CN107904304B - Purposes of the DNASE2 as parkinsonism diagnosis marker - Google Patents
Purposes of the DNASE2 as parkinsonism diagnosis marker Download PDFInfo
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- G—PHYSICS
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- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
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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
- G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
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- G—PHYSICS
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- 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
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
- G01N2333/922—Ribonucleases (RNAses); Deoxyribonucleases (DNAses)
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- G—PHYSICS
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- 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
The invention discloses DNASE2 genes and its expression product to prepare the application in diagnostic products.The diagnostic products can be used for the diagnosis of parkinsonism.Level by detecting DNASE2 gene and its expression product may determine that subject suffers from the risk of parkinsonism in the future or makes a definite diagnosis the state of parkinsonism.Diagnostic products of the invention are using subject's peripheral blood as test object, it can be achieved that noninvasive, quick, sensitive and accurate diagnosis effect.
Description
Technical field
The invention belongs to molecular diagnosis fields, are related to a kind of molecular marker for parkinsonism diagnosis, and in particular to
Application of the molecular marker-DNASE2 gene in the product of preparation diagnosis parkinsonism in blood.
Background technique
PD is second largest common, slow occur, the age-related nervus retrogression after Alzheimer after being ill
Disease, also known as fiber crops of trembling fight.Pathologically substantia nigra of midbrain dopaminergic neuron (dopaminergic neurons, DNs) selects
Property lose, Lewy body (Lewy body, LB) is formed in remaining neuron and Striatal Dopamine Content significantly reduces, lead
Caused static tremor, myotonia, bradykinesia, the main clinical symptoms of posture abnormal gait this 4 (Braak H,
Tredici K D, et al.Staging of brain pathology related to sporadic Parkinson,
sdisease.Neurobiology of Aging,2003,24(2)：197-211).In industrialized country, PD patient accounts for total people
The change 3% of mouth accounts for the 1% of 60 years old or more population, increases 8-18 PD patient (de Lau L newly in annual every 100000 people in the whole world
M,Breteler MM.Epidemiology of parkinson’s disease.The Lancet Neurology,2006,5
(6):525-535)。Stephen(K S.Incidence of parkinson’s Disease:Variation by age,
Gender,and Race/Ethnicity,2003:157,1015-1022) have studied PD incidence and age, gender and race
Relationship, discovery can quickly increase in 60 years old or more disease incidence, and the case of the right side of fifty only accounts for 4%；Male patient is women
1.9 times of patient；The incidence of not agnate PD is then variation, and generally speaking white man PD incidence is higher than Asian, Black people
It is then minimum.
At present other than SPECT PET image method has part auxiliary diagnostic effect, Parkinson's disease unified score amount
Hoehn the and Yahr Parkinson's disease of table (Unified Pakinson ' s Disease Rating Scale, UPDRS) and improvement
Scale (H-Y is by stages) is the common method of PD clinical diagnosis and treatment assessment by stages.In general, such as brain trauma, brain blood are excluded
Pipe disease, metal poisoning etc. can cause the cause of disease of secondary Parkinson's disease, also damage without ballet's disease, cerebellum sign, pyramidal system
Under the signs such as harmful and amyotrophia, according to the diagnosis of Parkinson disease standard of Britain's think-tank, patient has to be moved less, along with inactive shakes
It quivers, one of three kinds of myotonia, posture abnormal gait symptoms, PD can be diagnosed as.In addition, urine and cerebrospinal fluid in dopamine and
Its metabolite reduces, and well also contributes to distinguishing PD and other neurodegenerative diseases to the reaction of drug levodopa.
In fact there are clinical symptoms with top in degeneration of dopaminergic neurons missing 50%-70%, this often makes patient
The chance of early diagnosis, early treatment is missed, so just effective biomarker can help PD early diagnosis simultaneously to pole demander
Improve the correctness of diagnosis.
Neurodegenerative disease, including Parkinson's disease, Alzheimer disease, Huntington disease and amyotrophic lateral sclerosis funiculus lateralis medullae spinalis
Sclerosis etc., they are characterized in the storage for having paraprotein in the different parts of central nervous system and idiopathic nerve jointly
Member loses (Ross C A, Poirier M A.Protein aggregation and neurodegenerative
Disease.Nat Med, 2004,10Suppl：S10-S17) so as to cause the dysfunctions such as movement and cognition.Neurological
Property disease because pathological change occurs in brain, brain tissue acquirement be all in gained of after death dissecting a body, so pathomechanism study into
It postpones slow.Generally speaking, neurodegenerative disease is the result of various genes and environment joint effect.
Gene expression regulation network system obtains noticeable progress in cancer research field, in many tumor tissues
Such as lung cancer, breast cancer, gastric cancer, colorectal cancer, there are stable and bright with its source normal tissue for the expression of certain genes
Aobvious difference.The tumour of different tissue sources or even same tissue-derived different differentiation states plus tumor, gene expression profile is not
It is identical to the greatest extent, show that gene expression has apparent tissue specificity and stage of development specificity.Because neurodegenerative disease is sick
The overexpression for being directly characterized in disease specific protease matter in reason, so speculating the abnormal change of specific gene function
The expression of albumen, so as to cause disease.More and more research discovery specific genes can become diagnosis pa gold
Gloomy biomarker, such as the patent disclosure of following application number：201510713527.3,201510464905.9,
201510463593.X、201510463617.1、201510809837.5。
Most of disease is all that the disease of polygenes regulation joins lots of genes to improve the accuracy rate of diagnosis of disease
Closing diagnosis becomes inevitable, it is therefore desirable to excavate more molecular proportion marker relevant to Parkinson's disease so as to clinical application.
Summary of the invention
In order to make up for the deficiencies of the prior art, the purpose of the present invention is to provide one kind can be used for parkinsonism early diagnosis
Molecular marker.Compared to the diagnostic method of traditional parkinsonism, having for parkinsonism is diagnosed using gene marker
Timeliness, specificity and sensitivity, for risk height, are taken to make patient that can know disease risks in disease early stage
It is corresponding to prevent and treat measure.
To achieve the goals above, the present invention adopts the following technical scheme that：
The present invention provides application of the product of detection DNASE2 gene expression in the tool of preparation diagnosis parkinsonism.
Further, the product of detection DNASE2 gene expression mentioned above includes：Pass through RT-PCR, real-time quantitative
PCR, immune detection, in situ hybridization, chip or high-flux sequence detection of platform DNASE2 gene expression dose are to diagnose Parkinson
The product of disease.
Further, the product with RT-PCR diagnosis parkinsonism includes at least a pair of of specific amplified DNASE2 gene
Primer；The product with real-time quantitative PCR diagnosis parkinsonism includes at least the primer of a pair of of specific amplified DNASE2 gene；
It is described with immune detection diagnosis parkinsonism product include：Antibody in conjunction with DNASE2 protein-specific；The original position
Hybridization diagnosis parkinsonism product include：With the probe of the nucleic acid array hybridizing of DNASE2 gene；It is described to diagnose pa with chip
The product of the gloomy disease of gold includes：Protein chip and genetic chip；Wherein, protein chip includes in conjunction with DNASE2 protein-specific
Antibody, genetic chip include the probe with the nucleic acid array hybridizing of DNASE2 gene.
In specific embodiments of the present invention, the product with real-time quantitative PCR diagnosis parkinsonism is included at least
The sequence of the primer of a pair of of specific amplified DNASE2 gene is as shown in SEQ ID NO.1 and SEQ ID NO.2.
Preferably, the diagnostic tool includes chip, kit, test paper or high-flux sequence platform.Wherein, high pass measures
Sequence platform is a kind of special diagnostic tool, and the product of detection DNASE2 gene expression can be applied to platform realization pair
The detection of the expression of DNASE2 gene.With the development of high throughput sequencing technologies, to the structure of the gene expression profile of a people
Very easily work will be become by building.By comparing the gene expression profile of Disease and normal population, it is easy which is analyzed
The exception of gene is related to disease.Therefore, know that the exception of DNASE2 gene is related to parkinsonism in high-flux sequence
Belong to the purposes of DNASE2 gene, equally within protection scope of the present invention.
The present invention also provides a kind of tools for diagnosing parkinsonism, and the diagnostic tool includes chip, kit, examination
Paper or high-flux sequence platform.
Wherein, the chip includes genetic chip, protein-chip；The genetic chip includes solid phase carrier and fixation
In the oligonucleotide probe of solid phase carrier, the oligonucleotide probe includes for detecting being directed to for DNASE2 gene transcription level
The oligonucleotide probe of DNASE2 gene；The protein-chip includes solid phase carrier and the DNASE2 for being fixed on solid phase carrier
The specific antibody of albumen；The genetic chip can be used for detecting multiple genes including DNASE2 gene (for example, and pa
The relevant multiple genes of the gloomy disease of gold) expression.The protein-chip can be used for detecting including DNASE2 albumen
The expression of multiple protein (such as multiple protein relevant to parkinsonism).By by multiple with parkinsonism mark
Will object detects simultaneously, is greatly improved the accuracy rate of parkinsonism diagnosis.
Wherein, the kit includes gene detecting kit and protein immunization detection kit；The genetic test examination
Agent box includes the reagent for detecting DNASE2 gene transcription level；The protein immunization detection kit includes DNASE2 albumen
Specific antibody.Further, the reagent includes using RT-PCR, real-time quantitative PCR, immune detection, in situ hybridization or core
Piece method detects reagent needed for DNASE2 gene expression dose process.Preference, the reagent include being directed to DNASE2 base
The primer and/or probe of cause.Being easy to design according to the nucleotide sequence information of DNASE2 gene can be used for detecting DNASE2
The primer and probe of gene expression dose.
It can be DNA, RNA, DNA-RNA chimera, PNA or other with the probe of the nucleic acid array hybridizing of DNASE2 gene
Derivative.There is no limit for the length of the probe, as long as completing specific hybrid, being specifically bound with purpose nucleotide sequence,
Any length is ok.The length of the probe can be as short as 25,20,15,13 or 10 bases longs.Equally, the probe
Length can be grown to 60,80,100,150,300 base-pairs or longer or even whole genes.Since different probe lengths is to miscellaneous
Efficiency, signal specificity is handed over to have different influences, the length of the probe is typically at least 14 base-pairs, and longest does not surpass generally
30 base-pairs are crossed, the length complementary with purpose nucleotide sequence is best with 15-25 base-pair.The probe self-complementary sequence
Column are most preferably less than 4 base-pairs, in order to avoid influence hybridization efficiency.
The high-flux sequence platform includes the reagent for detecting DNASE2 gene expression dose.
The test paper includes test paper carrier and the oligonucleotides that is fixed on test paper carrier, and the oligonucleotides is able to detect
The transcriptional level of DNASE2 gene.
Further, the specific antibody of the DNASE2 albumen includes monoclonal antibody, polyclonal antibody.The DNASE2
The specific antibody of albumen include complete antibody molecule, antibody any segment or modification (for example, chimeric antibody, scFv,
Fab, F (ab ') 2, Fv etc..As long as the segment can retain the binding ability with DNASE2 albumen.For protein water
When the preparation of flat antibody well known to a person skilled in the art, and the present invention may use any method it is described anti-to prepare
Body.
In specific embodiments of the present invention, the primer sequence for DNASE2 gene is as follows：Forward primer sequence
Column are as shown in SEQ ID NO.1, and reverse primer is as shown in SEQ ID NO.2.
Source for the DNASE2 gene and its expression product that diagnose parkinsonism includes but is not limited to blood, tissue
Liquid, urine, saliva, spinal fluid etc. can obtain the body fluid of genomic DNA.In specific embodiments of the present invention, for examining
The DNASE2 gene of disconnected parkinsonism and its source of expression product are blood.
In the context of the present invention, " DNASE2 gene " includes any function of DNASE2 gene and DNASE2 gene
The polynucleotides of equivalent.DNASE2 gene (Chromosome 19, NC_000019.10 (12875211..12881521,
Complement)) sequence can inquire in international public GenBank GeneBank.
In the context of the present invention, DNASE2 gene expression product includes the portion of DNASE2 albumen and DNASE2 albumen
Divide peptide.The partial peptide of the DNASE2 albumen contains functional domain relevant to parkinsonism.
" DNASE2 albumen " includes any functional equivalent of DNASE2 albumen and DNASE2 albumen.The function is equivalent
Object includes DNASE2 albumen conservative variation protein or its active fragment or its reactive derivative, allelic variant, natural
Mutant, induced mutants, can be with the encoded protein of DNA of the DNA hybridization of DNASE2 under high or low stringent condition.
It is known that, conventionally, the modification of one or more amino acid will not influence the function of protein in a protein.
Those skilled in the art can approve the amino acid for changing single amino acids or small percentage or to amino acid sequence it is individual add,
Missing, insertion, replacement are conservative modifications, and wherein the change of protein generates the protein with identity function.Function phase is provided
As the Conservative substitution tables of amino acid be well known in the art.
Example by one amino acid of addition or the protein of more amino acid modification is melting for DNASE2 albumen
Hop protein.For the peptide or protein with DNASE2 protein fusion, there is no limit as long as resulting fusion protein retains
The biological activity of DNASE2 albumen.
In the context of the present invention, " diagnosis parkinsonism " both includes judging whether subject has suffered from Parkinson
Disease also includes the risk that judges subject and whether there is with parkinsonism.
The advantages of the present invention：
Present invention firstly discovers that DNASE2 gene expression is related to parkinsonism, pass through DNASE2 in detection subject
Expression, it can be determined that whether subject suffers from parkinsonism or judges that subject whether there is the risk with parkinsonism,
To instruct clinician to provide prevention scheme or therapeutic scheme to subject.
Present invention finds a kind of new molecular marked compound-DNASE2 genes, compared to traditional detection means, gene diagnosis
More in time, more special, sensitiveer, it can be realized the early diagnosis of parkinsonism, to reduce the death rate of parkinsonism.
Detailed description of the invention
Fig. 1, which is shown, utilizes differential expression of the genechip detection DNASE2 gene in Parkinson's disease patients and normal person；
Fig. 2 shows the differential expression using QPCR detection DNASE2 gene in Parkinson's disease patients and normal person.
Specific embodiment
The present invention is described in further detail with reference to the accompanying drawings and examples.Following embodiment is merely to illustrate this
It invents rather than limits the scope of the invention.Test method without specific conditions in embodiment, usually according to conventional strip
Part, such as Sambrook et al., molecular cloning：Laboratory manual (New York:Cold Spring HarborLaboratory
Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.
Embodiment 1 screens the gene of differential expression in Parkinson's disease patients and normal person
1, research object：
Primary PD patient 10 are collected, male 5, female 5, the age 32-85 years old, the course of disease -20 years 5 months.PD enters a group mark
It is quasi-：Diagnostic criteria meets PD clinical criteria (with reference to " Jiang Yuping, Wang Jian, Ding Zhengtong, etc. Primary ventricular hemorrhage is examined
Disconnected standard, 2005, Chinese Clinical Neuscience, 2006,14:40").Exclusion criteria：(1) essential tremor；(2) secondary pa
The gloomy syndrome of gold；(3) advanced dementia, dysarthrosis person；(4) other mental disease persons are suffered from.This studies oneself and passes through hospital's human relations
The approval of the reason committee and all patients signature informed consent form.
Normal group：Choose healthy volunteer 10, men and women each 5 of the age 32-80 years old.
Age, the not statistically significant (P of gender differences between two groups>0.10) it, is comparable.
2, in blood total serum IgE extraction
(1) homogenized (Homogenization)
Fresh blood (peripheral blood) is directly taken, 3 times of volume erythrocyte cracked liquids are added, 10 points are placed at room temperature for after mixing
Clock, 10,000rpm centrifugations 1 minute.It thoroughly inhales and abandons supernatant, collect leukocyte cell pellet.The leucocyte of every 100-200 μ l blood collection
1ml TRIzol is added in precipitating.
(2) it is layered (Phase Separation)
A. after TRIzol is added in sample, it is placed at room temperature for 5min, cracks sample sufficiently.
B. 200 μ l chloroforms are added in every 1ml TRIzol, and 3-5min is placed at room temperature for after shaking vigorously and mix well makes its natural split-phase.
(3) RNA precipitate (RNA Precipitation)
A.4 DEG C 12,000rpm is centrifuged 10-15min.Sample can be divided into three layers：The organic phase of yellow, middle layer and colourless
Water phase, RNA mainly in water phase, water phase (can usually draw 550 μ l) are transferred in new pipe.
B. isometric ice-cold isopropanol is added in supernatant, is placed at room temperature for 10-20min.4 DEG C of 12,000rpm centrifugations
10min abandons supernatant, and RNA precipitate is in tube bottom.
(4) RNA rinses (RNA Wash)
75% ethyl alcohol of 1ml (being prepared with RNase-free water) is added in a.RNA precipitating, mildly vibrates centrifuge tube, it is heavy to suspend
It forms sediment.75% ethyl alcohol of 1ml is added in every 1ml TRIzol.
B.4 DEG C 5,000-8,000rpm is centrifuged 1-2min, abandons supernatant；Of short duration rapid centrifugation is carefully inhaled in abandoning with pipettor
Clearly, 1-2 minutes are placed at room temperature for and dries precipitating.
(5) RNA (Redissolving the RNA) is dissolved
50-100 μ l RNase-free water is added in precipitating, flicks tube wall, sufficiently to dissolve RNA, -70 DEG C of preservations.
3, RNA mass and purity detecting
RNA mass：It is indicated by RNA integrality, plain agar sugar gel electrophoresis (deposition condition can be used：1.2% glue；
0.5 × TBE electrophoretic buffer；150v, 15 minutes) detection integrality.
RNA purity：OD260/OD280 ratio is the index for measuring protein contamination degree in RNA sample.High quality
RNA sample, OD260/OD280 value (10mM Tris, pH7.5) is 2.0 or so.
4, gene chip hybridization and scanning
After the linearized amplification of total serum IgE, cy3-UTP is marked, and the cRNAs after fluorescent marker uses RNEASY Mini Kit
Purifying, carries out fragmentation processing to the cRNAs marked with the RNA Fragmentation Reagents of Amhion.Using beauty
People's full genome chip of expression spectrum (4x 44K gene) of Agilent company of state, 65 DEG C of hybridization 17h in chip hybridization furnace, then
Elution, dyeing, finally use Agilent DNA MicroarrayScanner scanner scanning.
5, chip data processing and analysis
Chip after hybridization imports data to analysis software, for two groups of ratios after chip scanner reads data point
Natural logrithm absolute value be greater than 2.0 or the gene less than 0.5 as difference expression gene.
6, statistical procedures
Data analysis is carried out using 13.0 statistical software of SPSS, group difference compares using one-way analysis of variance method, P<
0.05 difference has significant.
7, result
(as shown in Figure 1) as the result is shown, is compared with normal people, the mRNA water of DNASE2 gene in Parkinson's disease patients blood
Head up display, which writes, to be increased, and difference has statistical significance (P<0.05).
The gene of differential expression in 2 QPCR experimental verification Parkinson's disease patients of embodiment and normal person
1, research object：
Screening criteria is with embodiment 1, Parkinson's disease patients and normal person each 50.
2, in blood total serum IgE extraction
Step is the same as embodiment 1.
3, reverse transcription
Reverse transcription synthesis cDNA is carried out to l μ g total serum IgE with RT Buffer.Using 25 μ l reaction systems, each sample
It takes 1 μ g total serum IgE as template ribonucleic acid, following components is separately added into PCR pipe：DEPC water, 5 × RT Buffer,
10mmol/L dNTP, 0.1mmol/l DTT, 30 μm of mol/l Oligo dT, 200U/ μ l M-MLV, template ribonucleic acid.42 DEG C of incubations
1h, 72 DEG C of 10min, of short duration centrifugation.
4、QPCR
(1) design of primers
QPCR amplimer is designed according to the coded sequence of DNASE2 gene and GAPDH gene in Genbank, is given birth to by Shanghai
The synthesis of work biotechnology Services Co., Ltd.Specific primer sequence is as follows：
DNASE2 gene：
Forward primer is 5 '-CAGCATCACACTCACATC-3 ' (SEQ ID NO.1)；
Reverse primer is 5 '-AGTACAGGTCATCTCCAA-3 ' (SEQ ID NO.2),
GAPDH gene：
Forward primer is 5 '-TTTAACTCTGGTAAAGTGGATAT-3 ' (SEQ ID NO.3)；
Reverse primer is 5 '-GGTGGAATCATATTGGAACA-3 ' (SEQ ID NO.4).
(2) PCR reaction system is prepared according to table 1：
Wherein, SYBR Green polymerase chain reaction system is purchased from Invitrogen company.
1 PCR reaction system of table
| Reagent | Volume |
| Forward primer | 1μl |
| Reverse primer | 1μl |
| SYBR Green polymerase chain reaction system | 12.5μl |
| Template | 2μl |
| Deionized water | Supply 25 μ l |
(3) PCR reaction condition：95 DEG C of 10min, (95 DEG C of 10s, 60 DEG C of 40s) * 45 circulations.Using SYBR Green as
Fluorescent marker carries out PCR reaction on Light Cycler fluorescence quantitative PCR instrument, true by melt curve analysis analysis and electrophoresis
Determine purpose band, Δ Δ CT method carries out relative quantification.
5, statistical method
Result data is indicated in a manner of mean+SD, is united using SPSS13.0 statistical software
Meter analysis, difference between the two is examined using t, it is believed that works as P<There is statistical significance when 0.05.
6, result
As a result as shown in Fig. 2, being compared with normal people, the mRNA level in-site of DNASE2 gene is significant in Parkinson's disease patients blood
Increase, difference has statistical significance (P<0.05), as a result homogenic array experiment.
3 immunoblot experiment of embodiment verifies the expression product of difference expression gene in Parkinson's disease patients and normal person
1, clinical subjects：With embodiment 2.
2, monocyte separates
Parkinson's disease patients and normal person extracting vein blood 10ml, injection are contained in the sterile vials of heparin, light immediately after capping
Jog is even.Isometric HBSS (NaCl 8.0g, Na is added with aseptic straw2HPO40.132g, KH2PO40.06g, KCl
0.4g, phenol red 1ml, NaHCO30.35g, D-Glucose 1.0g are dissolved in 1000ml distilled water), to reduce the cohesion of red blood cell.
It draws 8ml lymphocytes separating solution to set in 50ml centrifuge tube, dilute blood is slowly added to along tube wall, keep interface to understand, not
It mixes the two mutually, is centrifuged 30min, the careful canescence for drawing layering liquid with blood plasma handover position muddiness in 20 DEG C of 2000r/min
Layer, i.e. buffy coat are added in another centrifuge tube, wash 2 times with the HBSS of 5 times of volumes, successively with 2000r/min,
1500r/min is centrifuged 10min at room temperature, the blood platelet largely mixed to remove, with 10ml distilled water and cell mass
1min is mixed, residual red blood cells are cracked, is then rapidly added equivalent 1.8%NaCl solution, supernatant is removed in 2000r/min centrifugation,
Cell is adjusted to 1 × 10 with HBSS solution after cell count6A/ml is spare.
3, monocyte gross protein extracts
By cell suspension obtained by above-mentioned experiment, (concentration is 1 × 106A/ml) room temperature 1 000r/min be centrifuged 10min, in abandoning
It is added 100 μ l lysis buffers after clear, 4 DEG C of concussion 1h, with ultrasonoscope smudge cells, each 10s, totally 10 times, in 4 DEG C
12000r/min is centrifuged 1h；Supernatant Brandford standard measure albumen is taken, is distributed into 2.5 μ g/ μ l, -80 DEG C of refrigerators save standby
With.
4, Western blot is detected
Total protein of cell Brandford standard measure takes to mix with sample buffer in right amount and boils 5min, cooling 5min；
It takes 30pg albumen to be loaded to 15% polyacrylamide gel prepared, carries out electrophoresis, start to be set as 80V constant pressure, see
120V is increased to after Marker；Glue after electrophoresis is taken out, shifts 50min in 100V using the half-dried transferring system of Bio.Rad；Turn
It after film, is washed once with 1xPBS, immerses confining liquid, 40C is overnight；Confining liquid is outwelled, Western cleaning solution is added and washs 5-
10min is added primary antibody shaking table room temperature and hybridizes 2h；Block buffer is diluted in Western secondary antibody diluent according to proper proportion
In, it is incubated for 60min；Film washing liquid is washed 3 times, each 10min；Use the development of ECL reagent, fixing detection protein expression.
5, statistical procedures
The gray value of protein band is analyzed using Image J software, using β-actin as internal reference, by purpose informal voucher
The gray value of band is normalized.Result data is indicated in a manner of mean+SD, is used
SPSS13.0 statistical software is next for statistical analysis, and difference between the two is examined using t, it is believed that works as P<There is system when 0.05
Meter learns meaning.
6, result
The results show that with the albumen relative expression levels in normal person's group blood for 1, DNASE2 in parkinsonism group blood
Protein level is 5.13 ± 1.57, significant to increase, and difference has statistical significance (P<0.05).
The explanation of above-described embodiment is only intended to understand method and its core concept of the invention.It should be pointed out that for this
For the those of ordinary skill in field, without departing from the principle of the present invention, several improvement can also be carried out to the present invention
And modification, these improvement and modification will also be fallen into the protection scope of the claims in the present invention.
Sequence table
<110>Beijing Yang Shen biology information technology Co., Ltd
<120>Purposes of the DNASE2 as parkinsonism diagnosis marker
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<170> SIPOSequenceListing 1.0
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cagcatcaca ctcacatc 18
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<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
agtacaggtc atctccaa 18
<210> 3
<211> 23
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
tttaactctg gtaaagtgga tat 23
<210> 4
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
ggtggaatca tattggaaca 20
Claims (8)
1. detecting application of the product of DNASE2 gene expression in the tool of preparation diagnosis parkinsonism.
2. application according to claim 1, which is characterized in that the product includes：By RT-PCR, real-time quantitative PCR,
Immune detection, in situ hybridization, chip or high-flux sequence detection of platform DNASE2 gene expression dose are to diagnose parkinsonism
Product.
3. application according to claim 2, which is characterized in that the product with RT-PCR diagnosis parkinsonism at least wraps
Include the primer of a pair of of specific amplified DNASE2 gene；The product with real-time quantitative PCR diagnosis parkinsonism includes at least one
To the primer of specific amplified DNASE2 gene；It is described with immune detection diagnosis parkinsonism product include：With DNASE2 albumen
The antibody of specific binding；It is described in situ hybridization diagnosis parkinsonism product include：With the nucleic acid sequence of DNASE2 gene
The probe of hybridization；It is described with chip diagnosis parkinsonism product include：Protein chip and genetic chip；Wherein, protein chip
Including the antibody in conjunction with DNASE2 protein-specific, genetic chip includes the spy with the nucleic acid array hybridizing of DNASE2 gene
Needle.
4. application according to claim 3, which is characterized in that the product with real-time quantitative PCR diagnosis parkinsonism
The primer of a pair of of the specific amplified DNASE2 gene included at least is as shown in SEQ ID NO.1 and SEQ ID NO.2.
5. a kind of for diagnosing the tool of parkinsonism, which is characterized in that the tool can pass through DNASE2 in detection sample
The expression of gene diagnoses parkinsonism；The tool includes chip, kit, test paper or high-flux sequence platform；The core
Piece includes genetic chip, protein-chip；The genetic chip includes solid phase carrier and the few nucleosides for being fixed on solid phase carrier
Acid probe, the oligonucleotide probe include the few nucleosides for DNASE2 gene for detecting DNASE2 gene transcription level
Acid probe；The protein-chip include solid phase carrier and be fixed on solid phase carrier DNASE2 albumen specific antibody；
The kit includes gene detecting kit and protein immunization detection kit；The gene detecting kit includes for examining
Survey the reagent of DNASE2 gene transcription level；The protein immunization detection kit includes the specific antibody of DNASE2 albumen；
The test paper includes the reagent for detecting DNASE2 gene transcription level；The high-flux sequence platform includes for detecting
The reagent of DNASE2 gene transcription level.
6. tool according to claim 5, which is characterized in that it is described detection DNASE2 gene transcription level reagent include
For the primer or probe of DNASE2 gene.
7. tool according to claim 6, spy are characterized in that, the primer sequence for DNASE2 gene is as follows：
Forward primer sequence is as shown in SEQ ID NO.1, and reverse primer is as shown in SEQ ID NO.2.
8. the tool according to any one of claim 5-7, which is characterized in that the sample is blood.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711477253.8A CN107904304B (en) | 2017-12-29 | 2017-12-29 | Purposes of the DNASE2 as parkinsonism diagnosis marker |
| PCT/CN2018/123794 WO2019129029A1 (en) | 2017-12-29 | 2018-12-26 | Gene as marker for diagnosing and treating parkinson's disease and use thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711477253.8A CN107904304B (en) | 2017-12-29 | 2017-12-29 | Purposes of the DNASE2 as parkinsonism diagnosis marker |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107904304A CN107904304A (en) | 2018-04-13 |
| CN107904304B true CN107904304B (en) | 2018-11-20 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN201711477253.8A Active CN107904304B (en) | 2017-12-29 | 2017-12-29 | Purposes of the DNASE2 as parkinsonism diagnosis marker |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110055332A (en) * | 2019-05-16 | 2019-07-26 | 中国人民解放军联勤保障部队第九六0医院 | Marker relevant to thyroid cancer and its application |
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| WO2007046103A2 (en) * | 2005-10-19 | 2007-04-26 | Perf-Action Technologies Ltd. | System for iontophoretic transdermal delivery of polymeric agents and methods of use thereof |
| US8772468B2 (en) * | 2011-09-22 | 2014-07-08 | Rosalind Franklin University Of Medicine And Science | Splice variant specific messenger RNA transcripts as biomarkers of parkinson's disease |
| CN105385774B (en) * | 2015-12-24 | 2018-09-25 | 周丽丽 | Purposes of the TMF1 as parkinsonism diagnosis marker |
| CN105506083B (en) * | 2015-12-24 | 2018-10-19 | 孙梅芬 | CAPG is preparing the purposes in diagnosing parkinsonism product |
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2017
- 2017-12-29 CN CN201711477253.8A patent/CN107904304B/en active Active
Non-Patent Citations (1)
| Title |
|---|
| L-DNase II, a molecule that links proteases and endonucleases in apoptosis, derives from the ubiquitous serpin leukocyte elastase inhibitor;Alicia Torriglia et al.;《Molecular And Cellular Biology》;19980630;第18卷(第6期);第3612-3619、4947页 * |
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| CN107904304A (en) | 2018-04-13 |
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Effective date of registration: 20191210 Address after: Room 2503, Qianshan building, building D2, Qingdao International Innovation Park Phase II, No. 1, Keyuan Weiyi Road, Laoshan District, Qingdao, Shandong Province Patentee after: Qingdao Yangshen biomedical Co., Ltd Address before: 100080 Beijing city Haidian District Shanyuan Street No. 1 cubic court building room 3103 Patentee before: Beijing Yang Shen biology information technology company limited |