CN106596979B - A kind of kit and its detection method for parkinsonism detection - Google Patents

A kind of kit and its detection method for parkinsonism detection Download PDF

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CN106596979B
CN106596979B CN201710115042.3A CN201710115042A CN106596979B CN 106596979 B CN106596979 B CN 106596979B CN 201710115042 A CN201710115042 A CN 201710115042A CN 106596979 B CN106596979 B CN 106596979B
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angptl4
detection
disease
parkinsonism
aptamer
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CN106596979A (en
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张磊升
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Han's joint (Tianjin) Stem Cell Research Institute Co., Ltd.
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Han's Joint (tianjin) Stem Cell Research Institute Co Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/16—Aptamers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/475—Assays involving growth factors
    • G01N2333/515—Angiogenesic factors; Angiogenin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2835—Movement disorders, e.g. Parkinson, Huntington, Tourette

Abstract

This application involves a kind of kits and its detection method for parkinsonism detection.The kit includes the aptamer of energy specific detection parkinsonism.The aptamers of offer have preferable binding ability, have better detection accuracy than the kit of the prior art.

Description

A kind of kit and its detection method for parkinsonism detection
Technical field
The present invention relates to disease detection field, more particularly to a kind of kit for parkinsonism detection and its Detection method.
Background technology
Parkinson's disease (PD) also known as shaking plasy are one of most common neurodegenerative diseases.Epidemiology is shown, is suffered from Sick rate is 15~3,28/,100,000 populations, 65 years old crowd of ＞ about 1%；Incidence is 10~21/,100,000 populations/year.The PD causes of disease and hair Interpretation of the cause, onset and process of an illness system is not yet clear, may be related with social factor, drug factors, patients factors etc..PD pathological changes are：Substantia nigra of midbrain causes Compact part, Neurons of Locus Coeruleus depigmentation, black substance pigment is thin out and Lewy body occurs.PD nerve biochemical changes are：Substantia nigra of midbrain Compact part, Neurons of Locus Coeruleus depigmentation cause dopamine (DA) at above-mentioned position and its nerve endings to reduce, and (DA is produced when reducing >=70% Raw PD clinical manifestations), and acted on the acetylcholine of DA function antagonisms (ACH) in nigrostriatum system it is relatively hyperfunction, DA with ACH dysequilibriums.
So far, the cause of disease of pd is still unclear.Current research trend in age ageing, genetic predisposition and environment The composite factors such as the contact of toxin are related.
1) age ageing：
2) environmental factor：Epidemiological survey as a result, it has been found that, there are regional disparities for the illness rate of Parkinson's disease, so people Suspect in environment there may be some toxic substances, has damaged the neuron of brain.
3) Familial Occurrence：Physicians have found that Parkinson's disease seems there is Familial aggregation in long-term practice, There is the incidence of its relatives of the family of Parkinsonian higher compared with normal population.
4) genetic predisposition：Although the generation of Parkinson's disease is related with aging and environmental toxin, and not all old age People or exposure are in the people of same environment, or even Parkinson's disease can occur in the people for equally sucking a large amount of mptp.Although Parkinson Patient also has family aggregation, but does not also find specific Disease-causing gene in the Parkinsonian distributed so far, Illustrate that the cause of disease of Parkinson's disease is multifactor.
In conclusion any single factor cannot be satisfactory explanation pd the cause of disease.Most researchers tend to pa gold The cause of disease of gloomy disease is the above-mentioned coefficient result of each factor.I.e. after middle age, the individual susceptible to environmental toxin is touching After toxin, because of its function of detoxification obstacle, there is subclinical black substance damage, aggravate with advancing age, dopaminergic god Through the gradual continuous dead denaturation of member, there are the clinical symptoms of Parkinson's disease in final decompensation.
Currently, the biochemical markers research about Parkinson's disease early diagnosis is set and immunology, inflammatory reaction, oxidation The multiple fields such as stress reaction, natural death of cerebral cells, wherein W cynapses are total to nucleoprotein (a-Syn) most Research Prospects.Histopathology is ground Study carefully display, Parkinsonian's nigrostriatum cynapse is total to nucleoprotein abnormal aggregation, deposition and functional disturbance, and cynapse is total to core Albumen is the main component of Lewy body, if therefore detecting that cynapse is total to core egg in the body fluid such as cerebrospinal fluid, peripheral blood or saliva Albumen then judges that subject suffers from parkinsonism.But the detection method shortage that W albumen is disease marker is specific and sensitive Property.
Angiopoietin-like 4 albumen (ANGPTL4) is the member of the angiopoietin families of secretory protein.Angiogenin The conservative region of family includes helical region and C-terminal fibrinogen (FBN) spline structure domain.See for example, Kim et al., Biochem.J.346=603-610 (2000).Other members of the family include angiogenin I, angiopoietin 2 and blood Pipe generates element 3.Ang-1, angiopoietin 2 and 3/ angiogenin 4 of angiogenin combine Tie2 receptors.See example Such as, Davis et al., Ce 1187,1161-1169 (1996)；Maisonpierre et al.,Science277,55-60 (1997)；Valenzuela et al,Proc.Natl.Acad.Sc1.USA96,1904-1909(1999)；With United States Patent (USP) 5, 521,073；5,650,490；With 5,814,464.Angiogenin I and 4 is the agonist of Tie2 receptors, and angiopoietin 2 It is the antagonist (and possible agonist) of Tie receptors with 3.See for example, Folkman＆D ' Amore, Cell, 87:1153- 1155(1996)；Suri et al., Cell, 87:1171-1180(1996)；Masionpierre etal.,Science277: 55-60(1997)；With, Ward＆Dumont, Seminars in Cell＆DevelopmentalBiology, 13:19-27(20 02).Tie2 receptors belong to endothelial cell specific receptor tyrosine kinase family, also include Tiel orphan receptors.The family 3 protein binding Integrin ανβ3 of another member's angiopoietin-like is shown in for example, 20030215451 He of US patent applications Camenisch et al.,J.Biol.Chem.,277(19):17281-17290(2002)。
ANGPTL4 also is known as other terms.For example, ANGPTL4 also is known as liver fibrinogen/angiogenin- GAP-associated protein GAP (HFARP) (Kim et al., Biochem.J.346=603-610 (2000)), PPAR Y angiogenins are related Albumen (PGAR) (Yoon, et al., Mol.Cell Biol., 20:5343-5349 (2000)) and fasting induction moon purport The fat factor (fasting induced adiposefactor) (FIAF) (Kerten et al., J.Biol.Chem., 275: 28488-28493(2000))。
The in vitro and in vivo research of ANGPTL4 and it is qualitative for therapeutic agent and/or treatment provide it is valuable identification and It was found that the therapeutic agent and/or treatment can be used for preventing, alleviates or correct and ANGPTL4 activity and/or the relevant disease of expression Or dysfunction.For example, the mouse of Study on tissue culture and genetic modification have been found be and the relevant bioprocess of human disease Functional further investigation priceless tool, the disease includes immunology, Cancerous disease, Neurobiology, and angiocarpy is raw Object disease, fat and other diseases.Need to find and understand many biological functions of ANGPTL4.
Therefore it can be effectively urgently to solve in the gene marker of early stage i.e. diagnosable parkinsonism generation to find a kind of Certainly the problem of.
Invention content
In one aspect of the invention, the purposes for providing 4 its specific antibody of angiopoietin-like protein be used to make The diagnostic reagent or kit of standby detection parkinsonism.
The present invention additionally provides a kind of methods of relationship between identification angiopoietin-like protein 4 and parkinsonism.
The present invention additionally provides a kind of aptamers of angiopoietin-like protein 4, and the sequence of the aptamer is respectively such as SEQ ID NO：Shown in 1-18.
The present invention first detects parkinsonism using screening technique, the experimental results showed that angiogenin There is apparent up-regulated expression in parkinsonism in sample albumen 4.
In addition, the present invention determines expression of the angiopoietin-like protein 4 in parkinson's syndrome patient blood Amount detection.Research confirms that angiopoietin-like protein 4 can be as the diagnosis marker of parkinson's syndrome, while can also make To prevent the drug of parkinson's syndrome.
Advantageous effect:The present invention is screened by lot of experiments, the experimental results showed that angiopoietin-like protein 4 is in pa There is apparent up-regulated expression in the gloomy syndrome of gold, can be used as parkinson's syndrome disease clinical diagnosis marker.And the present invention is ground Study carefully the result shows that can be parkinson's syndrome preventive assessment or therapeutic scheme by the expression for detecting angiopoietin-like protein 4.
Description of the drawings
Fig. 1 is the statistical analysis figure of the concentration of ANGPTL4 in blood samples of patients and healthy control group blood.★ refers to P<0.05.
Fig. 2 is specificity and the sensitivity that ROC curve shows protein diagnostic parkinsonism.
Fig. 3 aptamer screening process figures
Log-Linear figure in Fig. 4 rat plasmas as complete-long aptamer residue percentage of the function of incubation time.
Specific implementation mode
It elaborates below in conjunction with the accompanying drawings to specific implementation mode provided by the invention.
Embodiment 1
It is compared by the batch of genomic data early period, passes through the protein expression data of 50 normal persons and 40 patients point Analysis, it is found by the applicant that ANGPTL4 albumen is overexpression in disturbances in patients with Parkinson disease.
The expression of mRNA and albumen in all patients and normal population blood are detected by the method for quantitative PCR, are led to It is as shown in table 1 below to cross measurement result：
MRNA (relative expression quantity) Albumen concentration (ng/mL)
Patient 1.7 110.2
Normal population 1 40.5
The above results show ANGPTL4 high level expressions in the blood of parkinsonism patient, prompt ANGPTL4 Expression and disturbances in patients with Parkinson disease the course of disease it is closely related.
ANGPTL4 concentration and disease association in 2 serum of embodiment
Using the monoclonal antibody ABB (heavy chain variable region of the antibody such as SEQ ID NO of anti-ANGPTL4：Shown in 19, The light chain variable region of the antibody such as SEQ ID NO：Shown in 20), have detected 100 by ELISA method (double antibody sandwich method) The concentration of ANGPTL4 in example parkinsonism and 40 healthy control group serum.The result shows that in patients serum A concentration of 110.4 ± 15.3ng/ml (average ± SD) of ANGPTL4；ANGPTL4's is a concentration of in healthy control group serum 40.6 ± 10.2ng/ml (average ± SD)；ANFPRL4 concentration is apparently higher than control group serum (P ＜ 0.05) in patients serum, Referring to Fig. 1.
In addition, the cut-off values that ROC curve sets ANGPTL4 according to fig. 2 are 10.25ng/ml, sensibility and spy The opposite sex is all higher than 90%, and positive rate is 92.5% (37/40).Statistic analysis result shows that ANGPTL4 concentration and pa are golden in blood Gloomy patient is closely related, neutralizes above-mentioned experimental study, it can be deduced that conclusion, secretory ANGPTL4 high scores in disturbances in patients with Parkinson disease It secretes, the particularly preferred marker that can be distinguished as parkinsonism and normal population.
The screening of embodiment 3ANGPTL4 albumen aptamers
ANGPTL4 recombinant protein 10mg are taken, according to aptamer screening technique, SELEX technologies screen aptamer：
It is pre-designed and synthesizes random single chain oligonucleotide library,
TTCGACAGTATGAAGCTCGG-N35-GGCACTGAAGTGACTAGACT
(1) single-stranded DNA banks are the random sequence of 35 length, each one section of the design in 5 ' ends and 3 ' ends among oligonucleotides Fixed sequence program is used for PCR amplification；In addition, there be the transcripting promoter of t7 rna polymerase at 5 ' ends.
(2) under optimum conditions, single stranded oligonucleotide library and target molecule are incubated, wherein can be with target substance specificity In conjunction with oligonucleotides just form DNA- target complexes, only a small number of sequences and target substance knot in first few cycle It closes.
(3) the unbonded oligonucleotides with specific binding is separated, separation method is gel blocking method；
(4) by affinity chromatography, the oligonucleotides of combination is made to be dissociated with target substance.
(5) isolated oligonucleotides is passed through into stringent washing, elution, PCR amplification generates secondary library, is used for down One wheel screening.
After screening by 14 wheels, 18 sequences are obtained by clone, sequencing in target sequence, respectively such as SEQ ID NO：(screening schematic diagram is shown in Fig. 3) shown in 1-18.
With balance osmosis combination HPLC, the dissociation constant Kd values of aptamers sequence are measured, it is as a result as follows：
Aptamer title Dissociation constant (nM)
ANGPTL4-1 47.6
ANGPTL4-2 86.5
ANGPTL4-3 89.6
ANGPTL4-4 70.5
ANGPTL4-5 90.9
ANGPTL4-6 100.1
ANGPTL4-7 98.4
ANGPTL4-8 97.6
ANGPTL4-9 87.6
ANGPTL4-10 76.8
ANGPTL4-11 102.3
ANGPTL4-12 77.6
ANGPTL4-13 88.7
ANGPTL4-14 478.5
ANGPTL4-15 104.7
ANGPTL4-16 456.5
ANGPTL4-17 97.4
ANGPTL4-18 356.4
As can be seen from the above table, the dissociation constant of the aptamers newly provided is all very low, especially ANGPTL4-1, has Better affinity.
Specific detection
This 18 aptamers of ANGPTL4-1~18 are taken to be combined respectively with human serum albumin, IgG and ANGPTL4 albumen Detection, found by analysis, this 18 aptamers also only with ANGPTL4 protein bindings, not with other protein bindings.Illustrate this hair Bright aptamer just has good specificity.
4 aptamer kit clinical sample of embodiment detects
-, clinical samples collect.
It collects and goes to a doctor in the specific parkinsonism of diagnosis (40 people) and participation health examination of West China Hospital Normal healthy people (50 people) the non-anti-freezing sample of peripheric venous blood, conventional centrifugal separation serum is standby after natural coagulation surveys.
The preparation of two, kits
Diagnostic kit, it is characterised in that kit is grouped as by the group of following independent packaging:(1)SEQ ID NO：1-18 Nucleic acid aptamer solution；(2) polyacrylamide gel mother liquor；(3) ammonium persulfate；(4) tetramethyl diethylamine (TEMED)；(5) 50% glycerite I；(6) 50% glycerite II；(7) 1.25M sodium chloride solutions；(8) GelRed dye solutions；(9) positive Control serum；(10) negative control sera；The 50% glycerite I is formulated with distilled water, 50% glycerite II It is formulated with combination buffer.
Three, interpretation of result
It is established by standard curve, judges the ANGPTL4 albumen concentration in corresponding blood, when positive threshold value takes 130ng/ When mL, it is disturbances in patients with Parkinson disease to have 39 evaluating samples, wherein 39 are passed through and verified, is patient, illustrates that accuracy is more increased.It says Bright accuracy is more increased.Show good diagnosis effect.
Degradability detects
Metabolic stability of the aptamer in rat plasma
Aptamer is tested in rat plasma to assess its stability, kinetic rate and degradation pathway.Use 5 ' ends- Radio-labeled (32P) aptamer and 95% blood plasma (citrate) are incubated 100 hours at 37 DEG C.At selection time point, equivalent is taken out The blood plasma for including aptamer, be rapidly frozen in liquid nitrogen, be stored in -80 DEG C.It is extracted using liquid-liquid (phenol chloroform), subsequent gel Electrophoresis (10% modacrylic acyl ammonia sequencing gel) and high-resolution autoradiograph measure and the aptamer in analysis blood plasma and Its metabolite.Fig. 4 shows the logarithm-of complete-long aptamer residue percentage of the function as incubation time in rat plasma Linear graph.Degradation characteristic in two species is all fully shown as single-phase, this is absolutely proved, aptamer tool provided by the invention There is preferable plasma stability, can be used for long-term blood plasma detection needs.
It these are only the preferred embodiment of the present invention, be not intended to restrict the invention, for those skilled in the art For member, any modification, equivalent substitution, improvement and etc. done all within the spirits and principles of the present invention should be included in this Within the protection domain of invention.
Sequence table
110 Shens the ＞ winters of ＜ are prosperous
A kind of kits and its detection method for parkinsonism detection of 120 ＞ of ＜
〈160〉20
〈210〉1
〈211〉75
〈212〉DNA
213 ＞ artificial sequences of ＜
〈400〉ANGPTL4-1
TTCGACAGTATGAAGCTCGGCCCCCAATTCAACTTAAAATACACCCTAATTCTTAGGCACTGAAGTGAC TAGACT
〈210〉2
〈211〉75
〈212〉DNA
213 ＞ artificial sequences of ＜
〈400〉ANGPTL4-2
TTCGACAGTATGAAGCTCGGTATAAAAATCCAACCCCTACACTACATCACACTCCGGCACTGAAGTGAC TAGACT
〈210〉3
〈211〉75
〈212〉DNA
213 ＞ artificial sequences of ＜
〈400〉ANGPTL4-3
TTCGACAGTATGAAGCTCGGTATCTTACCTATACTTACCTAATACTTAACATTATGGCACTGAAGTGAC TAGACT
〈210〉4
〈211〉75
〈212〉DNA
213 ＞ artificial sequences of ＜
〈400〉ANGPTL4-4
TTCGACAGTATGAAGCTCGGTTTAACACAATAATTAAATAACAACTCTATACAATGGCACTGAAGTGAC TAGACT
〈210〉5
〈211〉75
〈212〉DNA
213 ＞ artificial sequences of ＜
〈400〉ANGPTL4-5
TTCGACAGTATGAAGCTCGGCAACCTTACCTTCCAAACATCCCCCTATACCTTCCGGCACTGAAGTGAC TAGACT
〈210〉6
〈211〉75
〈212〉DNA
213 ＞ artificial sequences of ＜
〈400〉ANGPTL4-6
TTCGACAGTATGAAGCTCGGCATCTCCCCATCCCTAATAACTACTTTTACCACTTGGCACTGAAGTGAC TAGACT
〈210〉7
〈211〉75
〈212〉DNA
213 ＞ artificial sequences of ＜
〈400〉ANGPTL4-7
TTCGACAGTATGAAGCTCGGCCTCTTAAACAATCCTATATACTACCTCTACAAATGGCACTGAAGTGAC TAGACT
〈210〉8
〈211〉75
〈212〉DNA
213 ＞ artificial sequences of ＜
〈400〉ANGPTL4-8
TTCGACAGTATGAAGCTCGGCAACCAATCTTCCTTTTACTAATACATTTTACTCCGGCACTGAAGTGAC TAGACT
〈210〉9
〈211〉75
〈212〉DNA
213 ＞ artificial sequences of ＜
〈400〉ANGPTL4-9
TTCGACAGTATGAAGCTCGGCTCTCTCTATATCATTCCTCCTTTAATCAAAACCAGGCACTGAAGTGAC TAGACT
〈210〉10
〈211〉75
〈212〉DNA
213 ＞ artificial sequences of ＜
〈400〉ANGPTL4-10
TTCGACAGTATGAAGCTCGGCAATATTTATAATATACTTCTAATCTTTCCCAACCGGCACTGAAGTGAC TAGACT
〈210〉11
〈211〉75
〈212〉DNA
213 ＞ artificial sequences of ＜
〈400〉ANGPTL4-11
TTCGACAGTATGAAGCTCGGTCATAATCTTAAATCCAAAATTACCCCATCTATCCGGCACTGAAGTGAC TAGACT
〈210〉12
〈211〉75
〈212〉DNA
213 ＞ artificial sequences of ＜
〈400〉ANGPTL4-12
TTCGACAGTATGAAGCTCGGCTCTAACACTTTCCCCCCCTCATCACATCCCACACGGCACTGAAGTGAC TAGACT
〈210〉13
〈211〉75
〈212〉DNA
213 ＞ artificial sequences of ＜
〈400〉ANGPTL4-13
TTCGACAGTATGAAGCTCGGTACACTTACATTACCACAATCTCATTTTATACCATGGCACTGAAGTGAC TAGACT
〈210〉14
〈211〉75
〈212〉DNA
213 ＞ artificial sequences of ＜
〈400〉ANGPTL4-14
TTCGACAGTATGAAGCTCGGCTATTCCCCCTCCCTTCCTCTACTAATCAAACTCTGGCACTGAAGTGAC TAGACT
〈210〉15
〈211〉75
〈212〉DNA
213 ＞ artificial sequences of ＜
〈400〉ANGPTL4-15
TTCGACAGTATGAAGCTCGGTAAACAATCATTATTTTTCTATAATTCTATTATATGGCACTGAAGTGAC TAGACT
〈210〉16
〈211〉75
〈212〉DNA
213 ＞ artificial sequences of ＜
〈400〉ANGPTL4-16
TTCGACAGTATGAAGCTCGGCACAATACATTAACACTCCACTAACTCAAATCTACGGCACTGAAGTGAC TAGACT
〈210〉17
〈211〉75
〈212〉DNA
213 ＞ artificial sequences of ＜
〈400〉ANGPTL4-17
TTCGACAGTATGAAGCTCGGTATCTTCTATCATTTAACCTTTCCTCCCAACACACGGCACTGAAGTGAC TAGACT
〈210〉18
〈211〉75
〈212〉DNA
213 ＞ artificial sequences of ＜
〈400〉ANGPTL4-18
TTCGACAGTATGAAGCTCGGTTCTTCCTCCATACCTATACCTCTATCTCACTTTCGGCACTGAAGTGAC TAGACT
〈210〉19
〈211〉138
〈212〉PRT
213 ＞ artificial sequences of ＜
〈400〉ABB
MetGlyTrpSerTrpIlePheLeuPheLeuSerGluThrAlaGlyValLeuSerGluValGlnLeuGlnGlnSerGl yProGluLeuMetLysProGlyAlaSerValLysMetSerCysArgThrSerGlyTyrThrThrPheThrAspTyrS erIleHisTrpValLysGlnSerHisGlyLysArgLeuGluTrpIleGlyTyrIleAsnProTyrAsnGlyAspThr TyrCysAspGlnAsnPheLysGlyLysAlaThrLeuThrPheAsnLysAlaSerSerThrAlaTyrMetGluIlePr oArgLeuThrSerAspAspSerAlaValTyrTyrCysThrArgTrpLysThrIleGlnAlaProPheAlaTyrTrpG lyGlnGlyThrLeuValThrValSerAla
〈210〉20
〈211〉129
〈212〉PRT
213 ＞ artificial sequences of ＜
〈400〉ABB
MetAspMetArgAlaProAlaGlnPheLeuGlyIleLeuLeuLeuTrpPheProGlyAlaArgCysGluIleGlnMe tThrGlnSerProSerSerMetSerAlaSerLeuGlyAspArgIleThrIleThrCysGlnAlaThrGlnAspIleV alLysAsnLeuAsnTrpTyrGlnGlnLysProGlyLysProProSerPheLeuIleHisTyrAlaThrGluLeuAla GluGlyValProSerArgPheSerGlySerGlySerGlySerAspTyrSerLeuThrIleSerAsnLeuGluSerGl uAspPheAlaAspTyrTyrCysLeuGlnSerTyrAspPheProTyrThrPheGlyGlyGlyThrLysLeuGluIleA sn

Claims (1)

1. application of the aptamer in the kit for preparing detection parkinsonism, the sequence of the aptamer is respectively such as SEQ ID NO：Shown in 1-18.
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CN102641503A (en) * 2004-07-20 2012-08-22 健泰科生物技术公司 Inhibitors of angiopoietin-like 4 protein, combinations, and their use

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