CN105821152A - Biological marker for coronary heart disease - Google Patents
Biological marker for coronary heart disease Download PDFInfo
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- CN105821152A CN105821152A CN201610382179.0A CN201610382179A CN105821152A CN 105821152 A CN105821152 A CN 105821152A CN 201610382179 A CN201610382179 A CN 201610382179A CN 105821152 A CN105821152 A CN 105821152A
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- rad52
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- heart disease
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- albumen
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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 gene marker .The gene marker is RAD52 which can be used for judging whether an examinee has the risk of suffering from coronary heart disease or not, judging whether the examinee suffers from coronary heart disease or not at present or pre-judging prognostic risk of a coronary heart disease patient .By means of the biological marker for coronary heart disease, a new diagnosis method is provided for diagnosing coronary heart disease clinically on the molecular level.
Description
Technical field
The present invention relates to diagnosis of coronary heart disease, prediction prognosis field, more particularly it relates to detect the RAD52 diagnosis of coronary heart disease extremely as means, prediction method of prognosis.
Background technology
Coronary heart disease becomes one of principal disease affecting human health with high fatality rate and disability rate, accounts for the 40% of all mortality events.And the number of annual worldwide endogenous cause of ill cardiovascular disease death increases year by year.According to statistics, 500 ten thousand to the year two thousand thirties from 2000 will be increased to 60,000,000 by developed country's cardiovascular death number.Developing rapidly along with economy in China, living standards of the people are greatly improved, and resident living mode changes, and the incidence and mortality of cardiovascular disease increases the most year by year.Showing according to " China cardiovascular diseases report 2013 " survey result, the ill rate of Chinese urban and rural residents cardiovascular diseases is in rising trend, and mortality rate remains high, and the whole nation there are about 3,500,000 people every year and dies from cardiovascular diseases.Therefore, early screening, system diagnosis and treatment and the related basic research of the cardiovascular disease headed by coronary heart disease is become the great public health problem that current China faces.
People study and find that coronary heart disease is that many factors acts on different a kind of complex disease caused by link for many years.Except conventional risk factors such as age of early discovery, sex, dyslipidemia, obesity, hypertension, diabetes, bad life style, inherited genetic factors is also the principal element causing incidence of coronary heart disease.There is coronary heart disease, diabetes, hypertension, crowd's Incidence of CHD of dyslipidemia family history substantially increase.The most clone the susceptible or mutant gene relevant to coronary risk factor more than 200 kinds.
Summary of the invention
An object of the present invention is to provide a kind of method carrying out diagnosis of coronary heart disease by detection RAD52 gene or protein expression difference.
The two of the purpose of the present invention are to provide a kind of method predicting coronary heart disease prognosis by detection RAD52 gene or protein expression difference.
To achieve these goals, present invention employs following technical scheme:
The invention provides the product detecting RAD52 gene or RAD52 albumen purposes in preparation diagnosis of coronary heart disease instrument.
The product that present invention also offers detection RAD52 gene or RAD52 albumen is preparing the purposes predicted in coronary heart disease prognostic tool.
Further, the product of described detection RAD52 gene or RAD52 albumen includes the product detecting the expression of RAD52 gene or RAD52 albumen.Described product includes can be in conjunction with the nucleic acid of RAD52 gene or can be in conjunction with the material (such as antibody) of RAD52 albumen.Described nucleic acid can detect the expression of RAD52 gene；Described material can detect the expression of RAD52 albumen.
The product of the detection RAD52 gene of the present invention can play its function based on the known method using nucleic acid molecules: 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..This product is used can qualitatively, quantitatively or semi-quantitatively to implement to analyze.
The nucleic acid being included in the said goods can be obtained by chemosynthesis, or by from biomaterial preparation containing expectation nucleic acid gene, then use be designed for amplification expectation nucleic acid primer amplification it obtain.
Further, described PCR method is known method, such as, and ARMS (AmplificationRefractoryMutationSystem, abruptly-changing system is not answered in amplification) method, RT-PCR (reverse transcriptase-PCR) method, nested PCR method etc..The nucleic acid of amplification can detect by using dot blotting hybridization method, Surface Plasmon Resonance (SPR method), PCR-RFLP method, B by means of in situ RT PCR, 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.
Nucleic acid recited above includes the primer expanding RAD52 gene, and the primer that product includes can be by preparing by chemosynthesis, and the method that be those skilled in the art will know that by use is suitably designed with reference to Given information, and is prepared by chemosynthesis.
In specific embodiments of the present invention, described nucleic acid is the amplimer used in QPCR experiment, shown in the sequence of described primer such as SEQIDNO.1 (forward sequence) and SEQIDNO.2 (reverse sequence).
Nucleic acid recited above may also include probe, described probe can be prepared by chemosynthesis, the method that be those skilled in the art will know that by use is appropriately designed with reference to Given information, and prepared by chemosynthesis, or can by from biomaterial preparation containing expectation nucleotide sequence gene, and use be designed for amplification expectation nucleotide sequence primer amplification it prepare.
The product of the detection RAD52 albumen of the present invention can play its function based on the known method using antibody: for example, it is possible to include ELISA, radioimmunoassay, immunohistochemical method, western blot etc..
The product of the detection RAD52 albumen of the present invention includes antibody or its fragment of specific binding RAD52 albumen.Antibody or its fragment of any structure, size, immunoglobulin class, origin etc. can be used, as long as it combines target protein.Antibody or its fragment that the detection product of the present invention includes can be monoclonal or polyclonal.Antibody fragment refers to retain antibody to an antibody part (Partial Fragment) of the combination activity of antigen or the peptide containing an antibody part.Antibody fragment can include F (ab ')2, Fab ', Fab, scFv (scFv), the Fv (dsFv) of disulphide bonding or its polymer, dimerization V district (double antibody) or containing the peptide of CDR.The product of the detection RAD52 albumen of the present invention can include the nucleic acid of the separation of the aminoacid sequence of encoding antibody or Encoding Antibody Fragment, comprises the carrier of this nucleic acid, and carries the cell of this carrier.
Antibody can be by well known to a person skilled in the art that method obtains.Such as, preparation retains the polypeptide of target protein all or in part or integrates the mammalian cell expression vector of their polynucleotide of coding as antigen.After using antigen-immunized animal, from passing through immune animal adaptive immune cell fused bone myeloma cells to obtain hybridoma.Then antibody is collected from Hybridoma culture.Finally can be used as the RAD52 albumen of antigen or its part antibody to obtaining by use to implement antigenic specificity purification and obtain the monoclonal antibody for RAD52 albumen.Polyclonal antibody can be prepared as follows: with antigen-immunized animal same as above, collect blood sample from the animal through immunity, from blood, isolate serum, then use above-mentioned antigen that serum is implemented antigenic specificity purification.Antibody fragment can be obtained by the sequence information of the antibody obtained with ferment treatment or the antibody obtained by use.
The combination of label and antibody or its fragment can be implemented by method as commonly known in the art.For example, it is possible to following fluorescent marker protein or peptide: clean protein or peptide with phosphate buffer, add with DMSO, buffer agent, etc. the dyestuff of preparation, then mixed solution, place 10 minutes then at room temperature.It addition, labelling can the labelling kit of commodity in use, such as biotin labeling reagent box, such as biotin labeling reagent box-NH2, biotin labeling reagent box-SH (DojindoLaboratories)；Alkali phosphatase enzyme mark test kit such as alkali phosphatase enzyme mark test kit-NH2, alkali phosphatase enzyme mark test kit-SH (DojindoLaboratories)；Peroxidase labelling test kit such as peroxidase labelling test kit-NH2, peroxidase labelling test kit-NH2 (DojindoLaboratories)；Phycobniliprotein labelling kit 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, HiLyteFluor (TM) 555 labelling kit-NH2, HiLyteFluor (TM) 647 labelling kit-NH2 (DojindoLaboratories)；And DyLight547 and DyLight647 (TechnoChemicalCorp.), Zenon (TM), AlexaFluor (TM) antibody labeling test kit, Qdot (TM) antibody labeling test kit (InvitrogenCorporation) and EZ-label Protein Labeling Kit (FunakoshiCorporation).For correct labeling, it is possible to use suitable instrument detects the antibody through labelling or its fragment.
In the present invention, " prognosis " refer to that patients with coronary heart disease is by the suppression such as surgical procedure or the process after alleviating coronary heart disease or result.In this manual, life state when prognosis can be to be suppressed or alleviate after coronary heart disease 1,2,3,4,5,6,7,8,9,10,15,20 years or more long by surgical procedure.Prognosis can be by checking that the gene of biomarker i.e. RAD52 albumen or coding RAD52 albumen is predicted.Prognosis prediction can be performed such that according to biomarker with or without, or be raised and lowered, determine 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 that patient (such as 3,5,6,7,8,9,10,15,20 years or longer) over a long time does not has critical condition after being suppressed by surgical procedure etc. for patient or alleviate coronary heart disease.
In the present invention, " prognosis mala " refers to that patient occurs fatal condition within by the short-term (such as 1,2,3,4,5 years or shorter) after the suppression such as surgical procedure or alleviation coronary heart disease.
Prediction prognosis refers to predict process or the result of status of patient, is not meant to predict process or the result of status of patient with the accuracy of 100%.Prediction prognosis refers to whether the probability determining some process or result increases, and is not meant to be compared by situation about not occurring with some process or result and determines generation some process or the probability of result.For the present invention, in the patient that in the present invention, the level of RAD52 gene or RAD52 albumen is raised and lowered, compared with the patient not showing this feature, more likely observe particular procedure or result.
Further, the product of described detection RAD52 gene or RAD52 albumen can be detection RAD52 gene or the reagent of RAD52 albumen, can also be to comprise the test kit of described reagent, chip, reagent paper etc., it is also possible to be the high-flux sequence platform using described reagent.
Utilize the RAD52 gene in foregoing detection Product checking experimenter's sample or the expression of RAD52 albumen, compared with normal person, RAD52 gene or the expression of RAD52 albumen in experimenter's sample reduce, then diagnose the poor prognosis that this experimenter is patients with coronary heart disease or this experimenter.
Sample as the detection product according to the present invention, it is possible to use the tissue sample such as obtained from biopsy experimenter or fluid.Sample is not particularly limited, as long as it is suitable to the mensuration of the present invention；Such as, it can include tissue, blood, blood plasma, serum, lymph fluid, urine, serous cavity liquid, spinal fluid, synovial fluid, aqueous humor, tear, saliva or its fraction or treated material.
In specific embodiments of the present invention, described sample is from the blood of experimenter.
Present invention also offers the instrument of a kind of diagnosis of coronary heart disease, described instrument can detect RAD52 gene or the expression of RAD52 albumen in experimenter's sample.Described instrument includes can be in conjunction with the nucleic acid of RAD52 gene or can be in conjunction with the material (such as antibody) of RAD52 albumen.Described nucleic acid can detect the expression of RAD52 gene；Described material can detect the expression of RAD52 albumen.
Further, the character of described nucleic acid and described material is with noted earlier.
Further, the instrument of described diagnosis of coronary heart disease includes but not limited to chip, test kit, reagent paper or high-flux sequence platform；High-flux sequence platform is the instrument of a kind of special diagnosis of coronary heart disease, along with the development of high throughput sequencing technologies, will become the structure of the gene expression profile of a people and work the most easily.By contrast Disease and the gene expression profile of normal population, the exception easily analyzing which gene is relevant to disease.Therefore, high-flux sequence is known the exception purposes that fall within RAD52 gene relevant to coronary heart disease of RAD52 gene, equally within protection scope of the present invention.
Present invention also offers a kind of instrument predicting coronary heart disease prognosis, described instrument can detect RAD52 gene or the expression of RAD52 albumen in experimenter's sample, and described prediction coronary heart disease prognostic tool includes can be in conjunction with the nucleic acid of RAD52 gene or can be in conjunction with the material (such as antibody) of RAD52 albumen.Described nucleic acid can detect the mRNA level in-site of RAD52 gene；Described material can detect the expression of RAD52 albumen.
Further, the character of described nucleic acid and described material is with noted earlier.
Further, the instrument of described prediction coronary heart disease prognosis includes but not limited to chip, test kit, reagent paper or high-flux sequence platform；High-flux sequence platform is the instrument of a kind of special diagnosis of coronary heart disease, along with the development of high throughput sequencing technologies, will become the structure of the gene expression profile of a people and work the most easily.By contrast Disease and the gene expression profile of normal population, the exception easily analyzing which gene is relevant to disease.Therefore, high-flux sequence is known the exception purposes that fall within RAD52 gene relevant to coronary heart disease of RAD52 gene, equally within protection scope of the present invention.
The amino acid whose number that the anti-RAD52 antibody used in detection product, the diagnostic tool of the present invention or its fragment are identified is not particularly limited, as long as antibody can be in conjunction with RAD52.When antibody is as medicine, preferably it is capable of identify that aminoacid as much as possible, as long as it can suppress RAD52 function.The amino acid whose number of antibody or its fragment identification 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.
Further, described experimenter's sample can use the tissue sample or fluid such as obtained from biopsy experimenter.Sample is not particularly limited, as long as it is suitable to the mensuration of the present invention；Such as, it can include tissue, blood, blood plasma, serum, lymph fluid, urine, serous cavity liquid, spinal fluid, synovial fluid, aqueous humor, tear, saliva or its fraction or treated material.In specific embodiments of the present invention, described sample is from the blood of experimenter.
Present invention also offers a kind of diagnosis of coronary heart disease or the method for prediction coronary heart disease prognosis, described method comprises the steps:
(1) sample of experimenter is obtained；
(2) RAD52 gene or the expression of albumen in detection Samples subjects；
(3) the RAD52 gene recorded or the expression of albumen are associated with the whether ill of experimenter.
(4) compared with the control, the expression of RAD52 gene or albumen reduces, then this experimenter is diagnosed as coronary heart disease, or this experimenter is confirmed as prognosis mala.
In the context of the present invention, " diagnosis of coronary heart disease " both includes judging that experimenter has suffered from coronary heart disease, also included judging whether experimenter exists the risk suffering from coronary heart disease.
Advantages of the present invention and beneficial effect:
The molecular marker being found that a kind of diagnosis of coronary heart disease of the present invention, uses this molecular marker can be used as judging in coronary disease pathogenetic early stage, it is provided that the survival rate of patient.
It addition, by the prognosis predicting patient, the present invention can provide significant information to determine therapeutic scheme strategy for patient.
Accompanying drawing explanation
Fig. 1 shows the expression utilizing genechip detection RAD52 gene in patients with coronary heart disease with normal person；
Fig. 2 shows the expression utilizing QPCR detection RAD52 gene in patients with coronary heart disease with normal person；
Fig. 3 shows the expression utilizing Westernblot detection RAD52 albumen in patients with coronary heart disease with normal person.
Specific embodiment
The present invention is further detailed explanation with embodiment below in conjunction with the accompanying drawings.Following example are merely to illustrate the present invention rather than limit the scope of the present invention.The experimental technique of unreceipted actual conditions in embodiment, generally according to normal condition, such as Sambrook et al., molecular cloning: laboratory manual (NewYork:ColdSpringHarborLaboratoryPress, 1989) condition described in, or according to the condition proposed by manufacturer.
The differential expression of embodiment 1RAD52 gene
1, object of study:
Collect patients with coronary heart disease 5 example, normal person 5 example.
The inclusive criteria of CHD group: according to the diagnostic criteria of the ischemic heart desease that World Health Organization (WHO) formulates, selects underwent coronary radiography to confirm the patients with coronary heart disease having one or more angiostenosis degree >=50%.The inclusion criteria of matched group is: 1) when Epidemiological study, screening age, sex, nationality all match with CAD group, do not have the clinical manifestation of coronary heart disease and the examinee of Major Risk Factors through questionnaire survey, physical examination, Cardiac ultrasound and Electrocardioscopy and laboratory inspection；2) the parallel coronary artery revasualization of row health examination of being in hospital gets rid of coronary heart disease age, sex, national and CAD group matcher；Meet one person of any of the above to enter to elect matched group as.Informed Consent Form is signed for two groups before including research in.
Rejecting standard: the full person of CAD group-clinical data and merge the one of following disease simultaneously and rejected.
(such as: Congenital Heart patient, dissection of aorta, MOFE, rheumatic heart disease and there is mental disorder can not the person of cooperation).Matched group: spiritedness obstacle person or check that confirmation has carotid atherosclerotic plaque or narrow person through Doppler, is rejected.
2, the extraction of total serum IgE in blood
(1) homogenized (Homogenization)
Requiring object of study at least 12h on an empty stomach, under m seq 7:00～8:00 room temperature, extraction 10ml venous blood is in ethylenediaminetetraacetic acid (EDTA) anticoagulant tube, add 3 times of volume erythrocyte cracked liquids, after mixing, room temperature is placed 10 minutes, and 10,000rpm are centrifuged 1 minute.Thoroughly inhale and abandon supernatant, collect leukocyte cell pellet.The leukocyte cell pellet of every 100-200 μ l blood collecting adds 1mlTRIzol.
(2) layering (PhaseSeparation)
A., after sample adds TRIzol, room temperature places 5min, makes sample fully crack.
B. every 1mlTRIzol adds 200 μ l chloroforms, and acutely after vibration mixing, room temperature is placed 3-5min and made its natural split-phase.
(3) RNA precipitate (RNAPrecipitation)
A.4 a DEG C 12,000rpm is centrifuged 10-15min.Sample can be divided into three layers: the organic facies of yellow, intermediate layer and colourless aqueous phase, and RNA, mainly in aqueous phase, transfers to aqueous phase (generally can draw 550 μ l) in new pipe.
B. adding the isopropanol that equal-volume is ice-cold in supernatant, room temperature places 10-20min.4 DEG C 12,000rpm is centrifuged 10min, abandons supernatant, and RNA precipitate is at the bottom of pipe.
(4) RNA rinsing (RNAWash)
Adding 1ml75% ethanol (preparing with RNase-free water) in a.RNA precipitation, gentle vibration centrifuge tube, suspend precipitation.Every 1mlTRIzol adds 1ml75% ethanol.
B.4 DEG C 5,000-8,000rpm is centrifuged 1-2min, abandons supernatant；Of short duration the most centrifugal, carefully to inhale with pipettor and abandon supernatant, room temperature is placed and is dried precipitation in 1-2 minute.
(5) RNA (RedissolvingtheRNA) is dissolved
Precipitation adds 50-100 μ lRNase-free water, flicks tube wall, fully to dissolve RNA ,-70 DEG C of preservations.
3, RNA mass and purity detecting
RNA mass: represented by RNA integrity, available plain agar sugar gel electrophoresis (deposition condition: 1.2% glue；0.5 × TBE electrophoretic buffer；150v, 15 minutes) detection integrity.
RNA purity: OD260/OD280 ratio is to weigh the index of protein contamination degree in RNA sample.High-quality RNA sample, OD260/OD280 value (10mMTris, pH7.5) is about 2.0.
4, order-checking: above-mentioned RNA is delivered Shanghai Biotechnology Corporation and uses Solexa sequencing technologies that sample is checked order.
5, data analysis
5.1 original data processing
The raw image data that sequenator produces is converted into sequence data through BaseCalling, we term it original sequence data, result is with FASTQ stored in file format.The contaminating sequences data such as low quality sequence, joint sequence may be comprised due to original sequence data, cannot be directly used to analysis of biological information, so original sequence data must pass through data process and is converted to high-quality sequence data, the splicedmapping algorithm utilizing Tophat (version:2.0.6) software carries out genome alignment to high-quality sequence data, and using genome version is Sscrofa10.2.
The screening of 5.2 differential genes
The calculating of gene expression amount makes FPKM method.Expression (FPKM value) according to gene calculates this gene differential expression multiple between different samples.Difference expression gene is defined as FDR≤0.01 and fold difference at 2 times and above gene.
6, result
Result shows (as shown in Figure 1), and compared with normal person, in patients with coronary heart disease blood, the mRNA level in-site of RAD52 gene significantly reduces, and difference has statistical significance (P < 0.05).
The gene of differential expression in embodiment 2QPCR experimental verification patients with coronary heart disease and normal person
1, object of study:
Screening criteria is with each 50 examples of embodiment 1, patients with coronary heart disease and normal person.
2, the extraction of total serum IgE in blood
Step is with embodiment 1.
3, reverse transcription
With RT Buffer, l μ g total serum IgE is carried out reverse transcription and synthesize cDNA.Using 25 μ l reaction systems, each sample takes 1 μ g total serum IgE as template ribonucleic acid, is separately added into following components in PCR pipe: DEPC water, 5 × RT Buffer, 10mmol/LdNTP, 0.1mmol/lDTT, 30 μm mol/lOligodT, 200U/ μ lM-MLV, template ribonucleic acid.Hatch 1h for 42 DEG C, 72 DEG C of 10min, of short duration centrifugal.
4、QPCR
(1) design of primers
According to the coded sequence design QPCR amplimer of RAD52 gene and GAPDH gene in Genbank, Shanghai Sangon Biological Engineering Technology And Service Co., Ltd synthesize.Concrete primer sequence is as follows:
RAD52 gene:
Forward primer is 5 '-CAGTTGCCTCTTGAAGTG-3 ' (SEQIDNO.3)；
Reverse primer is 5 '-TGTTGTATCTTGCCTCCT-3 ' (SEQIDNO.4),
GAPDH gene:
Forward primer is 5 '-GAAGGTGAAGGTCGGAGT-3 ' (SEQIDNO.5)；
Reverse primer is 5 '-CATGGGTGGAATCATATTGGAA-3 ' (SEQIDNO.6).
(2) PCR reaction system is prepared according to table 1:
Wherein, SYBRGreen polymerase chain reaction system is purchased from Invitrogen company.
Table 1PCR reaction system
| 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 SYBRGreen as fluorescent marker, react at the enterprising performing PCR of LightCycler quantitative real time PCR Instrument, determine that purpose band, Δ Δ CT method carry out relative quantification by melt curve analysis analysis and electrophoresis.
5, statistical method
Result data is all to represent in the way of mean+SD, uses SPSS13.0 statistical software to carry out statistical analysis, and difference between the two uses t inspection, it is believed that when P < has statistical significance when 0.05.
6, result
Result is as in figure 2 it is shown, compared with normal person, in patients with coronary heart disease blood, the mRNA level in-site of RAD52 gene significantly reduces, and difference has statistical significance (P < 0.05), result is homogenic array experiment.
Embodiment 3RAD52 protein diversity detection of expression
1, object of study
With embodiment 2.
2, mononuclear cell separates
Aseptic collection venous blood 10ml, injects in the sterile vials containing heparin, shakes up the most gently after adding a cover.Isopyknic HBSS (NaCl8.0g, Na is added with aseptic straw2HPO40.132g, KH2PO40.06g, KCl0.4g, phenol red 1ml, NaHCO30.35g, D-Glucose 1.0g, be dissolved in 1000ml distilled water), to reduce erythrocytic cohesion.nullDraw 8ml lymphocytes separating solution to put in 50ml centrifuge tube，Dilute blood is slowly added to along tube wall，Interface is kept to understand，Both are not made to mix mutually，It is centrifuged 30min at 20 DEG C of 2000r/min，Careful layering liquid of drawing joins, with blood plasma, the buffy coat that position is muddy，I.e. buffy coat，Add in another centrifuge tube，Wash 2 times with the HBSS of 5 times of volumes，Successively with 2000r/min、1500r/min is at room temperature centrifuged 10min，To remove the platelet that major part mixes，With 10ml distilled water and cell mass mixing 1min，Residual red blood cells is made to crack，Then equivalent 1.8%NaCl solution it is rapidly added，2000r/min is centrifuged，Remove supernatant，Cell is adjusted to 1 × 10 with HBSS solution after cell counting6Individual/ml is standby.
3, mononuclear cell gross protein extracts
By above-mentioned experiment gained cell suspension, (concentration is 1 × 106Individual/ml) room temperature 1000r/min is centrifuged 10min, and add 100 μ l lysis buffers, 4 DEG C of concussion 1h after abandoning supernatant, use ultrasonoscope smudge cells, each 10s, totally 10 times, be centrifuged 1h in 4 DEG C of 12000r/min；Taking supernatant Brandford standard measure albumen, be distributed into 2.5 μ g/ μ l ,-80 DEG C of Refrigerator stores are standby.
4, Westernblot detection
The albumen of extraction is carried out SDS-PAGE electrophoresis, carries out transferring film, closing afterwards, one anti-hatch, two anti-hatch, develop the color.
5, statistical procedures
Use ImageJ software to be analyzed the gray value of protein band, with β-actin as internal reference, the gray value of purpose informal voucher band is normalized.Result data is all to represent in the way of mean+SD, uses SPSS13.0 statistical software to carry out statistical analysis, and difference between the two uses t inspection, it is believed that when P < has statistical significance when 0.05.
6, result
Result is as it is shown on figure 3, compared with normal person, in patients with coronary heart disease blood, RAD52 protein content is low, and difference has statistical significance (P < 0.05).
The explanation of above-described embodiment is only intended to understand the method for the present invention and core concept thereof.It should be pointed out that, for the person of ordinary skill of the art, under the premise without departing from the principles of the invention, it is also possible to the present invention is carried out some improvement and modification, these improve and modify also by the protection domain falling into the claims in the present invention.
Claims (10)
1. the product of detection RAD52 gene or RAD52 albumen application in the instrument preparing diagnosis of coronary heart disease or prediction coronary heart disease prognosis.
Application the most according to claim 1, it is characterised in that the product of described detection RAD52 gene or RAD52 albumen includes the product detecting the expression of RAD52 gene or RAD52 albumen.
Application the most according to claim 2, it is characterized in that, utilize the RAD52 gene in described Product checking experimenter's sample or the expression of RAD52 albumen, compared with normal person, RAD52 gene or the expression of RAD52 albumen in experimenter's sample reduce, then diagnose the poor prognosis that this experimenter is patients with coronary heart disease or this experimenter.
Application the most according to claim 3, it is characterised in that the source of described experimenter's sample is blood.
5. according to the application according to any one of claim 1-4, it is characterised in that described product includes can be in conjunction with the nucleic acid of RAD52 gene or can be in conjunction with the material of RAD52 albumen；Described nucleic acid can detect the expression of RAD52 gene；Described material can detect the expression of RAD52 albumen.
Application the most according to claim 5, it is characterised in that described nucleic acid is that the primer of the specific amplified RAD52 gene used in real-time quantitative PCR is as shown in SEQIDNO.1 and SEQIDNO.2.
7. a diagnosis of coronary heart disease or the instrument of prediction coronary heart disease prognosis, it is characterised in that described instrument includes the instrument of the expression of RAD52 gene or the RAD52 albumen that can detect in experimenter's sample.
Instrument the most according to claim 7, it is characterised in that described instrument includes can be in conjunction with the nucleic acid of RAD52 gene or can be in conjunction with the material of RAD52 albumen；Described nucleic acid can detect the expression of RAD52 gene；Described material can detect the expression of RAD52 albumen.
Instrument the most according to claim 8, it is characterised in that described nucleic acid is that the primer of the specific amplified RAD52 gene used in real-time quantitative PCR is as shown in SEQIDNO.1 and SEQIDNO.2.
10. according to the instrument according to any one of claim 7-9, it is characterised in that the source of described experimenter's sample is blood.
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