CN108048554B - The molecular marker that THBD gene is diagnosed as parkinsonism - Google Patents
The molecular marker that THBD gene is diagnosed as parkinsonism Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- 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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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
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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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/50—Determining the risk of developing a disease
Abstract
The invention discloses purposes of the THBD gene in diagnosis parkinsonism.There are significant differences before THBD gene content and normal person in the blood of Parkinson's disease patients, can distinguish Parkinson's disease patients and non-Parkinson's disease patients according to difference.The invention also discloses the diagnostic products of parkinsonism, which can realize the diagnostic purpose of parkinsonism by THBD gene in detection blood.
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-THBD gene in the product of preparation diagnosis parkinsonism in blood.
Background technique
Parkinson's disease (Parkinson ' s disease；It PD) is the Common neurologic for occurring mainly in mid-aged population
Degenerative disease.With the aggravation of global aging, the diseased colonies of Parkinson's disease constantly expand, it not only seriously affect patient and
The quality of life of household also brings increasingly heavier financial burden for society.
As most commonly seen neurodegenerative movement disorder disease, Parkinson's disease (PD) is characterized by several movements
In symptom, for example static tremor, myotonia, position are unstable, while including some non-motor symptoms, such as autonomic disorder,
Insanity, sensory disturbance, cognitive disorder and dementia (Volta M, Milnerwood A J, Farrer M J.Insights
from late-onset familial parkinsonism on the pathogenesis of idiopathic
Parkinson's disease[J].The Lancet Neurology,2015,14(10):1054-1064).PD is as a kind of
Complicated neurological disease and the neurodegenerative disease the most main for being only second to Alzheimer's disease, affect about 1% it is super
Spend 60 years old elderly population (Samii A, Nutt J G, Ransom B R.Parkinson's disease [J] .Lancet,
2004,363 (9423): 1783-1793), and in the world, cause every year more than 100,000 people death (Mortality G
B D,Causes of Death C.Global,regional,and national age-sex specific all-cause
and cause-specific mortality for 240 causes of death,1990-2013:a systematic
analysis for the Global Burden of Disease Study 2013[J].Lancet,2015,385
(9963):117-171).PD not only reduces the quality of life of patient and its care-giver, also causes very heavy warp to society
Ji burden.According to conservative estimation, only in the U.S., it is up to 23,000,000,000 dollars every year by the financial burden that PD causes.PD is as a kind of
Chronic, progressive disease, non-athletic property symptom, which typically precedes dyskinesia, to be occurred for many years.Although the pathological characteristics of PD are
Lewy body cynapse core egg in the generation of the nigrostriatal dopamine serotonergic neuron of degeneration and activity deficiency and neuronal cell
White and other protein accumulations, other neurotransmitters in addition to dopamine and other brain areas also assist in PD occurrence and development
In the process.Parkinson's disease is being considered mainly being caused and being influenced by environmental factor before, but it has recently been demonstrated that the disease
Derived from complicated interaction (Kalia L V, the Lang A E.Parkinson's disease of inherent cause and environmental factor
[J].Lancet,2015,386(9996):896-912).It is past during the decade, several researchs are from animal model, gene table
It reaches, the angle of whole-genome association (GWAS) and systems biology, be dedicated to understanding the pathogenic and molecule mechanism of PD and take
Obtain some achievements.The discovery of several potential core proteins, as leucine-rich repeat kinase 2 (LRRK2),
PTEN-induced putative kinase 1(PINK1)、parkin(PARK2)、PARK7、ubiquitin carboxyl-
Terminal esterase L1 (UCHL1), glucocerebrosidase (GBA) and alpha-synuclein (SNCA) are
Understand that its molecule mechanism has established important basis.Nevertheless, the pathogenesis of Parkinson's disease be still it is unilateral, local,
Distance thoroughly understands its pathogenesis, and there are also longer stretch.
The complexity of PD is also embodied in challenge clinically.Up to the present, there is not yet and PD early stage is determined
The diagnostic test of diagnosis.Gene expression regulation network system obtains noticeable progress in cancer research field, many swollen
Such as lung cancer, breast cancer, gastric cancer, colorectal cancer in tumor tissue, the expression of certain genes and its source normal tissue exist
Stable and apparent difference.The tumour of different tissue sources or even same tissue-derived different differentiation states plus tumor, gene table
It is all not quite similar up to spectrum, shows that gene expression has apparent tissue specificity and stage of development specificity.Because of neurological
Property disease pathology on be directly characterized in the overexpression of disease specific protease matter, so speculating that the exception of specific gene changes
The expression of functional protein is become, so as to cause disease.More and more research discovery specific genes can become
The biomarker for diagnosing Parkinson, 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:
The present invention provides application of the product of detection THBD gene expression in the tool of preparation diagnosis parkinsonism.
Further, the product of detection THBD gene expression mentioned above include: by RT-PCR, real-time quantitative PCR,
Immune detection, in situ hybridization, chip or high-flux sequence detection of platform THBD gene expression dose are to diagnose the production of parkinsonism
Product.
Further, the product with RT-PCR diagnosis parkinsonism includes at least drawing for a pair of of specific amplified THBD gene
Object；The product with real-time quantitative PCR diagnosis parkinsonism includes at least the primer of a pair of of specific amplified THBD gene；It is described
Product with immune detection diagnosis parkinsonism includes: the antibody in conjunction with THBD protein-specific；It is described to be examined in situ hybridization
The product of disconnected parkinsonism includes: the probe with the nucleic acid array hybridizing of THBD gene；It is described to diagnose parkinsonism with chip
Product includes: protein chip and genetic chip；Wherein, protein chip includes the antibody in conjunction with THBD protein-specific, gene
Chip includes the probe with the nucleic acid array hybridizing of THBD 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 THBD 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 THBD gene expression can be applied to the platform and realize to THBD base
The detection of the expression of cause.With the development of high throughput sequencing technologies, the building of the gene expression profile of a people will be become
Very easily work.Which by comparing the gene expression profile of Disease and normal population, it is easy to analyze the different of gene
It is often related to disease.Therefore, know that the exception of THBD gene is related to parkinsonism in high-flux sequence and also belong to THBD base
The purposes of cause, 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 THBD gene transcription level
The oligonucleotide probe of THBD gene；The protein-chip includes solid phase carrier and the THBD albumen for being fixed on solid phase carrier
Specific antibody；The genetic chip can be used for detecting multiple genes including THBD gene (for example, and parkinsonism
Relevant multiple genes) expression.The protein-chip can be used for detecting multiple albumen including THBD albumen
The expression of matter (such as multiple protein relevant to parkinsonism).By by multiple markers with parkinsonism simultaneously
Detection 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 THBD gene transcription level；The protein immunization detection kit includes the spy of THBD albumen
Heterogenetic antibody.Further, the reagent includes using RT-PCR, real-time quantitative PCR, immune detection, in situ hybridization or chip side
Method detects reagent needed for THBD gene expression dose process.Preference, the reagent include the primer for THBD gene
And/or probe.It is easy to design according to the nucleotide sequence information of THBD gene and can be used for detecting THBD gene expression dose
Primer and probe.
It can be DNA, RNA, DNA-RNA chimera with the probe of the nucleic acid array hybridizing of THBD gene, PNA or other spreads out
Biology.There is no limit appoint as long as completing specific hybrid, specifically binding with purpose nucleotide sequence the length of the probe
What length is ok.The length of the probe can be as short as 25,20,15,13 or 10 bases longs.Equally, the length of the probe
Degree can be grown to 60,80,100,150,300 base-pairs or longer or even whole genes.Since different probe lengths is to hybridization
Efficiency, signal specificity have different influences, and the length of the probe is typically at least 14 base-pairs, and longest is usually no more than
30 base-pairs, the length complementary with purpose nucleotide sequence are best with 15-25 base-pair.The probe self-complementary sequences
Most preferably less than 4 base-pairs, in order to avoid influence hybridization efficiency.
The high-flux sequence platform includes the reagent for detecting THBD 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 THBD gene.
Further, the specific antibody of the THBD albumen includes monoclonal antibody, polyclonal antibody.The THBD albumen
Specific antibody 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 THBD albumen.Antibody for protein level
Preparation when well known to a person skilled in the art and the present invention may use any method to prepare the antibody.
In specific embodiments of the present invention, the primer sequence for THBD gene is as follows: forward primer sequence
As shown in SEQ ID NO.1, reverse primer is as shown in SEQ ID NO.2.
Source for the THBD gene and its expression product that diagnose parkinsonism include but is not limited to blood, tissue fluid,
Urine, saliva, spinal fluid etc. can obtain the body fluid of genomic DNA.In specific embodiments of the present invention, for diagnosing pa
The THBD gene of the gloomy disease of gold and its source of expression product are blood.
In the context of the present invention, " THBD gene " includes any functional equivalent of THBD gene and THBD gene
Polynucleotides.THBD gene (Chromosome 20, NC_000020.11 (23045633..23049664,
Complement)) sequence can inquire in international public GenBank GeneBank.
In the context of the present invention, THBD gene expression product includes the partial peptide of THBD albumen and THBD albumen.
The partial peptide of the THBD albumen contains functional domain relevant to parkinsonism.
" THBD albumen " includes any functional equivalent of THBD albumen and THBD albumen.The functional equivalent includes
THBD albumen conservative variation protein or its active fragment or its reactive derivative, allelic variant, lure at natural mutation
Lead mutant, can be with the encoded protein of DNA of the DNA hybridization of THBD 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 the fusion of THBD albumen
Albumen.For the peptide or protein with THBD protein fusion, there is no limit as long as resulting fusion protein retains THBD albumen
Biological activity.
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 THBD gene expression is related to parkinsonism, pass through the table of THBD in detection subject
It reaches, it can be determined that whether subject suffers from parkinsonism or judges that subject whether there is the risk with parkinsonism, from
And clinician is instructed to provide prevention scheme or therapeutic scheme to subject.
Present invention finds a kind of new molecular marked compound-THBD genes, and compared to traditional detection means, gene diagnosis is 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 THBD gene in Parkinson's disease patients and normal person；Fig. 2
Differential expression of the display using QPCR detection THBD 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
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: choosing healthy volunteer 10, men and women each 5 of the age 32-80 years old.
Age, gender differences are not statistically significant (P > 0.10) between two groups, are 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: being indicated by RNA integrality, can use plain agar sugar gel electrophoresis (deposition condition: 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, 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 level in-site of THBD gene in Parkinson's disease patients blood
Significant to increase, 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 THBD gene and GAPDH gene in Genbank, by the raw work in Shanghai
The synthesis of biotechnology Services Co., Ltd.Specific primer sequence is as follows:
THBD gene:
Forward primer is 5 '-CTCAATGCCAGTCAGATC-3 ' (SEQ ID NO.1)；
Reverse primer is 5 '-GTTCAGTAGCAAGGAAATG-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 has statistical significance as P < 0.05.
6, result
As a result as shown in Fig. 2, being compared with normal people, the mRNA level in-site of THBD gene significantly increases in Parkinson's disease patients blood
Add, 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 has system as P < 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, THBD egg in parkinsonism group blood
White level is 4.14 ± 1.12, 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
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<120>molecular marker that THBD gene is diagnosed as parkinsonism
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Claims (10)
1. detecting application of the product of THBD gene expression in the tool of preparation diagnosis parkinsonism.
2. application according to claim 1, which is characterized in that the product include: by RT-PCR, real-time quantitative PCR,
Immune detection, in situ hybridization, chip or high-flux sequence detection of platform THBD 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 THBD gene；The product with real-time quantitative PCR diagnosis parkinsonism includes at least one
To the primer of specific amplified THBD gene；It is described with immune detection diagnosis parkinsonism product include: and THBD albumen
The antibody of specific binding；The product in situ hybridization diagnosis parkinsonism includes: the nucleic acid sequence with THBD gene
The probe of hybridization；The product with chip diagnosis parkinsonism includes: protein chip and genetic chip；Wherein, protein chip
Including the antibody in conjunction with THBD protein-specific, genetic chip includes the spy with the nucleic acid array hybridizing of THBD gene
Needle.
4. application according to claim 3, which is characterized in that the product with real-time quantitative PCR diagnosis parkinsonism
Including at least the primer of a pair of of specific amplified THBD gene, sequence is as shown in SEQ ID NO.1 and SEQ ID NO.2.
5. application according to claim 1, which is characterized in that the tool can pass through THBD gene in detection sample
Expression diagnose parkinsonism.
6. application according to claim 5, which is characterized in that the tool includes chip, kit, test paper or high throughput
Microarray dataset.
7. application according to claim 6, which is characterized in that the chip includes genetic chip, protein-chip；It is described
Genetic chip includes solid phase carrier and the oligonucleotide probe for being fixed on solid phase carrier, and the oligonucleotide probe includes being used for
Detect the oligonucleotide probe for THBD gene of THBD gene transcription level；The protein-chip includes that solid phase carries
Body and be fixed on solid phase carrier THBD albumen specific antibody；The kit includes gene detecting kit and egg
White immunity detection reagent；The gene detecting kit includes the reagent for detecting THBD gene transcription level；It is described
Protein immunization detection kit includes the specific antibody of THBD albumen；The test paper includes turning for detecting THBD gene
Record horizontal reagent；The high-flux sequence platform includes the reagent for detecting THBD gene transcription level.
8. application according to claim 7, which is characterized in that it is described detection THBD gene transcription level reagent include
For the primer and/or probe of THBD gene.
9. application according to claim 8, spy are characterized in that, the primer sequence for THBD 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.
10. the application according to any one of claim 5-9, which is characterized in that the sample is blood.
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| CN201711483559.4A CN108048554B (en) | 2017-12-29 | 2017-12-29 | The molecular marker that THBD gene is diagnosed as parkinsonism |
| PCT/CN2018/123794 WO2019129029A1 (en) | 2017-12-29 | 2018-12-26 | Gene as marker for diagnosing and treating parkinson's disease and use thereof |
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| CN201711483559.4A CN108048554B (en) | 2017-12-29 | 2017-12-29 | The molecular marker that THBD gene is diagnosed as parkinsonism |
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| WO2019129029A1 (en) * | 2017-12-29 | 2019-07-04 | 北京泱深生物信息技术有限公司 | Gene as marker for diagnosing and treating parkinson's disease and use thereof |
| CN109468373A (en) * | 2018-12-24 | 2019-03-15 | 潘伟 | A kind of biomarker relevant to Parkinson's occurrence and development |
Citations (1)
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| JPH101439A (en) * | 1996-06-11 | 1998-01-06 | Mochida Pharmaceut Co Ltd | Therapeutic agent for neurodegenerative disease |
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| EP2593567B1 (en) * | 2010-07-15 | 2016-04-20 | The Regents of The University of California | Biomarkers for diagnosis of stroke and its causes |
| CN103911438A (en) * | 2014-03-13 | 2014-07-09 | 眭维国 | Analyzing method of differential expression gene of uremia hydroxymethylation status and application thereof |
| EP3233115B1 (en) * | 2014-12-19 | 2021-02-17 | Children's Hospital Medical Center | Methods and compositions related to transplant-associated thrombotic microangiopathy |
| US9970056B2 (en) * | 2015-02-25 | 2018-05-15 | Rosalind Franklin University Of Medicine And Science | Methods and kits for diagnosing, prognosing and monitoring parkinson's disease |
| CN104975104B (en) * | 2015-07-31 | 2018-06-19 | 北京泱深生物信息技术有限公司 | Alzheimer disease diagnosis and treatment marker and its application |
| CN105018484B (en) * | 2015-07-31 | 2017-10-13 | 北京泱深生物信息技术有限公司 | CRTAP genes and its expression product as Alzheimer disease diagnosis and treatment target |
| CN105331690B (en) * | 2015-10-28 | 2019-10-11 | 北京泱深生物信息技术有限公司 | Application of the EPB41L4B gene in Parkinson's disease diagnosis and treatment |
| CN105506083B (en) * | 2015-12-24 | 2018-10-19 | 孙梅芬 | CAPG is preparing the purposes in diagnosing parkinsonism product |
| CN105385774B (en) * | 2015-12-24 | 2018-09-25 | 周丽丽 | Purposes of the TMF1 as parkinsonism diagnosis marker |
| CN106591461A (en) * | 2016-12-29 | 2017-04-26 | 天津协和华美医学诊断技术有限公司 | Detection kit for detecting hereditary thrombophilia related gene group |
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| JPH101439A (en) * | 1996-06-11 | 1998-01-06 | Mochida Pharmaceut Co Ltd | Therapeutic agent for neurodegenerative disease |
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| Identification of Neurodegenerative Factors Using Translatome-Regulatory Network Analysis;L.Brichta等;《Nat.Neurosci.》;20150930;第18卷(第9期);1325-1333 |
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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 |