CN106755563A - Application of the biomarker in Parkinson is diagnosed - Google Patents
Application of the biomarker in Parkinson is diagnosed Download PDFInfo
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Abstract
The invention discloses application of the biomarker in Parkinson is diagnosed, specifically the biomarker is UROD, present invention firstly discovers that UROD expresses in disturbances in patients with Parkinson disease downward, pointing out the expression of detection UROD can turn into one of index of Parkinson's early diagnosis.The invention provides a kind of product for realizing Parkinson's early diagnosis, find to intervene so as to early by early, and then improve existence and the quality of life of patient.
Description
Technical field
The invention belongs to biological technical field, it is related to a kind of application of biomarker in Parkinson is diagnosed, specifically
The biomarker is UROD.
Background technology
((Parkinson's disease, PD) is also called shaking plasy to Parkinson's, is a kind of maincenter of progressive development
Nervous system degeneration disease, the quality of life of people receives serious influence, and the sick major pathologic features show as black substance
Lewy body (Lewy in the degeneration of dopaminergic neurons missing and remaining neuron at the positions such as compact part, locus coeruleus, nuclei of median raphe
Body, LB) formation.Domestic and international epidemiological study has shown:The incidence of disease is 1~2%, more than 85 years old in over-65s crowd
4~5% are reached, PD subject populations are more than 1,700,000 people within more than 55 years old.With the natural aging of social population, the trouble of PD
Patient's number is continuously increased every year, and the number of patients of China is more than 2,000,000.Current PD has turned into and has been only second to alzheimer ' in the world
The second Common neurologic DD of disease of writing from memory.
The cognition dysfunction of Parkinson's is divided into Parkinson's mild cognition impairment (PD-MCI) and Parkinson
Sick dementia (PD-D) two class.The cognitive decrease that PD-D patient occurs is significantly affected and disturbs daily life function, and PD-
MCI patient only shows mild cognition impairment, although it is possible that slight difficult when performing complicated functional task, and
It is not enough to interfere significantly with functional independence.PD-MCI is often thought of as the performance of Parkinson's early stage decrease of cognitive function, is day
The laggard transition state for transforming into PD-D.Parkinsonian's decrease of cognitive function onset is hidden, and the course of disease is in progressive progression.
The cause of disease and pathogenesis of Parkinson's are unclear, are presently considered to be in the background of age ageing, it is hereditary because
Element and the coefficient result of environmental factor.Many research prompting PD patient black substance DA serotonergic neuron denaturation may be with mitochondria work(
A series of machines such as energy obstacle, oxidative stress, excitatory toxicity, neurotrophic factor shortage, immunoregulatory abnormality, natural death of cerebral cells
System, link are relevant, and epidemiological investigation shows, the Parkinsonian that there are about 10~15% has family history.By to losing
Study on mechanism of the biography factor in PD morbidities, has at least cloned 13 PD Disease-causing genes at present, shows the hair of Parkinson's
Disease is closely related with inherent cause, and inherent cause contains polygenes, polyfactorial interaction and influence.Thus from molecule
Level research Parkinson's develop, and prevention for it, control and treat significant.
The content of the invention
It is an object of the invention to provide a kind of application of biomarker in Parkinson diagnoses.
The invention provides the purposes of biomarker UROD, the product for preparing diagnosis Parkinson.
Further, the product is by determining the level of biomarker in sample and the reference water of corresponding biomarker
It is flat to determine compared to lowering.
Further, the biomarker includes the polynucleotides or its piece of the nucleotide sequence as shown in SEQ ID NO.1
Section, homologue, variant or derivative.
The invention provides a kind of product of UROD expressions in vitro detection sample, the product includes:By surveying
The method of sequence technology, nucleic acid hybridization technique, nucleic acid amplification technologies or immunoassays detects the expression of UROD genes.
Further, the nucleic acid amplification technologies are selected from PCR, reverse transcriptase polymerase chain reaction, transcription Jie
Amplification, ligase chain reaction, strand displacement amplification and the amplification based on nucleotide sequence led.
Further, the product includes preparation, chip or kit.
Further, the product includes the reagent of specific recognition UROD genes or the albumen of its coding.
Further, the reagent of the albumen of the specific recognition UROD genes or its coding is selected from:
The primer of specific amplification UROD genes;
The probe of specific recognition UROD genes;Or
The antibody or part of the albumen of specific binding UROD codings.
The invention provides a kind of kit for detecting Parkinson, the kit includes reagent recited above;And
Operation instructions or label.
The invention provides application of the said goods in the early diagnosis instrument for preparing Parkinson.
The advantages of the present invention:
Present invention firstly discovers that UROD genes are to the generation development of Parkinson related, for the pathogenesis of Parkinson
Research has important scientific research value and clinical meaning.
The invention provides diagnostic products, by detecting the expression of UROD in blood, so that disturbances in patients with Parkinson disease
Treatment is realized in early stage, the quality of life of patient is improved.
Brief description of the drawings
Fig. 1 is the expression figure in disturbances in patients with Parkinson disease using QPCR detection UROD genes
Specific embodiment
Present invention firstly discovers that UROD is to the generation development of Parkinson related, and the UROD in blood is demonstrated in handkerchief gold
Low expression in gloomy diagnosis.UROD can be used as the independentpredictor of Parkinson, it is also possible to and other biomarker joints should
With.
Biomarker
Term " biomarker " is its expression in tissue or cell and normal or healthy cell or tissue
Expression compares any gene or albumen for changing.
It would be recognized by those skilled in the art that practicality of the invention is not limited to marker gene of the invention
The gene expression of any specific variants is quantified.Used as nonrestrictive example, marker gene can have SEQ ID NO.1
Or the coded sequence or amino acid sequence that SEQ ID NO.2 are specified.In some embodiments, it has with listed sequence extremely
Few 85% same or analogous cDNA sequence or amino acid sequence, all sequences listed as described above at least 90%, 91%, 92%,
93%th, 94%, 95%, 96%, 97%, 98% or at least 99% same or analogous cDNA sequence or amino acid sequence.
So-called " fragment " refer to polynucleotides a part or amino acid sequence and thus coding albumen a part.For
The polynucleotides of the fragment of biomarker nucleotide sequence generally comprise at least 10,15,20,50,75,100,150,200,
250th, 300,350,400,450,500,550,600,650,700,800,900,1,000,1,100,1,200,1,300 or 1,
400 continuous nucleotides, or at most it is present in the nucleotides in total length biomarker polynucleotides disclosed herein
Number.The fragment of biomarker polynucleotides will generally encode at least 15,25,30,50,100,150,200 or 250 Continuance ammines
Base is sour, or is present in the sum of the amino acid in total length biomarker protein of the invention.
" variant " is intended to indicate that essentially similar sequence.In general, the variant of particular organisms mark of the invention
Will have by alignment programs measure with the biomarker at least about 40%, 45%, 50%, 55%, 60%,
65%th, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or
Sequence identity higher.
" polynucleotides " in the present invention include the polymer of RNA, cDNA, genomic DNA, synthesized form and mixing, have
Adopted chain and antisense strand, and can be chemistry or biochemical modification, or non-natural or derivative nucleosides soda acid can be contained
Base, this is easily for those skilled in the art are appreciated.It is this modification include for example mark, methylate, with analog replace one or
Modification is bonded (such as methyl phosphorodithioate, phosphotriester, phosphoric acid as uncharged between multiple natural nucleotides, nucleotides for occurring
Acid amides, carbamate etc.), electrically charged be bonded (such as thiophosphate, phosphorodithioate etc.), overhang
(pendent moieties) (such as polypeptide), insert (such as acridine, psoralen etc.), chelating agent, alkylating agent, and modification
Be bonded (such as α anomeric nucleic acids etc.).Also synthetic molecules are included, it has simulation polynucleotides by hydrogen bond and other changes
Learn to interact and combine the ability of specified sequence.This molecule is known in the art, including the peptide for example wherein in molecular backbone
Key replaces phosphate bond.
Gene expression detection technology
The present invention can determine gene expression using any method known in the art.Those skilled in the art should manage
Solution, the means for determining gene expression are not importances of the invention.Detected in (i.e. albumen) level transcribing or can be translated
The expression of biomarker.
In some embodiments, the expression of biomarker is detected on transcriptional level.Using nucleic acid hybridization skill
Various methods that art carries out specific DNA and RNA measurements are well known by persons skilled in the art.Certain methods are related to electrophoretic separation
(for example, Southern traces and Northern traces for detecting RNA for detecting DNA), but can also be unfavorable
With the measurement (for example, by Dot blot) that DNA and RNA is carried out in the case of electrophoretic separation.Genomic DNA is (for example, come from
People) Southern traces can be used to screen RFLP (RFLP), to detect influence polypeptide of the present invention
The presence of inherited disorder.Can detect the RNA of form of ownership, including but not limited to mRNA (mRNA), microRNA (miRNA),
RRNA (rRNA) and transfer RNA (tRNA).
The selection of nucleic acid hybridization formats is not crucial.Multiple nucleic acids hybrid versions include but is not limited to sandwich assay and competing
Strive or substitute measure.
The detection of hybridization complex can need to produce Signaling complex with target and the double helix of probe polynucleotide or nucleic acid
Combination.Generally, this combination is interacted and occurred by part and anti-ligand, and the probe of such as ligand coupling and coupling have
Interaction between the anti-ligand of signal.By the way that exposed to ultrasonic energy, the combination of signal generation compound is also easy to be added
Speed.
Mark can also allow indirect detection hybridization complex.If for example, mark is haptens or antigen, can pass through
Sample is detected using antibody.
Preparation, chip or kit
Term " chip " is also referred to as " array ", refers to the solid support of the nucleic acid comprising connection or peptide probes.Array is usual
Comprising the various different nucleic acid or peptide probes that substrate surface is connected to according to different known locations.These arrays, also referred to as
" microarray ", can generally produce these arrays using mechanical synthesis methods or light guiding synthetic method, and the light guiding is closed
The combination of photolithography method and solid phase synthesis process is incorporated into method.Array can include flat surface, or can be pearl
Nucleic acid or peptide in son, gel, polymer surfaces, the fiber of such as optical fiber, glass or any other suitable substrate.Can be with
Certain mode carrys out array of packages, so as to allow to carry out the diagnosis of global function device or the manipulation of other manner.
" microarray " is that hybridised arrays original paper is ordered in matrix, and the hybridised arrays original paper such as polynucleotide is visited
Pin (such as oligonucleotides) or bonding agent (such as antibody).The matrix can be solid matrix, for example, glass or silica
Slide, pearl, fibre optics binding agent or semi-solid matrix, such as nitrocellulose filter.Nucleotide sequence can be DNA, RNA or
Any arrangement therein.
Term " probe " refers to the molecule that can be combined with the particular sequence of another molecule or subsequence or other parts.Unless another
Point out, term " probe " is often referred to be matched and another polynucleotides (often referred to as " target polynucleotide ") by complementary base
With reference to polynucleotide probes.Lack sufficient sequence complementarity according to the preciseness of hybridization conditions, probe energy and with the probe
Target polynucleotide is combined.Probe can make direct or indirect mark, and its scope includes primer.Crossing system, including, but do not limit
In:Solution, solid phase, mixed phase or in situ hybridization determination method.
Probe is generally directly marked, such as with isotope, chromophore, illuminophore, chromogen, or by indirect labelling, example
Such as using biotin, Streptavidin compound can then be combined with biotin.Therefore, the present invention is used in determining can detect
Mark can be primary marker (wherein the mark comprising can direct detection element or can produce directly detectable element
Element) or two grades of marks (wherein detectable mark is combined with primary marker, for example, conventional in immune labeled).Generally, mark
The signal nucleic acid of note is used to detect hybridization.Can by be usually used in detect hybrid polynucleotide exist several methods in any
Plant mark complementary nucleic acid or signal nucleic acid.The most frequently used detection method is using use3H、125I、35S、14C or32The spy of P marks
The autoradiography of pin etc..Other marks include, for example, can the part of binding marker antibody, fluorogen, chemiluminescence agent,
Enzyme and can be used as the antibody of the specific binding pair members of tagged ligand.
The polynucleotides used as probe be preferably sized to 18 or more nucleotides, more preferably 20 or
More nucleotides, and coding region total length or less.When being used as primer, the polynucleotides are preferably sized to 18
Or more nucleotides, and 50 or more Oligonucleotide.
Above-mentioned probe has the base sequence with the specific base sequence complementary of target gene.Here, so-called " complementation ",
As long as hybridization, can not be complete complementary.These polynucleotides are commonly angled relative to the specific base sequence to be had
More than 80%, preferably more than 90%, more preferably more than 95%, particularly preferred 100% homology.These probes can be DNA,
Can also be RNA, furthermore it is possible to be artificial by PNA, LNA, ENA, GNA, TNA etc. in one part or whole nucleotides
The polynucleotides that replacement nucleic acid is obtained.
In the present invention, genetic chip can be used to detect including the multiple genes including UROD genes (for example, and Parkinson
Related multiple genes) expression.Protein-chip can be used to detect including the multiple protein (examples including UROD albumen
Multiple protein such as related to Parkinson) expression.
In the present invention, term " antibody " covering monoclonal antibody, polyclonal antibody, multi-specificity antibody (such as double spies
Heterogenetic antibody) and antibody fragment, combinatorial antibody etc., as long as they show desired BA." monoclonal resists
Body " refers to the antibody obtained from the antibody of a group substantially homogeneity, i.e. each antibody of composition colony is identical and/or combines identical table
Position, except production monoclonal antibody during it is issuable may become external, such variant is general with indivisible presence.This
Class monoclonal antibody is typically include the antibody comprising the polypeptide sequence for combining target, and wherein target Binding peptide sequence is to pass through
Including selecting what the process including single target Binding peptide sequence was obtained in many polypeptide sequences of comforming.
Monoclonal antibody also includes " chimeric " antibody, wherein a part for heavy chain and/or light chain and derived from particular species
Or the corresponding sequence that belongs in the antibody of specific antibodies classification or subclass is identical or homologous, and the remainder of chain with derived from another
One species or the corresponding sequence belonged in the antibody of another antibody isotype or subclass are identical or homologous, and this antibody-like piece
Section, as long as they show desired BA.
" antibody or antibody fragment " refers to no matter derive from any species for naturally occurring antibody, or by recombinant DNA skill
Art is created;No matter from the antibody of serum, B cell, hybridoma, transfectoma, yeast or bacteria distribution (such as IgG, IgM, IgA,
IgD or IgE) or fragment (such as Fab, F (ab ')2, Fv, disulphide connection Fv, scFv, closure conformation multispecific resist
Body, scFv, the double antibody of disulphide connection).
In the context of the present invention, " diagnosis Parkinson " both include judging subject whether suffered from Parkinson, or
Including judging subject with the presence or absence of the risk with Parkinson.
Term " sample " includes but is not limited to blood, saliva, tissue, the cell of cracking, biopsy of brain thing, the cell of culture
(for example, cell of primary culture, explant and conversion), excrement, urine etc..In specific embodiment of the invention, institute
Sample is stated for blood.
The present invention is further detailed explanation with reference to the accompanying drawings and examples.Following examples are merely to illustrate this
Invention rather than limitation the scope of the present invention.The experimental technique of unreceipted actual conditions in embodiment, generally according to conventional strip
Part, such as Sambrook et al., molecular cloning:Laboratory manual (New York:ColdSpring HarborLaboratory
Press, 1989) described in condition, or according to the condition proposed by manufacturer.
Embodiment 1 screens the biomarker related to Parkinson
1st, sample collection
Each to collect 10 normal human bloods and disturbances in patients with Parkinson disease blood sample, the acquirement of above-mentioned all samples passes through ethics
The agreement of the committee.
Normal group exclusion standard:(1) Parkinson's and Parkinson's family history;(2) Alzheimer disease.Epilepsy,
Other the nervous system disease histories such as cranial vascular disease;(3) there is the past psychiatric history.Such as schizophrenia, severe depression person, spirit
Developmental lag and physical disease caused by mental disorder person;(4) illiteracy, colour blindness, aphasis or other reasons cannot the persons of communication.
Parkinson's group exclusion standard:(1) illiteracy, colour blindness, aphasis or other reasons cannot the persons of communication;(2) exist
Other reasons such as drug poisoning, endocrine metabolism disease, severe depression cause cognition dysfunction or dement;(3) it is secondary
Property parkinson's syndrome, cranial vascular disease, encephalitis, craniocerebral trauma or operator;(4) malignant tumour and other serious disease persons.
2nd, the preparation and quality analysis of RNA sample
The preparation of 2.1 RNA samples
RNA is extracted using RNAliquid hypervelocity whole blood (liquid sample) total RNA extraction reagent boxes, kit is purchased from Beijing
Hui Tian east Science and Technology Ltd., comprises the following steps that:
1) 0.75mL lysate RLS are added per 0.25mL liquid samples, blows and beats fluid sample several times to help with sample loading gun
Cell in lysate sample.Every 5~10 × 106Individual cell at least adds 0.75ml lysates RLS.Lysate RLS and fluid sample
Final volume than always 3:1.
2) 0.75mL lysate RLS are added in EP pipes, 0.25mL blood samples are added, 30s is acutely shaken, are mixed,
10min is incubated under the conditions of 15-30 DEG C so that ribosome is decomposed completely.
3) add 0.2mL chloroforms per 0.75mL lysates RLS, acutely shake 15s and place 5min at room temperature.
4) in 4 DEG C of 12000rpm centrifugation 10min, sample can be divided into three layers:Lower floor's organic phase, intermediate layer and upper strata are colourless
Water phase, RNA is present in upper strata aqueous phase.The capacity of aqueous layer is about the 70% of added RLS volumes, water is mutually transferred to new pipe
In, carry out next step operation.
5) 1 times of ethanol of volume 70% is added, is overturned and is mixed.The solution and possible precipitation for obtaining are transferred in adsorption column RA together
(adsorption column is enclosed within collecting pipe).
6) 12000rpm centrifugations 45s, discards waste liquid, and adsorption column is recovered into collecting pipe again.
7) add 0.5mL protein liquid removals RE, 12000rpm centrifugation 45s, discard waste liquid.
8) 0.5mL rinsing liquids RW, 12000rpm centrifugation 45s is added, waste liquid is discarded.
9) repeat step 8).
10) adsorption column RA is put back in the collecting pipe of hole, 13000rpm centrifugation 2min remove rinsing liquid as far as possible, in order to avoid rinsing
Residual ethanol suppresses downstream reaction in liquid.
11) adsorption column RA is taken out, is put into a RNase free centrifuge tube, according to expected RNA yield in adsorbed film
Middle part adds the pre-heated RNase free wate of 30 μ L, and room temperature is placed 2min, 12000rpm centrifugation 1min, covers Sheng
There is the centrifuge tube of RNA, be stored in -80 DEG C.
The quality analysis (NanoDrop1000 spectrophotometers) of 2.2 RNA samples
NanoDrop1000 spectrophotometers detect RNA sample, the sample requirement of RNA-seq sequencings:OD260/OD280 is
1.8-2.2。
The quality analysis (Bioanalyzer of Agilent Technologies 2100) of 2.3 RNA samples
The RNA of said extracted is entered into row agarose gel electrophoresis, Agilent Technologies 2100
Bioanalyzer detects RNA sample quality, and observation 28S rRNA and 18S rRNA master tapes substantially, without degraded, RNA integralities refer to
The qualified, concentration of number reaches the requirement that RNA-seq sequencing cDNA libraries build that meets of requirement, can be used for library construction and survey
Sequence.
3rd, high flux transcript profile sequencing
3.1 RNA-seq reads are positioned
First by low-quality read removal obtain clean read, then using TopHat v1.3.1 will clean fragment and
UCSC H.sapiens reference genes group (hg19) is matched, the index of the advance structure of H.sapiens UCSC hg19 editions
Downloaded from TopHat homepages, and as reference gene group, when being matched with genome using TopHat, it is allowed to each read (acquiescence
Sites, most 2 mispairing are matched to 20) there is multiple.TopHat sets up possible according to exon region and GT-AG shear signals
Shearing site storehouse, navigates on genome the read for not navigating to genome according to these shearing site storehouses.We use
The system default parameter of TopHat methods.
3.2 transcript abundances are assessed
By Cufflinks v1.0.3 treatment, Cufflinks v1.0.3 are by RNA-seq pieces for the read file for matching
Hop count mesh is standardized the relative abundance for calculating transcript.FPKM values refer to it is every 1,000,000 sequencing fragment in match it is specific
The segment number of exon region gene 1kb long.The confidential interval of FPKM estimates is calculated by Bayesian inference method.
The GTF comment files of the reference that Cufflinks is used download (Homo_ from Ensembl databases
sapiens.GRCh37.63.gtf)。
The detection of 3.3 difference expression genes
Cuffdiff is transferred to by the Ensembl GTF files of download and by the original document that TopHat is matched,
Cuffdiff re-evaluates the gene expression abundance of the transcript listed in GTF files using original matching files, detects difference table
Reach.The only q values < 0.01 in Cuffidff outputs, test display is considered as successfully more just differential expression.
4th, result
RNA-seq results show that expression quantity of the UROD genes in disturbances in patients with Parkinson disease blood is substantially less than the table of normal person
Up to amount.
The differential expression of the QPCR sequence verification UROD genes of embodiment 2
1st, the testing result selection UROD genes according to high-flux sequence carry out large sample QPCR checkings.According to embodiment 1
In sample collection mode selection disturbances in patients with Parkinson disease blood and each 80 of normal human blood.
2nd, RNA extraction steps are with embodiment 1.
3rd, reverse transcription:Operated using the reverse transcription reagent box of TIANGEN companies, kit article No. is KR106.Specifically
Step is as follows:
Removal genomic DNA reaction, adds 5 × DNA Buffer 2.0,1 μ g of μ l, RNA in test tube, and RNase is removed in addition
ddH2O makes cumulative volume to 10 μ l, 42 DEG C of heating 3min in water-bath, then by 10 × Fast RT Buffer 2.0 μ L, RT
The μ L of 1.0 μ L, FQ-RT Primer Mix of Enzyme Mix 2.0, remove RNase ddH2The μ L of O 5.0, add above-mentioned test tube after mixing
In be mixed together, 42 DEG C of heating 15min, 95 DEG C of heating 3min in water-bath, when the cDNA of synthesis needs long-term preservation, please in-
20 DEG C or lower temperature preservation.
4th, QPCR amplifications
(1) design of primers
Coded sequence according to UROD genes and GAPDH genes in Genebank designs QPCR amplimers, by Bo Maide
Company synthesizes.Specific primer sequence is as follows:
UROD genes:
Forward primer is 5 '-TCTCCGACATCCTTGTTGTACC-3 ' (SEQ ID NO.3);
Reverse primer is 5 '-CCGTAGGCGTTCTAGGTCCT-3 ' (SEQ ID NO.4).
GAPDH genes:
Forward primer is 5 '-GGAGCGAGATCCCTCCAAAAT-3 ' (SEQ ID NO.5);
Reverse primer is 5 '-GGCTGTTGTCATACTTCTCATGG-3 ' (SEQ ID NO.6).
(2) PCR reaction systems are prepared according to table 1:
Wherein, SYBR Green PCRs system is purchased from TIANGEN companies (article No. FP205).
The PCR reaction systems of table 1
Reagent | Volume |
Forward primer | 0.6μl |
Reverse primer | 0.6μl |
2×SuperReal PreMix Plus | 10μl |
DNA profiling | 2μl |
50×Reference Dye△ | 2μl |
Distilled water | Complement to 20 μ l |
(3) PCR reaction conditions:95 ° of 15min, (95 DEG C of 10s, 55 DEG C of 30s, 72 DEG C of 32s) × 40 circulations.With SYBR
Green as fluorescent marker, in the enterprising performing PCR reaction of the type quantitative real time PCR Instruments of ABI 7300, analyzed by melt curve analysis and
Electrophoresis determines purpose band, and Δ Δ CT methods carry out relative quantification.
5th, statistical method
Experiment is tested using 3 repetitions, and result data is represented in the way of mean+SD, is used
SPSS13.0 statistical softwares carry out statistical analysis, and difference between the two is checked using t, it is believed that work as P<There is system when 0.05
Meter learns meaning.
6th, result
Result as shown in figure 1, compared with normal human blood, lower by expression of the UROD genes in disturbances in patients with Parkinson disease blood,
Difference has statistical significance (P<0.05) it is, consistent with RNA-sep results.
The explanation of above-described embodiment is only intended to understand the method for the present invention and its core concept.It should be pointed out that for this
For the those of ordinary skill in field, under the premise without departing from the principles of the invention, some improvement can also be carried out to the present invention
And modification, these are improved and modification will be also fallen into the protection domain of the claims in the present invention.
SEQUENCE LISTING
<110>General Hospital of Tianjin Medical Univ.
<120>Application of the biomarker in Parkinson is diagnosed
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 1104
<212> DNA
<213>People source
<400> 1
atggaagcga atgggttggg acctcagggt tttccggagc tgaagaatga cacattcctg 60
cgagcagcct ggggagagga aacagactac actcccgttt ggtgcatgcg ccaggcaggc 120
cgttacttac cagagtttag ggaaacccgg gctgcccagg actttttcag cacgtgtcgc 180
tctcctgagg cctgctgtga actgactctg cagccactgc gtcgcttccc tctggatgct 240
gccatcattt tctccgacat ccttgttgta ccccaggcac tgggcatgga ggtgaccatg 300
gtacctggca aaggacccag cttcccagag ccattaagag aagagcagga cctagaacgc 360
ctacgggatc cagaagtggt agcctctgag ctaggctatg tgttccaagc catcaccctt 420
acccgacaac gactggctgg acgtgtgccg ctgattggct ttgctggtgc cccatggacc 480
ctgatgacat acatggttga gggtggtggc tcaagcacca tggctcaggc caagcgctgg 540
ctctatcaga gacctcaggc tagtcaccag ctgcttcgca tcctcactga tgctctggtc 600
ccatatctgg taggacaagt ggtggctggt gcccaggcat tgcagctgtt tgagtcccat 660
gcagggcatc ttggcccaca gctcttcaac aagtttgcac tgccttacat ccgtgatgtg 720
gccaagcaag tgaaggccag gttgcgggag gcaggcctgg caccagtgcc catgatcatc 780
tttgctaagg atgggcattt tgccctggag gagctggccc aagctggcta tgaggtggtt 840
gggcttgact ggacagtggc cccaaagaaa gcccgggagt gtgtggggaa gacggtgaca 900
ttgcagggca acctggaccc ctgtgccttg tatgcatctg aggaggagat cgggcagttg 960
gtgaagcaga tgctggatga ctttggacca catcgctaca ttgccaacct gggccatggg 1020
ctttatcctg acatggaccc agaacatgtg ggcgcctttg tggatgctgt gcataaacac 1080
tcacgtctgc ttcgacagaa ctga 1104
<210> 2
<211> 367
<212> PRT
<213>People source
<400> 2
Met Glu Ala Asn Gly Leu Gly Pro Gln Gly Phe Pro Glu Leu Lys Asn
1 5 10 15
Asp Thr Phe Leu Arg Ala Ala Trp Gly Glu Glu Thr Asp Tyr Thr Pro
20 25 30
Val Trp Cys Met Arg Gln Ala Gly Arg Tyr Leu Pro Glu Phe Arg Glu
35 40 45
Thr Arg Ala Ala Gln Asp Phe Phe Ser Thr Cys Arg Ser Pro Glu Ala
50 55 60
Cys Cys Glu Leu Thr Leu Gln Pro Leu Arg Arg Phe Pro Leu Asp Ala
65 70 75 80
Ala Ile Ile Phe Ser Asp Ile Leu Val Val Pro Gln Ala Leu Gly Met
85 90 95
Glu Val Thr Met Val Pro Gly Lys Gly Pro Ser Phe Pro Glu Pro Leu
100 105 110
Arg Glu Glu Gln Asp Leu Glu Arg Leu Arg Asp Pro Glu Val Val Ala
115 120 125
Ser Glu Leu Gly Tyr Val Phe Gln Ala Ile Thr Leu Thr Arg Gln Arg
130 135 140
Leu Ala Gly Arg Val Pro Leu Ile Gly Phe Ala Gly Ala Pro Trp Thr
145 150 155 160
Leu Met Thr Tyr Met Val Glu Gly Gly Gly Ser Ser Thr Met Ala Gln
165 170 175
Ala Lys Arg Trp Leu Tyr Gln Arg Pro Gln Ala Ser His Gln Leu Leu
180 185 190
Arg Ile Leu Thr Asp Ala Leu Val Pro Tyr Leu Val Gly Gln Val Val
195 200 205
Ala Gly Ala Gln Ala Leu Gln Leu Phe Glu Ser His Ala Gly His Leu
210 215 220
Gly Pro Gln Leu Phe Asn Lys Phe Ala Leu Pro Tyr Ile Arg Asp Val
225 230 235 240
Ala Lys Gln Val Lys Ala Arg Leu Arg Glu Ala Gly Leu Ala Pro Val
245 250 255
Pro Met Ile Ile Phe Ala Lys Asp Gly His Phe Ala Leu Glu Glu Leu
260 265 270
Ala Gln Ala Gly Tyr Glu Val Val Gly Leu Asp Trp Thr Val Ala Pro
275 280 285
Lys Lys Ala Arg Glu Cys Val Gly Lys Thr Val Thr Leu Gln Gly Asn
290 295 300
Leu Asp Pro Cys Ala Leu Tyr Ala Ser Glu Glu Glu Ile Gly Gln Leu
305 310 315 320
Val Lys Gln Met Leu Asp Asp Phe Gly Pro His Arg Tyr Ile Ala Asn
325 330 335
Leu Gly His Gly Leu Tyr Pro Asp Met Asp Pro Glu His Val Gly Ala
340 345 350
Phe Val Asp Ala Val His Lys His Ser Arg Leu Leu Arg Gln Asn
355 360 365
<210> 3
<211> 22
<212> DNA
<213>Artificial sequence
<400> 3
tctccgacat ccttgttgta cc 22
<210> 4
<211> 20
<212> DNA
<213>Artificial sequence
<400> 4
ccgtaggcgt tctaggtcct 20
<210> 5
<211> 21
<212> DNA
<213>Artificial sequence
<400> 5
ggagcgagat ccctccaaaa t 21
<210> 6
<211> 23
<212> DNA
<213>Artificial sequence
<400> 6
ggctgttgtc atacttctca tgg 23
Claims (10)
1. the purposes of biomarker UROD, it is characterised in that the product for preparing diagnosis Parkinson.
2. purposes according to claim 1, it is characterised in that the product is by determining the water of biomarker in sample
Put down and lower and determine compared with the reference levels of corresponding biomarker.
3. purposes according to claim 2, it is characterised in that the biomarker is included as shown in SEQ ID NO.1
The polynucleotides of nucleotide sequence or its fragment, homologue, variant or derivative.
4. in a kind of vitro detection sample UROD expressions product, it is characterised in that the product includes:By the way that skill is sequenced
The method of art, nucleic acid hybridization technique, nucleic acid amplification technologies or immunoassays detects the expression of UROD genes.
5. product according to claim 4, it is characterised in that the nucleic acid amplification technologies be selected from PCR,
Reverse transcriptase polymerase chain reaction, the amplification of transcriptive intermediate, ligase chain reaction, strand displacement amplification and based on nucleotide sequence
Amplification.
6. product according to claim 4, it is characterised in that the product includes preparation, chip or kit.
7. product according to claim 6, it is characterised in that the product includes specific recognition UROD genes or its volume
The reagent of the albumen of code.
8. product according to claim 7, it is characterised in that albumen of the specific recognition UROD genes or its coding
Reagent be selected from:
The primer of specific amplification UROD genes;
The probe of specific recognition UROD genes;Or
The antibody or part of the albumen of specific binding UROD codings.
9. it is a kind of detect Parkinson kit, the kit include claim 7 or 8 described in reagent;And use is said
Bright book or label.
10. application of the product described in any one of claim 4-8 in the early diagnosis instrument for preparing Parkinson.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201710216455.0A CN106755563A (en) | 2017-04-05 | 2017-04-05 | Application of the biomarker in Parkinson is diagnosed |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201710216455.0A CN106755563A (en) | 2017-04-05 | 2017-04-05 | Application of the biomarker in Parkinson is diagnosed |
Publications (1)
Publication Number | Publication Date |
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CN106755563A true CN106755563A (en) | 2017-05-31 |
Family
ID=58965731
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201710216455.0A Pending CN106755563A (en) | 2017-04-05 | 2017-04-05 | Application of the biomarker in Parkinson is diagnosed |
Country Status (1)
Country | Link |
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CN (1) | CN106755563A (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111187827A (en) * | 2020-01-20 | 2020-05-22 | 厦门大学 | Parkinson disease biomarker and application thereof |
CN113166215A (en) * | 2018-11-29 | 2021-07-23 | 锐思沃株式会社 | Biomarker for diagnosing mental disease onset risk state |
CN113621614A (en) * | 2021-09-09 | 2021-11-09 | 天津医科大学总医院 | Long-chain non-coding RNA and application thereof as MDS molecular marker |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101796191A (en) * | 2007-09-04 | 2010-08-04 | 库瑞瓦格有限责任公司 | complexes of rna and cationic peptides for transfection and for immunostimulation |
CN102858985A (en) * | 2009-07-24 | 2013-01-02 | 西格马-奥尔德里奇有限责任公司 | Method for genome editing |
CN103492590A (en) * | 2011-02-22 | 2014-01-01 | 卡里斯生命科学卢森堡控股有限责任公司 | Circulating biomarkers |
CN105063194A (en) * | 2015-07-31 | 2015-11-18 | 北京泱深生物信息技术有限公司 | Parkinson diagnostic marker and application thereof |
-
2017
- 2017-04-05 CN CN201710216455.0A patent/CN106755563A/en active Pending
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101796191A (en) * | 2007-09-04 | 2010-08-04 | 库瑞瓦格有限责任公司 | complexes of rna and cationic peptides for transfection and for immunostimulation |
CN102858985A (en) * | 2009-07-24 | 2013-01-02 | 西格马-奥尔德里奇有限责任公司 | Method for genome editing |
CN103492590A (en) * | 2011-02-22 | 2014-01-01 | 卡里斯生命科学卢森堡控股有限责任公司 | Circulating biomarkers |
CN105063194A (en) * | 2015-07-31 | 2015-11-18 | 北京泱深生物信息技术有限公司 | Parkinson diagnostic marker and application thereof |
Non-Patent Citations (3)
Title |
---|
DA ROCHA FILHO等人: "Expression of Genes Involved in Porphyrin Biosynthesis Pathway in the Human Renal Cell Carcinoma", 《JOURNAL OF FLUORESCENCE》 * |
NIKOLAY PUSTOGAROV等人: "Hiding in the Shadows: CPOX Expression and 5-ALA Induced Fluorescence in Human Glioma Cells", 《MOLECULAR NEUROBIOLOGY》 * |
QIANG LIU等人: "Selection of PD Genes from Multiplex Three-Dimensional Brain Gene Expression Mapping", 《2007 1ST INTERNATIONAL CONFERENCE ON BIOINFORMATICS AND BIOMEDICAL ENGINEERING》 * |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113166215A (en) * | 2018-11-29 | 2021-07-23 | 锐思沃株式会社 | Biomarker for diagnosing mental disease onset risk state |
CN111187827A (en) * | 2020-01-20 | 2020-05-22 | 厦门大学 | Parkinson disease biomarker and application thereof |
CN113621614A (en) * | 2021-09-09 | 2021-11-09 | 天津医科大学总医院 | Long-chain non-coding RNA and application thereof as MDS molecular marker |
CN113621614B (en) * | 2021-09-09 | 2023-08-11 | 天津医科大学总医院 | Long-chain non-coding RNA and application thereof as MDS molecular marker |
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