage With administration mode to provide the reactive compound of enough levels, or keep required effect.The factor for being included into limit of consideration can wrap Include the severity of morbid state, the general health of subject, age, weight, gender, the opportunity of administration and frequency, medicine Compounds, reaction sensibility and therapeutic response.According to the half-life period of particular composition and clearance rate, depot drug product composition Administration frequency can be primary for every 3 days or 4 days, once a week, or biweekly.
Administration frequency of the invention will be dependent on the pharmacokinetic parameter of bonding agent in dosage form used.In general, applying Pharmaceutical composition is until drug reaches required effect.Therefore composition can be given in a single dose, or within a period it is more Secondary administration (with identical or different concentration/dosage) or continuous infusion.Accurately determine that suitable dosage will be regular works.It can Suitable dosage is estimated by using suitable administration response data.
Known way can be used in the administration route of pharmaceutical composition of the invention.Such as in oral, intravenous injection, peritonaeum, Intracerebral (in brain parenchym), the ventricles of the brain are interior, in intramuscular, intraocular, intra-arterial, portal vein, intralesional routes, marrow, in meninges, wear In skin, subcutaneous, peritonaeum, it is intranasal, enteral, local administration, sublingual and pass through sustained release system.If desired, vein can be used It injects or Continuous Perfusion.
In some cases, it can be possible to need to use pharmaceutical composition in a manner of first in vitro then in vivo.In this manner, from Patient takes out cell, tissue or organ and is then exposed under pharmaceutical composition, then transplants back patient's body.
[method for diagnosis of alzheimer's disease]
The eleventh aspect of the present invention provides the method for diagnosis of alzheimer's disease comprising measurement is from tested In the sample of person the step of circA β and/or circA β special peptide.
In certain embodiments, diagnostic method of the invention the following steps are included:
(1) amount of such as reagent measuring special peptide of circA β and/or circA β in the biological sample of subject is utilized The step of obtaining measured value；
(2) the step of measured value being compared with standard value, wherein standard value can be from the year with subject The value that the biological sample of age comparable normal subjects obtains；
(3) when the measured value is higher than the standard value, then the subject is diagnosed as with Alzheimer disease, Or the subject is predicted as the risk with Alzheimer disease.
In certain embodiments, diagnostic method of the invention the following steps are included:
(1) using such as reagent measuring first time point T1 from the biological sample that subject acquires circA β and/or The step of content of the special peptide of circA β obtains standard value；
(2) in the second time point T2, circA β and/or circA β from the biological sample that same subject acquires are special for measurement The step of content of different peptide is as measured value；
(3) when measured value is higher than standard value, then the subject is diagnosed as with Alzheimer disease, or by institute It states subject and is predicted as the risk with Alzheimer disease.
" reagent " herein, which refers to, can be used for showing the special peptide of circA β and/or circA β or their segment Content or level any reagent.In certain embodiments, reagent includes the primer and probe for expanding circA β.It is right In primer and probe, it is described in detail in position other herein, is not repeating herein.In certain embodiments, it tries Agent is the special peptide-binding proteins of circA β, preferred antibody.For antibody, it is described in detail in position other herein, Details are not described herein.
[kit for diagnosis of alzheimer's disease]
The twelveth aspect of the present invention provides the kit for diagnosis of alzheimer's disease, and it includes can be used for Show the content of the special peptide of circA β and/or circA β or their segment in such as biological sample from subject Or horizontal any reagent.In certain embodiments, reagent includes for expanding the primer of circA β, probe.In certain realities It applies in scheme, reagent is the special peptide-binding proteins of circA β, preferred antibody.
In certain embodiments, kit of the invention includes for showing in the biological sample from subject The primer of circA β.Primer of the invention is preferably divergence form primer pair, that is, under strict conditions, forward primer and app gene The site of hybridization is located at further downstream (that is, the 3 ' ends) in the site that reverse primer hybridizes with app gene, and divergence form primer pair expands The target sequence of increasing includes circA β or part thereof sequence.Preferably, the target sequence of divergence form primer of the invention includes app gene At least one of exons 14, exons 15, exon16 and exons 17 or part thereof sequence.It is highly preferred that this hair The target sequence of bright divergence form primer includes the exons 17 of app gene or part thereof sequence.In an exemplary embodiment, originally Shown in the primer sequence of invention such as SEQ ID No.64 (A β-VF2) and SEQ ID No.65 (A β-VR2).
In certain embodiments, kit of the invention includes for showing in the biological sample from subject The antibody of the special peptide of circA β.For antibody, it is described in detail in position other herein, details are not described herein.
Kit of the invention, which may also include, to be tried in the form of as defined in government organs with regulation manufacture, use or sale diagnosis The relevant points for attention of agent box.The detail specifications of use, storage and troubleshooting can also be provided in kit.Kit may be used also It is optionally located at suitable be preferred in the device of the robot manipulation of high throughput setting.
The component of kit of the invention can provide as dry powder.When reagent and/or component are provided as dry powder, powder can lead to The suitable solvent of addition is crossed to restore to the original state.It is expected that the solvent may also be disposed in another container.Container would generally include at least A kind of bottle, test tube, flask, bottle, syringe and/or other container means, wherein optional partially place solvent.Kit is also It may include the second container means to contain sterile, pharmaceutically acceptable buffer and/or other solvents.
In kit exist be more than a kind of component in the case where, the kit would generally also comprising can it is individually placed in addition Component second, third or other other containers.In addition, can in a reservoir include the combination of various ingredients.
Kit of the invention may also include holding or maintain the component of DNA, such as the reagent of anti-nucleolysis.Such group Divide to be the nuclease of the protection for example or without RNase or with anti-RNase.Any composition as described herein or reagent can be Component in kit.
[method for determining the treatment validity of Alzheimer disease]
The thirteenth aspect of the present invention provides the method for determining the treatment validity of Alzheimer disease comprising The step of measuring in the biological sample from subject the special peptide of circA β and/or circA β or their segment.
In certain embodiments, the method for the treatment validity of determination Alzheimer disease of the invention includes:
(1 ') using reagent measuring from during or after treatment subject acquire biological sample in circA β and/or The step of content of the special peptide of circA β obtains measured value；
The step of measured value is compared by (2 ') with standard value, it is preferable that same before measuring since treatment The content of the special peptide of circA β and/or circA β in the biological sample of subject's acquisition is as standard value；It is further preferred that the mark Quasi- value is the value obtained from the biological sample with the age of subject comparable normal subjects；
(3 ') are then judged to treating effectively, when the measured value is higher than the standard value when the measured value is lower than institute When stating standard value, then it is not effective for being judged to treating.
[for screening to the method for treating or slowing down the useful compound of Alzheimer disease]
The fourteenth aspect of the present invention provides a kind of for screening to treating or slow down the useful chemical combination of Alzheimer disease The method of object comprising the special peptide of circA β and/or circA β or the step of their segment in measurement sample.
In certain embodiments, of the invention for screening to treating or slow down the useful compound of Alzheimer disease Method include:
A. circA β and/or circA β from the biological sample that the non-human subject with Alzheimer disease acquires are measured The step of content of special peptide obtains the first measured value；
B. the step of untested compound being applied to the non-human subject；
C. measure from application untested compound after the non-human subject acquire biological sample in circA β and/or The step of content of the special peptide of circA β obtains the second measured value；
D. the step of comparing the first measured value and the second measured value；
E. when the second measured value is less than the first measured value, by untested compound screening for treating or slow down alzheimer ' The useful compound of silent disease, when the second measured value is greater than or equal to the first measured value, by untested compound screening for treatment Or slow down the useless compound of Alzheimer disease.
In certain embodiments, of the invention for screening to treating or slow down the useful compound of Alzheimer disease Method include:
A. measurement is overexpressed the cell (Alzheimer disease cell model preferably of the invention) of circA β or its cDNA The step of content of middle circA β and/or the special peptide of circA β obtains the first measured value；
B. the step of untested compound being applied to the cell；
C. the content for measuring the special peptide of circA β and/or circA β from the cell after application untested compound obtains second The step of measured value；
D. the step of comparing the first measured value and the second measured value；
E. when the second measured value is less than the first measured value, by untested compound screening for treating or slow down alzheimer ' The useful compound of silent disease, when the second measured value is greater than or equal to the first measured value, by untested compound screening for treatment Or slow down the useless compound of Alzheimer disease.
Embodiment 1
One, experimentation:
Pass through RT-PCR and sequencing identification circA β
The circular rna (being named as circA β) containing the code area A β from APP (amyloid P precursor albumen) gene It is to utilize to obtain by special " diverging " type RT-PCR amplification, that is, targets the protein coding exons 17 of app gene Primer is divergent orientation, and sequence is as follows:
Aβ-VF2 atataggatccGTGATCGTCATCACCTTGGTGATGC(SEQ ID No.64)
Aβ-VR2 tatatctcgagCACCATGAGTCCAATGATTGCACC(SEQ ID No.65)
The normal frontal lobe of user's adult and hippocampus two total serum IgEs (R1234051-50-BC, R1234052-10-BC, BioCat GmbH) it is used as template.According to the recommendation of manufacturer, with the SuperScript TM with random hexamers III First-Strand Synthesis SuperMix (18080400, Invitrogen) carries out cDNA synthesis.With PrimeSTAR GXL archaeal dna polymerase (R050A, TaKaRa) carries out PCR, extends 40 circulations at 68 DEG C.
In order to be enriched with circular rna, by 15 μ l people frontal lobes and the total serum IgE of hippocampus with 10 unit RNase R (RNR07250, Epicenter it) handles 1 hour at 37 DEG C, and is purified by phenol chloroform extraction.Obtained RNA sample is used for then CDNA synthesis and PCR amplification.With E.Z.N.A purified pcr product.Gel extraction kit (D2501-02, Omega Bio- Tek) according to the recommendation of manufacturer and with BamHI and XhoI endonuclease (NEB) digest and be connected to pCMV-MIR carrier (Origene) in.Confirmation positive colony is sequenced by Sanger.
Deep sequencing
According to the recommendation of manufacturer, the RT-PCR product TruSeq DNA Nano kit (FC- of circA β is used 121-4003, Illumina, Inc) prepare DNA sequencing library.All libraries use HiSeq4000 (Illumina, Inc) system System is sequenced.Each sample obtains about 1,000,000 readings, and (ultrafast general RNA-seq is aligned using STAR aligner Device) it is positioned, then circRNA detection is carried out using DCC (circRNA calculates detection and quantitative tool).
Plasmid construction and preparation
People hsa_circ_000755624,34-36 (circBase) is known as circA β-a in our current research.By circA β-a The cDNA of (GRCh37/hg19, chr21:27264033-27284274) is inserted into pCircRNA-BE or pCircRNA-DMo carrier In to generate pCircRNA-BE-A β-a or pCircRNA-DMo-A β-a.In order to which the positive controls 175 protein expression of A β, will contain Have in cDNA insertion pCMV-MIR carrier (OriGene) of its ORF (open reading frame).With EndoFree Plasmid Maxi Kit (QIAGEN) purifies recombinant plasmid.Pass through restriction endonuclease digestion and all plasmids of Sanger sequence verification.With EndoFree Plasmid Maxi kit (QIAGEN) plasmid DNA purification.
Cell culture and plasmid DNA transfection
The culture in the Eagle culture medium (DMEM, Invitrogen) that Dulbecco is improved of HEK293 cell line, is supplemented with 10% fetal calf serum (Gibco), 10mM Sodium Pyruvate (Sigma), 100 U/ml penicillin and 100 U/ml streptomysins (Gibco) At 37 DEG C, 5% (v/v) CO2In.
For transiently transfect, will in 150 μ l Opti-MEM (Invitrogen) diluted 2.5 μ g Plasmid DNA with Diluted 5 μ l lipofectamine2000 mixing in 150 μ l Opti-MEM；Obtained transfection mixture is added in 6 orifice plates In about 500,000 cells.After 24 hours, transfection mixture is replaced with fresh DMEM culture medium.After transfection 3 days, harvest cell is used In total serum IgE and Protein Extraction.
Primary neuronal culture and immunocytochemistry (ICC)
C57BL6N mouse is raised according to the guidance of federation, European Laboratory Animal Science association (FELASA).According to manufacture The recommendation (Miltenyi Biotec) of quotient separates primary mind from 13 day-old Mice embryos using nerve fiber dissociation kit Through member.According to standard scheme P3 Primary Cell 4D-Nucleofector TM X kit (V4XP-3024, Lonza Cologne GmbH) carry out the nuclear transfection of pCircRNA-DMo-A β-a carrier or empty vector control (pCircRNA-DMo).It will turn The neuron of dye is with every hole 5 × 105The density of a cell is inoculated on coated coverslip, and is being supplemented with 1 × MACS NeuroBrew-21 (130-093-566, Miltenyi Biotec) MACS neurobasal media (#130-093-570, Miltenyi Biotec) in culture.1 × glutamine (25030081, Gibco) and 1 × Pen .- Strep (15140122, Gibco).It is carried out with anti-GFP antibody (GFP-1020, Aveslab) and A β (6E10, BioLegend Inc.) 10th day progress ICC after nuclear transfection.488 goat anti-chicken IgGs (A-11039, thermo scientific) and Goat anti-mouse IgG (H+L) intersects absorption secondary antibody (M30010, thermo scientific) and is correspondingly used as secondary antibody.In room temperature Down by DAPI (D9542, Sigma) by nuclear targeting 2 hours.Image is recorded with Leica SP8 X Laser Scanning Confocal Microscope.
Total serum IgE separation and qRT-PCR
According to the recommended method of manufacturer, the total serum IgE from HEK293 cell is separated using TRIzol reagent (Ambion). Human brain frontal lobe and the total serum IgE of hippocampus are purchased from BioCat GmbH (R1234051-50-BC, R1234052-10-BC).With DNA enzymatic I (NEB) total serum IgE is handled, and with phenol chloroform purification.For usingIII the first chain synthesis system (Invitrogen) and the random hexamer for initiation carries out cDNA synthesis, and 0.5 μ g total serum IgE is used as template.Use Power SYBR Green PCR Master Mix (Applied Biosystems), with 7900HT Fast real-time PCR system (Applied Biosystems quantitative pcr amplification) is carried out.Use 2- Δ Δ CT method using beta-actin mRNA as internal contrast calculating at Manage the multiple differential expression between sample and control sample.
The details of table 7-RT-PCR Oligonucleolide primers
Rna blot analysis
As previously mentioned, carrying out RNA blot hybridization with NorthernMax TM kit (AM1940, Ambion).In short It, separates the 15 μ g total serum IgEs from HEK293 cell, and be transferred to band on 5% native polyacrylamide gel (Bio-Rad) On the nylon membrane of positive charge.Hybridized overnight (A β-NBR1:CCCACC is carried out at 42 DEG C with the DNA oligonucleotides that 5'P32 is marked ATGAGTCCAATGATTGCACCTTTGTTTGAACCCACATCTTCTGCAAAGAACACC).According to the recommendation of manufacturer, 42 Film is washed with the kit equipped with buffer at DEG C.For RNase R processing, by 15 μ g total serum IgEs with 10 unit RNase R (RNR07250, Epicenter) digests 1 hour at 37 DEG C；By the separating obtained RNA of gel electrophoresis, and pass through RNA as described above Engram analysis.
Western blot analysis
With RIPA buffer (50mM Tris-HCl pH 8.0,150mM NaCl, 1% (v/v) NP40,0.1% (w/v) SDS, 0.5% (w/v) Na- dexycholate, 1X prepare protein cracking, and Roche Protease inhibitor and phosphatase inhibit Agent).40 μ g are separated on 18% or 4-20%Criterion TM TGX Stain-Free TM protein gel (Bio-Rad) Gross protein, and be transferred on 0.2 μm of nitrocellulose filter (10600002, Amersham).With directed against amyloid-beta albumen (β-shallow lake Powder sample albumen [D54D2]Rabbit mAb, #8243, CST company), anti-alpha-tubulin (#2125, CST company) and anti- Beta-actin (#A5441, Sigma) carries out immunoblotting assay.Polyclonal antibody (the anti-A β of anti-A β 175 is cultivated in rabbit 175), using unique peptide (CFRKSKTIQMTSWPT) as antigen.Human brain sample is purchased from BioCat GmbH and BIOZOL Diagnostica Vertrieb GmbH.A β 42 (A9810, Sigma) is prepared in DMSO.It is quantified with ImageJ (NIH) Analysis.
Immunoprecipitation-mass spectral analysis (IP-MS) of CircA β-a derived peptide
PCircRNA-DMo-A β-a is transfected 24 hours in HEK293 cell, and then 50 nM alpha-secretase enzyme ADAM10 inhibit Agent GI254023X (SML0789 Sigma), beta-secretase inhibitor Begacestat (PZ0187, Sigma) are by gamma-secretase Inhibitor (SCP0004, Sigma) is added in cell culture other 24 hours.It collects cell and is cracked in RIPA buffer. With the anti-β-amyloid 6E10 combined with Dynabeads TM albumin A, G (10002D, 10004D, Invitrogen) Immunoprecipitation is carried out with 4G8 (803001,800701, BioLegend Inc.).By trypsase (V5280, Omega) in pearl The protein of immunoprecipitation is digested on son.It is analyzed by mass spectrometry 31 as previously described.Spread out for the circA β-a from human brain sample The IP-MS of raw peptide, by anti-A β 175 be used for in the immunoprecipitation of human brain sample (being prepared in RIPA buffer).Remaining step It carries out as described above.
Immunoprecipitation/Western blotting (IP-WB) of A β peptide
A β peptide detection is carried out by the immunoprecipitation of conditioned medium (CM), then carries out western blot analysis.Letter speech It, the HEK293 that will be transfected with pCircRNA-BE-A β-a, pCircRNA-DMo-A β-a or empty carrier (pCircRNA-DMo) is thin Born of the same parents' overnight incubation in serum free medium.Then prepare CM with protease and inhibitors of phosphatases (Roche), and with albumin A/ G (Dynabeads TM Protein A, 10002D, Dynabeads TM Protein G, 10004D, Invitrogen) is pre- clear Clean boss.Immunoprecipitation is carried out with the mixture of A β antibody (6E10,4G8, BioLegend Inc.).Then the peptide of precipitating is existed It separates in SDS sample-loading buffer, and is analyzed by Western blotting antibody (D54D2, CST) derived from anti-A β.
Two, result:
CircA β isotype is expressed by the app gene in human brain
The generation of circular rna for experimental analysis from the region app gene A β, using a pair of of specific amplification few nucleosides Acid carries out RT-PCR amplification (Fig. 1)；Using people's frontal lobe and the total serum IgE of hippocampus as template.The template representative that the design ensures to expand comes Circular rna from the area A β of app gene seat.The PCR product as obtained by the parsing of natural 5% polyacrylamide gel electrophoresis.Analysis It discloses from the region A β and generates a variety of different circular rnas (Figure 1A).In order to study the circular rna of this amplification in more detail, Using corresponding PCR product RNA deep sequencing and disclose 17 kinds of different isotypes (table 1).In these circular rnas, inspection It has measured Hsa_circ_0007556 (circbase, circular rna database) and has been referred to as circA β-a for convenience's sake (Figure 1B, table 1).Similarly, the other three circular rna very closely related with circA β-a is respectively designated circA β- B, circA β-c, circA β-d (Fig. 2 B, table 1).
CircA β is confirmed by RNA deep sequencing
In order to carry out individual authentication to sequencing analysis, circular rna identification is carried out with identical total serum IgE, these RNA quilts RNase R pre-processes (RNase R can digest linear rna and keep circular rna unaffected).In short, 16 in 17 circA β It is a resistant to RNase R processing, show that they represent circular rna really.Second wheel sequencing analysis, discloses another CircA β (table 1).Importantly, circA β-a is accredited as the most abundant copy (table 1)；The volume that the RNA is handled in RNase R 4.8/3.1 times is enriched in leaf and hippocampus RNA sample.Therefore, select circA β-a as analysis potential function relevant to circA β The model of energy.In order to analyze the full length sequence of circA β-a, the RT-PCR product cloning for being originated from the sample of RNase R enrichment is arrived In pCMV-MIR carrier.CircA β-a for containing recombinant clone with Sanger sequencing analysis, identifies circRNA by app gene Exons 14,15,16 and 17 form, but without the reservation intron sequences of any trace (data are not shown).
Finally, the expression in order to confirm circA β-a in selected human brain region, using designed for specific detection The Oligonucleolide primers of circA β-a carry out RT-PCR analysis to people's frontal lobe and hippocampus total serum IgE sample (details are shown in Figure 1B, D).It is real On border, circA β-a expresses (Fig. 1 D) in people's frontal lobe and hippocampus, shows that circA β-a may be acted as in memory and cognition With.
IME promotes overexpression of the circA β-a in human cell line
In order to promote the research of circRNA function, uses mediate the strategy of enhancing (IME) come real based on introne recently Existing steady circRNA expression.IME can promote the expression and translation of circRNA.CircA β-a should be enhanced in this way It expresses (Fig. 1 C).Compared with endogenous background level, as disclosed in control, pCircRNA-BE-A β-a and pCircRNA- Transient transfection of the DMo-A β-a in HEK293 cell leads to the overexpression 2185 and 3268 times (Fig. 1 D, E) of circA β-a.Due to PCircRNA-DMo-A β-a has IME introne, and the circA β-a expression ratio of enhancing does not have (pCircRNA-BE-A β-a) Expression is strong.In addition, monitoring the circA β-a expression in HEK293 cell using RNA blot hybridization.As shown in fig. 1F, circA β-a migrates faster than its correspondence linear rna object.Meanwhile the signal of circA β-a is not influenced by RNase processing, because What this was expressed in HEK293 is strictly circular rna.
Importantly, RNA hybridization display, pCircRNA-BE-A β-a and pCircRNA-DMo-A β-a do not generate maturation Linear mRNA variation, thus eliminate follow-up function analysis in linear rna pollution a possibility that.Finally, RT-PCR analysis and Sanger sequencing discloses the consistency (Fig. 1 G, H, I) of the circA β-a in transient expression and human brain.
In a word, it can be deduced that, circA β-a passes through IME effect strong expression in HEK293 cell.Since HEK293 is thin Born of the same parents represent the mature cell model of Alzheimer disease correlative study.Therefore it is expressed in HEK293 cell for circA β-a IME system provide the most suitable model for analyzing circA β-a function.
CircA β-a can translate into A beta related peptides in human cell line
The ORF of verified at least some circular rnas is interpretable.Detection circA β-a identifies opening for 19.2kDa Reading frame (ORF) (Fig. 2A, D) is put, therefore, carrying out Western blotting, whether to translate into A β relevant more to study circA β-a Peptide.Significant A β related polypeptide signal is detected by this research, size is about 15 to 20kDa, to confirm HEK293 The translation (Fig. 2 B) of circA β-a in system.CircA β-a translation product is known as circA β-a- derived peptide (circA β-a-DP or A β 175, because it has 175 amino acid, Fig. 2 D).Compared with the Endogenous level in HEK293 cell, pCircRNA-DMo- The protein expression of A β-a by force be about 3.3 times, and plasmid pCircRNA-BE-A β-a only have 1.4 times A β related polypeptide (Fig. 2 B, C)。
In order to further study the cyclic annular translation product from circA β-a, with anti-amyloid beta antibodies (6E10,4G8) immunoprecipitation A β 175, and the polypeptide obtained using subsequent analytical reagent composition.If Fig. 2A-D is shown, there is a section only in 175 polypeptide sequence of A β Special amino acid sequence, it is the result (Fig. 2 D, capitalization band underscore) of annular translation；This peptide sequence is complete in overall length APP It is not present entirely, because it represents the product of cyclisation.Obtain mass signal (SKTIOMTSWPT, the figure for corresponding to unique polypeptide 2D, E).Therefore, these are the result shows that circA β-a has translated into the relevant polypeptide of A β really.
A β 175 is further processed to form A β in human cell line
The translation of A β related polypeptide from circA β-a template highlights and the generation of the A beta polypeptides of circular rna dependence Significant potentiality.It is worth noting that, the primary structure of the A β 175 of prediction contains β and gamma-secretase cleavage site, showing can Potential A β associated products (Fig. 2 D) can be generated from A β 175 by the cutting that β and gamma-secretase mediate.Utilize the anti-A β-of specificity Antibody (6E10,4G8, mouse antibodies) Lai Jinhang immunoprecipitation and subsequent Western blotting (IP-WB) analyze circA β-a Whether A β expression is had in the conditioned medium (CM) of the HKE293 cell of overexpression.
Strikingly, it observed and correspond in Western blotting with another A β antibody (D54D2, rabbit antibody) The polypeptide signal of A β, to confirm the generation (Fig. 3 A, B) of the A β from circA β-a translation.With the sky for being used as negative control The HEK293 transfection of carrier is compared, and pCircRNA-BE-A β-a causes about 2.6 times of A beta polypeptides expression quantity increase in conditioned medium (Fig. 3 A, B).Strikingly, pCircRNA-DMo-A β-a makes more than 6 times (Fig. 3 A, B) of A beta polypeptides expression enhancing.It is worth note Meaning expresses the A β that three samples (control, pCircRNA-BE-A β-a, pCircRNA-DMo-A β-a) detect Difference is consistent with the horizontal variation of A β 175, shows that the A β of up-regulation is derived from A β 175.
CircA β-a overexpression develops into A β plaque block in primary neuronal culture
The present invention is investigated whether these A beta polypeptides (they are the results of circRNA translation) can develop into outside neuron A β plaque block.The latter is the mark of Alzheimer disease europathology development.In order to analyze, pCircRNA-DMo-A β-a is carried Body is transfected into mice embryonic neuron, and is screened with anti-amyloid beta antibodies 6E10, be incubated for 10 days after in neuron culture A β plaque block is had found in object.The signal that A β plaque block is formed specifically is observed in the neuronal cultures of expression circA β-a (in Fig. 3 D shown in arrow).It is used as negative control with the neuron cultures that empty carrier transfects, and does not form A β The significant signal (Fig. 3 C) of patch.Unloaded and circA β-a expression vector contains GFP expression cassette, the immunohistochemistry of GFP Allow to mark the neuronal cell transfected.The merging of GFP and A β plaque block signal shows that patch is specifically located at outside neuron, This (Fig. 3 D) consistent to the understanding of Alzheimer disease pathologic physiology at present with us.
In short, it could be assumed that, the A β plaque block that circA β-a expression results in primary neuronal cell culture is formed.
Three, it discusses:
Previous studies finally confirm that the biology of A β in familial Alzheimer disease generates, but sporadic alzheimer ' The generation of A β is still unknown in silent disease.Present invention finds 17 kinds from app gene different circA β.In introne With the help of the circRNA of mediation is expressed and translated enhancing technology, discovery circA β-a can translate into A β related polypeptide (A β 175).In addition, A β 175 is processed and develops into A β plaque block, this demonstrate find the potential new of Alzheimer disease molecular mechanism Approach and direction (Fig. 4).This mechanism has significant different (Fig. 4) from by A β caused by the processing of overall length APP proteolysis.Cause This, it provides the alternative route (Fig. 4) of A β biology generation.Mutation with known responsible familial Alzheimer disease form is not Together, the biology of circA β, which generates, does not need specific mutations.This shows that entire crowd may express circA β, therefore expresses its egg White matter product circA β-DP, this shows that it may play a crucial role in the pathogenesis of sporadic Alzheimer disease. The biological action of circA β may not only disclose the new mechanism for causing Alzheimer disease, and may be formulation A Erci The diagnosis of the silent disease in sea, prevents and treats new strategy and paves the way.In addition, crucial coding of the circA β-a in the nervous system disease Function shows that circA β-b and circA β-c may also generate the precursor of A β by protein coding, thus in Alzheimer Disease plays important pathogenic effects.
Embodiment 2
The present embodiment is the preparation of polyclonal antibody.Wherein immunizing antigen is the special peptide of circA β (circA β-DP-SP, figure 5).The special peptide of circA β-DP and KLH are coupled, obtain conjugate, amino acid sequence is respectively such as SEQ ID No.57,58,59 It is shown.Using any one in these conjugates as immunogene, pass through immune New Zealand White Rabbit (or mouse and other dynamic Object) obtain the polyclonal antibody of anti-circA β-DP-SP.
New Zealand White Rabbit is immunized with antigen after purification, collects rabbit blood serum, therefrom isolates and purifies out anti-circA β- The polyclonal antibody of DP-SP rabbit.The antibody serum after affinity chromatography purity up to 50% or more.The antibody of preparation is by indirect Method enzyme-linked immunosorbent assay and Western blot analysis experiment, surface feature with high specific and high-affinity. Indirect method enzyme-linked immunosorbent assay result:
Envelope antigen: free peptide
Envelope antigen concentration: 4 μ g/ml, 100 holes μ l/
Envelope antigen buffer: phosphate buffered saline (PBS), pH7.4
Secondary antibody: anti-rabbit IgG Fc monoclonal secondary antibody (HRP conjugate)
Antigen polypeptide sequence: CFRKSKTIQMTSWPT
Immunogene: Antigenic Peptide-carrier protein (KLH)
Immune animal: New Zealand White Rabbit
Table 8- antibody information
NC 1 2 3 4 5 Blank Titre
Extension rate 1:1,000 1:1,000 1:2,000 1:4,000 1:8,000 1:16,000
Animal #1 0.064 2.871 2.679 2.518 2.138 1.759 0.049 >1:16,000
Animal #2 0.077 2.764 2.509 2.188 1.834 1.237 0.049 >1:16,000
Titre be S/B (signal/blank) >=2.1 when highest dilution, the OD450 in blank is technology repetition twice Average value.NC is negative control (preimmune serum).
The antibody of table 9- purifying
Western blot analysis has been carried out using the purified antibody from animal 1, it is shown that adult brain Middle strong expression A β 175.
Expression of the circA β-a translated polypeptide in human brain
Present invention demonstrates that circA β-a is translated into A beta related peptides in cell line and primary neuronal culture.These numbers According to showing latent effect of these circRNA in Alzheimer disease pathologic.For potential in Alzheimer disease The A β 175 (circA β-a-DP) in human brain sample has been screened in the internal analysis of circA β-a correlation function.For this purpose, Produce the specific antibody (anti-A β 175, Fig. 6 A) of the C-terminal structural domain for A β 175.The C-terminal of A β 175 contains by 17 kinds The unique sequences of amino acid composition.The structural domain can distinguish A β peptide and APP albumen from A β 175；With anti-175 antibody of A β into Row Western blot finally confirms the expression from the A β peptide translated of circA β-a in human brain.In fact, this research is also It was found that several signal specific ranges in protein blot are 20 to 40 kDa (Fig. 6 B).The HEK293 cell for expressing circA β-a is used Make the positive control and marker (Fig. 6 A) that A β 175 is migrated in gel electrophoresis.Note that secretase is to HEK293 cell in order to prevent α, β and inhibitors of gamma-secretase is added to allow part to cut in the complete cutting of middle A β 175.Have found a little species specificity letters Number, the proteolysis elaboration of secretion enzymatic is reflected in A β 175 (Fig. 6 A, B).In addition, in order to clearly prove in albumen The characteristic of these peptides detected in matter blot hybridization carries out human brain extract (in RIPA with the specific antibody (anti-A β 175) In buffer) immunoprecipitation/mass spectrum (IP-MS) analysis.Detect two peptides for being located at the end A β 175N- (peptide in Fig. 6 A _ 1, Fig. 6 C) and another peptide for being located at C- terminal region, cover the differentiated part (peptide 2 in Fig. 6 A, Fig. 6 D) of A β 175.This The amino acid sequence (Fig. 6 A) and western blot analysis (Fig. 6 A, B) that a little results and computer are inferred are consistent.
Being established according to the research achievement will be world head based on the AD diagnostic method detected in blood or cerebrospinal fluid The reliable and stable minimally invasive detection method of the efficient and sensible of wound.It very big will must simplify detection program and cost, reduction pair from now on The injury of patient.Application in clinic will also assess therapeutic effect more accurately and really anticipate for drug test screening Health and patient AD in justice, to establish and improve the comprehensive diagnostic system of AD.
Embodiment 3
The present embodiment passes through the specific antisense oligonucleotides (ASO) of directed against amyloid-beta albumen ring-type ribonucleic acid (circA β) Inhibit and the circA β that degrades, thus to prevent and treat the application of Alzheimer disease.
As shown in fig. 7, passing through the antisense oligonucleotides of directed against amyloid-beta albumen ring-type ribonucleic acid-a (circA β-a) (anti-circA β-a-ASO) reduces the level of the intracellular circular rna.
As shown, the antisense of the anti-beta amyloid ring-type ribonucleic acid (circA β) containing SEQ ID No.41-57 Oligonucleotides.Antisense oligonucleotides identifies amyloid beta ring-type ribonucleic acid by base pair complementarity.Combine target spot The antisense oligonucleotides of RNA can be by activating RNase H enzymatic activity, come target RNA of degrading (Fig. 7).Antisense oligonucleotides It can be by regulating and controlling (for example inhibition) its protein translation in conjunction with target RNA.
Embodiment 4
The present embodiment is the building of cell model.The expression system of circA β and its cDNA are imported into cell, established Stable cell lines, to obtain Alzheimer disease novel cell model.
As shown in figure 8, the expression system of building circA β and its cDNA.Wherein pCircRNA-BE-A β expression passes through ring-type Rna expression plasmid pCircRNA-BE expresses the plasmid of circA β (a, b, c-q).PCircRNA-DMo-A β expression passes through ring-type Rna expression plasmid pCircRNA-DMo expresses the plasmid of circA β (a, b, c-q).PCMV-circA β-ORF indicates to be overexpressed CircA β encodes the plasmid of the ORF cDNA of polypeptide.PCMV-circA β-SP indicates the plasmid for being overexpressed circA β specific polypeptides. PCMV-circA β-(SP) n indicates the plasmid for being overexpressed multiple circA β specific polypeptides；N is indicated 1 time, 2 times or is repeated several times. CircA β includes being not limited to circA β-a, circA β-b, circA β-c.
Embodiment 5
The present embodiment is the specific biological synthetic method of circA β, is described in detail as follows:
1. generating pCircRNA-BE-Rtn4
For construct circRtn4 expression plasmid, containing circRtn4 exon (chr11:29,704,497-29,708, 881, mouse GRCm38/mm10) genome area include part 5' and 3' flanking intron sequence (1014bp and 111bp). Using the Oligonucleolide primers listed in table 10, expanded by PCR from the genomic DNA template for being isolated from mouse N2a cell.It will For expressing in pCMV-MIR (OriGene) of the products therefrom insertion containing CMV promoter.Obtained plasmid is known as compareing -2. Inverted repeat facilitates the efficiency replied, and generates circular rna in turn.For this purpose, the Regional Representative of 800 nucleotide of selection The 5' of control -2 includes subdivision (corresponding to chr11:29,704,521-29,705,320).The region is mixed in 3' flank Containing the downstream part for generating inverted repeat in son.Therefore, opposite in gained box compared with its 5' introne counterpart Orientation is reversed.Since flanking intron lacks 5' and 3' splice site, they cannot support specification montage to react and generate line Property mRNA.Obtained plasmid is named as pCircRNA-BE-Rtn4.
2. generating pCircRNA-DMo-Rtn4
PCircRNA-DMo-Rtn4 is generated by pCircRNA-BE-Rtn4 carrier, is inserted into from the embedding of pCI-neo-FLAG Introne is closed, in the upstream of circRNA structural domain.The primer used is as shown in the following table 10.
Table 10- primer information
3. generating pCircRNA-BE and pCircRNA-DMo
In order to construct circRNA expression general carrier, by multiple restriction endonuclease sites (BglII, NheI, BmtI, EcoRV, NotI, SacII, XbaI) it is added to the original of pCircRNA-BE-Rtn4 or pCircRNA-DMo-Rtn4 In circRtn4 exon, lead to carrier pCircRNA-BE or pCircRNA-DMo.The Oligonucleolide primers used such as 10 institute of table Show.
4. generating pCircRNA-BE-A β-a, b, c-q and pCircRNA-DMo-A β-a, b, c-q:
By circA β-a, circA β-b, circA β-c ... the DNA sequences encoding of circA β-q is inserted into pCircRNA-BE In the two carriers of pCircRNA-DM, to form expression circA β-a, circA β-b, circA β-c ... circA β-q's Plasmid.
The external synthetic method of 5.circA β:
As shown in figure 9, the circA β exon DNA fragmentation of PCR or plasmid synthesis comprising T7 RNA polymerase is first passed through, Then linear circA β exon RNA is transcribed by T7 RNA polymerase；Again by T4 RNA ligase linear RNA Connect into cricoid circA β RNA；CircA β includes and is not limited to circA β-a, circA β-b, circA β-c.
Without departing substantially from the scope or spirit of the invention, the specific embodiment of description of the invention can be done more Kind improvements and changes, this will be apparent to those skilled in the art.Other realities obtained by specification of the invention Applying mode for technical personnel is apparent obtain.Present specification and embodiment are merely exemplary.
Sequence table
<110>Mo Dingding
<120>beta-amyloid protein ring-type ribonucleic acid, polypeptide and its application
<130> BH1900100-1
<141> 2019-07-19
<160> 59
<170> SIPOSequenceListing 1.0
<210> 1
<211> 524
<212> DNA
<213> Homo sapiens
<400> 1
atgagctgct tcagaaagag caaaactatt cagatgacgt cttggccaac atgattagtg 60
aaccaaggat cagttacgga aacgatgctc tcatgccatc tttgaccgaa acgaaaacca 120
ccgtggagct ccttcccgtg aatggagagt tcagcctgga cgatctccag ccgtggcatt 180
cttttggggc tgactctgtg ccagccaaca cagaaaacga agttgagcct gttgatgccc 240
gccctgctgc cgaccgagga ctgaccactc gaccaggttc tgggttgaca aatatcaaga 300
cggaggagat ctctgaagtg aagatggatg cagaattccg acatgactca ggatatgaag 360
ttcatcatca aaaattggtg ttctttgcag aagatgtggg ttcaaacaaa ggtgcaatca 420
ttggactcat ggtgggcggt gttgtcatag cgacagtgat cgtcatcacc ttggtgatgc 480
tgaagaagaa acagtacaca tccattcatc atggtgtggt ggag 524
<210> 2
<211> 302
<212> DNA
<213> Homo sapiens
<400> 2
ttgagcctgt tgatgcccgc cctgctgccg accgaggact gaccactcga ccaggttctg 60
ggttgacaaa tatcaagacg gaggagatct ctgaagtgaa gatggatgca gaattccgac 120
atgactcagg atatgaagtt catcatcaaa aattggtgtt ctttgcagaa gatgtgggtt 180
caaacaaagg tgcaatcatt ggactcatgg tgggcggtgt tgtcatagcg acagtgatcg 240
tcatcacctt ggtgatgctg aagaagaaac agtacacatc cattcatcat ggtgtggtgg 300
ag 302
<210> 3
<211> 248
<212> DNA
<213> Homo sapiens
<400> 3
gttctgggtt gacaaatatc aagacggagg agatctctga agtgaagatg gatgcagaat 60
tccgacatga ctcaggatat gaagttcatc atcaaaaatt ggtgttcttt gcagaagatg 120
tgggttcaaa caaaggtgca atcattggac tcatggtggg cggtgttgtc atagcgacag 180
tgatcgtcat caccttggtg atgctgaaga agaaacagta cacatccatt catcatggtg 240
tggtggag 248
<210> 4
<211> 147
<212> DNA
<213> Homo sapiens
<400> 4
gtgttctttg cagaagatgt gggttcaaac aaaggtgcaa tcattggact catggtgggc 60
ggtgttgtca tagcgacagt gatcgtcatc accttggtga tgctgaagaa gaaacagtac 120
acatccattc atcatggtgt ggtggag 147
<210> 5
<211> 488
<212> DNA
<213> Homo sapiens
<400> 5
gtgcaatcat tggactcatg gtgggcggtg ttgtcatagc gacagtgatc gtcatcacct 60
tggtgatgct gaagaagaaa cagtacacat ccattcatca tggtgtggtg gaggttgacg 120
ccgctgtcac cccagaggag cgccacctgt ccaagatgca gcagaacggc tacgaaaatc 180
caacctacaa gttctttgag cagatgcaga actagacccc cgccacagca gcctctgaag 240
ttggacagca aaaccattgc ttcactaccc atcggtgtcc atttatagaa taatgtggga 300
agaaacaaac ccgttttatg atttactcat tatcgccttt tgacagctgt gctgtaacac 360
aagtagatgc ctgaacttga attaatccac acatcagtaa tgtattctat ctctctttac 420
attttggtct ctatactaca ttattaatgg gttttgtgta ctgtaaagaa tttagctgta 480
tcaaacta 488
<210> 6
<211> 460
<212> DNA
<213> Homo sapiens
<400> 6
gtgttctttg cagaagatgt gggttcaaac aaaggtgcaa tcattggact catggtgggc 60
ggtgttgtca tagcgacagt gatcgtcatc accttggtga tgctgaagaa gaaacagtac 120
acatccattc atcatggtgt ggtggaggtt gacgccgctg tcaccccaga ggagcgccac 180
ctgtccaaga tgcagcagaa cggctacgaa aatccaacct acaagttctt tgagcagatg 240
cagaactaga cccccgccac agcagcctct gaagttggac agcaaaacca ttgcttcact 300
acccatcggt gtccatttat agaataatgt gggaagaaac aaacccgttt tatgatttac 360
tcattatcgc cttttgacag ctgtgctgta acacaagtag atgcctgaac ttgaattaat 420
ccacacatca gtaatgtatt ctatctctct ttacattttg 460
<210> 7
<211> 531
<212> DNA
<213> Homo sapiens
<400> 7
gttctgggtt gacaaatatc aagacggagg agatctctga agtgaagatg gatgcagaat 60
tccgacatga ctcaggatat gaagttcatc atcaaaaatt ggtgttcttt gcagaagatg 120
tgggttcaaa caaaggtgca atcattggac tcatggtggg cggtgttgtc atagcgacag 180
tgatcgtcat caccttggtg atgctgaaga agaaacagta cacatccatt catcatggtg 240
tggtggaggt tgacgccgct gtcaccccag aggagcgcca cctgtccaag atgcagcaga 300
acggctacga aaatccaacc tacaagttct ttgagcagat gcagaactag acccccgcca 360
cagcagcctc tgaagttgga cagcaaaacc attgcttcac tacccatcgg tgtccattta 420
tagaataatg tgggaagaaa caaacccgtt ttatgattta ctcattatcg ccttttgaca 480
gctgtgctgt aacacaagta gatgcctgaa cttgaattaa tccacacatc a 531
<210> 8
<211> 450
<212> DNA
<213> Homo sapiens
<400> 8
atggatgcag aattccgaca tgactcagga tatgaagttc atcatcaaaa attggtgttc 60
tttgcagaag atgtgggttc aaacaaaggt gcaatcattg gactcatggt gggcggtgtt 120
gtcatagcga cagtgatcgt catcaccttg gtgatgctga agaagaaaca gtacacatcc 180
attcatcatg gtgtggtgga ggttgacgcc gctgtcaccc cagaggagcg ccacctgtcc 240
aagatgcagc agaacggcta cgaaaatcca acctacaagt tctttgagca gatgcagaac 300
tagacccccg ccacagcagc ctctgaagtt ggacagcaaa accattgctt cactacccat 360
cggtgtccat ttatagaata atgtgggaag aaacaaaccc gttttatgat ttactcatta 420
tcgccttttg acagctgtgc tgtaacacaa 450
<210> 9
<211> 626
<212> DNA
<213> Homo sapiens
<400> 9
agttcagcct ggacgatctc cagccgtggc attcttttgg ggctgactct gtgccagcca 60
acacagaaaa cgaagttgag cctgttgatg cccgccctgc tgccgaccga ggactgacca 120
ctcgaccagg ttctgggttg acaaatatca agacggagga gatctctgaa gtgaagatgg 180
atgcagaatt ccgacatgac tcaggatatg aagttcatca tcaaaaattg gtgttctttg 240
cagaagatgt gggttcaaac aaaggtgcaa tcattggact catggtgggc ggtgttgtca 300
tagcgacagt gatcgtcatc accttggtga tgctgaagaa gaaacagtac acatccattc 360
atcatggtgt ggtggaggtt gacgccgctg tcaccccaga ggagcgccac ctgtccaaga 420
tgcagcagaa cggctacgaa aatccaacct acaagttctt tgagcagatg cagaactaga 480
cccccgccac agcagcctct gaagttggac agcaaaacca ttgcttcact acccatcggt 540
gtccatttat agaataatgt gggaagaaac aaacccgttt tatgatttac tcattatcgc 600
cttttgacag ctgtgctgta acacaa 626
<210> 10
<211> 348
<212> DNA
<213> Homo sapiens
<400> 10
gtgcaatcat tggactcatg gtgggcggtg ttgtcatagc gacagtgatc gtcatcacct 60
tggtgatgct gaagaagaaa cagtacacat ccattcatca tggtgtggtg gaggttgacg 120
ccgctgtcac cccagaggag cgccacctgt ccaagatgca gcagaacggc tacgaaaatc 180
caacctacaa gttctttgag cagatgcaga actagacccc cgccacagca gcctctgaag 240
ttggacagca aaaccattgc ttcactaccc atcggtgtcc atttatagaa taatgtggga 300
agaaacaaac ccgttttatg atttactcat tatcgccttt tgacagct 348
<210> 11
<211> 274
<212> DNA
<213> Homo sapiens
<400> 11
gtgcaatcat tggactcatg gtgggcggtg ttgtcatagc gacagtgatc gtcatcacct 60
tggtgatgct gaagaagaaa cagtacacat ccattcatca tggtgtggtg gaggttgacg 120
ccgctgtcac cccagaggag cgccacctgt ccaagatgca gcagaacggc tacgaaaatc 180
caacctacaa gttctttgag cagatgcaga actagacccc cgccacagca gcctctgaag 240
ttggacagca aaaccattgc ttcactaccc atcg 274
<210> 12
<211> 308
<212> DNA
<213> Homo sapiens
<400> 12
gtgttctttg cagaagatgt gggttcaaac aaaggtgcaa tcattggact catggtgggc 60
ggtgttgtca tagcgacagt gatcgtcatc accttggtga tgctgaagaa gaaacagtac 120
acatccattc atcatggtgt ggtggaggtt gacgccgctg tcaccccaga ggagcgccac 180
ctgtccaaga tgcagcagaa cggctacgaa aatccaacct acaagttctt tgagcagatg 240
cagaactaga cccccgccac agcagcctct gaagttggac agcaaaacca ttgcttcact 300
acccatcg 308
<210> 13
<211> 211
<212> DNA
<213> Homo sapiens
<400> 13
aagatgtggg ttcaaacaaa ggtgcaatca ttggactcat ggtgggcggt gttgtcatag 60
cgacagtgat cgtcatcacc ttggtgatgc tgaagaagaa acagtacaca tccattcatc 120
atggtgtggt ggaggttgac gccgctgtca ccccagagga gcgccacctg tccaagatgc 180
agcagaacgg ctacgaaaat ccaacctaca a 211
<210> 14
<211> 325
<212> DNA
<213> Homo sapiens
<400> 14
gttctgggtt gacaaatatc aagacggagg agatctctga agtgaagatg gatgcagaat 60
tccgacatga ctcaggatat gaagttcatc atcaaaaatt ggtgttcttt gcagaagatg 120
tgggttcaaa caaaggtgca atcattggac tcatggtggg cggtgttgtc atagcgacag 180
tgatcgtcat caccttggtg atgctgaaga agaaacagta cacatccatt catcatggtg 240
tggtggaggt tgacgccgct gtcaccccag aggagcgcca cctgtccaag atgcagcaga 300
acggctacga aaatccaacc tacaa 325
<210> 15
<211> 214
<212> DNA
<213> Homo sapiens
<400> 15
acggaggaga tctctgaagt gaagatggat gcagaattcc gacatgactc aggatatgaa 60
gttcatcatc aaaaattggt gttctttgca gaagatgtgg gttcaaacaa aggtgcaatc 120
attggactca tggtgggcgg tgttgtcata gcgacagtga tcgtcatcac cttggtgatg 180
ctgaagaaga aacagtacac atccattcat catg 214
<210> 16
<211> 82
<212> DNA
<213> Homo sapiens
<400> 16
gtgcaatcat tggactcatg gtgggcggtg ttgtcatagc gacagtgatc gtcatcacct 60
tggtgatgct gaagaagaaa ca 82
<210> 17
<211> 170
<212> DNA
<213> Homo sapiens
<400> 17
atggatgcag aattccgaca tgactcagga tatgaagttc atcatcaaaa attggtgttc 60
tttgcagaag atgtgggttc aaacaaaggt gcaatcattg gactcatggt gggcggtgtt 120
gtcatagcga cagtgatcgt catcaccttg gtgatgctga agaagaaaca 170
<210> 18
<211> 17
<212> PRT
<213> Homo sapiens
<400> 18
Met Ser Cys Phe Arg Lys Ser Lys Thr Ile Gln Met Thr Ser Trp Pro
1 5 10 15
Thr
<210> 19
<211> 13
<212> PRT
<213> Homo sapiens
<400> 19
Leu Ser Leu Leu Met Pro Ala Leu Leu Pro Thr Glu Asp
1 5 10
<210> 20
<211> 3
<212> PRT
<213> Homo sapiens
<400> 20
Val Leu Gly
1
<210> 21
<211> 15
<212> PRT
<213> Homo sapiens
<400> 21
Met Ile Tyr Ser Leu Ser Pro Phe Asp Ser Cys Ala Val Thr Gln
1 5 10 15
<210> 22
<211> 11
<212> PRT
<213> Homo sapiens
<400> 22
Trp Val Asp Lys Tyr Gln Asp Gly Gly Asp Leu
1 5 10
<210> 23
<211> 41
<212> PRT
<213> Homo sapiens
<400> 23
Trp Met Gln Asn Ser Asp Met Thr Gln Asp Met Lys Phe Ile Ile Lys
1 5 10 15
Asn Trp Cys Ser Leu Gln Lys Met Trp Val Gln Thr Lys Val Gln Ser
20 25 30
Leu Asp Ser Trp Trp Ala Val Leu Ser
35 40
<210> 24
<211> 175
<212> PRT
<213> Homo sapiens
<400> 24
Met Ile Ser Glu Pro Arg Ile Ser Tyr Gly Asn Asp Ala Leu Met Pro
1 5 10 15
Ser Leu Thr Glu Thr Lys Thr Thr Val Glu Leu Leu Pro Val Asn Gly
20 25 30
Glu Phe Ser Leu Asp Asp Leu Gln Pro Trp His Ser Phe Gly Ala Asp
35 40 45
Ser Val Pro Ala Asn Thr Glu Asn Glu Val Glu Pro Val Asp Ala Arg
50 55 60
Pro Ala Ala Asp Arg Gly Leu Thr Thr Arg Pro Gly Ser Gly Leu Thr
65 70 75 80
Asn Ile Lys Thr Glu Glu Ile Ser Glu Val Lys Met Asp Ala Glu Phe
85 90 95
Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys Leu Val Phe Phe
100 105 110
Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly Leu Met Val
115 120 125
Gly Gly Val Val Ile Ala Thr Val Ile Val Ile Thr Leu Val Met Leu
130 135 140
Lys Lys Lys Gln Tyr Thr Ser Ile His His Gly Val Val Glu Met Ser
145 150 155 160
Cys Phe Arg Lys Ser Lys Thr Ile Gln Met Thr Ser Trp Pro Thr
165 170 175
<210> 25
<211> 80
<212> PRT
<213> Homo sapiens
<400> 25
Met Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln
1 5 10 15
Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile
20 25 30
Ile Gly Leu Met Val Gly Gly Val Val Ile Ala Thr Val Ile Val Ile
35 40 45
Thr Leu Val Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile His His Gly
50 55 60
Val Val Glu Leu Ser Leu Leu Met Pro Ala Leu Leu Pro Thr Glu Asp
65 70 75 80
<210> 26
<211> 70
<212> PRT
<213> Homo sapiens
<400> 26
Met Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln
1 5 10 15
Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile
20 25 30
Ile Gly Leu Met Val Gly Gly Val Val Ile Ala Thr Val Ile Val Ile
35 40 45
Thr Leu Val Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile His His Gly
50 55 60
Val Val Glu Val Leu Gly
65 70
<210> 27
<211> 49
<212> PRT
<213> Homo sapiens
<400> 27
Met Val Gly Gly Val Val Ile Ala Thr Val Ile Val Ile Thr Leu Val
1 5 10 15
Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile His His Gly Val Val Glu
20 25 30
Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly
35 40 45
Leu
<210> 28
<211> 65
<212> PRT
<213> Homo sapiens
<400> 28
Met Val Gly Gly Val Val Ile Ala Thr Val Ile Val Ile Thr Leu Val
1 5 10 15
Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile His His Gly Val Val Glu
20 25 30
Val Asp Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser Lys Met Gln
35 40 45
Gln Asn Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu Gln Met Gln
50 55 60
Asn
65
<210> 29
<211> 102
<212> PRT
<213> Homo sapiens
<400> 29
Met Pro Glu Leu Glu Leu Ile His Thr Ser Val Met Tyr Ser Ile Ser
1 5 10 15
Leu Tyr Ile Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly
20 25 30
Ala Ile Ile Gly Leu Met Val Gly Gly Val Val Ile Ala Thr Val Ile
35 40 45
Val Ile Thr Leu Val Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile His
50 55 60
His Gly Val Val Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg His
65 70 75 80
Leu Ser Lys Met Gln Gln Asn Gly Tyr Glu Asn Pro Thr Tyr Lys Phe
85 90 95
Phe Glu Gln Met Gln Asn
100
<210> 30
<211> 100
<212> PRT
<213> Homo sapiens
<400> 30
Met Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln
1 5 10 15
Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile
20 25 30
Ile Gly Leu Met Val Gly Gly Val Val Ile Ala Thr Val Ile Val Ile
35 40 45
Thr Leu Val Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile His His Gly
50 55 60
Val Val Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser
65 70 75 80
Lys Met Gln Gln Asn Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu
85 90 95
Gln Met Gln Asn
100
<210> 31
<211> 115
<212> PRT
<213> Homo sapiens
<400> 31
Met Ile Tyr Ser Leu Ser Pro Phe Asp Ser Cys Ala Val Thr Gln Met
1 5 10 15
Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys
20 25 30
Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile
35 40 45
Gly Leu Met Val Gly Gly Val Val Ile Ala Thr Val Ile Val Ile Thr
50 55 60
Leu Val Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile His His Gly Val
65 70 75 80
Val Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser Lys
85 90 95
Met Gln Gln Asn Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu Gln
100 105 110
Met Gln Asn
115
<210> 32
<211> 100
<212> PRT
<213> Homo sapiens
<400> 32
Met Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln
1 5 10 15
Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile
20 25 30
Ile Gly Leu Met Val Gly Gly Val Val Ile Ala Thr Val Ile Val Ile
35 40 45
Thr Leu Val Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile His His Gly
50 55 60
Val Val Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser
65 70 75 80
Lys Met Gln Gln Asn Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu
85 90 95
Gln Met Gln Asn
100
<210> 33
<211> 81
<212> PRT
<213> Homo sapiens
<400> 33
Met Ile Tyr Ser Leu Ser Pro Phe Asp Ser Cys Ala Ile Ile Gly Leu
1 5 10 15
Met Val Gly Gly Val Val Ile Ala Thr Val Ile Val Ile Thr Leu Val
20 25 30
Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile His His Gly Val Val Glu
35 40 45
Val Asp Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser Lys Met Gln
50 55 60
Gln Asn Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu Gln Met Gln
65 70 75 80
Asn
<210> 34
<211> 129
<212> PRT
<213> Homo sapiens
<400> 34
Met Val Trp Trp Arg Leu Thr Pro Leu Ser Pro Gln Arg Ser Ala Thr
1 5 10 15
Cys Pro Arg Cys Ser Arg Thr Ala Thr Lys Ile Gln Pro Thr Ser Ser
20 25 30
Leu Ser Arg Cys Arg Thr Arg Pro Pro Pro Gln Gln Pro Leu Lys Leu
35 40 45
Asp Ser Lys Thr Ile Ala Ser Leu Pro Ile Gly Ala Ile Ile Gly Leu
50 55 60
Met Val Gly Gly Val Val Ile Ala Thr Val Ile Val Ile Thr Leu Val
65 70 75 80
Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile His His Gly Val Val Glu
85 90 95
Val Asp Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser Lys Met Gln
100 105 110
Gln Asn Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu Gln Met Gln
115 120 125
Asn
<210> 35
<211> 65
<212> PRT
<213> Homo sapiens
<400> 35
Met Val Gly Gly Val Val Ile Ala Thr Val Ile Val Ile Thr Leu Val
1 5 10 15
Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile His His Gly Val Val Glu
20 25 30
Val Asp Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser Lys Met Gln
35 40 45
Gln Asn Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu Gln Met Gln
50 55 60
Asn
65
<210> 36
<211> 103
<212> PRT
<213> Homo sapiens
<400> 36
Met Val Gly Gly Val Val Ile Ala Thr Val Ile Val Ile Thr Leu Val
1 5 10 15
Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile His His Gly Val Val Glu
20 25 30
Val Asp Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser Lys Met Gln
35 40 45
Gln Asn Gly Tyr Glu Asn Pro Thr Tyr Lys Arg Cys Gly Phe Lys Gln
50 55 60
Arg Cys Asn His Trp Thr His Gly Gly Arg Cys Cys His Ser Asp Ser
65 70 75 80
Asp Arg His His Leu Gly Asp Ala Glu Glu Glu Thr Val His Ile His
85 90 95
Ser Ser Trp Cys Gly Gly Gly
100
<210> 37
<211> 105
<212> PRT
<213> Homo sapiens
<400> 37
Met Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln
1 5 10 15
Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile
20 25 30
Ile Gly Leu Met Val Gly Gly Val Val Ile Ala Thr Val Ile Val Ile
35 40 45
Thr Leu Val Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile His His Gly
50 55 60
Val Val Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser
65 70 75 80
Lys Met Gln Gln Asn Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Trp Val
85 90 95
Asp Lys Tyr Gln Asp Gly Gly Asp Leu
100 105
<210> 38
<211> 68
<212> PRT
<213> Homo sapiens
<400> 38
Met Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln
1 5 10 15
Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile
20 25 30
Ile Gly Leu Met Val Gly Gly Val Val Ile Ala Thr Val Ile Val Ile
35 40 45
Thr Leu Val Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile His His Asp
50 55 60
Gly Gly Asp Leu
65
<210> 39
<211> 61
<212> PRT
<213> Homo sapiens
<400> 39
Met Val Gly Gly Val Val Ile Ala Thr Val Ile Val Ile Thr Leu Val
1 5 10 15
Met Leu Lys Lys Lys Gln Cys Asn His Trp Thr His Gly Gly Arg Cys
20 25 30
Cys His Ser Asp Ser Asp Arg His His Leu Gly Asp Ala Glu Glu Glu
35 40 45
Thr Val Gln Ser Leu Asp Ser Trp Trp Ala Val Leu Ser
50 55 60
<210> 40
<211> 98
<212> PRT
<213> Homo sapiens
<400> 40
Met Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln
1 5 10 15
Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile
20 25 30
Ile Gly Leu Met Val Gly Gly Val Val Ile Ala Thr Val Ile Val Ile
35 40 45
Thr Leu Val Met Leu Lys Lys Lys Gln Trp Met Gln Asn Ser Asp Met
50 55 60
Thr Gln Asp Met Lys Phe Ile Ile Lys Asn Trp Cys Ser Leu Gln Lys
65 70 75 80
Met Trp Val Gln Thr Lys Val Gln Ser Leu Asp Ser Trp Trp Ala Val
85 90 95
Leu Ser
<210> 41
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 41
aagcagctca tctccaccac ac 22
<210> 42
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 42
aacaggctca actccaccac ac 22
<210> 43
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 43
caacccagaa cctccaccac ac 22
<210> 44
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 44
gcaaagaaca cctccaccac a 21
<210> 45
<211> 26
<212> DNA
<213> Artificial Sequence
<400> 45
ccaatgattg cactagtttg atacag 26
<210> 46
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 46
tgcaaagaac accaaaatgt aaag 24
<210> 47
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 47
caacccagaa ctgatgtgtg ga 22
<210> 48
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 48
tctgcatcca tttgtgttac ag 22
<210> 49
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 49
caggctgaac tttgtgttac ag 22
<210> 50
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 50
caatgattgc acagctgtca aaag 24
<210> 51
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 51
caatgattgc accgatgggt agtg 24
<210> 52
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 52
gcaaagaaca ccgatgggta gt 22
<210> 53
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 53
acccacatct tttgtaggtt gg 22
<210> 54
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 54
caacccagaa cttgtaggtt gg 22
<210> 55
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 55
atctcctccg tcatgatgaa tg 22
<210> 56
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 56
caatgattgc actgtttctt cttc 24
<210> 57
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 57
ttctgcatcc attgtttctt cttc 24
<210> 58
<211> 36
<212> DNA
<213> Artificial Sequence
<400> 58
atataggatc cgtgatcgtc atcaccttgg tgatgc 36
<210> 59
<211> 35
<212> DNA
<213> Artificial Sequence
<400> 59
tatatctcga gcaccatgag tccaatgatt gcacc 35

Claims (13)

1. a kind of separation or synthesis beta-amyloid protein ring-type ribonucleic acid circA β, it includes the Amyloid Precursors of cross-film Base sequence of at least one exon of protein gene or part thereof sequence, or with these substantial sequence homologies and from same The sequence of one species；
Preferably, A β 40 or A β 42 or its segment can be expressed or be generated to the beta-amyloid protein ring-type ribonucleic acid, or Beta-amyloid protein ring-type ribonucleic acid described in person includes the base sequence of coding A β 40 or A β 42 or their segment；
It is further preferred that the beta-amyloid protein ring-type ribonucleic acid includes the amyloid precursor protein gene China and foreign countries selected from cross-film The base sequence of at least one of aobvious son 14, exons 15, exon16 and exons 17 or part thereof sequence, or and these Substantial sequence homology and the sequence for deriving from same species, wherein the partial sequence includes at least the alkali of coding A β 40 or A β 42 Basic sequence.
It is further preferred that the beta-amyloid protein ring-type ribonucleic acid is selected from circA β-a, circA β-b, circA β-c, circA β-d、circAβ-e、circAβ-f、circAβ-g、circAβ-h、circAβ-i、circAβ-j、circAβ-k、circAβ-l、 CircA β-m, circA β-n, circA β-o, circA β-p and circA β-q, or comprising with these substantial sequence homologies and come Derived from the sequence of same species；
It is further preferred that the beta-amyloid protein ring-type ribonucleic acid include in sequence shown in SEQ ID NO.1-17 extremely It is one of few；Or with these substantial sequence homologies and from the sequence of same species.
2. a kind of carrier can express circA β according to claim 1 or generate its cDNA；
Preferably, the carrier is selected from pCircRNA-BE-A β, pCircRNA-DMo-A β, pCMV-circA β-ORF, pCMV- At least one of circA β-SP and pCMV-circA β-(SP) n.
3. a kind of cell is overexpressed circA β or its cDNA according to claim 1 in the cell.
4. a kind of special peptide of separation or synthesis circA β generates to be encoded by circA β according to claim 1 , but the polypeptide of any continuous amino acid sequence in APP cannot be corresponded to；
Preferably, the special peptide of circA β includes the sequence shown in the SEQ ID No.18-23, or basic with these sequences Sequence that is homologous and deriving from same species.
5. a kind of separation or synthesis A beta related peptides, to be generated or being encoded by circA β according to claim 1 The polypeptide arrived.
Preferably, the A beta related peptides include basic sequence and particular sequence, in which: the basic sequence include with APP or its The identical sequence being made of multiple continuous amino acid of segment, the particular sequence are according to circA as claimed in claim 4 β specific peptide sequences；
It is further preferred that the A beta related peptides include basic sequence and particular sequence, in which: the basic sequence includes A β 40 or A β 42 amino acid sequence or its segment, the particular sequence are according to circA β specific peptide sequences as claimed in claim 4；
It is further preferred that the A beta related peptides include the sequence shown in the SEQ ID No.24-40, or substantially same with these sequences Source and the sequence for deriving from same species.
6. a kind of antisense oligonucleotides, it includes the oligonucleotides that is complementary to target sequence and can hybridize with target sequence, wherein The target sequence be according in circA β described in claim 1 by the sequence of multiple base compositions of arbitrary continuation；
Preferably, the antisense oligonucleotides includes the sequence shown in the SEQ ID No.41-57, or comprising with these sequences Substantially homologous sequence.
7. a kind of inhibition ribonucleic acid targets circA β according to claim 1 or part thereof sequence；
Preferably, the inhibition ribonucleic acid is siRNA, miRNA or sgRNA；
Preferably, the inhibition ribonucleic acid includes antisense oligonucleotides according to claim 6.
8. a kind of special peptide-binding proteins of circA β, can be with the special peptide of circA β according to claim 4 or its piece Section specific binding；
Preferably, the special peptide-binding proteins of circA β are the special peptide antibody of circA β or its modifier or its conjugate, Using the special peptide of the circA β or its segment as epitope, wherein the antibody include polyclonal antibody, monoclonal antibody, Single-chain antibody and nano antibody.
9. a kind of for preventing or treating the pharmaceutical composition and method of Alzheimer disease, in which:
Described pharmaceutical composition includes circA beta inhibitor and/or the special inhibitor peptides of circA β and/or A beta related peptides inhibitor. Optionally, pharmacological-acceptable carrier is further included；
The method includes to subject with this need apply prevention or therapeutically effective amount circA beta inhibitor and/or The special inhibitor peptides of circA β and/or A beta related peptides inhibitor；
Preferably, the circA beta inhibitor includes according to claim 6 antisense oligonucleotides or according to claim 7 Inhibition ribonucleic acid；The special inhibitor peptides of the circA β and/or A beta related peptides inhibitor include according to claim 8 The special peptide-binding proteins of circA β.
10. a kind of method for diagnosis of alzheimer's disease comprising measurement the sample from subject in circA β and/or The step of special peptide of circA β or their segment；
Preferably, it the described method comprises the following steps:
(1) the step of measuring measured value of the special peptide of circA β and/or circA β in the biological sample from subject is measured；
(2) the step of measured value being compared with standard value, wherein the standard value is from the age phase with subject When normal subjects biological sample obtain value；
(3) when the measured value is higher than the standard value, then the subject is diagnosed as with Alzheimer disease, or The subject is predicted as the risk with Alzheimer disease；
Preferably, it the described method comprises the following steps:
(1) measure that the circA β and/or circA special peptide of β contains from the biological sample that subject acquires in first time point T1 The step of measuring standard value；
(2) measurement in the second time point T2 from the biological sample that same subject acquires the special peptide of circA β and/or circA β Content as measured value the step of；
(3) when measured value is higher than standard value, then the subject is diagnosed as with Alzheimer disease, or will it is described by Examination person is predicted as the risk with Alzheimer disease.
11. a kind of kit for diagnosis of alzheimer's disease, it includes can be used for display for example from subject's Any reagent of the content or level of the special peptide of circA β and/or circA β in biological sample or their segment；
Preferably, the reagent includes primer, the probe for expanding circA β or its segment, or includes the special peptide of circA β Binding protein；
Preferably, the reagent includes the divergence form primer pair that is made of forward primer and reverse primer, wherein forward primer with The site of app gene hybridization is located at the further downstream of the site that reverse primer hybridizes with app gene, and the divergence form primer pair The target sequence of amplification includes circA β or part thereof sequence.
12. a kind of method for determining the treatment validity of Alzheimer disease comprising biology of the measurement from subject The special peptide of circA β and/or circA β or the step of their segment in sample；
Preferably, which comprises
(1 ') the special peptide of circA β and/or circA β from the biological sample that the subject during or after treatment acquires is measured Content obtains the step of measured value；
The step of measured value is compared by (2 ') with standard value, it is preferable that same tested before measuring since treatment The content of the special peptide of circA β and/or circA β in the biological sample of person's acquisition is as standard value；It is further preferred that the standard value For the value obtained from the biological sample with the age of subject comparable normal subjects；
(3 ') are then judged to treating effectively, when the measured value is higher than the standard value when the measured value is lower than the mark When quasi- value, then it is not effective for being judged to treating.
13. a kind of for screening to the method for treating or slowing down the useful compound of Alzheimer disease comprising measurement sample The step of middle circA β and/or special peptide of circA β or their segment；
Preferably, which comprises
A. it is special that circA β and/or circA β from the biological sample that the non-human subject with Alzheimer disease acquires are measured The step of content of peptide obtains the first measured value；
B. the step of untested compound being applied to the non-human subject；
C. it is special that circA β and/or circA β from the biological sample that the non-human subject after application untested compound acquires are measured The step of content of different peptide obtains the second measured value；
D. the step of comparing the first measured value and the second measured value；
E. when the second measured value is less than the first measured value, by untested compound screening for treating or slow down Alzheimer disease Useful compound, when the second measured value is greater than or equal to the first measured value, by untested compound screening for treating or subtract The useless compound of slow Alzheimer disease；
Preferably, which comprises
A. the content that measurement is overexpressed the special peptide of circA β and/or circA β in the cell of circA β or its cDNA obtains first The step of measured value；
B. the step of untested compound being applied to the cell；
C. the content for measuring the special peptide of circA β and/or circA β from the cell after application untested compound obtains the second measurement The step of value；
D. the step of comparing the first measured value and the second measured value；
E. when the second measured value is less than the first measured value, by untested compound screening for treating or slow down Alzheimer disease Useful compound, when the second measured value is greater than or equal to the first measured value, by untested compound screening for treating or subtract The useless compound of slow Alzheimer disease.
CN201910656135.6A 2019-07-19 2019-07-19 Beta-amyloid protein ring-type ribonucleic acid, polypeptide and its application Pending CN110317814A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113344076A (en) * 2021-06-08 2021-09-03 汕头大学 Integrated learning-based circRNA-miRNA interaction relation prediction method

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101802007A (en) * 2007-06-12 2010-08-11 Ac免疫有限公司 Monoclonal anti-beta amyloid antibody
CN102596221A (en) * 2009-06-10 2012-07-18 纽约大学 Immunological targeting of pathological TAU proteins

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101802007A (en) * 2007-06-12 2010-08-11 Ac免疫有限公司 Monoclonal anti-beta amyloid antibody
CN102596221A (en) * 2009-06-10 2012-07-18 纽约大学 Immunological targeting of pathological TAU proteins

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DINGDING MO ET AL: "The role of Aβ circRNA in Alzheimer’s disease: alternative mechanism of Aβ biogenesis from Aβ circRNA translation", 《BIORXIV》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113344076A (en) * 2021-06-08 2021-09-03 汕头大学 Integrated learning-based circRNA-miRNA interaction relation prediction method
CN113344076B (en) * 2021-06-08 2022-03-22 汕头大学 Integrated learning-based circRNA-miRNA interaction relation prediction method

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