CN105821152B - Coronary heart disease biomarker - Google Patents
Coronary heart disease biomarker Download PDFInfo
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- CN105821152B CN105821152B CN201610382179.0A CN201610382179A CN105821152B CN 105821152 B CN105821152 B CN 105821152B CN 201610382179 A CN201610382179 A CN 201610382179A CN 105821152 B CN105821152 B CN 105821152B
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- rad52
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- albumen
- heart disease
- coronary heart
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- 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
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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
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- 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
- C12Q2600/118—Prognosis of disease development
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- 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
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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
- G01N2800/32—Cardiovascular disorders
- G01N2800/324—Coronary artery diseases, e.g. angina pectoris, myocardial infarction
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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
- G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
Abstract
The invention discloses a kind of gene marker, which is RAD52.RAD52 can be used for judging that subject whether there is the risk with coronary heart disease, judge whether subject has suffered from coronary heart disease or prejudged the prognostic risk of patients with coronary heart disease.The present invention provides new diagnostic method for clinical diagnosis of coronary heart disease on a molecular scale.
Description
Technical field
The present invention relates to diagnosiss of coronary heart disease, prediction prognosis field, more particularly it relates to detect RAD52 exception
Diagnosis of coronary heart disease, prediction method of prognosis for means.
Background technique
Coronary heart disease becomes one of the principal disease for influencing human health with high lethality and disability rate, accounts for all fatal
The 40% of sexual behavior part.And the number of annual worldwide internal cause cardiovascular disease death increases year by year.According to statistics, flourishing state
Family's cardiovascular death number will will increase to 60,000,000 from 5,000,000 in 2000 to the year two thousand thirty.In China with economic rapidly hair
Exhibition, living standards of the people greatly improve, and resident living mode changes, and the incidence and mortality of cardiovascular disease also increases year by year
It is high.Show that Chinese urban and rural residents' cardiovascular disease illness rate becomes in rising according to " Chinese cardiovascular disease report 2013 " investigation result
Gesture, the death rate is high, and there are about 3,500,000 people to die of cardiovascular disease every year in the whole nation.Therefore, to the angiocarpy headed by coronary heart disease
Early screening, system diagnosis and treatment and the related basic research of disease become the great public health problem that current China faces.
It is that many factors act on a kind of complex disease caused by different links that people, which study discovery coronary heart disease, for many years.It removes
The conventional risk factors such as the age of early detection, gender, dyslipidemia, obesity, hypertension, diabetes, bad life style,
Inherent cause is also to lead to the principal element of incidence of coronary heart disease.There are coronary heart disease, diabetes, hypertension, dyslipidemia family histories
Crowd's Incidence of CHD obviously increase.Susceptible or mutated gene relevant to coronary risk factor has been cloned in recent years
200 kinds or more.
Summary of the invention
One of the objects of the present invention is to provide one kind to diagnose coronary disease by detection RAD52 gene or protein expression difference
The method of disease.
The second object of the present invention is to provide one kind by detection RAD52 gene or protein expression difference to predict coronary disease
The method of sick prognosis.
To achieve the goals above, present invention employs following technical solutions:
The present invention provides use of the product of detection RAD52 gene or RAD52 albumen in preparation diagnosis of coronary heart disease tool
On the way.
The present invention also provides the products of detection RAD52 gene or RAD52 albumen to predict coronary heart disease prognostic tool in preparation
In purposes.
Further, the product of the detection RAD52 gene or RAD52 albumen includes detection RAD52 gene or RAD52 albumen
Expression product.The product includes the nucleic acid that can combine RAD52 gene or the object that can combine RAD52 albumen
Matter (such as antibody).The nucleic acid is able to detect the expression of RAD52 gene；The substance is able to detect RAD52 albumen
Expression.
The product of detection RAD52 gene of the invention can play its function based on the known method of nucleic acid molecules is used:
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.
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.
Nucleic acid recited above includes the primer for expanding RAD52 gene, and the primer for including in product can be by passing through
Synthesis is learned to prepare, by using those skilled in the art will know that method be suitably designed with reference to Given information, and pass through
It is prepared by chemical synthesis.
In specific embodiments of the present invention, the nucleic acid is amplimer used in QPCR experiment, 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 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 detection RAD52 albumen of the invention can play its function based on the known method of antibody is used: for example,
It may include ELISA, radioimmunoassay, immunohistochemical method, western blot etc..
The product of detection RAD52 albumen of the invention includes the antibody or its segment for specifically binding RAD52 albumen.It can be with
Using the antibody or its segment of any structure and size, immunoglobulin class, origin etc., as long as it combines target protein.
The antibody or its segment for including in testing product of the invention can be monoclonal or polyclonal.Antibody fragment refers to that reservation is anti-
Peptide of the body to the active antibody of the combination of antigen a part of (Partial Fragment) or containing antibody a part.Antibody fragment may include
F(ab′)2, Fab ', Fab, scFv (scFv), the Fv (dsFv) of disulphide bonding or its polymer, the area dimerization V it is (dual anti-
Body) or peptide containing CDR.The product of detection RAD52 albumen of the invention may include encoding antibody or Encoding Antibody Fragment
The isolated nucleic acid of amino acid sequence, 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 RAD52 albumen for being used as antigen or part thereof to acquisition
Antigentic specificity purifying is applied to obtain the monoclonal antibody for RAD52 albumen.Polyclonal antibody can be prepared as follows: with it is upper
The identical antigen-immunized animal of text collects blood sample from by immune animal, serum is isolated from blood, is then used
Above-mentioned antigen implements antigentic specificity purifying to serum.It can the antibody by being obtained with enzymatic treatment or resisting by using acquisition
The sequence information of body 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.
In the present invention, " prognosis " refers to mistake of the patients with coronary heart disease after inhibiting by surgical procedure etc. or alleviating coronary heart disease
Journey or result.In the present specification, prognosis can be by surgical procedure inhibit or alleviate coronary heart disease after 1,2,3,4,5,6,7,
8,9,10,15,20 years or more long when life state.Prognosis can be by checking biomarker, that is, RAD52 albumen or coding
The gene of RAD52 albumen is predicted.Prognosis prediction can be performed such that according to biomarker with or without, or increase or
It reduces, determines that the prognosis of patient is good or bad, or determine the probability of good prognosis or poor prognosis.
In the present invention, " prognosis bona " refers to after inhibiting for patient by surgical procedure etc. or alleviating coronary heart disease,
Patient's long-term (such as 3,5,6,7,8,9,10,15,20 years or longer) does not have critical condition.
In the present invention, " prognosis mala " refers to patient after inhibiting or alleviating coronary heart disease by surgical procedure etc. in short-term
Fatal condition occurs in phase (such as 1,2,3,4,5 year or shorter).
Prediction prognosis is referred to the process of prediction status of patient or as a result, is not meant to be predicted with 100% accuracy
The process or result of status of patient.Prediction prognosis refers to whether a possibility that determining certain processes or result increases, and simultaneously unexpectedly
Taste by determining a possibility that certain processes or result occurs or not compared with certain processes or result.Such as this
For invention, in the present invention in the patient that is raised and lowered of the level of RAD52 gene or RAD52 albumen, and this feature is not shown
Patient compare, more likely observe particular procedure or result.
Further, the product of the detection RAD52 gene or RAD52 albumen can be detection RAD52 gene or RAD52 egg
White reagent is also possible to kit, chip, test paper comprising the reagent etc., is also possible to the high pass using the reagent
Measure microarray dataset.
Utilize the expression water of RAD52 gene or RAD52 albumen in mentioned-above testing product detection subject's sample
It is flat, it is compared with normal people, the expression of RAD52 gene or RAD52 albumen in subject's sample reduces, then it is tested to diagnose this
Person is patients with coronary heart disease or the poor prognosis of the subject.
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, blood of the sample from subject.
The present invention also provides a kind of tool of diagnosis of coronary heart disease, the tool is able to detect RAD52 in subject's sample
The expression of gene or RAD52 albumen.The tool includes that can combine the nucleic acid of RAD52 gene or can combine
The substance (such as antibody) of RAD52 albumen.The nucleic acid is able to detect the expression of RAD52 gene；The substance can be examined
Survey the expression of RAD52 albumen.
Further, the property of the nucleic acid and the substance is the same as noted earlier.
Further, the tool of the diagnosis of coronary heart disease includes but is not limited to chip, kit, test paper or high-flux sequence
Platform；High-flux sequence platform is a kind of tool of special diagnosis of coronary heart disease, with the development of high throughput sequencing technologies, to one
The building of personal gene expression profile will become very easily work.By the gene table for comparing Disease and normal population
Up to spectrum, the exception for being easy to analyze which gene is related to disease.Therefore, the different of RAD52 gene is known in high-flux sequence
The often purposes for also belonging to RAD52 gene related to coronary heart disease, equally within protection scope of the present invention.
The present invention also provides a kind of tool for predicting coronary heart disease prognosis, the tool is able to detect in subject's sample
The expression of RAD52 gene or RAD52 albumen, the prediction coronary heart disease prognostic tool include that can combine RAD52 gene
Nucleic acid can be in conjunction with the substance (such as antibody) of RAD52 albumen.The nucleic acid is able to detect the mRNA water of RAD52 gene
It is flat；The substance is able to detect the expression of RAD52 albumen.
Further, the property of the nucleic acid and the substance is the same as noted earlier.
Further, the tool of the prediction coronary heart disease prognosis 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 of coronary heart disease, with the development of high throughput sequencing technologies,
Very easily work will be become to the building of the gene expression profile of a people.By the base for comparing Disease and normal population
Because of express spectra, the exception for being easy to analyze which gene is related to disease.Therefore, RAD52 gene is known in high-flux sequence
The exception purposes for also belonging to RAD52 gene related to coronary heart disease, equally within protection scope of the present invention.
The amino acid that anti-RAD52 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 RAD52.When antibody is as therapeutic agent, preferably its energy
Amino acid as much as possible is enough identified, as long as it can inhibit RAD52 function.Antibody or the number of amino acid of its segment identification are
At least one, more preferably at least three.The immunoglobulin class of antibody is unrestricted, can be IgG, IgM, IgA, IgE,
IgD or IgY.
Further, the tissue sample or fluid for example obtained from biopsy subject can be used in subject's sample.Sample
Originally it 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
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 diagnosis of coronary heart disease or the methods for predicting coronary heart disease prognosis, and the method includes walking as follows
It is rapid:
(1) sample of subject is obtained；
(2) expression of RAD52 gene or albumen in Samples subjects is detected；
(3) it associates whether by the expression of the RAD52 gene or albumen that measure with the illness of subject.
(4) compared with the control, the expression of RAD52 gene or albumen reduces, then the subject is diagnosed as coronary heart disease,
Or the subject is confirmed as prognosis mala.
In the context of the present invention, " diagnosis of coronary heart disease " both include judge subject whether suffered from coronary heart disease or
Including judging that subject whether there is the risk with coronary heart disease.
The advantages of the present invention:
The molecular marker for having found a kind of diagnosis of coronary heart disease of the invention, can be in coronary heart disease using the molecular marker
The early stage of generation can be used as judging, provide the survival rate of patient.
In addition, the present invention is capable of providing significant information to determine treatment side for patient by the prognosis of prediction patient
Case strategy.
Detailed description of the invention
Fig. 1 shows the expression using genechip detection RAD52 gene in patients with coronary heart disease and normal person；
Fig. 2 shows the expression using QPCR detection RAD52 gene in patients with coronary heart disease and normal person；
Fig. 3 shows the expression using Western blot detection RAD52 albumen in patients with coronary heart disease 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.
The differential expression of 1 RAD52 gene of embodiment
1, research object:
Collect patients with coronary heart disease 5, normal person 5.
CHD group is included in standard: according to the diagnostic criteria for the ischemic heart disease that the World Health Organization formulates, selection
Underwent coronary radiography confirms the patients with coronary heart disease for having one or more hemadostewnosis degree >=50%.The inclusion criteria of control group
Are as follows: 1) screened in epidemiological survey the age, gender, it is national match with CAD group, by questionnaire survey, physical examination,
Cardiac ultrasound and ECG examination and laboratory check the not clinical manifestation of coronary heart disease and Major Risk Factors
Examinee；2) the parallel coronary artery revasualization of row health examination of being hospitalized excludes coronary heart disease and age, gender, national and CAD group
Matcher；Meet one person of any of the above to enter to be selected as control group.Two groups are signed informed consent form before being included in research.
Rejecting standard: the infull person of CAD group-clinical data and the one for merging following disease simultaneously are rejected.
(such as: Congenital Heart patient, dissection of aorta, multiple organ failure, rheumatic heart disease and have spirit
Obstacle is not able to cooperate person).Control group: having mentally disturbed or checking through Doppler confirms there is carotid plaques or narrow person, gives
To reject.
2, in blood total serum IgE extraction
(1) homogenized (Homogenization)
It is required that at least 12h at room temperature in next morning 7:00~8:00 extracts 10ml venous blood in second to research object on an empty stomach
Ethylenediamine tetraacetic acid (EDTA) (EDTA) anticoagulant tube, is added 3 times of volume erythrocyte cracked liquids, and 10 minutes are placed at room temperature for after mixing, and 10,000rpm
Centrifugation 1 minute.It thoroughly inhales and abandons supernatant, collect leukocyte cell pellet.1ml is added in the leukocyte cell pellet of every 100-200 μ l blood collection
TRIzol。
(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, it is sequenced: above-mentioned RNA is delivered into Shanghai Biotechnology Corporation using Solexa sequencing technologies to sample
It is sequenced.
5, data are analyzed
5.1 original data processing
The raw image data that sequenator generates is converted into sequence data through Base Calling, and we term it original sequences
Column data, as a result with FASTQ stored in file format.Since original sequence data may include low quality sequence, joint sequence etc.
Contaminating sequences data, cannot be directly used to analysis of biological information, so original sequence data must pass through data processing and be converted to
High quality sequence data, using the spliced mapping algorithm of Tophat (version:2.0.6) software to high quality sequence
Data carry out genome alignment, use genome version for Sscrofa10.2.
The screening of 5.2 differential genes
The calculating of gene expression amount makes FPKM method.The gene is calculated in different samples according to the expression quantity (FPKM value) of gene
Between differential expression multiple.Difference expression gene is defined as FDR≤0.01 and gene of the fold difference at 2 times or more.
6, result
(as shown in Figure 1) as the result is shown, is compared with normal people, the mRNA level in-site of RAD52 gene in patients with coronary heart disease blood
It significantly reduces, difference has statistical significance (P < 0.05).
The gene of differential expression in 2 QPCR experimental verification patients with coronary heart disease of embodiment and normal person
1, research object:
Screening criteria is with embodiment 1, patients with coronary heart disease 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 RAD52 gene and GAPDH gene in Genbank, is given birth to by Shanghai
The synthesis of work biotechnology Services Co., Ltd.Specific primer sequence is as follows:
RAD52 gene:
Forward primer is 5 '-CAGTTGCCTCTTGAAGTG-3 ' (SEQ ID NO.3)；
Reverse primer is 5 '-TGTTGTATCTTGCCTCCT-3 ' (SEQ ID NO.4),
GAPDH gene:
Forward primer is 5 '-GAAGGTGAAGGTCGGAGT-3 ' (SEQ ID NO.5)；
Reverse primer is 5 '-CATGGGTGGAATCATATTGGAA-3 ' (SEQ ID NO.6).
(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 5min, (95 DEG C of 10s, 60 DEG C of 40s) * 45 circulations.Using SYBR Green as glimmering
Signal object carries out PCR reaction on Light Cycler fluorescence quantitative PCR instrument, is determined by melt curve analysis analysis and electrophoresis
Purpose band, Δ Δ CT method carry 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 RAD52 gene significantly drops in patients with coronary heart disease blood
Low, difference has statistical significance (P < 0.05), as a result homogenic array experiment.
3 RAD52 protein diversity detection of expression of embodiment
1, research object
With embodiment 2.
2, monocyte separates
Aseptic collection venous blood 10ml, injection are contained in the sterile vials of heparin, are gently shaken up immediately after capping.With sterile suction
Isometric HBSS (NaCl 8.0g, Na is added in pipe2HPO40.132g, 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 is thin to draw 8ml lymph
Born of the same parents are layered liquid and set in 50ml centrifuge tube, and dilute blood is slowly added to along tube wall, and interface is kept to understand, mix the two mutually,
20 DEG C of 2 000r/min is centrifuged 30min, the careful buffy coat for drawing layering liquid and blood plasma handover position muddiness, i.e. lymphocyte
Layer is added in another centrifuge tube, is washed 2 times with the HBSS of 5 times of volumes, successively with 2 000r/min, 1 500r/min in room
Temperature is lower to be centrifuged 10min, and the blood platelet largely mixed to remove is made residual with 10ml distilled water and cell mass mixing 1min
Remaining erythrocyte splitting, is then rapidly added equivalent 1.8%NaCl solution, and 2 000r/min centrifugation removes supernatant, after cell count
Cell is adjusted to 1 × 10 with HBSS solution6A/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 12
000r/min is centrifuged 1h；Supernatant Brandford standard measure albumen is taken, 2.5 μ g/ μ l are distributed into, -80 DEG C of refrigerators save backup.
4, Western blot is detected
The albumen of extraction is subjected to SDS-PAGE electrophoresis, transferring film is carried out later, closing, primary antibody incubation, secondary antibody incubation, shows
Color.
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
As a result as shown in figure 3, being compared with normal people, RAD52 protein content is low in patients with coronary heart disease blood, and difference has system
Meter learns meaning (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.
Claims (10)
1. application of the product of detection RAD52 gene or RAD52 albumen in the tool for preparing diagnosis of coronary heart disease.
2. application according to claim 1, which is characterized in that the product packet of the detection RAD52 gene or RAD52 albumen
Include the product of the expression of detection RAD52 gene or RAD52 albumen.
3. application according to claim 2, which is characterized in that utilize the RAD52 in the product testing subject sample
The expression of gene or RAD52 albumen, is compared with normal people, the table of RAD52 gene or RAD52 albumen in subject's sample
It is reduced up to level, then diagnosing the subject is patients with coronary heart disease.
4. application according to claim 3, which is characterized in that the source of subject's sample is blood.
5. application described in any one of -4 according to claim 1, which is characterized in that the product includes that can combine RAD52
The nucleic acid of gene can be in conjunction with the substance of RAD52 albumen；The nucleic acid is able to detect the expression of RAD52 gene；Institute
State the expression that substance is able to detect RAD52 albumen.
6. application according to claim 5, which is characterized in that the nucleic acid is specifically expanded used in real-time quantitative PCR
Increase the primer of RAD52 gene, the primer sequence is as shown in SEQ ID NO.1 and SEQ ID NO.2.
7. application according to claim 1, which is characterized in that the tool includes being able to detect in subject's sample
The tool of the expression of RAD52 gene or RAD52 albumen.
8. application according to claim 7, which is characterized in that the tool includes can be in conjunction with the nucleic acid of RAD52 gene
Or it can be in conjunction with the substance of RAD52 albumen；The nucleic acid is able to detect the expression of RAD52 gene；The substance can
Detect the expression of RAD52 albumen.
9. application according to claim 8, which is characterized in that the nucleic acid is specifically expanded used in real-time quantitative PCR
Increase the primer of RAD52 gene, the primer sequence is as shown in SEQ ID NO.1 and SEQ ID NO.2.
10. the application according to any one of claim 7-9, which is characterized in that the source of subject's sample is blood
Liquid.
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| CN107012236A (en) * | 2017-05-03 | 2017-08-04 | 程琦 | SKIL as diagnosis of coronary heart disease molecular marker |
| CN107796946B (en) * | 2017-10-20 | 2019-10-25 | 中国医学科学院基础医学研究所 | The protein marker and application thereof of coronary heart disease in urine |
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|---|---|---|---|---|
| CN105543408A (en) * | 2016-03-16 | 2016-05-04 | 宫蕊 | Marker for early diagnosis of coronary heart disease |
| CN105793437A (en) * | 2013-09-23 | 2016-07-20 | 芝加哥大学 | Methods and compositions relating to cancer therapy with DNA damaging agents |
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| CN105793437A (en) * | 2013-09-23 | 2016-07-20 | 芝加哥大学 | Methods and compositions relating to cancer therapy with DNA damaging agents |
| CN105543408A (en) * | 2016-03-16 | 2016-05-04 | 宫蕊 | Marker for early diagnosis of coronary heart disease |
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