CN106048006A - Application of SMCHD1 to preparation of product for diagnosing coronary heart disease - Google Patents
Application of SMCHD1 to preparation of product for diagnosing coronary heart disease Download PDFInfo
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- CN106048006A CN106048006A CN201610383238.6A CN201610383238A CN106048006A CN 106048006 A CN106048006 A CN 106048006A CN 201610383238 A CN201610383238 A CN 201610383238A CN 106048006 A CN106048006 A CN 106048006A
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- smchd1
- gene
- heart disease
- albumen
- 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 an SMCHD1 gene and encoding protein thereof, wherein the SMCHD1 gene and the encoding protein can serve as molecular markers for diagnosing the coronary heart disease. By detecting the content of the SMCHD1 gene and the expression product in blood of a subject, whether the subject suffers from the coronary heart disease or has the risk of the coronary heart disease can be judged. The research result provides a noninvasive, specific and sensitive coronary heart disease diagnosing method for clinic.
Description
Technical field
The present invention relates to diagnosis of coronary heart disease, prediction prognosis field, more particularly it relates to abnormal with detection SMCHD1
Diagnosis of coronary heart disease, prediction method of prognosis for means.
Background technology
Coronary atherosclerotic heart disease (abbreviation coronary heart disease), is modal one in heart disease, cardiovascular disease
Disease is one of whole world major disease causing patient death, occupies second in numerous lethal causes of disease, and sickness rate is about 319/
100000.In China owing to lacking disease registration and the taxonomic hierarchies of standard, so the most Chinese stream cannot be obtained
Row disease learns data.Started from 1980, the domestic register system beginning to use international standard.Dead people in 2004 1 year
Number has accounted for 9% that urban population is dead, the 22% of cardiovascular disease death toll；Rural area corresponding data is respectively 4% He
13%.In the same year, there are the patients with coronary artery disease death of 400,000, the new diagnosed SARS case of 650,000.In world wide, the most nearly 2,500,000
Patient dies from coronary heart disease relevant disease.Along with the most accurate of statistics and urbanization process, the patients with coronary artery disease of China is just
More and more.Expected centroid angiopathy death toll can arrive 1/3rd of all dead people to the year two thousand twenty.Problem is more come
The severeest.
The pathogenesis of coronary heart disease is sufficiently complex, existing environmental factors, also has inherited characteristic, numerous perspective or return
Find on the basis of the analysis and research of Gu property, hypertension, smoking, obesity (hypercholesterolemia and diabetes and coronary heart disease in Chinese
Dependency is the highest, is not only because the risk factor that these factors are, what is more important hypertension, smoking and overweight
The most important change that compatriots bring due to lifestyle change during China Modernization Process.In the research that the sixth of the twelve Earthly Branches has
Verified, that Abnormal Lipid Metabolism, smoking, diabetes and hypertension are main pathogenic, accounts in morbidity's factor
Left and right.Additionally, be exactly genetic factors impact.In recent years, molecular diagnosis becomes study hotspot, because molecular diagnosis can be in disease
Sick early stage can suffer from the risk probability of coronary heart disease to patient as diagnosis or provide for experimenter, to remind risky person
The generation of the prevention coronary heart disease that takes appropriate measures.Therefore, a kind of new specificity and the high coronary heart disease molecule of susceptiveness are found
Diagnosis marker is the research emphasis of the present invention.
Summary of the invention
An object of the present invention is that providing a kind of diagnoses hat by detection SMCHD1 gene or protein expression difference
The method of cardiopathia.
The two of the purpose of the present invention are that providing a kind of predicts hat by detection SMCHD1 gene or protein expression difference
The method of cardiopathia prognosis.
To achieve these goals, present invention employs following technical scheme:
The invention provides the product of detection SMCHD1 gene or SMCHD1 albumen in preparation diagnosis of coronary heart disease instrument
Purposes.
Present invention also offers the product of detection SMCHD1 gene or SMCHD1 albumen in preparation prediction coronary heart disease prognosis work
Purposes in tool.
Further, the product of described detection SMCHD1 gene or SMCHD1 albumen includes detecting SMCHD1 gene or SMCHD1
The product of the expression of albumen.Described product includes can be in conjunction with the nucleic acid of SMCHD1 gene or can be in conjunction with SMCHD1 egg
White material (such as antibody).Described nucleic acid can detect the expression of SMCHD1 gene；Described material can detect
The expression of SMCHD1 albumen.
The product of the detection SMCHD1 gene of the present invention can play its function based on the known method using nucleic acid molecules:
As PCR, 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 containing from biomaterial preparation
Expect the gene of nucleic acid, then use be designed for amplification expectation nucleic acid primer amplification it obtain.
Further, described PCR method is known method, such as, and ARMS (Amplification Refractory
Mutation System, abruptly-changing system is not answered in amplification) method, RT-PCR (reverse transcriptase-PCR) method, nested PCR method etc..Amplification
Nucleic acid can by use dot blotting hybridization method, Surface Plasmon Resonance (SPR method), PCR-RFLP method, in situ 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 expanding SMCHD1 gene, and the primer that product includes can be by by changing
Learning synthesis to prepare, the method that be those skilled in the art will know that by use is suitably designed with reference to Given information, and passes through
Prepared by chemosynthesis.
In specific embodiments of the present invention, described nucleic acid is the amplimer used in QPCR experiment, described primer
Sequence such as SEQ ID NO.1 (forward sequence) and SEQ ID NO.2 (reverse sequence) shown in.
Nucleic acid recited above may also include probe, and described probe can be prepared by chemosynthesis, by using this
The method that skilled person knows appropriately designs with reference to Given information, and is prepared by chemosynthesis, or can lead to
Cross the gene containing expectation nucleotide sequence from biomaterial preparation, and use the primer expansion being designed for amplification expectation nucleotide sequence
Increase it to prepare.
The product of the detection SMCHD1 albumen of the present invention can play its function based on the known method using antibody: example
As, ELISA, radioimmunoassay, immunohistochemical method, western blot etc. can be included.
The product of the detection SMCHD1 albumen of the present invention includes antibody or its fragment of specific binding SMCHD1 albumen.Can
To use antibody or its fragment of any structure, size, immunoglobulin class, origin etc., as long as it combines target protein i.e.
Can.Antibody or its fragment that the detection product of the present invention includes can be monoclonal or polyclonal.Antibody fragment refers to protect
Stay antibody to an antibody part (Partial Fragment) of the combination activity of antigen or the peptide containing an antibody part.Antibody fragment is permissible
Including F (ab ')2, Fab ', Fab, scFv (scFv), disulphide bonding Fv (dsFv) or its polymer, dimerization V district
(double antibody) or the peptide containing CDR.The product of the detection SMCHD1 albumen of the present invention can include encoding antibody or encoding antibody
The nucleic acid of the separation of the aminoacid sequence of 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 target all or in part
The polypeptide of protein or the mammalian cell expression vector of integration their polynucleotide of coding are as antigen.Use antigen is exempted from
After epidemic disease animal, from passing through immune animal adaptive immune cell fused bone myeloma cells to obtain hybridoma.Then from hybridization
Antibody collected by tumor culture.SMCHD1 albumen or its part antibody reality to obtaining of antigen finally can be used as by use
Execute antigenic specificity purification and obtain the monoclonal antibody for SMCHD1 albumen.Can prepare polyclonal antibody as follows: with
The most identical antigen-immunized animal, collects blood sample from the animal through immunity, isolates serum, then make from blood
With above-mentioned antigen, serum is implemented antigenic specificity to purify.By the antibody obtained with ferment treatment or can be obtained by use
The sequence information of antibody obtains antibody fragment.
The combination of label and antibody or its fragment can be implemented by method as commonly known in the art.Such as, may be used
With following fluorescent marker protein or peptide: clean protein or peptide with phosphate buffer, add with DMSO, buffer agent, etc. standard
Standby dyestuff, then mixed solution, place 10 minutes then at room temperature.It addition, labelling can the labelling kit of commodity in use, all
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, alkalescence phosphorus
Acid enzyme labelling test kit-SH (Dojindo Laboratories)；Peroxidase labelling test kit such as peroxidase mark
Note test kit-NH2, peroxidase labelling test 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, HiLyte Fluor (TM) 647 labelling kit-NH2 (Dojindo Laboratories)；And
DyLight 547 and DyLight647 (Techno Chemical Corp.), Zenon (TM), Alexa Fluor (TM) antibody
Labelling kit, Qdot (TM) antibody labeling test kit (Invitrogen Corporation) and EZ-label protein mark
Note test kit (Funakoshi Corporation).For correct labeling, it is possible to use suitable instrument detects through labelling
Antibody 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 mistake after alleviating coronary heart disease
Journey or result.In this manual, prognosis can be suppressed or alleviate after coronary heart disease 1 by surgical procedure, 2,3,4,5,6,7,
8,9,10,15,20 years or more long time life state.Prognosis can be by checking biomarker i.e. SMCHD1 albumen or coding
The gene of SMCHD1 albumen is predicted.Prognosis prediction can be performed such that according to biomarker with or without, or raise or
Reduce, 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 after being suppressed by surgical procedure etc. for patient or alleviate coronary heart disease,
Patient (such as 3,5,6,7,8,9,10,15,20 years or longer) over a long time does not has critical condition.The most preferred state of prognosis bona
It it is the survival for a long time without disease.
In the present invention, " prognosis mala " refer to patient by the suppression such as surgical procedure or after alleviating coronary heart disease in short-term
Fatal condition is there is in phase (such as 1,2,3,4,5 years or shorter).
Prediction prognosis refers to predict process or the result of status of patient, is not meant to predict with the accuracy of 100%
The process of status of patient or result.Prediction prognosis refers to whether the probability determining some process or result increases, and the most unexpectedly
Taste to be compared by situation about not occurring with some process or result and is determined some process of generation or the probability of result.Such as this
For invention, in the patient that in the present invention, the level of SMCHD1 gene or SMCHD1 albumen is raised and lowered, and do not show this spy
The patient levied compares, and more likely observes particular procedure or result.
Further, the product of described detection SMCHD1 gene or SMCHD1 albumen can be detection SMCHD1 gene or
The reagent of SMCHD1 albumen, can also be to comprise the test kit of described reagent, chip, reagent paper etc., it is also possible to be to use described examination
The high-flux sequence platform of agent.
Utilize the SMCHD1 gene in foregoing detection Product checking experimenter's sample or the expression of SMCHD1 albumen
Level, compared with normal person, SMCHD1 gene or the expression of SMCHD1 albumen in experimenter's sample raise, then diagnosis should
Experimenter is the poor prognosis of 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
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 SMCHD1 in experimenter's sample
Gene or the expression of SMCHD1 albumen.Described instrument includes can be in conjunction with the nucleic acid of SMCHD1 gene or can be in conjunction with
The material (such as antibody) of SMCHD1 albumen.Described nucleic acid can detect the expression of SMCHD1 gene；Described material can
The expression of detection SMCHD1 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, to one
The structure of gene expression profile of individual will become and work the most easily.By contrast Disease and the gene table of normal population
Reaching spectrum, the exception easily analyzing which gene is relevant to disease.Therefore, high-flux sequence is known the different of SMCHD1 gene
The often purposes that fall within SMCHD1 gene relevant to coronary heart disease, 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 in experimenter's sample
SMCHD1 gene or the expression of SMCHD1 albumen, described prediction coronary heart disease prognostic tool includes can be in conjunction with SMCHD1 base
The nucleic acid of cause or can be in conjunction with the material (such as antibody) of SMCHD1 albumen.Described nucleic acid can detect SMCHD1 gene
MRNA level in-site；Described material can detect the expression of SMCHD1 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
Order-checking 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,
The structure of the gene expression profile of one people will be become and work the most easily.By contrast Disease and the base of normal population
Because of express spectra, the exception easily analyzing which gene is relevant to disease.Therefore, high-flux sequence is known SMCHD1 gene
The exception purposes that fall within SMCHD1 gene relevant to coronary heart disease, equally within protection scope of the present invention.
It is amino acid whose that the anti-SMCHD1 antibody used in detection product, the diagnostic tool of the present invention or its fragment are identified
Number is not particularly limited, as long as antibody can be in conjunction with SMCHD1.When antibody is as medicine, preferably its energy
Enough identify aminoacid as much as possible, as long as it can suppress SMCHD1 function.Antibody or the amino acid whose number of its fragment identification
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.
Other character of the anti-SMCHD1 antibody used in detection product, the diagnostic tool of the present invention are with noted earlier.
Further, described experimenter's sample can use the tissue sample or fluid such as obtained from biopsy experimenter.Sample
Originally it 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
Liquid, urine, serous cavity liquid, spinal fluid, synovial fluid, aqueous humor, tear, saliva or its fraction or treated material.In the present invention
Specific embodiments in, 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 includes walking as follows
Rapid:
(1) sample of coronary heart disease experimenter is obtained；
(2) SMCHD1 gene or the expression of albumen in detection Samples subjects；
(3) the SMCHD1 gene recorded or the expression of albumen are associated with the whether ill of experimenter.
(4) compared with the control, the expression of SMCHD1 gene or albumen raises, then this experimenter is diagnosed as coronary disease
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
Including judging whether experimenter exists and suffer from the risk of 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 in coronary heart disease
The early stage occurred can be used as judging, 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 treatment side for patient
Case strategy.
Accompanying drawing explanation
Fig. 1 shows the expression utilizing genechip detection SMCHD1 gene in patients with coronary heart disease with normal person；
Fig. 2 shows the expression utilizing QPCR detection SMCHD1 gene in patients with coronary heart disease with normal person；
Fig. 3 shows the expression feelings utilizing Western blot detection SMCHD1 albumen in patients with coronary heart disease with normal person
Condition.
Specific embodiment
The present invention is further detailed explanation with embodiment below in conjunction with the accompanying drawings.Following example are merely to illustrate this
Invention rather than restriction the scope of the present invention.The experimental technique of unreceipted actual conditions in embodiment, generally 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 condition proposed by manufacturer.
The differential expression of embodiment 1 SMCHD1 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, select
Underwent coronary radiography confirms the patients with coronary heart disease having one or more angiostenosis degree >=50%.The inclusion criteria of matched group
For: 1) screen age, sex when Epidemiological study, national all match with CAD group, through questionnaire survey, physical examination,
Cardiac ultrasound and Electrocardioscopy and laboratory inspection do not have the clinical manifestation of coronary heart disease and Major Risk Factors
Examinee；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 spirit
Obstacle can not the person of cooperation).Matched group: spiritedness obstacle person or check that confirmation has carotid atherosclerotic plaque or narrow person through Doppler, gives
To reject.
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 second
Ethylenediamine tetraacetic acid (EDTA) (EDTA) anticoagulant tube, adds 3 times of volume erythrocyte cracked liquids, room temperature placement 10 minutes after mixing, and 10,000rpm
Centrifugal 1 minute.Thoroughly inhale and abandon supernatant, collect leukocyte cell pellet.The leukocyte cell pellet of every 100-200 μ l blood collecting adds 1ml
TRIzol。
(2) layering (Phase Separation)
A., after sample adds TRIzol, room temperature places 5min, makes sample fully crack.
B. every 1ml TRIzol 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 (RNA Precipitation)
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, 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 of 12,000rpm are centrifuged
10min, abandons supernatant, and RNA precipitate is at the bottom of pipe.
(4) RNA rinsing (RNA Wash)
A.RNA precipitation adds 1ml 75% ethanol (preparing with RNase-free water), gentle vibration centrifuge tube, suspends heavy
Form sediment.Every 1ml TRIzol adds 1ml 75% ethanol.
B.4 DEG C 5,000-8,000rpm is centrifuged 1-2min, abandons supernatant；Of short duration the most centrifugal, carefully inhale with pipettor and abandon
Clearly, room temperature is placed and is dried precipitation in 1-2 minute.
(5) RNA (Redissolving the RNA) is dissolved
Precipitation adds 50-100 μ l RNase-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 (10mM Tris, pH7.5) is about 2.0.
4, order-checking: above-mentioned RNA is delivered Shanghai Biotechnology Corporation and uses Solexa sequencing technologies to sample
Check order.
5, data analysis
5.1 original data processing
The raw image data that sequenator produces is converted into sequence data through Base Calling, we term it original sequence
Column data, result is with FASTQ stored in file format.Owing to original sequence data may comprise low quality sequence, joint sequence etc.
Contaminating sequences data, it is impossible to be directly used in analysis of biological information, so original sequence data must pass through data process and is converted to
High-quality sequence data, utilizes the spliced mapping algorithm of Tophat (version:2.0.6) software to high-quality sequence
Data carry out genome alignment, 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 at different samples
Between differential expression multiple.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), compared with normal person, and the mRNA level in-site of SMCHD1 gene in patients with coronary heart disease blood
Notable rising, difference has statistical significance (P < 0.05).
The gene of differential expression in embodiment 2 QPCR 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.Use 25 μ l reaction systems, each sample
Take 1 μ g total serum IgE as template ribonucleic acid, PCR pipe is separately added into following components: DEPC water, 5 × RT Buffer,
10mmol/L dNTP, 0.1mmol/l DTT, 30 μm mol/l Oligo dT, 200U/ μ l M-MLV, template ribonucleic acid.Hatch for 42 DEG C
1h, 72 DEG C of 10min, of short duration centrifugal.
4、QPCR
(1) design of primers
According to the coded sequence design QPCR amplimer of SMCHD1 gene and GAPDH gene in Genbank, give birth in Shanghai
The synthesis of work biotechnology Services Co., Ltd.Concrete primer sequence is as follows:
SMCHD1 gene:
Forward primer is 5 '-ATTCATTGTCTGCTACTTCTC-3 ' (SEQ ID NO.3)；
Reverse primer is 5 '-TCCTCTTCCATTATCTATCACT-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.
Table 1 PCR reaction system
(3) PCR reaction condition: 95 DEG C of 5min, (95 DEG C of 5s, 60 DEG C of 45s) * 45 circulations.Using SYBR Green as glimmering
Signal thing, is reacted at the Light enterprising performing PCR of Cycler quantitative real time PCR Instrument, is determined by melt curve analysis analysis and electrophoresis
Purpose band, Δ Δ CT method carries out relative quantification.
5, statistical method
Result data is all to represent in the way of mean+SD, uses SPSS13.0 statistical software to unite
Meter is analyzed, 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 SMCHD1 gene significantly increases
Adding, difference has statistical significance (P < 0.05), result is homogenic array experiment.
Embodiment 3 SMCHD1 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.Use aseptic suction
Pipe adds isopyknic HBSS (NaCl 8.0g, Na2HPO40.132g, KH2PO40.06g, KCl 0.4g, phenol red 1ml,
NaHCO30.35g, D-Glucose 1.0g, be dissolved in 1000ml distilled water), to reduce erythrocytic cohesion.Draw 8ml lymph thin
Born of the same parents are layered liquid and put in 50ml centrifuge tube, are slowly added to along tube wall by dilute blood, keep interface to understand, do not make both mix mutually,
20 DEG C of 2 000r/min is centrifuged 30min, and careful layering liquid of drawing joins the buffy coat that position is muddy, i.e. lymphocyte with blood plasma
Layer, adds in another centrifuge tube, washs 2 times with the HBSS of 5 times of volumes, successively with 2 000r/min, 1 500r/min in room
The lower centrifugal 10min of temperature, in order to remove the platelet that major part mixes, with 10ml distilled water and cell mass mixing 1min, make residual
Remaining erythrocyte splitting, is then rapidly added equivalent 1.8%NaCl solution, and 2 000r/min are centrifuged, and remove supernatant, after cell counting
Cell is adjusted to 1 × 10 with HBSS solution6Individual/ml is standby.
3, mononuclear cell gross protein extracts
By above-mentioned experiment gained cell suspension, (concentration is 1 × 106Individual/ml) room temperature 1 000r/min is centrifuged 10min, abandons
Add 100 μ l lysis buffers, 4 DEG C of concussion 1h after clear, use ultrasonoscope smudge cells, each 10s, totally 10 times, in 4 DEG C 12
000r/min is centrifuged 1h；Taking supernatant Brandford standard measure albumen, be distributed into 2.5 μ g/ μ l ,-80 DEG C of Refrigerator stores are standby.
4, Western blot detection
The albumen of extraction is carried out SDS-PAGE electrophoresis, carry out afterwards transferring film, closing, one anti-hatch, two anti-hatch, aobvious
Color.
5, statistical procedures
Image J software is used to be analyzed, with β-actin as internal reference, by purpose informal voucher the gray value of protein band
The gray value of band is normalized.Result data is all to represent in the way of mean+SD, uses
SPSS13.0 statistical software carries out statistical analysis, and difference between the two uses t inspection, it is believed that when P < has system when 0.05
Meaning learned by meter.
6, result
Result is as it is shown on figure 3, compared with normal person, in patients with coronary heart disease blood, SMCHD1 protein content is high, 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 this
For the those of ordinary skill in field, 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 SMCHD1 gene or SMCHD1 albumen is preparing diagnosis of coronary heart disease or the instrument of prediction coronary heart disease prognosis
In application.
Application the most according to claim 1, it is characterised in that described detection SMCHD1 gene or the product of SMCHD1 albumen
Including detection SMCHD1 gene or the product of the expression of SMCHD1 albumen.
Application the most according to claim 2, it is characterised in that utilize the SMCHD1 in described Product checking experimenter's sample
Gene or the expression of SMCHD1 albumen, compared with normal person, the SMCHD1 gene in experimenter's sample or SMCHD1 albumen
Expression raise, 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 SMCHD1
The nucleic acid of gene or can be in conjunction with the material of SMCHD1 albumen；Described nucleic acid can detect the expression of SMCHD1 gene；
Described material can detect the expression of SMCHD1 albumen.
Application the most according to claim 5, it is characterised in that described nucleic acid is the special expansion used in real-time quantitative PCR
Increase the primer of SMCHD1 gene as shown in SEQ ID NO.1 and SEQ ID NO.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 detecting and is subject to
SMCHD1 gene in examination person's sample or the instrument of the expression of SMCHD1 albumen.
Instrument the most according to claim 7, it is characterised in that described instrument includes can be in conjunction with the nucleic acid of SMCHD1 gene
Or can be in conjunction with the material of SMCHD1 albumen；Described nucleic acid can detect the expression of SMCHD1 gene；Described material energy
Enough detect the expression of SMCHD1 albumen.
Instrument the most according to claim 8, it is characterised in that described nucleic acid is the special expansion used in real-time quantitative PCR
Increase the primer of SMCHD1 gene as shown in SEQ ID NO.1 and SEQ ID NO.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
Liquid.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013192185A1 (en) * | 2012-06-18 | 2013-12-27 | Fred Hutchinson Cancer Research Center | Methods compositions related to the smchd1 gene |
| CN105543408A (en) * | 2016-03-16 | 2016-05-04 | 宫蕊 | Marker for early diagnosis of coronary heart disease |
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2016
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013192185A1 (en) * | 2012-06-18 | 2013-12-27 | Fred Hutchinson Cancer Research Center | Methods compositions related to the smchd1 gene |
| CN105543408A (en) * | 2016-03-16 | 2016-05-04 | 宫蕊 | Marker for early diagnosis of coronary heart disease |
Non-Patent Citations (1)
| Title |
|---|
| 高阅春等: "冠心病患者冠状动脉病变严重程度与冠心病危险因素的相关分析", 《中国循环杂志》 * |
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