CN106086182A - PCYOX1 albumen is as the application in cerebral infarction biomarker - Google Patents
PCYOX1 albumen is as the application in cerebral infarction biomarker Download PDFInfo
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Abstract
The invention discloses PCYOX1 albumen as the application in cerebral infarction biomarker.Specifically disclose PCYOX1 albumen or its active fragment or detection PCYOX1 albumen or the application in diagnosing ischemia apoplexy and/or assessment suffer from the product of ischemic cerebral apoplexy risk of the material of its active fragment.The invention discloses a kind of albumen PCYOX1 relevant to apoplexy, and be further characterized by this PCYOX1 albumen or its active fragment down-regulated expression in apoplexy tissue.Utilize this Protein Detection apoplexy can not only accomplish fast and effectively in early days to detect, and provide therapy target and important evidence for the clinical practice such as gene therapy, Drug therapy.
Description
Technical field
The present invention relates to biomedicine field, be specifically related to PCYOX1 albumen as cerebral infarction biomarker
In application.
Background technology
Apoplexy is also known as " apoplexy ", " cerebrovas-cularaccident " (cerebralvascular accident, CVA).It is a kind of anxious
Property cerebrovascular disease, is to rupture suddenly due to cerebral vessels or cause brain group because angiemphraxis causes blood to cannot flow into brain
Knit one group of disease of damage, including ischemic and hemorrhagic apoplexy.
Cerebral infarction is referred to as apoplexy also known as cerebral infarction, the traditional Chinese medical science.Primary disease system is by the local cerebral group caused by a variety of causes
Tissue region blood supply obstacle, causes brain tissue ischemia Hypoxic pathological changes downright bad, and then produces function of nervous system corresponding clinically
Disappearance performance.Cerebral infarction is divided into cerebral thrombosis, cerebral embolism and lacunar to wait indefinitely according to pathogenetic difference
Main Types.Wherein cerebral thrombosis is the modal type of cerebral infarction, accounts for the 60% of whole cerebral infarction, thus usually said
' cerebral infarction ' actually refer to cerebral thrombosis.
The cerebral infarction cause of disease: due to the cause of disease foundation main of the classification cerebral thrombosis of cerebral infarction
Atherosclerosis to be, thus producing atherosclerotic factor is that the modal cause of disease of cerebral infarction occurs.Due to tremulous pulse
The atherosis crotch being apt to occur in big blood vessel and knee, thus the predilection site of cerebral thrombosis be carotid initial part and
Hypomere etc. in siphon portion, middle cerebral artery initial part, vertebral artery and basilar artery.When the speckle in the tunica intima at these positions
After rupturing, in the blood such as platelet and cellulose visible component stick subsequently, assemble, formation of deposits thrombosis, and thrombus breaks loose shape
Become embolus can cause cerebral infarction by blocking remote tremulous pulse.
The INTERSTROKE result of study carried out in the world in the recent period shows: 90% in cerebral infarction risk can be returned
Censure in 10 simple risk factors, they are that hypertension, smoking, waist-to-hipratio be excessive successively, improper diet, shortage physical culture forging
Refining, diabetes, excessive consumption of alcohol, excessive stress and depression, there are basal cardiac disease and hyperlipemia.
Apoplexy is the China's the third-largest fatal disease after cardiovascular disease, malignancy disease, falls ill every year
Rate is 1,50/,100,000, and mortality rate is 1,20/,100,000.Apoplexy is divided into ischemia apoplexy and hemorrhagic apoplexy.Wherein, ischemia clinically
Property apoplexy is the most common, accounts for the 70% of whole apoplexy.There are about 50%～75% apoplexy relevant with hypertension, hypertension
History makes the risk of apoplexy increase by more than 2.5 times.If applying tightened up hypertension definition blood pressure ＞ 160/90mm
Hg, the dependency of this risk factor can increase.The case fatality rate of cerebral infarction is about 10%, disability rate up to 50% with
On.The relapse rate of survivor is up to 40%, and ischemic stroke recurrence can seriously undermine daily life and the social function of patient,
And can substantially increase mortality rate.
At present, identify that the reason causing apoplexy is main according to skull CT scanning, nmr imaging technique and tremulous pulse
Radiography.But CT, MRI scan are not complete believable method, because cerebral infarction being only had 16% at diagnosis CT in early days
Sensitivity, and hemorrhagic apoplexy is had to the sensitivity of 89%.20% cerebral infarction can not be by MRI scan
Technology detects.And in the minds of video diagnostic technology is not present in all diagnosis and treatment, these all hinder quickly examining of apoplexy
Disconnected and the timely of medicine is applied.
Biomarker is the effective tool in drug development process, it provides the relevant of pharmaceutical properties and disease process
Information, reflects concrete medication effect.The multiple disease such as diabetes and immune disease is the most according to biological marker
Thing is treated.But, in cerebrovascular, biomarker lacks the most relatively.Therefore, the biology of cerebral infarction is explored
Mark has great significance.
Owing to conventional diagnostic is not enough, the novel biological marker of diagnosis apoplexy is found to become a kind of urgent demand.
Carrying out relative quantification for identification biological markers iTRAQ and high-resolution mass spectrum is a up-and-coming skill
Art.Identify that the cerebral infarction biological markers in body fluid has complementary effect for imaging diagnosis.
Summary of the invention
In order to realize the early discovery of apoplexy, early intervention, it is an object of the invention to provide a kind of iso-amylene half Guang
Amino acid oxidase 1 (Prenylcysteine oxidase 1 (PCYOX1)) albumen is as cerebral infarction biomarker
In application.
For achieving the above object, present invention firstly provides PCYOX1 albumen or its active fragment or detection PCYOX1
The material of albumen or its active fragment is in diagnosing ischemia apoplexy and/or assessment suffer from the product of ischemic cerebral apoplexy risk
Application.
A second aspect of the present invention provides PCYOX1 albumen or its active fragment or detection PCYOX1 albumen or it is lived
Property fragment material in the therapeutic effect of assessment apoplexy or the application in judging the prognosis product of apoplexy.
Preferably, described product includes chip or test kit.
Preferably, described PCYOX1 albumen down-regulated expression in cerebral infarction biological sample.
Preferably, described detection includes the detection of gene level and the detection of protein level.As, the inspection of described gene level
Survey includes that real-time PCR method, Northern blot, Southern blot, gene chip, in situ hybridization or RNase are protected
Protect experiment etc..The detection of described protein level includes immune detection, Western blot or protein chip etc..In order to simplify experiment
Program, the detection of described protein level also includes protein detection kit, such as ELISA detection kit, gold-immunochromatographyreagent reagent for assay
Box, co-immunoprecipitation test kit, chemical luminescence reagent kit, immunofluorescent reagent box etc..Described test kit generally is equipped with phase
The operation instructions answered, description generally comprises corporate logo and title, test kit title, test kit composition, shelf-life, use
The project such as field, using method, user is not required to specifications or only needs a small amount of optimization i.e. can obtain satisfied result.
Preferably, described immunization method is ELISA method detection and/or gold colloidal detection.
Described ELISA method is for using ELISA detection kit, and described test kit includes: be coated PCYOX1 monoclonal anti
The solid phase carrier of body, enzyme labelled antibody, the substrate of enzyme, protein standard substance, negative controls, diluent, cleaning mixture, enzyme reaction terminates
Liquid etc..
A third aspect of the present invention provides PCYOX1 albumen or its active fragment or detection PCYOX1 albumen or it is lived
Property fragment material for screening prevention or the application of medicine for the treatment of apoplexy.
Preferably, described PCYOX1 albumen down-regulated expression in cerebral infarction biological sample.
Preferably, described sample is blood, such as serum, blood plasma or whole blood.
A fourth aspect of the present invention provides and can improve in blood the material of PCYOX1 protein content for preparing prevention
Or the application of the medicine for the treatment of apoplexy.
Containing the reagent promoting PCYOX1 protein expression in described medicine；The reagent bag of described promotion PCYOX1 protein expression
Include the reagent promoting PCYOX1 gene expression stability, promote the reagent etc. of PCYOX1 expression activity.
Those skilled in the art know and promote that protein expression generally can use the one in following method and/or several: activate
The promoter of molecular marker, the albumen of activating molecules marker expression or the factor, importing promote that molecular marker is transcribed or table
The carrier reached.
The medicine of the treatment apoplexy that the present invention provides, on the one hand for supplementing disappearance or the deficiency of PCYOX1 albumen, passes through
Improve the expression of PCYOX1 albumen, thus treat the apoplexy that PCYOX1 protein delation causes.On the other hand may be used for promoting
The activity of PCYOX1 albumen or function, thus treat apoplexy.
A fifth aspect of the present invention provides for detecting the system that apoplexy occurs, and this system includes detecting PCYOX1 egg
White or the material of its active fragment.
Preferably, described system includes data processing equipment, sets collection comparison module a and place in described data processing equipment
Reason module b, the function of described collection comparison module a is: gather and relatively described in the person to be measured tissue samples with collator
PCYOX1 albumen or the expression of its active fragment；The function of described processing module b is according to such as lower section according to result of the comparison
Method determines whether person to be measured is patients with cerebral apoplexy: if PCYOX1 albumen or its active tablet described in the tissue samples of described person to be measured
The expression of section is substantially less than PCYOX1 albumen or the expression of its active fragment described in the tissue samples of described matched group, then
Described person to be measured is or candidate is patients with cerebral apoplexy；Otherwise, the most described person to be measured is not or candidate is not for patients with cerebral apoplexy；Described
Collator is Healthy People.
Preferably, processing module b judges lower standard be person to be measured tissue samples described in PCYOX1 albumen or
The expression of its active fragment at least reduces by 10% than collator.
Preferably, processing module b judges lower standard be person to be measured tissue samples described in PCYOX1 albumen or
The expression of its active fragment at least reduces by 60% than collator.
Preferably, processing module b judges lower standard be person to be measured tissue samples described in PCYOX1 albumen or
The expression of its active fragment at least reduces by 90% than collator.
Beneficial effects of the present invention is as follows:
The invention discloses a kind of albumen PCYOX1 relevant to apoplexy, and be further characterized by this PCYOX1 albumen or its
Active fragment is down-regulated expression in apoplexy tissue.Utilize this Protein Detection apoplexy can not only accomplish fast and effectively in early days
Detection, and provide therapy target and important evidence for the clinical practice such as gene therapy, Drug therapy.
Accompanying drawing explanation
Chromatogram in Fig. 1 embodiment of the present invention 2；
PCYOX1 expressing quantity standard curve in Fig. 2 embodiment of the present invention 3.
Detailed description of the invention
Following example are used for illustrating the present invention, but are not limited to the scope of the present invention.If not specializing, embodiment
The conventional means that technological means used by is well known to those skilled in the art, reagent used can be commercially available.
The experimental technique of unreceipted actual conditions, usually this area conventional method in embodiment.
The present inventor carries out high-flux sequence, in conjunction with bio information to 6 example apoplexy samples and 6 example check samples
Method is screened, and picks out candidate albumen PCYOX1, and in existing research, PCYOX1 albumen is not relevant with apoplexy
Report, further, inventor has carried out molecular biology method and has verified it was confirmed PCYOX1 albumen is at cerebral infarction tissue
Middle down-regulated expression, its Related product can be used for diagnosing, treating cerebral infarction.
The PCYOX1 albumen of the present invention is known albumen before making the present invention, and its essential information is as follows:
NCBI Reference Sequence:NP_057381.3.
The present invention also uses ELISA detection kit to detect above-mentioned albumen table in ischemic cerebral apoplexy and in normal population
Reach, and to demonstrate this albumen be down-regulated expression in cerebral infarction.
Present invention application iTRAQ combines in mass spectrographic method qualification patients with acute ischemic cerebral stroke serum and causes endothelium merit
The protein marker that energy is disorderly.Isotope labelling is relatively and absolute quantitation (isobaric tags for relative and
Absolute quantitation, iTRAQ) technology is external of the same race isotope-labeled by the one of AB SCIEX company research and development
Relatively and absolute quantitation technology.This technology utilizes multiple isotope reagent labelled protein polypeptide N-terminal or lysine side-chain group,
Through high accuracy mass spectrograph Tandem analysis, the expressing quantity between up to 8 kinds samples can be compared simultaneously, be Quantitative Western in recent years
The High Throughput Screening Assay that matter group is conventional.ITRAQ quantitative proteomics is formation polypeptide after proteolytic cleavage, same with iTRAQ
Position element reagent labeling polypeptide N-terminal or lysine side-chain group.Peptide fragment after labelling through liquid phase separation, carry out first mass spectrometric and
Second mass analysis, before second order ms, the labeled same peptide fragment in different samples show as identical mass-to-charge ratio and its
His physicochemical property.And in second order ms, signal ion shows as the peak of different mass-to-charge ratio (114～121), according to the height of crest
Degree and area, can identify protein and analyze the quantitative information of same protein different disposal.
ITRAQ reagent includes three parts: report section, reactive polypeptide part, balance portion.(1) report section has eight kinds:
113-121 (without 120), therefore iTRAQ reagent can 8 groups of samples of labelling simultaneously.(2) reactive polypeptide part: can be with peptide fragment N end and bad ammonia
There is covalently bound and peptide fragment on labelling in acid side-chain amino group.(3) balance portion: ensure the mass-to-charge ratio phase of labeled same peptide fragment
With.Compared with traditional two-dimensional electrophoresis quantitative analysis, iTRAQ has techniques below service advantage: (1) is highly sensitive, detection limit
Low, can detect that low-abundance protein；(2) separating power is strong, and analyst coverage is wide, and any kind of protein can be entered by iTRAQ
Row isolation identification, including high molecular weight protein, acidic protein and basic protein, memebrane protein and insoluble protein；(3) high pass
Amount: be analyzed 8 samples simultaneously, improve experiment flux, can be simultaneously to multiple time points or the protein of different disposal
It is analyzed；(4) reliable results: quantification and qualification result is relatively reliable；(5) automaticity is high: liquid phase is with mass spectrum even
With, automation mechanized operation, analyze speed fast, good separating effect.
Nucleotide full length sequence or its fragment of the PCYOX1 gene of the present invention generally can use PCR TRAP, restructuring
The method of method or synthetic obtains.For PCR TRAP, can be the most open according to published relevant nucleotide sequence
Reading frame sequence designs primer, and with commercially available cDNA storehouse or as prepared by conventional method well known by persons skilled in the art
CDNA, as template, expands and obtains relevant sequence.When sequence is longer, it is often necessary to carry out twice or repeatedly expand, then
Again the fragment repeatedly amplified is stitched together by proper order.
Once obtain relevant sequence, it is possible to obtain relevant sequence in large quantity with recombination method.This is typically will
It is cloned into carrier, then proceeds to cell, then by conventional method relevant sequence of isolated from the host cell after propagation.
Additionally, can also be used with the method for synthetic to synthesize relevant sequence, when especially fragment length is shorter.Generally, by first synthesizing
Multiple small fragments, are attached obtaining the fragment that sequence is the longest the most again.
At present, it is already possible to carried out the albumen (or its fragment, or derivatives thereof) of code book invention completely by chemosynthesis
DNA sequence.Then this DNA sequence can be introduced in the various DNA moleculars (such as carrier) in this area and cell.Additionally, also
By chemosynthesis, sudden change can be introduced in protein sequence of the present invention.
The fragment of albumen of the present invention, in addition to available recombination method produces, can also be used with solid phase technique by being directly synthesized peptide
Produced (Stewart et al., (1969) Soliod-Phase Peptide Synthesis, WH Freeman Co., San
Francisco；Merrifield J. (1963) J.Am Chem.Soc85:2149-2154).Synthetic protein is permissible in vitro
By hand or automatically carry out.For example, it is possible to the 431A type peptide synthesizer of Applied Biosystems (Foster City,
CA) it is automatically synthesized peptide.Each fragment of chemosynthesis albumen of the present invention can be distinguished, the most chemically connected to produce
The molecule of raw total length.
Terms used herein " down-regulated expression " refers to the sequence corresponding to expressed gene, wherein the measurement card of sequence amount
Bright, separate from from normal individual or from being determined the individuality identifying morbid state different with apoplexy by apoplexy by stages
Biological sample in same albumen compare, described gene is from suffering from apoplexy or the apoplexy determined by apoplexy by stages
Identify that the expression in the biological sample separated in the individuality of morbid state reduces.According to the present invention, " down-regulated expression "
Refer at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% measured by the inventive method intensity for hybridization
Or more express reduction, such as 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or lower.
Terms used herein " expression " refers to be measured by known to those skilled in the art and method described herein
Given nucleic acid or the measuring of protein.Relate to RNA, hnRNA, mRNA or mRNA montage that biomarker of the present invention is corresponding
During variant, expression can be measured by hybridization or more quantitative measurement, such as include using that SYBR is green, TaqMan and point
The RT PCR the most in real time of sub-beacon technique.
" comparison " used herein refers to do not show any OA symptom and have not been diagnosed as apoplexy or the individuality of OA or individuality
Group.Preferably, described comparison individuality does not uses the medicine affecting OA, and has not been diagnosed as suffering from any other disease.More preferably
Ground, comparison individuality has sex, age and Body Mass Index (BMI) similar compared with test sample.According to the present invention, " comparison "
Also refer to be isolatable from the sample of normal individual, including the total serum IgE or the mRNA that are isolatable from normal individual.The sample taking from comparison individual can
Including being isolatable from the RNA of sample tissue, wherein RNA is isolatable from sample tissue, when described sample tissue is isolatable from tissue displacement not
It is diagnosed as OA and does not shows the individuality of any OA symptom.
The biological sample of the present invention
Blood
In one aspect of the invention, from experimenter, blood sample is obtained according to method well known in the art.Can use
Technology well known by persons skilled in the art (particularly known blood-sampling method) from any body part of experimenter (as finger, hands,
Wrist, arm, lower limb, foot, ankle, harmonization of the stomach neck) take blood.In a specific embodiment, obtain venous blood and according to this from experimenter
Inventive method uses.Vacuum tube, syringe or butterfly needle can be used to take blood.
Blood sampling volume will depend upon which that amount and the comfort level of experimenter that blood sampling site, the inventive method need change.As
In multiple specific embodiments, from experimenter collect 0.001mL, 0.005mL, 0.01mL, 0.05mL, 0.1mL, 0.15mL,
0.2mL, 0.25mL, 0.5mL, 0.75mL, 1mL, 1.5mL, 2mL, 3mL, 4mL, 5mL, 10mL, 15mL or more blood.Blood is protected
Exist in K3/EDTA pipe.
In an aspect, according to blood-sampling method well known in the art from normal individual or from be diagnosed as suffering from apoplexy or
Suspect that the individuality suffering from apoplexy takes whole blood.Whole blood includes the blood that can be used directly, and includes according to side well known in the art
Method (being the most preferably centrifuged 5-10 minute in 300-800 × g gentleness) removes serum or blood plasma and has divided from residue blood sample
Blood from RNA or mRNA.In a specific embodiment, whole blood (the most non-classification blood) and the cracking of experimenter will be derived from
Buffer is (such as lysis buffer (1L): 0.6g EDTA；1.0g KHCO2,8.2g NH4Cl, adjusts to pH7.4 with NaOH)
Mixing, Centrifuge A sample also retains cell precipitation, extracts RNA or mRNA (" cracking blood ") according to method well known in the art and (refer to
Such as Sambrook etc.).Whole blood is preferably used, because it avoids the step of cell type in costly and time consuming separation blood
(Kimoto, 1998, Mol.Gen.Genet258:233-239；Chelly J etc., 1989,
Proc.Nat.Acad.Sci.USA86:2617-2621；Chelly J etc., 1988, Nature333:858-860).
In some embodiments of the present invention, the whole blood being collected from experimenter is carried out classification (being i.e. separated into component).
In one embodiment of the invention, use techniques known in the art thin from the whole blood separation blood being collected from experimenter
Born of the same parents.For example, it is possible to the blood being collected from experimenter is carried out Ficoll-Hypaque (Pharmacia) gradient centrifugation.This centrifugal
Erythrocyte (erythrocyte) is separated with various types of nucleated cell and blood plasma.Specifically, Ficoll-Hypaque gradient
Centrifugal can be used for separates peripheral blood leucocyte (PBL), and they can use according to the inventive method.Other whole blood separation methods according to
Method well-known to those skilled in the art operates.
Before using according to the inventive method, optionally the biological sample of collection is saved in low temperature such as 4 by (but preferred)
At DEG C.In some embodiments, use a part for sample in the very first time according to the inventive method, and by one or more
Remaining sample part preserves a period of time for future use.This period can be 1 hour or longer, 1 day or longer, 1 week or
Longer, January or longer, 1 year longer or uncertain.For long-term preservation, it is possible to use store method well known in the art,
Such as preserve in cryogenic temperature (as less than-80 DEG C).In some embodiments, as preserving supplementing or substituting of sample, will
Separate nucleic acid or protein preserve a period of time (as 1 hour or longer, 1 day or longer, 1 week or longer, January or longer, 1
Year is longer or uncertain) for future use.
The collection of embodiment 1 sample
Go to a doctor in hospital general of rocket army of PLA during taking 2015 06 month in December, 2015 ischemic cerebral stroke patients,
Case group collects 6 examples altogether, and comparison derives from same time PLA's rocket army hospital general's health check-up but indices is good for the most normally
Health volunteer, collects 6 examples altogether, and specifying information is as shown in table 1.Obtain the median cubital vein blood of all object of study, 5ml, 4 degree of ice
Case stands 1h, centrifugal 1500rpm, 10min, takes supernatant, and centrifugal 1500rpm, 10min take supernatant, and subpackage is stored in-80 degree ice
Case.
Table 1 sample information
| Factor | Case group | Matched group |
| Mean age | 61.67 | 60.67 |
| Sex | 6 men | 3 men, 3 female |
| Average BMI | 25.22684 | 23.14447 |
| Smoking | 3 are, 2 is no, and 1 guards against | 6 is no |
| Drink | 2 are, 3 is no, and 1 guards against | 6 is no |
| Diabetes | 6 is no | 6 is no |
| Hypertension | 6 are | 6 is no |
| Coronary heart disease | 6 is no | 6 is no |
| Blood coagulation | 1 Gao Ning, 5 is normal | 6 is normal |
Embodiment 2 iTRAQ experiment screening differential protein
One, instrument and reagent
Turbula shaker (Haimen City its woods Bel instrument manufacturing company limited, model: QL-901)
Constant temperature incubates bath (PVG Rong Feng scientific instrument company limited, model: HH.S4)
Vacuum freeze drier (Thermo, model: SPD2010-230)
RIGOL L-3000 highly effective liquid phase chromatographic system (BeiJing PuYuanJing power Science Co., Ltd), mobile phase A: 98%
DdH2O, 2% acetonitrile (pH 10)；Mobile phase B: 98% acetonitrile, 2%ddH2O (pH 10) high performance liquid chromatograph: (Thermo
Scientic EASY-nLC 1000 System (Nano HPLC)), mobile phase A: 100% ultra-pure water, 0.1% formic acid；Flowing
Phase B:100% acetonitrile, 0.1% formic acid
Mass spectrometer system (Thermo, model: Q-Exactive)
Blue albumin
IgG removes test kit (Sigma-Aldrich, article No.: PROTBA-1KT)
DTT (Bio-Rad, article No.: 161-0611, the U.S.)
Iodoacetamide (Bio-Rad, article No.: 163-2109, the U.S.)
Dissolution Buffer (AB Sciex, PN:4381664) in test kit
Pancreatin (Promega, article No.: V5111, the U.S.)
10K super filter tube (milipore, PN:UFC5010BK)
8 marksTest kit (AB Sciex, PN:4390812, PN:4381664)
Ziptip (Millipore, PN:ZTC18M096 (2 μ g))
Chromatographic column: Durashell-C18,4.6mm × 250mm, 5 μm,(Agela, article No.: DC952505-0)
Acetonitrile (Merck, article No.: 100030, Germany)
Ammonia (Sigma-Aldrich, article No.: 17837, the U.S.)
Two, iTRAQ quantitative experiment flow process
2.1 sample albumen process
2.11, balance pillar
1) culture fluid is slowly overturned, vortex mixing culture fluid；
2) 0.4ml culture fluid is taken in pillar, it is ensured that culture fluid is mixing；
3) pillar is put in 2ml collecting pipe, with the centrifugation 5-10s of 10000rpm (8000g)；
4) add 0.4ml level pad in pillar, with the centrifugation 5-10s of 10000rpm (8000g), 2ml is received
Buffer in collector is outwelled, and is again put into by pillar in original 2ml centrifuge tube；
5) 0.4ml level pad is added again in pillar, with the centrifugation 20-40s of 10000rpm (8000g), by 2ml
Buffer in collecting pipe is outwelled, and is put into by pillar in a new 2ml centrifuge tube.
2.12, the albumin in serum and immunoglobulin G (IgG) are removed
1) add 25-100ul serum sample in the pillar processed, and at room temperature hatch 5-10min；
2) centrifugal pillar and collecting pipe, 10000-12000rpm (8000-12000g) * 60s；
3) again the eluate in collecting pipe is joined in pillar, incubated at room temperature 5-10min；This step eliminates and exceedes
The albumin of 5%；
4) centrifugal pillar 10000-12000rpm (8000-12000g) * 60s in same collecting pipe；
5) serum in collecting pipe and the liquid mixing of the 6th step cleaning step are stayed through twice eluting；
6) remaining unbound albumen in eluting pillar: add 100ul level pad in pillar, centrifugal；10000-
12000rpm (8000-12000g) * 60s, the liquid mixing that will obtain in the 4th, 6 liang of collection step pipes；
7) serum removing albumin and IgG can be-20 DEG C long-term storages.
2.2Bradford method protein quantification
Employing Bradford method [Marion M.Bradford.Analytical Biochemistry, 1976,72:248-
254] protein concentration of sample extraction is measured.Obtain each sample protein concentration (being shown in Table 2)
Table 2 protein sample concentration
2.3 proteolysiss (Filter Aided Sample Preparation, FASP)
1) take 200 μ g protein solutions after protein quantification to be placed in centrifuge tube；
2) adding final concentration 25mM DTT, 60 degree are reacted 1 hour；
3) final concentration 50mM iodoacetamide, room temperature 10 minutes are added；
4) being added by the protein solution after reductive alkylation in the super filter tube of 10K, 12,000 leave the heart 20 minutes, discard receipts
Collector bottom solution；
5) adding the Dissolution Buffer 100 μ l in iTRAQ test kit, 12,000 leave the heart 20 minutes, discard
Collecting pipe bottom solution, is repeated 3 times；
6) collecting pipe more renewed, adds trypsin, total amount 4 μ g (with albumen quality ratio 1: 50), body in super filter tube
Long-pending 50 μ l, 37 degrees Celsius of reactions are overnight；
7) next day, 12,000 leave the heart 20 minutes, and the peptide fragment solution centrifugal after enzymolysis, digestion is bottom collecting pipe；
8) adding 50 μ l Dissolution Buffer in super filter tube, 12,000 turns of recentrifuge 20 minutes, with upper step
Merge, bottom collecting pipe, there are the sample after 100 μ l enzymolysis.
2.4iTRAQ labelling
1) from refrigerator, take out iTRAQ reagent, equilibrate to room temperature, willReagent is centrifuged at the bottom of pipe；
2) to often managingReagent adds 150 μ l isopropanols, vortex oscillation, is centrifuged at the bottom of pipe；
2) take 50 μ l samples (100 μ g enzymatic hydrolysate) to transfer in new centrifuge tube；
4) iTRAQ reagent is dosed in sample, vortex oscillation, it is centrifuged at the bottom of pipe, room temperature reaction 2 hours；
5) add 100 μ l water and terminate reaction；
6) in order to detect labeling effciency and dosing accuracy, from 4 groups of samples, each 1ul of taking-up mixes, and uses Ziptip desalination
After carry out MALDI-TOF-TOF (AB SCIEX 4800Plus) and identify, confirmation flag reaction is good；
7) sample after mixed mark, vortex oscillation, is centrifuged at the bottom of pipe；
8) vacuum freezing centrifugal drying；
9) the sample freezen protective after draining is stand-by；
10) pre-separation of peptide hydrolysis off-line and LC-MS/MS mass spectral analysis.
Reversed phase chromatography separation under the conditions of 2.5 high pH
1) sample after mixed mark dissolves by 100 μ l mobile phase A, and 14000g is centrifuged 20min, takes supernatant stand-by；
2) BSA using 400 μ g enzymolysis good carries out separating (column temperature 45 DEG C detects wavelength 214nm), detecting system situation；
3) 100 μ l ready sample loading is taken；
4) flow velocity 0.7ml/min, separates gradient as shown in table 3, and chromatogram is as shown in Figure 1.
Table 3 reversed phase chromatography separation gradient
2.6 nanoliters of level reversed phase chromatography-QExactive carry out protein analysis
1) component high pH reverse phase separation obtained 20 μ l 2% methanol, 0.1% formic acid redissolves；
2) 12,000 leaves the heart 10 minutes, draws supernatant loading；
3) loading volume 10 μ l, takes sandwich assay loading；
4) Loading Pump flow velocity 350nl/min, 15 minutes；
5) separate flow velocity 300nl/min, separate gradient as shown in table 4.
4 nanoliters of level reversed phase chromatography separation gradients of table
Three, MASS SPECTRAL DATA ANALYSIS and result
The selection of data base is with required species, database annotation completeness and sequence reliability as reference frame.?
The data base selected in this experiment is from UniProt (http://www.uniprot.org/), and this version of data base is:
UniProt_Sus scrofa_201510.fasta.The mass spectral analysis of iTRAQ is complete by Thermo Q-Exactive type mass spectrum
Becoming, the mass spectrum original document * .RAW of generation uses the supporting business software Proteome Discoverer1.4 of Thermo company
Process.This iTRAQ experiment identifies 272 total proteins altogether, and it analyzes differential protein 14.
In order to be better understood from the function of differential protein, we have carried out Gene Onlogy to differential protein and signal leads to
Road is analyzed, and differential protein carries out functional annotation and protein interaction analysis of network, the knot analyzed in view of data above
Really, in conjunction with document, we have screened differential protein PCYOX1, and this albumen is down-regulated expression in cerebral infarction sample.
The expression of embodiment 3 large sample checking differential protein PCYOX1
1, sampling
Go to a doctor in hospital general of rocket army of PLA during taking 2015 06 month in December, 2015 ischemic cerebral stroke patients,
Case group collects 20 examples altogether, is numbered A1-A20 respectively, comparison derive from same time PLA's rocket army hospital general's health check-up but
The all normal healthy volunteer of indices, collects 10 examples altogether, is numbered B1-B10 respectively.Just obtain the elbow of all object of study
Medium-sized vein blood, 5ml, 4 degree of refrigerators stand 1h, centrifugal 1500rpm, 10min, take supernatant, and centrifugal 1500rpm, 10min take supernatant,
Subpackage is stored in-80 degree refrigerators.
2, experimentation
Experiment selects people's iso-amylene cysteine oxidase 1 (PCYOX1) ELISA kit (to buy from upper Haikang nangzan thing
Science and Technology Ltd.)
This people's iso-amylene cysteine oxidase 1 (PCYOX1) ELISA detection kit operating procedure is as follows:
(1) the dilution every bottle standard substance of standard substance add standard dilutions 1mL, build that rear chamber is gentle and quiet puts about 10 points
Clock, the most reverse/rubbing is with hydrotropy solution, and its concentration is 20ng/mL.Preparing the EP pipe of 7 dilution standard product, each EP manages
The standard dilutions of middle addition 150 μ L, is diluted to different gradients, and standard dilutions (0pg/mL) is directly as blank
Hole.
(2) sample-adding: set respectively blank well (blank control wells is not added with sample and enzyme marking reagent, remaining respectively step operation identical),
Testing sample hole.It is coated on plate at enzyme mark and testing sample hole first adds sample diluting liquid 40 μ l, add testing sample 10 μ l the most again
(the final dilution factor of sample is 5 times).Sample is added on bottom ELISA Plate hole by sample-adding, does not touch hole wall as far as possible, rocks mixing gently.
(3) incubation: use the rearmounted 37 DEG C of incubations of shrouding film shrouding 30 minutes.
(4) dosing: by standby after 30 (20 times of 48T) times concentrated cleaning solution distilled water 30 (20 times of 48T) times dilution.
(5) washing: carefully take shrouding film off, discard liquid, dries, and every hole is filled it up with cleaning mixture, discarded after standing 30 seconds, as
This is repeated 5 times, and pats dry.
(6) enzyme-added: every hole adds enzyme marking reagent 50 μ l, except blank well.
(7) incubation: operation is with 3.
(8) washing: operation is with 5.
(9) colour developing: every hole is initially charged developer A50 μ l, adds developer B50 μ l, shakes mixing, 37 DEG C of lucifuges gently
Develop the color 15 minutes.
(10) terminate: every hole adds stop buffer 50 μ l, terminate reaction (now blueness is vertical turns yellow).
(11) measuring: with blank air-conditioning zero, 450nm wavelength sequentially measures the absorbance (OD value) in each hole, measures Ying Jia
Carry out within 15 minutes after stop buffer.
(12) standard curve processed, calculates the content of PCYOX1 albumen in sample.
3 results
Obtaining the OD value of standard substance, as shown in table 5, the standard curve of making is as shown in Figure 2.Experiment has obtained 20 samples
OD value, according to standard curve, calculate the concentration of PCYOX1 in sample, its result is as shown in table 6.Wherein, the OD value of sample is
Three secondary orifices go the OD average after background.Data in statistical analysis table 6, it is known that the concentration value of 20 samples 6-7ng/ml it
Between, the concentration value of check sample is at more than 8ng/ml, and above-mentioned difference has statistical significance (P ＜ 0.05), and the above results shows,
PCYOX1 albumen expression in cerebral infarction sample reduces.
The OD value of 57 standard substance of table
| Standard substance | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
| Concentration (ng/ml) | 0 | 1.12 | 2.24 | 4.48 | 6.72 | 8.96 | 11.2 |
| OD value | 0.065 | 0.097 | 0.152 | 0.252 | 0.346 | 0.423 | 0.572 |
The OD value of table 6 sample and comparison and concentration value
| Sample number into spectrum | A1 | A2 | A3 | A4 | A5 | A6 | A7 | A8 | A9 | A10 |
| OD average | 0.340 | 0.342 | 0.347 | 0.341 | 0.339 | 0.346 | 0.350 | 0.351 | 0.338 | 0.347 |
| Concentration (ng/ml) | 6.739 | 6.782 | 6.891 | 6.760 | 6.717 | 6.870 | 6.957 | 6.978 | 6.695 | 6.891 |
| Sample number into spectrum | A11 | A12 | A13 | A14 | A15 | A16 | A17 | A18 | A19 | A20 |
| OD average | 0.346 | 0.336 | 0.352 | 0.349 | 0.350 | 0.339 | 0.353 | 0.346 | 0.348 | 0.347 |
| Concentration (ng/ml) | 6.870 | 6.651 | 7.000 | 6.935 | 6.957 | 6.717 | 7.022 | 6.870 | 6.913 | 6.891 |
| Comparison numbering | B1 | B2 | B3 | B4 | B5 | B6 | B7 | B8 | B9 | B10 |
| OD average | 0.418 | 0.420 | 0.425 | 0.423 | 0.428 | 0.429 | 0.431 | 0.423 | 0.421 | 0.440 |
| Concentration (ng/ml) | 8.393 | 8.434 | 8.536 | 8.495 | 8.597 | 8.617 | 8.658 | 8.495 | 8.454 | 8.840 |
Embodiment 4 ELISA evaluates the therapeutic effect of apoplexy
6 example Ischemic Strokes in embodiment 1 selected by sample, and the ELISA of PCYOX1 albumen operates same embodiment
3, before treatment, PCYOX1 protein level in detection serum, follows the tracks of and follows up a case by regular visits to this 6 patients, detect in serum after terminating the course for the treatment of again
PCYOX1 protein level, PCYOX1 protein content change in contrast serum, the expression of concrete PCYOX1 albumen sees table 7.
From table 7, the comparison of PCYOX1 changes of contents and therapeutic effect is it can be seen that the most significant patient of clinical therapeutic efficacy, its serum
It is the fastest that middle PCYOX1 protein level raises；The unconspicuous patient of clinical therapeutic efficacy, in its serum PCYOX1 protein level without
Significant change.This result shows, in detection serum, cerebral infarction therapeutic effect can be estimated by PCYOX1 protein level,
I.e. PCYOX1 albumen has application prospect at the therapeutic effect of assessment apoplexy or in judging the prognosis product of apoplexy.
PCYOX1 level before and after the treatment of table 7 Clinical detection cerebral infarction
Although, the present invention is described in detail the most with a general description of the specific embodiments, but
On the basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to the scope of protection of present invention.
Claims (10)
- The material of 1.PCYOX1 albumen or its active fragment or detection PCYOX1 albumen or its active fragment is at diagnosing ischemia The application in the product of ischemic cerebral apoplexy risk is suffered from apoplexy and/or assessment.
- The material of 2.PCYOX1 albumen or its active fragment or detection PCYOX1 albumen or its active fragment is at assessment apoplexy Therapeutic effect or application in judging the prognosis product of apoplexy.
- Apply the most as claimed in claim 1 or 2, it is characterised in that described product includes chip or test kit.
- Apply the most as claimed in claim 1 or 2, it is characterised in that described PCYOX1 albumen is at cerebral infarction biology sample Down-regulated expression in product.
- The material of 5.PCYOX1 albumen or its active fragment or detection PCYOX1 albumen or its active fragment is used for screening prevention Or the application of the medicine for the treatment of apoplexy.
- Apply the most as claimed in claim 5, it is characterised in that described PCYOX1 albumen is in cerebral infarction biological sample Down-regulated expression.
- Apply the most as claimed in claim 6, it is characterised in that described sample is blood.
- 8. can improve the material of PCYOX1 protein content in blood for preparing the application of the medicine of prevention or treatment apoplexy.
- 9. for detecting the system that apoplexy occurs, it is characterised in that this system includes detecting PCYOX1 albumen or its active tablet The material of section.
- 10. system as claimed in claim 9, it is characterised in that described system includes data processing equipment, and described data process Setting collection comparison module and processing module in device, the function of described collection comparison module is: gather and relatively person to be measured is with right PCYOX1 albumen described in tissue samples according to person or the expression of its active fragment；Compare according to the function of described processing module Result relatively determines whether person to be measured is patients with cerebral apoplexy as follows: if described in the tissue samples of described person to be measured The expression of PCYOX1 albumen or its active fragment be substantially less than PCYOX1 albumen described in the tissue samples of described matched group or The expression of its active fragment, the most described person to be measured is or candidate is patients with cerebral apoplexy；Otherwise, the most described person to be measured is not or waits Choosing is not patients with cerebral apoplexy；Described collator is Healthy People.
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| CN106434982A (en) * | 2016-11-24 | 2017-02-22 | 汕头大学医学院第附属医院 | Relevant molecular markers for cerebral ischemic stroke and application of molecular markers |
| CN107151703A (en) * | 2016-06-17 | 2017-09-12 | 北京致成生物医学科技有限公司 | A kind of molecular marker for detecting cerebral arterial thrombosis and application thereof |
| CN109576361A (en) * | 2018-12-24 | 2019-04-05 | 河北医科大学第三医院 | A kind of biomarker relevant to ischemic cardiomyopathy occurrence and development |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106337089A (en) * | 2016-11-24 | 2017-01-18 | 汕头大学医学院第附属医院 | LncRNA for diagnosing cerebral arterial thrombosis |
| CN106434982A (en) * | 2016-11-24 | 2017-02-22 | 汕头大学医学院第附属医院 | Relevant molecular markers for cerebral ischemic stroke and application of molecular markers |
| CN109576361A (en) * | 2018-12-24 | 2019-04-05 | 河北医科大学第三医院 | A kind of biomarker relevant to ischemic cardiomyopathy occurrence and development |
| CN111443210A (en) * | 2020-04-10 | 2020-07-24 | 青海大学附属医院 | Early diagnosis and treatment biomarker for alveolar echinococcosis and application thereof |
| CN111443210B (en) * | 2020-04-10 | 2023-09-22 | 青海大学附属医院 | Bubble type echinococcosis early diagnosis and treatment biomarker and application thereof |
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