CN107034275A - A kind of purposes of the gene related to cerebral apoplexy generation development - Google Patents

Abstract

The invention discloses a kind of purposes of the gene related to cerebral apoplexy generation development, specifically the gene is LOC103611081.LncRNA unconventionality expression is related in the generation evolution of disease, and research lncRNA unconventionality expression provides new approach for the diagnosis of disease.The present invention is experimentally confirmed LOC103611081 up-regulated expressions in patients with cerebral apoplexy, points out LOC103611081 to be applied to as auxiliary characteristics in the clinical diagnosis of early stage cerebral arterial thrombosis.

Description

A kind of purposes of the gene related to cerebral apoplexy generation development

Technical field

The invention belongs to biomedicine field, it is related to a kind of purposes of the gene related to cerebral apoplexy generation development, specifically The ground gene is lncRNA LOC103611081.

Background technology

Cerebral apoplexy is the refractory disease for seriously endangering human life's safety, dead with high incidence, high disability rate and height The characteristics of dying rate.Epidemiology survey shows the incidence of disease more and more higher of young stroke in recent years, wherein less than 45 years old is grown up The cerebral apoplexy of generation, is respectively 5% and 13.44% in the ratio of western countries and domestic report.Cerebral apoplexy is divided into by its property In ischemic cerebral apoplexy and hemorrhagic apoplexy, wherein cerebral arterial thrombosis accounts for the 75%~85% of patients with stroke sum.Ischemic Cerebral apoplexy also known as cerebral infarction/dead, refer to because brain blood supplies obstacle, and limitation brain tissue caused by ischemic, anoxic lacks Courageous and upright necrosis or cerebromalacia.

The treatment clinically for cerebral apoplexy is broadly divided into thrombolysis class drug therapy and neuroprotection class drug therapy at present Two methods.Wherein, thrombolysis class drug therapy is uniquely to be considered as definite effective method so far, but because of therapeutic time window Shorter and bleeding risk is high, only about 5% patient can obtain effective thromboembolism treatment at present.The research and development of neuroprotection class medicine are then Make slow progress, clinic is used for the auxiliary treatment of cerebral apoplexy.In addition, for excitatory amino acid sample neurotransmitter, active oxygen certainly Antagonist (such as glycine inhibitor GV150526, free radical scavenger NXY059) treatment designed by the cause of disease such as base hypothesis is equal Obvious clinical efficacy is not yet obtained, points out us to be goed deep into from new angle and direction to the pathomechanism of acute cerebral ischemia Inquire into, to seek the new breakthrough of clinical treatment.

With the development of biology techniques, it has been found that gene plays an important role in cerebral apoplexy develops, It is found that lncRNA is a variety of different in DNA methylation, histone modification, post-transcriptional control, RNA interference, imprinted genes etc. Mechanism, the expression of gene is influenceed in a variety of aspects, adjust various cell processes, such as cell growth, propagation and apoptosis. The generation development of lncRNA unconventionality expression and disease is closely related, with to stroke onset reason and disease mechanisms research Deeply, our understanding to therapy target are also gradually clear, comprehensively, and this research and Clinical practice for Treatment of Cerebral Stroke medicine is carried More research directions are supplied.In the prior art as patent 201510853891.X and 201510853894.3 has been disclosed for base Because of the application in stroke clinical diagnosis, it is believed that with being deepened continuously to cerebral apoplexy basic research, study lncRNA and brain The relation that palsy develops provides new possibility for the diagnosis and treatment of cerebral apoplexy.

The content of the invention

In order to make up the deficiencies in the prior art, the invention provides a kind of gene related to cerebral apoplexy generation development, it is The early diagnosis of cerebral apoplexy provides a kind of product and means, compared to the diagnostic method of traditional cerebral apoplexy, uses mark Diagnosis cerebral apoplexy has promptness, sensitivity, so that patient just can know disease risks in disease early stage, so as to take corresponding Prevention and treatment measure.

To achieve these goals, the present invention is adopted the following technical scheme that：

The invention provides a kind of LOC103611081 purposes, the product for preparing diagnosing ischemia cerebral apoplexy.

Further, the product is by determining expression and ginseng in sample by determining LOC103611081 in sample Level is examined to determine compared to up-regulation.

Further, the product is detected by the method for sequencing technologies, nucleic acid hybridization technique, nucleic acid amplification technologies The expression of LOC103611081 genes.

Further, the nucleic acid amplification technologies are selected from PCR (PCR), reverse transcriptase polymerase chain reaction (RT-PCR), the amplification (TMA) of transcriptive intermediate, ligase chain reaction (LCR), strand displacement amplification (SDA) and based on nucleic acid sequence The amplification (NASBA) of row.Wherein, PCR is needed RNA reverse transcriptions before amplification into DNA (RT-PCR), and TMA and NASBA directly expand Increase RNA.

Further, the LOC103611081 include as shown in SEQ ID NO.1 the polynucleotides of nucleotide sequence or its Fragment, homologue, variant or derivative.

The invention provides a kind of product of LOC103611081 expressions in vitro detection sample, the product includes Preparation, chip or kit.Wherein, the chip includes solid phase carrier；And the oligonucleotides being fixed on solid phase carrier is visited Pin.The kit includes genetic chip or SYBR Green PCR systems.

Further, the product of LOC103611081 expressions includes in the vitro detection sample：

Specific recognition LOC103611081 probe；Or

Specific amplification LOC103611081 primer.

Further, the primer sequence of the specific amplification LOC103611081 such as SEQ ID NO.2 and SEQ ID NO.3 It is shown.

Diagnosis cerebral apoplexy is being prepared the invention provides the product of LOC103611081 expressions in vitro detection sample Purposes in instrument.

The invention provides a kind of kit of diagnosing ischemia cerebral apoplexy, including：

Detect LOC103611081 one or more reagents；With

The one or more materials being selected from the group：Container, operation instructions, positive control, negative control thing, buffering Agent, auxiliary agent or solvent.

Further, the kit is used to detect LOC103611081 by the method for the following group：QRT-PCR, biochip Detection method, southern blotting technique method or RNA blotting hybridization in situ.

Brief description of the drawings

Fig. 1 is the difference expression gene figure using cDNA microarray ischemic cerebral stroke patients；

Fig. 2 is to detect expression figures of the LOC103611081 in ischemic cerebral stroke patients using QPCR.

Embodiment

The present invention is most wide using current covering database by high throughput method by in-depth study extensively LncRNA chips, transcriptional levels of the detection lncRNA in patients with cerebral apoplexy and Healthy People sample, find wherein there is obvious table Up to the lncRNA fragments of difference, its relation between the generation of cerebral apoplexy is inquired into, so as to be found for the early detection of cerebral apoplexy More preferable approaches and methods.By screening, present invention firstly discovers that LOC103611081 conspicuousnesses are raised in patients with cerebral apoplexy, LOC103611081 is pointed out to be applied to the clinical diagnosis of cerebral apoplexy as Testing index.

LOC103611081 genes

LOC103611081 genes are located on No. 11 chromosome long arms of people 2nd area 4 subzone of band the 3rd, a kind of representational The nucleotide sequence of LOC103611081 genes is as shown in SEQ ID NO.1.LOC103611081 genes are included such as in the present invention The polynucleotides or its fragment, homologue, variant or derivative of nucleotide sequence shown in SEQ ID NO.1.

It would be recognized by those skilled in the art that the practicality of the present invention is not limited to appointing to the target gene of the present invention The gene expression of what specific variants is quantified.If when nucleic acid or its fragment and other nucleic acid (or its complementary strand) optimal comparison When (have appropriate nucleotides inserted or missing), nucleotide base at least about 60%, usually at least about 70%, more Usually at least about 80%, it is preferably at least about 90% and more preferably at least about there is nucleosides in 95-98% nucleotide bases The acid sequence phase same sex, then the two sequences are " substantially homologous " (or substantially similar).

Therefore, polynucleotides of the invention and SEQ ID NO.1 preferably have at least 75%, more preferably at least 85%, more Preferably at least 90% homology.It is highly preferred that in the presence of at least 95%, more preferably at least 98% homology.

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 the importances of the present invention.The table of biomarker can be detected on transcriptional level Up to level.

Detection technique

The present invention lncRNA detected using multiple nucleic acids technology known to persons of ordinary skill in the art, these skills Art includes but is not limited to：Nucleic acid sequencing, nucleic acid hybridization and nucleic acid amplification technologies.

The present invention can simultaneously be expanded before detection or with detection to nucleic acid (for example, ncRNA).Nucleic acid amplification technologies Exemplary, non-limitative example include but is not limited to：PCR (PCR), reverse transcriptase polymerase chain reaction (RT- PCR), the amplification (TMA) of transcriptive intermediate, ligase chain reaction (LCR), strand displacement amplification (SDA) and based on nucleotide sequence Expand (NASBA).One of ordinary skill in the art will be it will be recognized that some amplification techniques (for example, PCR) needs will before amplification RNA reverse transcriptions are into DNA (for example, RT-PCR), and other amplification techniques then direct cloning RNA (for example, TMA and NASBA).

Generally, PCR uses the annealing and multiple circulations of primer extend of denaturation, primer pair and opposite strand, with index side Formula increases the copy number of target nucleic acid sequence；Reverse transcriptase (RT) is then used to prepare complementary DNA (cDNA) from mRNA by RT-PCR, Then cDNA is expanded by PCR to produce DNA multiple copies；Temperature, ionic strength and pH of the TMA in substantial constant Under the conditions of autocatalytically synthesize multiple copies of target nucleic acid sequence, multiple RNA copies of wherein target sequence are autocatalytically given birth to Into other copy, TMA is optionally including the use of blocking, part, dwell section and other modified parts, to improve TMA processes Sensitivity and the degree of accuracy；LCR uses two groups of complementary DNA oligonucleotides that the adjacent area with target nucleic acid hybridizes.DNA few nucleosides Acid is covalently attached in the multiple circulations of repetition of thermal denaturation, hybridization and connection by DNA ligase, to produce detectable double-strand Connect oligonucleotide product；SDA uses multiple circulations of following steps：The opposite strand of primer sequence pair and target sequence is moved back Fire, primer extend is carried out in the case where there is dNTP α S to produce (hemiphosphorothioated) of the thiophosphorylation of double-strand half Primer extension product, the nicking for the endonuclease mediation that semi-modified restriction enzyme enzyme recognition site is carried out, and from cutting The polymerase-mediated thing drawn that mouth 3' ends are carried out extends to be put with replacing existing chain and producing for next round primer annealing, nicking and chain The chain changed, so as to cause the geometry of product to expand.

The nucleic acid of non-amplification or amplification can be detected by any conventional means in the present invention.

Nucleic acid hybridization technique in the present invention include but is not limited in situ hybridization (ISH), microarray and Southern or Northern traces.In situ hybridization (ISH) be it is a kind of use mark complementary DNA or RNA chains as probe with position tissue one Part or section (original position) or if organize it is sufficiently small if for entirely organize (full organization embedding ISH) in specific DNA or The hybridization of RNA sequence.DNA ISH can be used for the structure for determining chromosome.RNA ISH are used to measure and position tissue section or complete MRNA and other transcripts (for example, ncRNA) in organization embedding.Solid with original position generally is handled to sample cell and tissue Targeting transcript, and increase the entrance of probe.Probe hybridizes with target sequence at high temperature, then washes unnecessary probe off.Point Shi Yong not autoradiograph, fluorescence microscopy or immunohistochemistry, the base to using radiation, fluorescence or antigenic mark in tissue The probe of mark is positioned and quantified.ISH can also be used two or more to pass through radioactivity or other nonradioactive labelings The probe of substance markers, to detect two or more transcripts simultaneously.

Southern and Northern traces are respectively used to detection specific DNA or RNA sequence.Make to extract from sample DNA or RNA fracture, it is separated by electrophoresis on matrix gel, be then transferred on molecular filter.Make filter combine DNA or RNA and the label probe hybridization complementary with sequence of interest.Detection is attached to the hybridization probe of filter.A kind of change of the program Change form is reverse northern trace, wherein set of the substrate nucleic acid fixed to film for the DNA fragmentation of separation, and probe is From tissue extraction and the RNA that is marked.

Chip, kit

Chip of the present invention includes：Solid phase carrier；And the oligonucleotides on the solid phase carrier is fixed in order Probe, described oligonucleotide probe specifically corresponds to the part or all of sequence shown in LOC103611081.

Specifically, it can design suitable probe according to lncRNA of the present invention, be fixed on solid phase carrier, shape Into " oligonucleotide arrays ".Described " oligonucleotide arrays " refer to (addressable i.e. with distinctive with addressable point The position that address is characterized) array, each addressable point is containing a coupled characteristic oligonucleotides.According to Need, oligonucleotide arrays can be divided into multiple sub- battle arrays.

In the present invention, the solid phase carrier includes plastic products, microparticle, membrane carrier etc..The plastic products can lead to Non-covalent or physical absorption mechanism is crossed to be combined with antibody or proteantigen, what the most frequently used plastic products were made for polystyrene Small test tube, globule and micro-reaction plate；The microparticle is the microballoon or particle aggregated into by high polymer monomer, and its diameter is generally Micron, because with the functional group that can be combined with protein, easily with antibody (antigen) formation chemical coupling, binding capacity is big；Institute Stating membrane carrier includes nitrocellulose filter, the plain miillpore filter such as film and nylon membrane of glass fibre.

" probe " refers to the molecule that can be combined with the particular sequence or subsequence or other parts of another molecule.Unless otherwise finger Go out, term " probe " is often referred to combine with another polynucleotides (often referred to as " target polynucleotide ") by complementary base pairing Polynucleotide probes.Lack the complementary target of sufficient sequence according to the preciseness of hybridization conditions, probe energy and with the probe many Nucleotides is combined.Probe can make direct or indirect mark, and its scope includes primer.Crossing system, includes, but are not limited to：It is molten Liquid phase, solid phase, mixed phase or in situ hybridization determination method.

" probe " refers to the molecule that can be combined with the particular sequence or subsequence or other parts of another molecule.Unless otherwise finger Go out, term " probe " is often referred to combine with another polynucleotides (often referred to as " target polynucleotide ") by complementary base pairing Polynucleotide probes.Lack the complementary target of sufficient sequence according to the preciseness of hybridization conditions, probe energy and with the probe many Nucleotides is combined.Probe can make direct or indirect mark, and its scope includes primer.Crossing system, includes, but are not limited to：It is molten Liquid phase, solid phase, mixed phase or in situ hybridization determination method.

The 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, which 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 pass through PNA (Polyamide nucleic in one part or whole nucleotides Acid, peptide nucleic acid), LNA (registration mark, locked nucleic acid, Bridged Nucleic Acid, Cross-linked core Acid), ENA (registration mark, 2 '-O, 4 '-C-Ethylene-bridged nucleic acids), GNA (Glycerol Nucleic acid, glycerine nucleic acid), the artificial replacement nucleic acid such as TNA (Threose nucleic acid, threose nucleic acid) obtains Polynucleotides.

Term " homology " refers to the degree of complementarity.There may be Homoeology or complete homology is (that is, same Property).The sequence of partial complementarity is that the nucleic acid molecules and " substantially homologous " target nucleic acid of suppression complete complementary at least in part are miscellaneous The nucleic acid molecules of friendship.The suppression of the hybridization of fully-complementary sequence and target sequence can be surveyed by using hybridization under low stringency Fixed (Southern or Northern traces, solution hybridization etc.) is checked.Substantially homologous sequence or probe will be competed simultaneously Suppress homologous nucleic acid molecules completely under low stringency and target combination (that is, hybridizing).This is not to say, low strict Property condition be so that allow non-specific binding condition；Spy is combined between two sequences of low stringency requirement The interaction of the opposite sex (that is, selectivity).Being not present for non-specific binding can be by using substantially incomplementarity (for example, low In about 30% homogeneity) the second target tested；In the case of in the absence of non-specific binding, probe will not be with second Incomplementarity target hybridization.

Term " hybridization " in the present invention is used for the pairing for referring to complementary nucleic acid.Hybridization and intensity for hybridization are (that is, between nucleic acid Association intensity) influenceed by such as following factor：Complementarity between nucleic acid, the stringency of involved condition, formed Crossbred Tm and nucleic acid in G:C ratio.The individual molecule of the pairing containing complementary nucleic acid is referred to as " self in its structure Hybridization ".

The present invention for LOC103611081 genes oligonucleotide probe can be DNA, RNA, DNA-RNA chimera, PNA or other derivatives.The length of the probe is not limited, as long as completing specific hybrid and purpose nucleotide sequence spy The opposite sex is combined, and any length can.The length of the probe can be as short as 25,20,15,13 or 10 bases longs.Equally, institute State the length of probe and can grow to 60,80,100,150,300 base-pairs or longer, or even whole gene.Due to different probes Length has different influences to hybridization efficiency, signal specificity, and the length of the probe is typically at least 14 base-pairs, most long 30 base-pairs are usually no more than, complementary length is optimal with 15-25 base-pair with purpose nucleotide sequence.The probe is certainly Body complementary series is most preferably less than 4 base-pairs, in order to avoid influence hybridization efficiency.

The kit of the present invention includes the reagent of the detection LOC103611081 genes of detection effective dose, one be selected from the group Plant or many kinds of substance：Container, operation instructions, positive control, negative control thing, buffer, auxiliary agent or solvent.For example for The solution of cell is suspended or fixes, detectable label or tag makes nucleic acid be easy to the solution of hybridization, for the molten of cell lysis Liquid, or the solution for nucleic acid purification.

Can also have the operation instructions of kit in the kit of the present invention, how be entered wherein describing using kit Row detection, and how disease development to be judged using testing result.

Using the kit of the present invention, it can be detected by the various methods (including but is not limited to) being selected from the group LOC103611081：Real Time RT-PCR, biochip test method, southern blotting technique method or RNA blottings or in situ hybridization Method.Those of ordinary skill in the art can be according to physical condition and needing to be adjusted detection mode and change.

The chip or kit of the present invention can be used for multiple genes of the detection including LOC103611081 genes (for example The multiple genes related to cerebral arterial thrombosis) expression.

Sample

In the present invention, " sample " includes but is not limited to, can be blood, tissue, urine, serum, blood plasma, amniotic fluid, Cerebrospinal fluid, placenta cells or tissue, endothelial cell, leucocyte or monocyte.Sample can derive from patient or object is direct Use, or pre-processed, such as by filtering, distilling, extract, concentrate, centrifuge, inactivating interference component, addition reagent side Formula processing, the characteristic of sample is modified in some modes as described herein or known in the art.In the specific implementation of the present invention In example, " sample " is blood.

Term " diagnosis " in the present invention refers to by the S＆S of disease or genetic analysis, pathological analysis, tissue The identification disease such as credit analysis.

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 method of unreceipted actual conditions in embodiment, generally according to conventional strip Part, such as Sambrook et al., molecular cloning：Laboratory manual (New York:Cold Spring HarborLaboratory Press, 1989) described in condition, or according to the condition proposed by manufacturer.

Embodiment 1 screens the gene marker related to cerebral arterial thrombosis

1st, sample collection

10 normal human bloods and the blood of ischemic cerebral stroke patients are respectively collected, the equal informed consent of patient is above-mentioned all The acquirement of sample passes through the agreement of the committee of organizational ethics.

2nd, the preparation of RNA sample

1) 12000rpm high speed centrifugation 10min after clinical serum sample is collected, are repeated 2 times.

2) 250 μ 1 serum sample is drawn to 1.5m1EP pipes, plus 750 μ 1Trizol LS Reagent, and piping and druming is mixed, quiet Put 5min.

3) chlorination imitates (CHCl₃：trizol 250μ1:750 μ 1), overturn mix, stand 15min, 4 DEG C, 12000rpm, from Heart 15min.

4) careful supernatant liquid of drawing is to a new EP pipes, plus appropriate isopropanol (isopropanol：trizo1 500μ1：750 μ 1), It is reverse to mix, stand 10min；4 DEG C, 12000rpm centrifugations 10min.

5) upper strata centrifugate, plus 75% ethanol (RNase inactivation) are abandoned；4 DEG C, 8000rpm centrifugations 5min；

6) retain precipitation, abandon upper strata centrifugate, be stored at room temperature 5-10min；Appropriate DEPC water dissolving precipitation is added, is taken appropriate RNA solution surveys concentration and observation RNA extracting situations.

3rd, reverse transcription and mark

With Low RNA Input Linear Amplification Kit by mRNA reverse transcriptions into cDNA, while using Cy3 Difference labelling experiment group and control group.

4th, hybridize

Genetic chip uses Kang Cheng biology-Human lncRNA Array, carries out the step of by chip operation instructions miscellaneous Hand over.

5th, data processing

Chip Agilent scanner scannings after hybridization, resolution ratio is 5 μm, and scanner is automatically with 100% and 10%PMT Each to scan 1 time, 2 times result Agilent softwares merge automatically.Scan image data uses Feature

Extraction carries out Treatment Analysis, and obtained initial data application Bioconductor program bags are subsequently counted According to processing.Last Ratio values are experimental group and control group.Differential gene screening criteria：Ratio >=4 are up-regulated gene, ratio ≤ 0.25 is down-regulated gene.

6th, result

As a result as shown in figure 1, compared with healthy human blood, expression of the LOC103611081 in ischemic cerebral stroke patients Level is significantly higher than the expression of Healthy People.

The differential expression of the QPCR sequence verification LOC103611081 genes of embodiment 2

1st, large sample QPCR checkings are carried out to LOC103611081 gene differential expressions.Received according to the sample in embodiment 1 Mode set selects each 60 of the blood sample sample of normal person and ischemic cerebral stroke patients.

2nd, RNA extraction steps be the same as Example 1.

3rd, reverse transcription：

(1) reverse transcription reaction：

Configure 25 μ l reaction systems：DEPC water, 5 × RT Buffer, 10mM dNTP, 0.1mM DTT, 30 μM Oligo dT, 200U/ μ l M-MLV, 1 μ g template ribonucleic acids.

Above-mentioned system is blown and beaten and mixed, 42 DEG C are incubated 1h, 72 DEG C of 10min, of short duration centrifugation.

(2) design of primers：

The primer sequence of LOC103611081 genes is：

Forward primer：5’-AACTGAAGAACCACAAGAGAAG-3’(SEQ ID NO.2)

Reverse primer：5’-TTGCTGTCCAGGAGAAGG-3’(SEQ ID NO.3)

House-keeping gene GAPDH primer sequence is：

Forward primer：5’-CCGGGAAACTGTGGCGTGATGG-3’(SEQ ID NO.4)

Reverse primer：5’-AGGTGGAGGAGTGGGTGTCGCTGTT-3’(SEQ ID NO.5)

(3) QPCR amplifications are examined：

Prepare following reaction system：The μ l of SYBR Green PCRs system 12.5, forward and reverse primer (5 μM) is each 1 μ l, template cDNA2.0 μ l, no μ l of enzyme water 8.5；Each sample sets 3 parallel pipes, and operations are carried out on ice.

Amplification program is：95 DEG C of 60s, (95 DEG C of 15s, 60 DEG C of 15s, 72 DEG C of 45s) × 40 circulations.

It is anti-in the enterprising performing PCR of Light Cycler fluorescence real-time quantitative PCR instrument using SYBR Green as fluorescent marker Should, purpose band is determined by melt curve analysis analysis and electrophoresis, Δ Δ CT methods carry out relative quantification.

3rd, statistical method

Experiment is all completed according to being repeated 3 times, and result data is represented in the way of mean+SD, Statistical analysis is carried out using SPSS18.0 statistical softwares, difference between the two is examined using t, it is believed that work as P<Have when 0.05 It is statistically significant.

4th, result

As a result as shown in Fig. 2 compared with Healthy People, on LOC103611081 genes are expressed in ischemic cerebral stroke patients Adjust, 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 and its core concept of the present invention.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 improve 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>Wang Si Kui Liu will state grandson's regular way

<120>A kind of purposes of the gene related to cerebral apoplexy generation development

<160> 5

<170> PatentIn version 3.5

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<211> 3999

<212> DNA

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ttagcaatct ggagaggact gaggcaagag cttgtattga aacacaagca agctccacgt 60

atggttttgg acatggaaga aagagtactg ttggggtgga atgacctaag tttttaatag 120

gattaggatt tctgtggatt tttcccaagt gatagaagtt ttcttcatct agcagggagc 180

ctcacccaat tgcttctcta tgttcatttc tgaaactcct ttgttaaaac taaatagcaa 240

cagagaagaa actttcttca aaaaactctt catatctgtc atttctccta ttcttatgga 300

acttcacaat tgctttttct ttattccttt ttttttttct atatcagctc atcagtcttt 360

gtgggtgact tggctacccc actaatctaa ttgccttttt gatagttaaa tcaatatgga 420

catcaactta aggctatatg tgaaccttaa atgcatgata ttccctatac ttgattccac 480

aattgatatt ctgtgaggaa agttggccag atattccctt ctgtgaagtc agaataacta 540

ccgaccctaa gaataaacat tttgaccaat tctttatatg ttgcaaacta ttcacacaac 600

tagaaagttt agccattcac atctcttccc ctgattttaa gtcccagatt ctgagactca 660

gaaagtttag tgacttgttg agctttctgt ggaaaatctg ttgtaaaaac agttcagaca 720

ttggaaaata taaaatagcc cctagagatc ccaataaagc ttcctctccc caaatccctc 780

tacaagttta tcctcgaaaa actttctgaa aactgatgag aaaatactga gtaattaatg 840

ctcaaatggg aaagatgaca tgtaaaagca aaaacctgtt aatggcatct gttcatatag 900

ttccatgtgc ctttttactt aactgcactg ataggcaatg taatgaagaa gtttcctttt 960

aaaacgtaaa atgtaattaa acagagttta tcttaaattg gctggacccg cacagcttga 1020

ctgagacata cacagctgtc ttcattttga agcacctaca ctagtcaagc ttctttctaa 1080

cccgccctta gggtttgcct gttggtctgt cttccctact gggctgttaa tcccttggag 1140

gcagattgtc ttgtttccca gagtttttcc agcatatacc atggcagaca attttcacct 1200

tatctattct aactcgtaag cctcagctag tgcctccatc ctcgggtgca tagcccagtg 1260

acaggcatgt aacagacttt gcctacaagc agttgctgat ccataaatga cgttggcaac 1320

cttcttgtgg cagtgagtaa catatttctt tcctcctgat aggcttggaa tcctcatccc 1380

catctggcaa ttttctttcc atctttccta cactcacaat ggagcccaca cgcctcacaa 1440

cccccttgac taaaccagat gacagcaaga atgcctagtt ccaagtgagc aagggtgaag 1500

ccttaggccc ttgtctcctc cagacctcct ctctgctcta agagtatact gggcatgggg 1560

tgtggtcggg gggttggggg cggaggagta cttaacctca aactgagaaa aagggtgtcc 1620

tgagcaagtt tcatagcgta gcagcttcca tttgaaatgt gaagctttga aggcctgttg 1680

ctacagtaac cagctagctc ctgactccaa tgcctgtcta atctaaggtg gaaatgtccc 1740

agaaacagac cccaggctgg cagctcttaa ctatgaccca tctcataagc acatatccag 1800

ctgtagcctc ttggttgctg gggtgaaggg gcctctgtat cgccacagga aaaaaaatct 1860

gataaattac gggctcaaga agggtgttgc ttttttttcc actaacagac tttgcaaccc 1920

agttcctggt gaaagggctc cgcacccacc cgttttgtgt gtataatgct gagctaaagt 1980

gggaaggagc agtaacacca aaggaggcta attctcttcc tgtcagtagg agagagtgtc 2040

tgcccccagt gtgtgaaaag ggaaggcatg agaagtgaaa aatcagcctt ctggaagcac 2100

cttcttacca cgagaaaaca ggtgattctt actatctttg agactcatcc agattgtgtc 2160

tgtttaactc ttcaggcaat atgaaggcag agaagccatt tcagagaaga gattttttaa 2220

aaaatcattt cgttctttgg cattgtcaca ggattatgat gtgggttctc ataaaaattt 2280

cactcccccc aaagccaccc aaaggaaagg cagtgaaatg gggaggaaca cacatttgcc 2340

tggcatcgca agagctgcct tgtagtcatg ggttccagtg atcttggacc gatctcttca 2400

cttccctgga cctcagtttc ttccatacaa agaagttgaa ctggatcata gaaaagatgc 2460

tgtccagctt ggtaatatca gatagggagc agaagctgtc tcttagcaca tggccagatt 2520

ccggaacaac gatgatggtc tccatgtttg tcctaaaaat gcatctcatc tcagtagaga 2580

ttttcttcca cctcatttgg gtctcaattt aaggaaggaa gaaaggaaag aaacccgggg 2640

attctcttct tgaaaggaat atacttcact ttctctaggc ttctaggata aaaacttcct 2700

atccttcatt tttcctcttt aaacctgcca aaatcaagtt gagtaaagat accatacaga 2760

tatgaaaaag aagccacatc cttaaatttt catggagtcc aagagcctgc tcaccgccag 2820

ttagcttttc ccaggatggt ggtggcactg atcatctagg ttgcactttt atgttcatgt 2880

cccagagcgt ttcccatcag ccatctcatt tcatcttttc atcattcctc tgaggaaaag 2940

acttcacaca tgtataggac tttctatatc acaaagtatt tcatatccaa tgtcttattt 3000

agatcttgac aatcactgtt taaggtaatt tggcaagtta ttaatactca tgtcctcatt 3060

ttactgaaga ggaaactgaa atgcagagag cttgtctttt gcccaggatc actcacccag 3120

tttattggga ggcagcagca cggggattaa gaacatgggt tatgccgaca accaaccagg 3180

cagaattcca gctctgtcag tagttagctg tctggcctta ggaaagggat ttcacacctc 3240

tgtggcttag tttccttgtc cattaaatgc tcataagaat agagctttcc tcacagagat 3300

tgggtgggca cgggagctaa atgagctcat acgtttaaga acaacctcta gcacacagtg 3360

cataataaat gtcattatta ttagcaaagc caggacctgg gtcttccagc ttctagctca 3420

gtggcatttc tgctgcccca caccaggact atccttcgta tacaggtatg aacctaaagc 3480

cttcagtgtt tcaatgaaaa gttcacccaa ggtctttctg ttcagtaagt ctagagctaa 3540

gggctcctgg cactctgttc agtcctttcg ctggtaaacc acgctgccct ccgttgctct 3600

gggcagaatg tcaaaccttc ttatgttttt aaaatttata aatcaagcac tcttatttaa 3660

tattactgac agcccgaagg cagacgctcc cactctcctg gggaagaggg tcttctaatc 3720

tgccatctta aggaggtaaa agggcttgat tgggtaactc attgcaatgg gcacgtattt 3780

attactgtct cccggccttt tctcatgctg tttgtctgct ggaattacca ctagtgccag 3840

gccatttgaa tatttgacca tttgtcttag aattctctga atacaaatgt ttttcatacc 3900

tttactatgt actttttaag aatgaagact atgttgcatg ttggtttgta ctctctcctg 3960

cacataaaaa atgcttagta aatagataca caaatgttt 3999

<210> 2

<211> 22

<212> DNA

<213>Artificial sequence

<400> 2

aactgaagaa ccacaagaga ag 22

<210> 3

<211> 18

<212> DNA

<213>Artificial sequence

<400> 3

ttgctgtcca ggagaagg 18

<210> 4

<211> 22

<212> DNA

<213>Artificial sequence

<400> 4

ccgggaaact gtggcgtgat gg 22

<210> 5

<211> 25

<212> DNA

<213>Artificial sequence

<400> 5

aggtggagga gtgggtgtcg ctgtt 25

Claims (10)

1.LOC103611081 purposes, it is characterised in that the product for preparing diagnosing ischemia cerebral apoplexy.

2. purposes according to claim 1, it is characterised in that the product is by determining in sample by determining in sample LOC103611081 expression is raised and determined compared with reference levels.

3. purposes according to claim 2, it is characterised in that the product passes through sequencing technologies, nucleic acid hybridization technique, core The method of sour amplification technique detects the expression of LOC103611081 genes.

4. the purposes according to claim any one of 1-3, it is characterised in that the LOC103611081 includes such as SEQ ID The polynucleotides of nucleotide sequence shown in NO.1 or its fragment, homologue, variant or derivative.

5. the product of LOC103611081 expressions in a kind of vitro detection sample, it is characterised in that the product includes system Agent, chip or kit.

6. product according to claim 5, it is characterised in that including：

Specific recognition LOC103611081 probe；Or

Specific amplification LOC103611081 primer.

7. product according to claim 6, it is characterised in that the primer sequence of the specific amplification LOC103611081 As shown in SEQ ID NO.2 and SEQ ID NO.3.

8. purposes of the product in diagnosis cerebral apoplexy instrument is prepared described in claim any one of 5-7.

9. a kind of kit of diagnosing ischemia cerebral apoplexy, it is characterised in that including：

Detect LOC103611081 one or more reagents；With

The one or more materials being selected from the group：Container, operation instructions, positive control, negative control thing, buffer, help Agent or solvent.

10. kit according to claim 9, it is characterised in that the kit is used to detect by the method for the following group LOC103611081：QRT-PCR, biochip test method, southern blotting technique method or RNA blotting hybridization in situ.