CN108315405A - CNTNAP2 genes as diagnosis of chronic obstructive pulmonary disease marker - Google Patents
CNTNAP2 genes as diagnosis of chronic obstructive pulmonary disease marker Download PDFInfo
- Publication number
- CN108315405A CN108315405A CN201810120650.8A CN201810120650A CN108315405A CN 108315405 A CN108315405 A CN 108315405A CN 201810120650 A CN201810120650 A CN 201810120650A CN 108315405 A CN108315405 A CN 108315405A
- Authority
- CN
- China
- Prior art keywords
- cntnap2
- genes
- albumen
- chronic obstructive
- expression
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- 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
-
- 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
-
- C—CHEMISTRY; METALLURGY
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/12—Pulmonary diseases
- G01N2800/122—Chronic or obstructive airway disorders, e.g. asthma COPD
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Microbiology (AREA)
- Pathology (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Hematology (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Cell Biology (AREA)
- General Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses the application of CNTNAP2 genes and its expression product in preparing diagnostic products.The diagnostic products can be used for the diagnosis of Chronic Obstructive Pulmonary Disease.Level by detecting CNTNAP2 genes and its expression product may determine that subject suffers from the risk of Chronic Obstructive Pulmonary Disease or makes a definite diagnosis the state of Chronic Obstructive Pulmonary Disease in the future.The diagnostic products of the present invention are to detect object, it can be achieved that noninvasive, quick, sensitive and accurate diagnosis effect with subject's peripheral blood.
Description
Technical field
The present invention relates to diagnosis of chronic obstructive pulmonary disease fields, more particularly it relates to which CNTNAP2 genes are made
For the diagnostic tool of Chronic Obstructive Pulmonary Disease.
Background technology
Chronic Obstructive Pulmonary Disease (COPD) is the common disease of division of respiratory disease, frequently-occurring disease, and serious prestige generally acknowledged in the world
The chronic lung disease of health of people is coerced, incidence and the death rate are high, the serious financial burden for increasing people and society.According to generation
Boundary's health organization statistics, COPD in 2000 account for the 4th (Aibek E Mirrakhimov.Chronic of the global cause of death
obstructive pulmonary disease and glucose metabolism:a bitter sweet
symphony.Cardiovasc Diabetol, 2012,11:132), it is contemplated that arrive the year two thousand twenty, COPD will rise to the cause of third position
Dead reason, and as the 5th (development of Yu Guohui, Chen Min pathogenesis of chronic obstructive pulmonary disease mechanism of global economy burden
Situation [J] clinical lungs section magazine, 2010,15 (1):72-74).In China, COPD has occupied first of Disease Spectrum sequence.Its
Continue chronic progress and seriously endangers patient's labour capacity and quality of life.
Chronic Obstructive Pulmonary Disease is a kind of lung's chronic disease, it is since chronic bronchitis and pulmonary emphysema lead to gas
The limited a kind of pulmonary disease of stream, while the outer organ of lung can also be caused to be damaged.Flow limitation is not fully reversible, is in progressive
Development, can be with airway hyperreactivity (Beasley V, Joshi PV, Singanayagam A, et al.microbiology
and exacerbations in COPD[J].Int J Chron Obstruct Puhnon Dis,2012,7:555-569)。
It is now recognized that the occurrence and development of Chronic Obstructive Pulmonary Disease are related with factors, such as infection, intolerance factors, smoking, occupational
The long-term sucking of dust, atmosphere pollution, noxious material etc. and the internal factor of body, nutritional status, autonomic nervous function are disorderly
Disorderly etc., they can lead to bronchial chronic inflammation, make tunica mucosa bronchiorum epithelial cell that denaturation, necrosis occur, cilium occur and fall
It lies prostrate, shorten, adhesion or even partial exfoliation, so that mucous epithelium unicorn columnar epithelium metaplasia occurs and granulation tissue, fibr tissue increase
It is raw that tracheae and bronchus luminal stenosis, the remodeling of air flue wall construction and disease trace is caused to be formed, to cause airway hyperreactivity, caused
Flow limitation (Chih-Hsin Lee, Ming-Chia Lee, Hsien-Ho Lin, et a1.Puhnonary
Tuberculosis and Delay in Anti-Tuberculous Treatment Are Important Risk
Factors for Chronic Obstructive Pulmonary Disease.PLoS One,2012,7(5):e37978)。
The pathogenesis of COPD not yet illustrates completely at present, is one of the hot issue of medical field research.In recent years, the morbidity of COPD
Mechanism theory is broadly divided into following several：Oxidation/it is anti-oxidant it is unbalance (Eirini Neofytou, Eleni G.Tzortzaki,
Argiro Chatziantoniou,et al.DNA Damage Due to Oxidative Stress in Chronic
Obstructive Pulmonary Disease(COPD)[J].Int J Mol Sci, 2012,13(12):16853-
16864), chronic inflammation, Apoptosis (Jeffrey T J Huang, Rekha Chaudhuri, Osarna
Albarbarawi,et a1.Clinical validity of plasma and urinary desmosine as
biomarkers for chronic obstructive pulmonary disease.Thorax,2012,67(6):502-
508), protease antiprotease is unbalance etc., and the chronic airway inflammation reaction of the wherein mediations such as inflammatory mediator, cell factor is COPD
The key link of morbidity.When body encounters intolerance factors or sucking dust, pernicious gas and particle, macrophage is thin in lung tissue
The inflammatory cells such as born of the same parents, lymphocyte, neutrophil leucocyte start hyperplasia, and discharge inflammatory mediator, chemotactic factor (CF) and cell factor etc.,
A series of cascade reactions are triggered, effector cell is acted on, cause the destruction of alveolar structure, Air way mucus glandular hyperplasia loose and viscous
Liquid is exceedingly secreted, so as to cause airway remodeling (Stockley RA.Progression of chronic obstructive
pulmonary disease:impact of inflammation,comorbidities and therapeutic
intervention.Curr Med Res Opin,2009,25(5):1235-1245)。
GOLD and China《Chronic Obstructive Pulmonary Disease diagnosis and treatment guide》Using FEV1/FVC ＜ 0.70 as COPD's
Diagnostic criteria, with the extensive publicity of this diagnostic mode, it is by more and more division of respiratory disease doctor and lung work(both at home and abroad in recent years
Energy Examined effect personnel are received.But more and more demonstrate,prove the diagnosis mark it has been found that using FEV1/FVC ＜ 0.70 as COPD
It will definitely lead to a large amount of clinical misdiagnosis, this current diagnostic criteria is just by unprecedented challenge.Therefore exploitation one kind can be used for
The method of Accurate Diagnosis COPD is a problem to be solved.
Invention content
The purpose of the present invention is to provide a kind of molecular marker for diagnosing chronic obstructive disease of lung, the molecules
Marker is CNTNAP2 genes.
To achieve the goals above, present invention employs following technical solutions：
The present invention provides the products of detection CNTNAP2 genes or CNTNAP2 albumen to prepare Chronic Obstructive Pulmonary Disease
Purposes in diagnostic tool.
Further, it is described detection CNTNAP2 genes or CNTNAP2 albumen product include detection CNTNAP2 genes or
The product of the expression of CNTNAP2 albumen.The product includes that can combine the nucleic acid of CNTNAP2 genes or can tie
Close the substance (such as antibody) of CNTNAP2 albumen.The nucleic acid can detect the expression of CNTNAP2 genes；The substance
The expression of CNTNAP2 albumen can be detected.
The product of the detection CNTNAP2 genes of the present invention can play its work(based on the known method of nucleic acid molecules is used
Energy：Such as PCR, such as Southern hybridization, Northern hybridization, dot blot, fluorescence in situ hybridization (FISH), DNA microarray, ASO
Method, high-flux sequence platform etc..It can qualitatively, quantitatively or semi-quantitatively implement analysis using the product.
Nucleic acid included 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 it is expected designed for amplification nucleic acid primer amplification it obtain.
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 methods etc..Expand
The nucleic acid of increasing can be by using dot blotting hybridization method, Surface Plasmon Resonance (SPR methods), PCR-RFLP methods, original position RT-
PCR methods, PCR-SSO (sequence specific oligonucleotide) method, PCR-SSP methods, AMPFLP (amplifiable fragment length polymorphism)
Method, MVR-PCR methods and PCR-SSCP (single-strand conformation polymorphism) methods detect.
Nucleic acid recited above includes the primer for expanding CNTNAP2 genes, and the primer that product includes can be by passing through
It is prepared by chemical synthesis, by using those skilled in the art will know that method be suitably designed with reference to Given information, and lead to
Chemical synthesis is crossed to prepare.
In specific embodiments of the present invention, the nucleic acid is the amplimer used in QPCR experiments, the primer
Sequence such as SEQ ID NO.1 (positive sequence) and SEQ ID NO.2 (reverse sequence) shown in.
Nucleic acid recited above may also include probe, and the probe can be prepared by chemical synthesis, by using this
The method that field technology personnel know appropriately is designed with reference to Given information, and is prepared by chemical synthesis, or can be led to
It crosses and prepares the gene containing desired nucleic acid sequence from biomaterial, and it is expected that the primer of nucleic acid sequence expands using designed for expanding
Increase it to prepare.
The product of the detection CNTNAP2 albumen of the present invention can play its function based on the known method of antibody is used：Example
Such as, it may include ELISA, radioimmunoassay, immunohistochemical method, western blot etc..
The product of the detection CNTNAP2 albumen of the present invention includes the antibody or its segment for specifically binding CNTNAP2 albumen.
The antibody or its segment of any structure and size, immunoglobulin class, origin etc. can be used, as long as it combines target protein
.The antibody or its segment that the detection product of the present invention includes can be monoclonals or polyclonal.Antibody fragment refers to
Retain peptide of the antibody to the active antibody of the combination of antigen a part of (Partial Fragment) or containing an antibody part.Antibody fragment can
To include F (ab ')2, Fab ', Fab, scFv (scFv), disulphide bonding Fv (dsFv) or its polymer, dimerization V
Area's (double antibody) or the peptide containing CDR.The product of the detection CNTNAP2 albumen of the present invention may include encoding antibody or coding
The nucleic acid of the separation of the amino acid sequence of antibody fragment, includes the carrier of the nucleic acid, and carries the cell of the carrier.
Antibody can be obtained by the way that well known to a person skilled in the art methods.Retain target all or in part for example, preparing
The polypeptide of protein integrates the mammalian cell expression vector for encoding their polynucleotides 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 by using CNTNAP2 albumen for being used as antigen or part thereof to the antibody of acquisition
Implement antigentic specificity purifying to obtain the monoclonal antibody for CNTNAP2 albumen.Polyclonal antibody can be prepared as follows：
With antigen-immunized animal same as above, blood sample is collected from by immune animal, serum is isolated from blood, so
Antigentic specificity purifying is implemented to serum using above-mentioned antigen afterwards.It can be by the antibody that is obtained with enzymatic treatment or by using obtaining
The sequence information of antibody obtain 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 kits-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 marks
Remember kit (Funakoshi Corporation).For correct labeling, can be detected by label using suitable instrument
Antibody or its segment.
Further, it is described detection CNTNAP2 genes or CNTNAP2 albumen product can be detection CNTNAP2 genes or
The reagent of CNTNAP2 albumen can also be the kit comprising the reagent, chip, test paper etc., can also be to use the examination
The high-flux sequence platform of agent.
Utilize the table of CNTNAP2 genes or CNTNAP2 albumen in foregoing detection product testing subject's sample
Up to level, compared with normal person, the expression of CNTNAP2 genes or CNTNAP2 albumen in subject's sample reduces, then
The subject is diagnosed as Patients with Chronic Obstructive Pulmonary Disease or diagnoses risk height of the subject with Chronic Obstructive Pulmonary Disease.
As the sample of the detection product according to the present 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 the measurement of the present 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, blood of the sample from subject.
The present invention also provides a kind of tool of diagnosing chronic obstructive disease of lung, the tool can detect subject's sample
The expression of CNTNAP2 genes or CNTNAP2 albumen in this.The tool includes can be in conjunction with the nucleic acid of CNTNAP2 genes
Or it can be in conjunction with the substance (such as antibody) of CNTNAP2 albumen.The nucleic acid can detect the expression water of CNTNAP2 genes
It is flat；The substance can detect the expression of CNTNAP2 albumen.
Further, the property of the nucleic acid and the substance is the same as noted earlier.
Further, the tool of the diagnosing chronic obstructive disease of lung include but not limited to chip, kit, test paper or
High-flux sequence platform；High-flux sequence platform is a kind of tool of special diagnosing chronic obstructive disease of lung, with high pass
The development for measuring sequencing technologies will become the structure of the gene expression profile of a people and very easily work.By comparing disease
The gene expression profile of patient and normal population, the exception for being easy to analyze which gene are related to disease.Therefore, it is measured in high pass
The exception of the CNTNAP2 genes purposes for also belonging to CNTNAP2 genes related to Chronic Obstructive Pulmonary Disease is known in sequence, is equally existed
Within protection scope of the present invention.
The amino acid that the detection product of the present invention, the anti-CNTNAP2 antibody used in diagnostic tool or its segment are identified
Number be not particularly limited, as long as antibody can combine CNTNAP2.When antibody is as medicine, preferably
It can identify amino acid as much as possible, as long as it can inhibit CNTNAP2 functions.The amino acid that antibody or its segment identify
Number be it is at least one, more preferably at least three.The immunoglobulin class of antibody is unrestricted, can be IgG, IgM, IgA,
IgE, IgD or IgY.
The detection product of the present invention, the anti-CNTNAP2 antibody used in diagnostic tool other properties with noted earlier.
Further, subject's sample can use the tissue sample or fluid for example obtained from biopsy subject.Sample
Originally it is not particularly limited, as long as it is suitable for the measurement of the present invention；For example, it may include tissue, blood, blood plasma, serum, lymph
Liquid, urine, serous cavity liquid, spinal fluid, synovia, aqueous humor, tear, saliva or its fraction or treated material.In the present invention
Specific embodiment in, blood of the sample from subject.
The present invention also provides a kind of methods of diagnosing chronic obstructive disease of lung, and described method includes following steps：
(1) sample of Chronic Obstructive Pulmonary Disease subject is obtained；
(2) expression of CNTNAP2 genes or albumen in Samples subjects is detected；
(3) it is associated whether by the expression of the CNTNAP2 genes or albumen that measure with the illness of subject.
(4) compared with normal control, the expression of CNTNAP2 genes or albumen reduces, then the subject is diagnosed as
It is high that Chronic Obstructive Pulmonary Disease or the subject are diagnosed as the risk with Chronic Obstructive Pulmonary Disease in the future.
In the context of the present invention, " diagnosing chronic obstructive disease of lung " had both included judging whether subject has suffered from
Chronic Obstructive Pulmonary Disease also includes the risk that judges subject and whether there is with Chronic Obstructive Pulmonary Disease.
The advantages of the present invention：
The present invention's is found that a kind of molecular marker of diagnosing chronic obstructive disease of lung, can using the molecular marker
Can be used as judging early stage Chronic Obstructive Pulmonary Disease occurs, the survival rate of patient is provided.
Description of the drawings
Fig. 1 shows the expression in Patients with Chronic Obstructive Pulmonary Disease and normal person using QPCR detection CNTNAP2 genes
Difference.
Fig. 2 is shown detects CNTNAP2 albumen in Patients with Chronic Obstructive Pulmonary Disease and normal person using Western blot
In differential expression.
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 Patients with Chronic Obstructive Pulmonary Disease and normal person
1, clinical study：
Choose Patients with Chronic Obstructive Pulmonary Disease 5, wherein male 2, women 3, the range of age 50-79 Sui, diagnosis
Standard meets China's revision in 2007《Chronic Obstructive Pulmonary Disease diagnosis and treatment specification》.
Diagnostic criteria：Any patient with expiratory dyspnea, chronic cough or more phlegm, and have and be exposed to risk factor
Medical history, row pulmonary function test are shown, after sucking bronchodilators, are shown there are flow limitation, can be diagnosed as COPD.
Exclusion criteria：1. merging other pulmonary disease persons, such as bronchial asthma, pulmonary interstitial fibrosis, lung cancer；2. there is it
Its site infection person；3. declining with serious cardiovascular and cerebrovascular disease, diabetes, disease in the blood system, malignant tumour, organ function
It exhausts, hepatitis person；4. suffering from disease of immune system or using immunosuppressor person in the recent period.
Normal control：Choose 6 people of Healthy People of physical examination, wherein 3 people of male, 3 people of women, the range of age 50-79.
Inclusion criteria：The medical histories such as no chronic cough, expectoration, end breath；In the recent period without the infection of the upper respiratory tract, pulmonary infection history；Nothing
Whole body other site infections person；Without other pulmonary diseases person；Immunosuppressor is not used in the recent period or without systemic immune system disease
Patient；Without organ failure or serious cardiovascular and cerebrovascular disease, tumour person；Without anaphylactia person.Selected object row lung work(
Can check exclude simultaneously with Chronic Obstructive Pulmonary Disease group compare, gender, on the age difference it is not statistically significant have can
Compare property.
All research objects endorsed informed consent form.
2, sample collection
All research objects extract peripheric venous blood 10ml, EDTA anti-freezing under early morning fasting state.
3, blood sample Total RNAs extraction
The extraction of blood total serum IgE is carried out using hundred Tyke blood rna extracts kits：
(1) it takes in 250 μ l (or 0.25g) to RNase-Free Filter columns of whole blood, 13000rpm is centrifuged 2 minutes, under collection
0.75ml lysates RLS is added in liquid.
(2) homogenised sample is acutely shaken into mixing, 5 minutes is incubated under the conditions of 15-30 DEG C so that ribosome divides completely
Solution.
(3) optional step：12,000rpm is centrifuged 10 minutes under conditions of 4 DEG C, and supernatant is carefully taken to be transferred to a new nothing
In the centrifuge tube of RNA enzyme.
(4) add 0.2ml chloroforms per 1ml RLS.Sample tube cover is covered tightly, acutely vibrate 15 seconds and is incubated at room temperature 3
Minute.
(5) it is centrifuged 10 minutes in 4 DEG C of 12,000rpm, sample can be divided into three layers：Lower layer's organic phase, middle layer and upper layer without
The water phase of color, RNA are present in water phase.The capacity of aqueous layer is about the 60% of added RLS volumes, and water phase is transferred to new pipe
In, carry out next step operation.
(6) 1 times of 70% ethyl alcohol of volume is added, overturns mixing (at this time it is possible that precipitation), obtained solution and possibility
Precipitation is transferred in adsorption column RA (adsorption column is sleeved in collecting pipe) together.
(7) 10,000rpm are centrifuged 45 seconds, discard waste liquid, adsorption column is recovered collecting pipe again.
(8) plus 500 μ l protein liquid removals RE, 12,000rpm centrifugations 45 seconds discard waste liquid.
(9) 700 μ l rinsing liquids RW are added, 12,000rpm centrifugations 60 seconds discard waste liquid.
(10) 500 μ l rinsing liquids RW are added, 12,000rpm centrifugations 60 seconds discard waste liquid.
(11) adsorption column RA to be put back in sky collecting pipe, 12,000rpm centrifugations 2 minutes remove rinsing liquid as possible, in order to avoid drift
Residual ethanol inhibits downstream reaction in washing lotion.
(12) adsorption column RA is taken out, is put into a centrifuge tube without RNA enzyme, according to expected RNA yield in adsorbed film
Intermediate position adds water of the 50-80 μ l without RNA enzyme, is placed at room temperature for 2 minutes, and eluent is collected in 12,000rpm centrifugations 1 minute.
3, the quality analysis of RNA sample
The concentration and purity of carried RNA are detected using Nanodrop2000, it is complete that agarose gel electrophoresis detects RNA
Whole property, Agilent2100 measure RIN values.Single requirement for construction data base RNA total amounts 5 μ g, concentration >=200ng/ μ L, OD260/280 between
Between 1.8~2.2.
4, fragmentation RNA
Illumina platforms are sequenced for short sequence fragment, and mRNA average lengths may reach several kb, it is therefore desirable to
It is interrupted at random.It, can be by RNA random fractures at the small fragment of 200bp or so using metal ion.
5, reversion synthesis cDNA
Under the action of reverse transcriptase, using random primer, one chain cDNA of synthesis is inverted by template of mRNA, carries out two chains
When synthesis, dTTP is replaced with dUTP in dNTPs reagents, it includes A/U/C/G to make base in the second chains of cDNA.
6, adaptor is connected
The cDNA structures of double-strand are cohesive end, and End Repair Mix are added and are mended into flat end, then at 3 ' ends
End adds an A base, the connector for connecting Y-shaped.
7, bis- chains of UNG enzymic digestions cDNA
Before PCR amplification, the second chains of cDNA are digested with UNG enzymes, to make only to include the first chains of cDNA in library.
8, machine is sequenced on Illumina x-ten
Illumina x-ten microarray datasets carry out 2*150bp sequencings.
9, bioinformatic analysis
It is as follows that sequencing data obtains later rawdata analytic processes：
(1) trim is carried out to the 5 ' of reads and 3 ' sections with cutadapt, trim falls quality<20 base, and delete N
Reads more than 10%；
(2) tophat is compared onto reference gene group.Reference gene group version used be GRCh38.p7, fasta and
Gff file downloads are from NCBI；
(3) expression quantity of cuffquant quantification of mrna and normalization output；
(4) compare differential expression of the control group with disease group mRNA with DEGseq packets under R environment.Significant difference mRNA sieves
Select condition：p-value<0.05.
10, result
Difference expression gene 3296, the wherein gene of up-regulated expression 1428 are obtained with the above standard screening, under expression
The gene of tune has 1868.
The relationship of embodiment 2 QPCR verification candidate gene and Chronic Obstructive Pulmonary Disease
It is based on high-flux sequence early period as a result, according to the size of P value, we select CNTNAP2 genes, and (it is expressed
Lowered in Patients with Chronic Obstructive Pulmonary Disease) it is verified.
1, research object：
According to the method choice Patients with Chronic Obstructive Pulmonary Disease 45 of embodiment 1, normal person 35.
2, blood Total RNAs extraction
The extraction of blood total serum IgE is carried out using hundred Tyke blood rna extracts kits：
(1) it takes in 250 μ l (or 0.25g) to RNase-Free Filter columns of whole blood, 13000rpm is centrifuged 2 minutes, under collection
0.75ml lysates RLS is added in liquid.
(2) homogenised sample is acutely shaken into mixing, 5 minutes is incubated under the conditions of 15-30 DEG C so that ribosome divides completely
Solution.
(3) optional step：12,000rpm is centrifuged 10 minutes under conditions of 4 DEG C, and supernatant is carefully taken to be transferred to a new nothing
In the centrifuge tube of RNA enzyme.
(4) add 0.2ml chloroforms per 1ml RLS.Sample tube cover is covered tightly, acutely vibrate 15 seconds and is incubated at room temperature 3
Minute.
(5) it is centrifuged 10 minutes in 4 DEG C of 12,000rpm, sample can be divided into three layers：Lower layer's organic phase, middle layer and upper layer without
The water phase of color, RNA are present in water phase.The capacity of aqueous layer is about the 60% of added RLS volumes, and water phase is transferred to new pipe
In, carry out next step operation.
(6) 1 times of 70% ethyl alcohol of volume is added, overturns mixing (at this time it is possible that precipitation), obtained solution and possibility
Precipitation is transferred in adsorption column RA (adsorption column is sleeved in collecting pipe) together.
(7) 10,000rpm are centrifuged 45 seconds, discard waste liquid, adsorption column is recovered collecting pipe again.
(8) plus 500 μ l protein liquid removals RE, 12,000rpm centrifugations 45 seconds discard waste liquid.
(9) 700 μ l rinsing liquids RW are added, 12,000rpm centrifugations 60 seconds discard waste liquid.
(10) 500 μ l rinsing liquids RW are added, 12,000rpm centrifugations 60 seconds discard waste liquid.
(11) adsorption column RA to be put back in sky collecting pipe, 12,000rpm centrifugations 2 minutes remove rinsing liquid as possible, in order to avoid drift
Residual ethanol inhibits downstream reaction in washing lotion.
(12) adsorption column RA is taken out, is put into a centrifuge tube without RNA enzyme, according to expected RNA yield in adsorbed film
Intermediate position adds water of the 50-80 μ l without RNA enzyme, is placed at room temperature for 2 minutes, and eluent is collected in 12,000rpm centrifugations 1 minute.
3, total rna concentration and purity are measured
With the concentration and purity of NanoVue Plus apparatus measures sample rnas.
4, reverse transcription
Reverse transcription is carried out to l μ g total serum IgEs with RT Buffer and synthesizes cDNA.Using 25 μ l reaction systems, each sample
It takes 1 μ g total serum IgEs 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, 200 U/ μ l M-MLV, template ribonucleic acid.42 DEG C incubate
Educate 1h, 72 DEG C of 10min, of short duration centrifugation.
5, QPCR amplifications are examined
Using 25 μ l reaction systems, 3 parallel pipes are arranged in each sample, and all amplified reactions are repeated three times above to protect
Demonstrate,prove the reliability of result.Prepare following reaction system：12.5 μ l of SYBR Green PCRs system, forward primer (5
μM/μ l) 1 μ l, reverse primer (5 μM/μ l) 1 μ l, template cDNA 2.0 μ l, 8.5 μ l of no enzyme water；Expand CNTNAP2 genes just
To sequence 5 '-TACAGCATCCGATTATTG -3 ' (SEQ ID NO.1), reverse sequence 5 '-CAGTAAGAACAGCCATAA-3 '
(SEQ ID NO.2)；The preferred GAPDH of house-keeping gene, the forward primer sequence for expanding the gene are 5 '-
ATGTTCCAATATGATTCCA-3 ' (SEQ ID NO.3), reverse primer sequences 5 '-GATTTCCATTGATGACAAG-3 '
(SEQ ID NO.4).Operations are carried out on ice.Amplification program is：95 DEG C of 5min, (95 DEG C of 10s, 60 DEG C of 60s) * 45
Cycle.Using SYBR Green as fluorescent marker, it is anti-that PCR is carried out on Light Cycler fluorescence real-time quantitative PCR instrument
It answers, purpose band is determined by melt curve analysis analysis and electrophoresis, Δ Δ CT methods carry out relative quantification, and the results are shown in Figure 1, and just
Ordinary person compares, and CNTNAP2 genes are lowered in Patients with Chronic Obstructive Pulmonary Disease blood, and difference has statistical significance (* P<
0.05)。
3 immunoblot experiment of embodiment verifies the table of difference expression gene in Patients with Chronic Obstructive Pulmonary Disease and normal person
Up to product
1, clinical subjects：With embodiment 2.
2, monocyte detaches
Patients with Chronic Obstructive Pulmonary Disease and normal person extracting vein blood 10ml, injection are contained in the sterile vials of heparin, capping
It gently shakes up immediately afterwards.Isometric HBSS (NaCl 8.0g, Na is added with aseptic straw2HPO40.132g, KH2PO4
0.06g, KCl 0.4g, phenol red 1ml, NaHCO30.35g, D-Glucose 1.0g are dissolved in 1000ml distilled waters), it is red thin to reduce
The cohesion of born of the same parents.It draws 8ml lymphocytes separating solutions to set in 50ml centrifuge tubes, dilute blood is slowly added to along tube wall, keep boundary
Face understands, the two is not made mutually to mix, and centrifuges 30min in 20 DEG C of 2000r/min, careful absorption layering liquid and blood plasma handing-over position are mixed
Turbid buffy coat, i.e. buffy coat are added in another centrifuge tube, and 2 times are washed with the HBSS of 5 times of volumes, successively with
2000r/min, 1500r/min centrifuge 10min at room temperature, the blood platelet largely mixed to remove, with 10ml distilled waters
With cell mass mixing 1min, residual red blood cells is made to crack, is then rapidly added equivalent 1.8%NaCl solution, 2000r/min from
The heart removes supernatant, and cell is adjusted to 1 × 10 with HBSS solution after cell count6A/ml is spare.
3, monocyte gross protein extracts
By cell suspension (a concentration of 1 × 10 obtained by above-mentioned experiment6A/ml) room temperature 1 000r/min centrifuge 10min, abandon
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 centrifuges 1h；Supernatant Brandford standard measure albumen is taken, is distributed into 2.5 μ g/ μ l, -80 DEG C of refrigerators preserve standby
With.
4, Western blot are detected
Total protein of cell Brandford standard measures take to mix with sample buffer in right amount and boil 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 pressures, see
120V is increased to after Marker；Glue after electrophoresis is taken out, 50min is shifted in 100V using the half-dried transferring systems of Bio.Rad；
It after transferring film, is washed once with 1xPBS, immerses confining liquid, 40C is overnight；Confining liquid is outwelled, the washing of Western cleaning solutions is added
5-10min is added primary antibody shaking table room temperature and hybridizes 2h；According to proper proportion closing buffering is diluted in Western secondary antibody diluents
In liquid, it is incubated 60min；Film washing liquid is washed 3 times, each 10min；Use the development of ECL reagents, fixing detection protein expression.
5, statistical procedures
The gray value of protein band is analyzed using Image J softwares, 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 softwares are 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
As a result such as Fig. 2 is shown, compared with normal person, CNTNAP2 protein levels in Patients with Chronic Obstructive Pulmonary Disease blood
It significantly reduces, difference has statistical significance (P<0.05).
The explanation of above-described embodiment is only intended to understand the method and its core concept of the present invention.It should be pointed out that for this
For the those of ordinary skill in field, 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 domain of the claims in the present invention.
Sequence table
<110>Beijing Yang Shen biology information technologies Co., Ltd
<120>Purposes of the CNTNAP2 genes as diagnosis of chronic obstructive pulmonary disease marker
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
tacagcatcc gattattg 18
<210> 2
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
cagtaagaac agccataa 18
<210> 3
<211> 19
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
atgttccaat atgattcca 19
<210> 4
<211> 19
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
gatttccatt gatgacaag 19
Claims (10)
1. detecting product the answering in preparing diagnosis of chronic obstructive pulmonary disease tool of CNTNAP2 genes or CNTNAP2 albumen
With.
2. application according to claim 1, which is characterized in that the production of the detection CNTNAP2 genes or CNTNAP2 albumen
Product include the product for the expression for detecting CNTNAP2 genes or CNTNAP2 albumen.
3. application according to claim 2, which is characterized in that using in the product testing subject sample
The expression of CNTNAP2 genes or CNTNAP2 albumen, compared with normal person, CNTNAP2 genes in subject's sample or
The expression of CNTNAP2 albumen reduces, then diagnoses that the subject is Patients with Chronic Obstructive Pulmonary Disease or to diagnose this tested
Risk of the person with Chronic Obstructive Pulmonary Disease is high.
4. application according to claim 3, which is characterized in that the source of subject's sample is blood.
5. according to the application described in any one of claim 1-4, which is characterized in that the product includes that can combine
The nucleic acid of CNTNAP2 genes can be in conjunction with the substance of CNTNAP2 albumen；The nucleic acid can detect CNTNAP2 genes
Expression；The substance can detect the expression of CNTNAP2 albumen.
6. application according to claim 5, which is characterized in that the nucleic acid is the special expansion used in real-time quantitative PCR
Increase the primer of CNTNAP2 genes as shown in SEQ ID NO.1 and SEQ ID NO.2.
7. a kind of tool of diagnosing chronic obstructive disease of lung, which is characterized in that the tool includes that can detect subject's sample
The tool of the expression of CNTNAP2 genes or CNTNAP2 albumen in this.
8. tool according to claim 7, which is characterized in that the tool includes can be in conjunction with the core of CNTNAP2 genes
Acid can be in conjunction with the substance of CNTNAP2 albumen；The nucleic acid can detect the expression of CNTNAP2 genes；The object
Matter can detect the expression of CNTNAP2 albumen.
9. tool according to claim 8, which is characterized in that the nucleic acid is the special expansion used in real-time quantitative PCR
Increase the primer of CNTNAP2 genes as shown in SEQ ID NO.1 and SEQ ID NO.2.
10. according to the tool described in any one of claim 7-9, which is characterized in that the source of subject's sample is blood
Liquid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810120650.8A CN108315405A (en) | 2018-02-07 | 2018-02-07 | CNTNAP2 genes as diagnosis of chronic obstructive pulmonary disease marker |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810120650.8A CN108315405A (en) | 2018-02-07 | 2018-02-07 | CNTNAP2 genes as diagnosis of chronic obstructive pulmonary disease marker |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108315405A true CN108315405A (en) | 2018-07-24 |
Family
ID=62902069
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810120650.8A Pending CN108315405A (en) | 2018-02-07 | 2018-02-07 | CNTNAP2 genes as diagnosis of chronic obstructive pulmonary disease marker |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108315405A (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105907870A (en) * | 2016-06-01 | 2016-08-31 | 北京泱深生物信息技术有限公司 | Use of DHX36 as coronary heart disease diagnostic marker |
| CN105950768A (en) * | 2016-07-01 | 2016-09-21 | 江苏医诺万细胞诊疗有限公司 | Kit for auxiliary diagnosis of multiple tumors by taking micro ribonucleic acid (RNA) combination as tumor marker, and detection method of kit |
-
2018
- 2018-02-07 CN CN201810120650.8A patent/CN108315405A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105907870A (en) * | 2016-06-01 | 2016-08-31 | 北京泱深生物信息技术有限公司 | Use of DHX36 as coronary heart disease diagnostic marker |
| CN105950768A (en) * | 2016-07-01 | 2016-09-21 | 江苏医诺万细胞诊疗有限公司 | Kit for auxiliary diagnosis of multiple tumors by taking micro ribonucleic acid (RNA) combination as tumor marker, and detection method of kit |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP3218521B1 (en) | Method for diagnosing organ injury | |
| CN113528653B (en) | Biomarker related to chronic thromboembolic pulmonary hypertension and application thereof | |
| CN108303547A (en) | A kind of molecular marker for diagnosing chronic obstructive disease of lung | |
| EP3346270B1 (en) | Composition for diagnosing infectious diseases or infectious complications by using tryptophanyl-trna synthetase and method for detecting diagnostic marker | |
| CN107267649A (en) | Applications of the STRADB in asthma early diagnosis | |
| CN108384847B (en) | Diagnosis marker of the RNF182 gene as Chronic Obstructive Pulmonary Disease | |
| CN110656169B (en) | Diagnostic markers for atrial fibrillation | |
| CN107227358A (en) | Purposes of the NUMB in postmenopausal women's diagnosis of primary osteoporosis or prognosis | |
| CN116298310A (en) | Use of a reagent for detecting markers for the preparation of a product for the diagnosis and/or prognosis of chronic obstructive pulmonary disease | |
| CN108315405A (en) | CNTNAP2 genes as diagnosis of chronic obstructive pulmonary disease marker | |
| CN105821152B (en) | Coronary heart disease biomarker | |
| CN108034718A (en) | Biomarkers of the SHISA4 as early diagnosis Chronic Obstructive Pulmonary Disease | |
| KR102627788B1 (en) | Gastric cancer biomarkers and their uses | |
| CN113631723B (en) | Application of tuberculosis marker in tuberculosis diagnosis and efficacy evaluation | |
| CN113969312A (en) | Marker for predicting susceptibility of ulcerative colitis patient to golimumab drug | |
| CN106048005B (en) | A kind of molecular marker of diagnosis of coronary heart disease | |
| CN108300780A (en) | TMTC1 genes are as a kind of new molecular marker | |
| CN107022633A (en) | Gene marker for diagnosing ankylosing spondylitis | |
| EP3775282A1 (en) | Biomarkers for diabetes therapy | |
| CN108486243A (en) | Purposes of the DEFA1B genes in the diagnostic tool for preparing Chronic Obstructive Pulmonary Disease | |
| CN115011681B (en) | Marker molecule related to hypertrophic cardiomyopathy and application of marker molecule in diagnosis of hypertrophic cardiomyopathy | |
| CN108300781A (en) | The Blood diagnosis marker of Chronic Obstructive Pulmonary Disease | |
| CN107164551A (en) | Purposes of the UBAP2L in type I diabetes of children diagnosis or prognosis | |
| CN106048006B (en) | SMCHD1 is preparing the application in diagnosis of coronary heart disease product | |
| CN105907871B (en) | Biomarker for diagnosis of coronary heart disease |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB02 | Change of applicant information |
Address after: Room 1210, Building 3, Ronghua Xintai Building, 10 Ronghua South Road, Yizhuang Economic and Technological Development Zone, Daxing District, Beijing Applicant after: BEIJING MEDINTELL BIOMED Co.,Ltd. Address before: 100080 Beijing city Haidian District Shanyuan Street No. 1 cubic court building room 3103 Applicant before: BEIJING MEDINTELL BIOMED Co.,Ltd. |
|
| CB02 | Change of applicant information | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20200226 Address after: Room 2503, Qianshan building, building D2, Qingdao International Innovation Park Phase II, No. 1, Keyuan Weiyi Road, Laoshan District, Qingdao, Shandong Province Applicant after: Qingdao Yangshen biomedical Co.,Ltd. Address before: Room 1210, Building 3, Ronghua Xintai Building, 10 Ronghua South Road, Yizhuang Economic and Technological Development Zone, Daxing District, Beijing Applicant before: BEIJING MEDINTELL BIOMED Co.,Ltd. |
|
| TA01 | Transfer of patent application right | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180724 |
|
| RJ01 | Rejection of invention patent application after publication |