CN106435009B - Purposes of the TUBB2A as Blood diagnosis marker - Google Patents
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Abstract
Purposes the invention discloses TUBB2A as Blood diagnosis marker, more specifically, the purposes the invention discloses TUBB2A as the marker of diagnosis Bgudd-Chiari Syndrome.The present invention demonstrates TUBB2A gene by high-flux sequence and PCR experiment and has differences expression in the blood of Bgudd-Chiari Syndrome patient and normal person, accordingly using TUBB2A gene as the molecular marker of diagnosis Bgudd-Chiari Syndrome.Present invention finds a kind of method of new diagnosis Bgudd-Chiari Syndrome, the diagnostic method is noninvasive and can judge in disease early stage, is conducive to the survival rate for improving patient.
Description
Technical field
The present invention relates to medical diagnosis on disease fields, more particularly it relates to which TUBB2A is in Bgudd-Chiari Syndrome diagnosis
Using.
Background technique
Budd-Chiari syndrome (Budd-Chiari syndrome, BCS) is by vena hepatica and (or) its opening with upper section cavity of resorption
Property portal hypertension after a kind of liver with the characteristics of inferior vena cava syndrome is often accompanied by caused by vein occlusion lesion.From global model
From the point of view of enclosing, BCS disease incidence is lower, and the cause of disease has apparent regional difference, and western countries are common with hepatic venous obstruction type BCS, greatly
There are the specific cause of disease, such as oral contraceptive, gestation, blood disease, it has been reported that vena hepatica type BCS and gene more
High blood coagulation state caused by being mutated, Hepatic venous thrombosis are related；And in Asia and Southern African region then with IVC obstructive type BCS
Common, pathogenic factor is mostly unclear.
BCS can cause liver damage, portal hypertension, and it is serious to even result in cirrhosis, hepatic failure, upper gastrointestinal bleeding etc.
Complication, Nature prognosis is very poor, and survival rate is very low within 5 years, and the non-operative treatment death rate is high.The clinical manifestation of BCS patient's early stage is hidden
Hide, no specificity, and lack the effective ways of early stage diagnosis and treatment, patient when medical more belong to advanced stage, complicated clinical manifestation multiplicity is held
Easily cause mistaken diagnosis, misdiagnosis.The country focuses mostly in terms of clinical treatment to the research of BCS, in contrast epidemiology and teiology
Research lag, lack the whole nation generaI investigation and Study of Etiology.Therefore, it carries out BCS Study of Etiology in a deep going way, improves the early stage of BCS
Diagnosis is the critical issue for improving BCS survival of patients and prognosis.
Summary of the invention
The present invention relates to a kind of diagnosis marker of Bgudd-Chiari Syndrome, the present invention is experimentally confirmed to be suffered from Bgudd-Chiari Syndrome
TUBB2A gene mRNA content is significantly higher than normal population in the blood of person, therefore the property of the differential expression of TUBB2A gene makes
It, which becomes, can be used to diagnose the molecular marker whether subject suffers from Bgudd-Chiari Syndrome.
Specifically, the present invention provides the products of detection TUBB2A to prepare the application in Bgudd-Chiari Syndrome diagnostic tool.
Further, the product of the detection TUBB2A includes the product of TUBB2A gene expression amount in test sample.
Further, the product of the detection TUBB2A includes the product that can quantify TUBB2A gene mRNA in sample,
And/or the product of TUBB2A albumen in sample can be quantified.
In quantitative sample of the invention the product of TUBB2A gene mRNA can based on use the known method of nucleic acid molecules come
Play its function: as PCR, such as Southern hybridization, Northern hybridization, dot blot, fluorescence in situ hybridization (FISH), DNA are micro-
Array, ASO method, high-flux sequence platform etc..It can qualitatively, quantitatively or semi-quantitatively implement analysis using the product.
Include that nucleic acid in the said goods can be obtained by chemical synthesis, or by containing from biomaterial preparation
It is expected that the gene of nucleic acid, then using primer amplification designed for amplification expectation nucleic acid, it is obtained.
Further, the PCR method is known method, for example, ARMS (Amplification Refractory
Abruptly-changing system is not answered in Mutation System, amplification) method, RT-PCR (reverse transcriptase-PCR) method, nesting PCR method etc..Amplification
Nucleic acid can be by using dot blotting hybridization method, Surface Plasmon Resonance (SPR method), PCR-RFLP method, original position RT-PCR
Method, PCR-SSO (sequence specific oligonucleotide) method, PCR-SSP method, AMPFLP (amplifiable fragment length polymorphism) method,
MVR-PCR method and PCR-SSCP (single-strand conformation polymorphism) method detect.
The product that TUBB2A gene mRNA in sample can be quantified includes specific amplified used in real-time quantitative PCR
The primer of TUBB2A gene, the primer sequence is as shown in SEQ ID NO.1 and SEQ ID NO.2.
The primer for including in product can be prepared by chemical synthesis, by using those skilled in the art will know that side
Method is suitably designed with reference to Given information, and is prepared by chemical synthesis.
Nucleic acid recited above may also include probe, and the probe can be prepared by chemical synthesis, by using this
The method that field technical staff knows appropriately is designed with reference to Given information, and is prepared by chemical synthesis, or can lead to
The gene for containing desired nucleic acid sequence from biomaterial preparation is crossed, and is expanded using the primer designed for amplification expectation nucleic acid sequence
Increase it to prepare.
The product of TUBB2A albumen can play its function based on the known method of antibody is used in quantitative sample of the invention
Can: for example, may include ELISA, radioimmunoassay, immunohistochemical method, western blot etc..
In quantitative sample of the invention the product of TUBB2A albumen include specifically bind TUBB2A albumen antibody or its
Segment.The antibody or its segment of any structure and size, immunoglobulin class, origin etc. can be used, as long as it combines target egg
White matter.The antibody or its segment for including in testing product of the invention can be monoclonal or polyclonal.Antibody piece
Section, which refers to, retains peptide of the antibody to the active antibody of the combination of antigen a part of (Partial Fragment) or containing antibody a part.Antibody piece
Section may include F (ab ')2, Fab ', Fab, scFv (scFv), disulphide bonding Fv (dsFv) or its polymer, dimerization
Change the area V (double antibody) or the peptide containing CDR.The product of TUBB2A albumen may include that coding is anti-in quantitative sample of the invention
The isolated nucleic acid of the amino acid sequence of body or Encoding Antibody Fragment, the carrier comprising the nucleic acid, and carry the cell of the carrier.
Antibody can be obtained by the way that well known to a person skilled in the art methods.For example, preparation retains target all or in part
The mammalian cell expression vector of their polynucleotides of the polypeptide or integration coding of protein is as antigen.Exempted from using antigen
After epidemic disease animal, from the immune animal adaptive immune cell of process and myeloma cell is merged to obtain hybridoma.Then from hybridization
Tumor culture collects antibody.It finally can be real by using antibody of the TUBB2A albumen for being used as antigen or part thereof to acquisition
Antigentic specificity purifying is applied to obtain the monoclonal antibody for TUBB2A albumen.Polyclonal antibody can be prepared as follows: with
Identical antigen-immunized animal above collects blood sample from by immune animal, serum is isolated from blood, is then made
Implement antigentic specificity to serum with above-mentioned antigen to purify.It can be by the antibody that is obtained with enzymatic treatment or by using acquisition
The sequence information of antibody obtains antibody fragment.
The combination of marker and antibody or its segment can be implemented by method as commonly known in the art.For example, can
With following fluorescent marker protein or peptide: clean protein or peptide with phosphate buffer, addition DMSO, buffer, etc. standards
Standby dyestuff, then mixed solution, then at being placed at room temperature for 10 minutes.In addition, the labelling kit of commercialization can be used in label, it is all
Such as biotin labeling reagent box, such as biotin labeling reagent box-NH2, biotin labeling reagent box-SH
(DojindoLaboratories)；Alkali phosphatase enzyme mark kit such as alkali phosphatase enzyme mark kit-NH2, alkaline phosphorus
Sour enzyme labelling kit-SH (Dojindo Laboratories)；Peroxidase labelling kit such as peroxidase mark
Remember kit-NH2, peroxidase labelling kit-NH2 (Dojindo Laboratories)；Phycobniliprotein labelled reagent
Box such as phycobniliprotein labelling kit-NH2, phycobniliprotein labelling kit-SH, B- phycoerythrin labelling kit-NH2,
B- phycoerythrin labelling kit-SH, R-PE labelling kit-NH2, R-PE labelling kit SH
(DojindoLaboratories)；Fluorescent labeling reagent box such as fluorescein labelling kit-NH2, HiLyte Fluor (TM)
555 labelling kit-NH2,647 labelling kit-NH2 of HiLyte Fluor (TM) (Dojindo Laboratories)；And
DyLight 547 and DyLight647 (Techno Chemical Corp.), Zenon (TM), Alexa Fluor (TM) antibody
Labelling kit, Qdot (TM) antibody labeling kit (Invitrogen Corporation) and EZ- marker protein mark
Remember kit (Funakoshi Corporation).For correct labeling, suitable instrument can be used to detect by label
Antibody or its segment.
As the sample according to testing product of the invention, the tissue sample for example obtained from biopsy subject can be used
Or fluid.Sample is not particularly limited, as long as it is suitable for measurement of the invention；For example, it may include tissue, blood, blood plasma,
Serum, lymph, urine, serous cavity liquid, spinal fluid, synovia, aqueous humor, tear, saliva or its fraction or treated material
Material.
In specific embodiments of the present invention, the sample source is in blood.
Further, the product of the quantitative TUBB2A gene or TUBB2A albumen can be detection TUBB2A gene or
The reagent of TUBB2A albumen is also possible to include kit, chip, test paper of the reagent etc., is also possible to using the examination
The high-flux sequence platform of agent.
The present invention also provides a kind of tool for diagnosing Bgudd-Chiari Syndrome, the tool is able to detect TUBB2A.
Further, the tool is able to detect TUBB2A gene expression amount in sample.
Further, the tool includes the reagent that can quantify TUBB2A gene mRNA in sample, and/or can quantify sample
The reagent of TUBB2A albumen in product.
Further, the reagent that can quantify TUBB2A gene mRNA in sample is used in real-time quantitative PCR
The primer of specific amplified TUBB2A gene, the primer sequence is as shown in SEQ ID NO.1 and SEQ ID NO.2.It is described can
The reagent of TUBB2A albumen includes the antibody in conjunction with TUBB2A protein-specific in quantitative sample.
Further, the tool of the diagnosis Bgudd-Chiari Syndrome includes but is not limited to chip, kit, test paper or high throughput
Microarray dataset；High-flux sequence platform is a kind of tool of special diagnosis Bgudd-Chiari Syndrome, with high throughput sequencing technologies
Development will become very easily work to the building of the gene expression profile of a people.Pass through comparison Disease and normal person
The gene expression profile of group, the exception for being easy to analyze which gene are related to disease.Therefore, know in high-flux sequence
The exception of the TUBB2A gene purposes for also belonging to TUBB2A related to Bgudd-Chiari Syndrome, equally protection scope of the present invention it
It is interior.
The amino acid that anti-TUBB2A antibody used in testing product of the invention, diagnostic tool or its segment are identified
Number is not particularly limited, as long as antibody can combine TUBB2A.
The present invention also provides a kind of methods for diagnosing Bgudd-Chiari Syndrome, and described method includes following steps:
(1) sample of subject is obtained；
(2) expression of TUBB2A gene or albumen in Samples subjects is detected；
(3) it associates whether by the expression of the TUBB2A gene or albumen that measure with the illness of subject.
(4) compared with normal control, the expression of TUBB2A gene or albumen is increased, then the subject is judged and has
Tendency with Bgudd-Chiari Syndrome or suffered from Bgudd-Chiari Syndrome or Bgudd-Chiari Syndrome patient be judged as recurrence or
Person Bgudd-Chiari Syndrome patient is judged as prognosis mala.
In the context of the present invention, " diagnosis Bgudd-Chiari Syndrome " includes judging whether subject has suffered from cloth and added synthesis
Sign, judge subject with the presence or absence of suffer from Bgudd-Chiari Syndrome risk, judge whether Bgudd-Chiari Syndrome patient has been recurred, judged
The prognosis of Bgudd-Chiari Syndrome patient.
The advantages of the present invention:
Of the invention has found a kind of molecular marker for diagnosing Bgudd-Chiari Syndrome, can be in cloth using the molecular marker
The early stage for adding syndrome to occur can judge, and provide the survival rate of patient.
Detailed description of the invention
Fig. 1 shows the statistical chart for detecting TUBB2A gene differential expression situation in mRNA level in-site using QPCR.
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 difference expression gene
1, research object and sample collection
(1) research object: 3 Bgudd-Chiari Syndrome patients and 5 Healthy Volunteers.
(2) sample collection: it is required that at least 12h at room temperature in next morning 7:00~8:00 is extracted research object on an empty stomach
10ml venous blood is added 3 times of volume erythrocyte cracked liquids, is placed at room temperature for 10 after mixing in ethylenediamine tetra-acetic acid (EDTA) anticoagulant tube
Minute, 10,000rpm centrifugations 1 minute.It thoroughly inhales and abandons supernatant, collect leukocyte cell pellet.Every 100-200 μ l blood collection it is white thin
Born of the same parents precipitate be added 1ml TRIzol, -80 DEG C freeze it is spare.
2, the acquisition of total serum IgE
The total serum IgE in blood leucocyte is conventionally extracted using TRIzol.
3, RNA concentration and purity testing
NanoDrop1000 spectrophotometer detects RNA sample, the sample requirement of RNA-seq sequencing, and: OD260/OD280 is
1.8-2.2。
4, the quality analysis (Agilent Technologies 2100Bioanalyzer) of RNA sample
Agilent Technologies 2100Bioanalyzer detects RNA sample quality, observes 28S rRNA and 18S
RRNA master tape is obvious, without degradation, RNA Perfection Index is qualified, concentration reaches requirement meets wanting for sequencing cDNA library building
It asks, can be used for library construction and sequencing.
5, high-throughput transcript profile sequencing
(1) RNA-seq read positions
Low-quality read is removed to obtain cleaning read first, then using TopHat v1.3.1 will clean segment and
UCSC H.sapiens is matched with reference to genome (hg19), the H.sapiens UCSC hg19 editions indexes constructed in advance
It is downloaded from TopHat homepage, and as reference genome, when matching using TopHat with genome, allows each read (default
To 20) having multiple matching sites, most 2 mispairing.TopHat establishes possible according to exon region and GT-AG shear signal
Shearing site library navigates to the read for not navigating to genome on genome according to these shearing site libraries.We use
The system default parameter of TopHat method.
(2) transcript abundance is assessed
The read file matched is handled by Cufflinks v1.0.3, and Cufflinks v1.0.3 is by RNA-seq piece
Number of segment mesh is standardized the relative abundance for calculating transcript.FPKM value refers to being matched in every 1,000,000 sequencing fragment specific
The segment number of the exon region of gene 1kb long.The confidence interval of FPKM estimated value is calculated by Bayesian inference method.
The GTF comment file for the reference that Cufflinks is used downloads (Homo_ from Ensembl database
sapiens.GRCh37.63.gtf)。
(3) detection of difference expression gene
It is transferred to Cuffdiff by the Ensembl GTF file of downloading and by the matched original document of TopHat,
Cuffdiff re-evaluates the gene expression abundance for the transcript listed in GTF file using original matching files, detects difference table
It reaches.The only q value < 0.01 in Cuffidff output, test display is more just considered as successfully differential expression.
6, result
Compare the gene expression difference in Bgudd-Chiari Syndrome patient and normal human blood, share 405 difference expression genes,
317 up-regulations, 88 downwards.Wherein, it is compared with normal people, on TUBB2A gene is expressed in Bgudd-Chiari Syndrome blood samples of patients
It adjusts, mRNA relative expression quantity is 4.71 ± 0.93, and difference has statistical significance (P < 0.05).
2 large sample of embodiment verifies the difference expression gene filtered out
Selection TUBB2A gene is verified.
1, sample collection
Peripheral blood sample each 50 for collecting Bgudd-Chiari Syndrome patient and normal population according to the method for embodiment 1.
2, it is verified in mRNA level in-site
2.1 extract blood total serum IgE
Step is the same as embodiment 1.
2.2 reverse transcription
Reverse transcription uses Primescript 1stStrand cDNA synthesis kit kit, operating procedure are as follows
It carries out:
(1) following reaction liquid is added in microcentrifugal tube, as shown in table 1:
1 reaction liquid of table
| Reagent | Dosage |
| RNA | 2.0μg |
| dNTP | 1.0μl |
| Oligo(dT) | 2.0μl |
| Rnase free dH2O | Add to 10.0 μ l |
(2) 70 DEG C of incubation 5min, are rapidly cooled to 4 DEG C；
Following reaction reagent is added in microcentrifugal tube, reaction system is made:
The preparation of 2 reaction system of table
| Reagent | Dosage |
| 5x1st Strand Synthesis Buffer | 4.0μl |
| PrimeScript RTase | 1.0μl |
| RNase Inhibitor | 1.0μl |
| Rnase free dH2O | 4.0μl |
It gently shakes, after rapid centrifugation, 42 DEG C of reactions 1h, 70 DEG C of 10min terminate reaction, 4 DEG C of coolings, -20 DEG C of preservations.
Using SYBP Premix Ex TapTMII kit is carried out in Eppendorf Real-time PCR analyzer,
Concrete operations are as follows:
(1) following PCR reaction solution is prepared on ice:
The preparation of table 3PCR reaction solution
| Reagent | Dosage |
| SYBR | 10.0μl |
| Forward primer | 1.0μl |
| Reverse primer | 1.0μl |
| cDNA | 2.0μl |
| ddH2O | 6.0μl |
| Total amount | 20.0μl |
Primer sequence design is as follows:
TUBB2A gene:
5'-GCATCCTTAGTGAACTTCT-3'(SEQ ID NO.1)；
5’-CCACATCATTACATCAACAG-3’(SEQ ID NO.2)
β-actin:
5'-GTGGGGCGCCCCAGGCACCA-3'(SEQ ID NO.3)；
5’-CTCCTTAATGTCACGCACGATTT-3’(SEQ ID NO.4)
(2) machine on executes following programs: 95 DEG C of initial denaturation 3min；95 DEG C of denaturation 15s.60 DEG C of annealing 15s, 72 DEG C of extensions
20s, totally 40 recycle.
As a result relative quantification method, formula 2 are used-△△ctIt calculates.Experiment is repeated 3 times.
△ ct=ct (A)-ct (β-actin)
△ △ ct=△ ct (experimental group)-△ ct (control group)
As a result as shown in Figure 1, compared with normal control population, the mRNA of TUBB2A gene in Bgudd-Chiari Syndrome blood samples of patients
Horizontal obvious up-regulation, difference have statistical significance (P < 0.05).
The preparation of 2 Bgudd-Chiari Syndrome diagnostic kit of embodiment
According to the correlation of TUBB2A gene and Bgudd-Chiari Syndrome, can by detect the expression of TUBB2A gene come
Bgudd-Chiari Syndrome is diagnosed, the present invention provides a kind of examinations that Bgudd-Chiari Syndrome is diagnosed based on detection TUBB2A gene expression accordingly
Agent box, the component in the diagnostic kit are as follows: SYBR Green polymerase chain reaction system；Expand TUBB2A gene and β-
The primer pair of actin gene.The forward primer sequence for expanding TUBB2A gene is 5 '-GCATCCTTAGTGAACTTCT-3 ', instead
It is 5 '-CCACATCATTACATCAACAG-3 ' to primer sequence；The forward primer sequence for expanding β-actin is 5 '-
GTGGGGCGCCCCAGGCACCA-3 ', reverse primer sequences 5 '-CTCCTTAATGTCACGCACGATTT-3 '.SYBR
Green polymerase chain reaction system includes PCR buffer, dNTPs, SYBR Green fluorescent dye.PCR buffer components
Are as follows: 25mM KCL, 2.5mM MgCL2、200mM(NH4)2SO4。
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 LISTING
<110>Beijing Yang Shen biology information technology Co., Ltd
<120>purposes of the TUBB2A as Blood diagnosis marker
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 19
<212> DNA
<213>artificial sequence
<400> 1
gcatccttag tgaacttct 19
<210> 2
<211> 20
<212> DNA
<213>artificial sequence
<400> 2
ccacatcatt acatcaacag 20
<210> 3
<211> 20
<212> DNA
<213>artificial sequence
<400> 3
gtggggcgcc ccaggcacca 20
<210> 4
<211> 23
<212> DNA
<213>artificial sequence
<400> 4
ctccttaatg tcacgcacga ttt 23
Claims (10)
1. detecting application of the product of TUBB2A in preparation diagnosis Bgudd-Chiari Syndrome tool.
2. application according to claim 1, which is characterized in that the product of the detection TUBB2A includes in test sample
The product of TUBB2A gene expression amount.
3. application according to claim 2, which is characterized in that the product of the detection TUBB2A includes that can quantify sample
The product of middle TUBB2A gene mRNA, and/or the product of TUBB2A albumen in sample can be quantified.
4. application according to claim 3, which is characterized in that the production that TUBB2A gene mRNA in sample can be quantified
Product include the primer of specific amplified TUBB2A gene used in real-time quantitative PCR, the primer sequence such as SEQ ID NO.1 and
Shown in SEQ ID NO.2；The product that TUBB2A albumen in sample can be quantified includes specific binding TUBB2A albumen
Antibody.
5. the application according to any one of claim 2-4, which is characterized in that the sample source is in blood.
6. application according to claim 1, which is characterized in that the tool includes the work for being able to detect TUBB2A in sample
Tool.
7. application according to claim 6, which is characterized in that the tool includes being able to detect TUBB2A gene in sample
The tool of expression quantity.
8. application according to claim 7, which is characterized in that the tool includes including that can quantify TUBB2A in sample
The reagent of gene mRNA, and/or the reagent of TUBB2A albumen in sample can be quantified.
9. application according to claim 8, which is characterized in that the examination that TUBB2A gene mRNA in sample can be quantified
Agent is the primer of specific amplified TUBB2A gene used in real-time quantitative PCR, the primer sequence such as SEQ ID NO.1 and
Shown in SEQ ID NO.2；The reagent that TUBB2A albumen in sample can be quantified includes specific binding TUBB2A albumen
Antibody.
10. the application according to any one of claim 6-9, which is characterized in that the sample source is in blood.
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| CN201611222765.5A CN106435009B (en) | 2016-12-27 | 2016-12-27 | Purposes of the TUBB2A as Blood diagnosis marker |
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|---|---|---|---|
| CN201611222765.5A CN106435009B (en) | 2016-12-27 | 2016-12-27 | Purposes of the TUBB2A as Blood diagnosis marker |
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| Publication Number | Publication Date |
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| CN106435009A CN106435009A (en) | 2017-02-22 |
| CN106435009B true CN106435009B (en) | 2019-08-23 |
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| CN101778637A (en) * | 2007-03-05 | 2010-07-14 | 新南创新私人有限公司 | methods for detecting and modulating the sensitivity of tumour cells to anti-mitotic agents |
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