CN105988007A - Use of urea apolipoprotein C-II - Google Patents
Use of urea apolipoprotein C-II Download PDFInfo
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- CN105988007A CN105988007A CN201510067730.8A CN201510067730A CN105988007A CN 105988007 A CN105988007 A CN 105988007A CN 201510067730 A CN201510067730 A CN 201510067730A CN 105988007 A CN105988007 A CN 105988007A
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- G—PHYSICS
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- 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
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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Abstract
The invention provides a use of urea apolipoprotein C-II, Apo C-II and concretely relates to expression of the urea apolipoprotein C-II in urea and a use of the urea apolipoprotein C-II in urea content detection. A research proves that the urea apolipoprotein C-II has high expression so that disease diagnosis information can be obtained through the urea apolipoprotein C-II expression amount detection. The urea apolipoprotein C-II utilizes advantages of urea sample large-scale repeated sampling and utilizes a random urine sample to detect apolipoprotein C-II.
Description
Technical field
The present invention relates to the new application of urine apoC-II, be specifically related to apoC-II in urine
Express and urine content detect in application.
Background technology
Apo C-II gene arranges with ApoC-I, ApoE gene cluster, is positioned at No. 19 chromosome long arm q13
District.Apo C-II gene length 3320bp, containing 4 exons and 3 introns.ApoC-II is by 79 ammonia
The single chain polypeptide of base acid residue composition, without cysteine and serine, has two kinds of polymorphisies, isoelectric point, IP
Being respectively 4.86 and 4.69, in its secondary structure, alpha-helix accounts for 23%.Apo C-II is CM, VLDL
One of structural protein with HDL, account for the 14% of its Proteins, 7%-10% and 1%-3% respectively.Apo C-II is
One of most important hypotype in Apo C family, it is the cofactor of LPL, plays important in lipid metabolism
Effect, the lipoprotein lipase (LPL) in multiple source can be activated.55-78 amino acids residue in its structure
It is to maintain its shortest required region to LPL activation.C-terminus 43-50 amino acids residue is a spiral knot
The lipid binding of structure.Apo C-II has various biological function: (1) activates LPL.Apo C-II is LPL
Cofactor, it can activate the LPL in multiple source.Its activation mechanism is probably LP L and is typically found in periphery
Circulation is combined with heparin-like molecule, and is attached on Ink vessel transfusing.As LPL contact Chylomicron (CM) or extremely low
During density lipoprotein (VLDL), LPL just has an effect with the phospholipid on hdl particle surface and then is incorporated into fat
On protein body.(2) stability of lipoprotein is maintained.Application fluorescent spectrometry research Apo C-II c-terminus
19-39 amino acids residue, finds that its alpha-helix can not only be combined with lipid sur by mediating protein, and it
It not self-association, but by associating with other bipolarity spiral in Apo C-II and apolipoprotein, thus tie up
Hold the stability of lipoprotein.(3) protection vascular endothelial cell, in terms of stoping atherosclerotic formation
Play an important role.
Shortage or the exception of Apo C-II can cause Lipoprotein Disorders.Apo C-II with LPL has an effect, can
With change LPL structure, thus can catalyzing hydrolysis triglyceride, only gene appropriateness express just can activate LPL
Activity, overexpression or disappearance all can cause triglyceride.Studies have found that Apo C-II defect is permissible
Cause plasma TG, VLDL and the rising of Chylomicron level and the reduction of LDL, IDL and HDL level.Apo
C-II defect patient is varied, but major part it has been determined that.Its Apo C-II gene intron montage position
The defect of point, can cause the low-level of plasma A po C-II.Additionally, the synthesis of Apo C-II defect or generation
Non-functional Apo C-II be considered as to be replaced by various single aminoacid to cause.Apo C-II gene is many
State property is common in some Senile disease patient such as coronary heart disease, diabetes.Studies have found that cerebral infarction group is fallen ill
Initial stage serum Apo C-II content is the highest, along with the state of an illness recovery cerebral infarction group serum Apo C-II content in
The trend being gradually lowered.Apo C-II horizontal abnormality occurs closely related with acute cerebral infarction, and and acute brain
Infarction coincident with severity degree of condition also exists obvious dependency, monitor its level change for cerebral infarction prevention,
The state of an illness is improved significant.In dynamic measurement Patients with Cerebral Infarction serum, the change of Apo C-II level contributes to brain
The early diagnosis of infarction and prognosis speculate, provide certain foundation for the prevention of Patients with Cerebral Infarction and change of illness state.
There are some researches show, the Apo C-II of high concentration is one of risk factor of CHD, and with the TG phase of high concentration
Ratio, the increase of Apo C-II concentration is that CHD occurs more sensitive index.Researcher is had to find and without CHD
Type 2 diabetes mellitus patient compare, type 2 diabetes mellitus merges in patients with coronary heart disease body the Apo C-that there is higher concentration
II.Clinical experiment shows that chronic viral hepatitis B, liver cirrhosis patient HDL, LDL, cholesterol are all in being remarkably decreased
Gesture, measure Apo C-II change can accurately, directly, the lipid generation in reflection hepatopathy each period rapidly
Thank to situation.Cholesterol calculus patients serum's Apo C-II level is proportionate with biliary cholesterol content.Therefore,
By the change of the detection Apo indexs such as Apo C-II can reflect cholesterol calculus patient's lipid metabolism change and
Its status in the cholelithiasis origin cause of formation and effect.
Summary of the invention
It is an object of the invention to provide a kind of urine apoC-II expression in urine and for urinating
Application in liquid hold-up detection.
Preferably, the aminoacid sequence of described urine apoC-II is as shown in SEQ ID NO:1
TQQPQQDEMP SPTFLTQVKE SLSSYWESAK TAAQNLYEKT YLPAVDEKLR DLYSKSTAAM
STYTGTFTDQ VLSVLKGEE
Preferably, described preparation is urine apoC-II detection kit.Described test kit is that antibody resists
Former reaction.
Preferably, described antigen antibody reaction is coated by urine apoC-II or polypeptide and its antibody
Or it is marked at solid phase or liquid phase carrier.
First inventor have collected the random urine specimen of normal health check-up, centrifugal after take supernatant, utilize weak sun from
Son exchange magnetic beads for purifying and separated urine specimen.By 1 μ l specimen and the 10 μ l substrate (alpha-cyano-4-of 0.3%
Hydroxycinnamic acid, HCCA) after mixing, take 1 μ l point Anchorchip (Autoflex MALDI TOF,
Bruker-Dalton) on target plate, after specimen ionizing, carry out mass spectral analysis, gather 1000-10000Da scope
Interior data, it is thus achieved that the mass spectrum polypeptide figure being made up of the protein peak of different mass-to-charge ratioes.Application ClinProTools2.1
Analyze all mass spectruies of software to be analyzed, filter out the high-abundance proteins polypeptide of stable expression.Then invent
People utilizes Liquid Chromatography-Tandem Mass Spectrometry instrument to identify, these protein polypeptides filtered out at International
Protein Index (IPI human v3.45fasta with 71983entries) database retrieval obtains carrying fat egg
White C-II (Apolipoprotein C-II, Apo C-II).
By research, the present invention confirms that apoC-II can stable in the urine of the people of normal health check-up occur.
Thus propose to detect urine apoC-II and can be used for the application in urine coherence check.
The present invention plays urine specimen and obtains noninvasive advantage, utilizes random urine Samples detection apoC-II
Or polypeptide.
For above and other objects of the present invention, feature and advantage can be become apparent, cited below particularly preferably
Embodiment, and coordinate accompanying drawing, it is described in detail below.
Accompanying drawing explanation
Fig. 1 be mass-to-charge ratio be between 1000-10000 a little meansigma methods in 30 example normal health check-up specimen.
Fig. 2 be mass-to-charge ratio be the scatterplot expressed o'clock in 30 example normal health check-up specimen of 1735.7.
Fig. 3 is the mass spectrum of apoC-II.
Detailed description of the invention
Embodiment 1The collection of urine specimen and process
Collect the 30 normal health check-ups of example (Beijing Shijitan Hospital, CMU's MEC) the most clear
Clean mud-stream urine specimen, in 2h centrifugal (1500rpm, 5min), retains supernatant.After subpackage ,-80 DEG C of refrigerators freeze
Deposit.
Embodiment 2Polypeptide in magnetic beads for purifying and separated urine specimen
Taking out urine specimen from-80 DEG C of refrigerators, 4 DEG C melt again, take supernatant after centrifugal (3000rpm, 10min)
Standby.Balance weak cation magnetic bead (MB-WCX) under room temperature, and manually mix bead suspension.At sample
Adding 10ul MB-WCX in pipe and 10ul magnetic bead combines buffer, sample loading gun blows and beats mixing up and down, it is to avoid
Bubble.In sample cell, add 5ul urine supernatant, fully stand 1 minute on magnetic frame after mixing, magnetic bead with
The liquid suspended separates.Remove the clear liquid suspended with sample loading gun, rifle head should avoid contact to magnetic bead, keep away
Exempt to siphon away magnetic bead.100ul magnetic bead cleaning buffer solution is added, by sample cell at magnetic after fully mixing in sample cell
Standing 1 minute on power frame, magnetic bead is adherent, separates with the liquid suspended, and removes the liquid suspended with sample loading gun.
It is repeated 3 times, discards suspension.In sample cell, add 5ul magnetic bead elution buffer, repeatedly inhale and make a call to 10 times
Above, magnetic bead and elution buffer mixing are made, it is to avoid bubble.Sample cell is positioned on magnetic frame, stands
2rmin, makes magnetic bead be sufficiently separated with suspension, and supernatant (eluent) is moved into marked new 0.5ml
Sample cell.Adding 5ul stabilizing buffer, sample loading gun carefully blows and beats mixing.
Embodiment 3The point target of urine specimen and polypeptide spectrum map generalization
After rectifying an instrument with standard substance, by 1 μ l eluent and the 10 μ l substrate (alpha-cyano-4-hydroxyl meat of 0.3%
Cinnamic acid, HCCA) mixing, take 1 μ l point Anchorchip (Autoflex MALDI TOF,
Bruker-Dalton) on target plate, drying at room temperature.Irradiated by nitrogen laser and carry out mass spectrum after making specimen ionizing
Analyze, gather the data in the range of 1000-10000Da, it is thus achieved that the matter being made up of the protein peak of different mass-to-charge ratioes
Spectrogram.For each MALDI crystalline temperature, concurrent irradiation 400 laser (8 of each crystalline temperature
Different positions respectively irradiates 50 times), meansigma methods represents a specimen, thus obtains the polypeptide figure of all samples
Spectrum.Application ClinProTools2.1 analyzes software to Normal group, type 2 diabetes mellitus without complication and complication
Group and type 2 diabetes mellitus merge the mass spectrum of albuminuria group and are analyzed, and screen diversity polypeptide.Screening
Condition: mass range 1000-10000Da, signal to noise ratio (S/N) is more than 5, and Mass Drift is less than 0.1%,
All mass spectruies are normalized according to total ion current.Specifically refer to Fig. 1, it shows in 30 example urine specimens
The meansigma methods of the point of all mass-to-charge ratioes between 1000-10000Da；Peak area shows as quantitative standard, Fig. 2
Going out apoC-II expression in all Urine specimens, as can be seen from the figure m/z 1735.7 is at all marks
Peak area in Ben is both greater than 600.
Embodiment 4The qualification of protein polypeptide
Magnetic bead eluent in sample cell is rotated and is evaporated, add 20ul mobile phase A (5% acetonitrile, 0.1% formic acid
Aqueous solution) dissolve, be transferred in sample injection bottle.Sampling volume 18ul, first enters with the speed of 15 μ l/min
Enter trapping column desalination, trap time 3min.Then carry out gradient with the flow velocity entrance analytical column of 400nl/min to wash
De-, gradient be 5%B-50%B-80%B-80%B-50%B-5%B (Mobile phase B: 95% acetonitrile, 0.1%
First aqueous acid, is shown in Table 1).Analysis time 60min, chromatogram column temperature 35 DEG C, all eluting compositions enter
Enter spectrometer analysis.Nano ion source, spray voltage 1.8kV；Mass spectrum pattern is data dependence and dynamically arranges
Remove, sweep limits 400-2000m/z；One-level scanning (MS) uses Obitrap, and resolution setting is 100000；
CID and two grades of scannings use LTQ；The single coordination of 10 the strongest ions of intensity is chosen in MS spectrogram
Element carries out MS/MS (single electric charge is got rid of, not as parent ion) as parent ion.Scanning of the mass spectrum time 60min.
Application data analysis software BioworksBrowser 3.3.1SP1 carries out SequestTMRetrieval.Searching database
For International Protein Index (IPI human v3.45fasta with 71983entries).Parent ion
Error is set as that 100ppm, fragment ion error are set to 1Da, and enzyme action mode is non-enzyme action, variable is modified to
Methionine oxidized.Retrieval result parameter is set as deltacn >=0.10, two electric charge Xcorr 2.6, tricharged
Xcorr 3.1, the above Xcorr of tricharged 3.5.In data base, retrieval obtains albumen apoC-II, carries
The mass spectrum of lipoprotein C-II refer to Fig. 3.
The program of table 1 analytical column gradient elution
Embodiment 5The preparation of test kit
1. coated antibody and the selection of enzyme labelled antibody working concentration
Operate according to the BCA determination of protein concentration test kit description of Pierce company, measure antibody and antigen
Concentration, then use the chessboard assay method of standard, with being coated buffer by rabbit anti-human apoC-II
It is 10.0ng/ml, 1.0ng/ml and 0.1ng/ml that polyclonal antibody (Abcam company) is diluted to concentration, point
Not being coated on solid phase elisa plate and liquid phase magnetic bead, each concentration includes three stringers, and 4 DEG C overnight, washing
3 times.Wherein one walk crosswise be coated in hole addition strong positive antigen liquid, another walks crosswise the weak positive of middle addition
Antigen liquid, the third line adds negative control.Hatch 2 hours for 37 DEG C, wash 3 times.Add mouse-anti people and carry fat
PROTEIN C-II monoclonal antibody (Abcam company), hatches 1 hour for 37 DEG C, washs 3 times.Add labelling
Two resist, hatch 30 minutes for 37 DEG C, wash 4 times, add substrate, room temperature lucifuge placement 20 minutes, add
Enter and stop liquid, reading.Select coated antibody optium concentration.
2. the preparation of test kit
Rabbit anti-human apoC-II polyclonal antibody is diluted with being coated buffer, adds it to solid
In phase microwell plate and liquid phase magnetic bead, 4 DEG C are coated jog overnight.Remove the most coated liquid, wash 3 times, add
Enter to block liquid and stop nonspecific binding site, hatch 1 hour for 37 DEG C, wash 3 times.Put into 4 DEG C of preservations standby
With.Test kit subpackage add mouse-anti human apolipoprotein C-II monoclonal antibody, labelling two anti-etc..
Embodiment 7The detection of test kit sensitivity
By apoC-II recombiant protein (Germany OriGene company) with PBS be diluted to 200ng/ml,
100ng/ml、50ng/ml、25ng/ml、10ng/ml、2ng/ml、0.5ng/ml、0.05ng/ml、0.01ng/ml、
0ng/ml, every hole 100ul join in the above-mentioned ELISA Plate being coated and in liquid phase magnetic bead, and 37 DEG C to hatch 2 little
Time, wash 3 times.Mouse-anti apoC-II monoclonal antibody being diluted by 1: 2000, every hole adds 100ul,
Hatch 1 hour, wash 3 times for 37 DEG C.Add labelling two to resist, hatch 30 minutes, wash 3 times for 37 DEG C.Add the end
Thing room temperature is placed 15 minutes, adds stop buffer, reading.Detecting minimum apoC-II amount, result shows
Showing, this reagent can detect the concentration of 0.01ng/ml apoC-II, illustrates have higher detection spirit
Sensitivity.The content of apoC-II in above-mentioned experiment shows the test kit detection urine specimen of the present invention,
Having the highest sensitivity, lowest detectable limit 0.01ng/ml of sample, the response rate is 90% ± 13%.This reagent
Needed for box, instrument is less, it is only necessary to microplate reader, agitator, centrifuge, pipettor etc., required low cost.
Embodiment 8The specificity of test kit, the detection of stability
Take normal health check-up (Beijing Shijitan Hospital, CMU) cleaning to be measured stage casing random urine
30-50ml, loads the urinary catheter of cleaning, should avoid menstrual period, should prevent vaginal secretions during women preserving urine sample
Being mixed in urine, under room temperature, 1500rpm is centrifuged 5 minutes, takes supernatant to be checked.
Quantitative apoC-II the recombiant protein of purification is as standard substance, by antigen (Urine specimens after Li Xin)
After 1: 3 dilution, join before in the most coated good ELISA Plate, hatch 2 hours, wash away not for 37 DEG C
In conjunction with antigen, blot residual liquid.Add mouse-anti human apolipoprotein C-II monoclonal antibody 37 DEG C and hatch 1
Hour, wash away unconjugated antibody, blot residual liquid.Add the two of labelling to resist, hatch 30 minutes for 37 DEG C,
Wash 4 times, blot residual liquid.Adding chromogenic substrate, room temperature is placed 10 minutes, adds stop buffer and terminates
Reaction, microplate reader reading, calculate the content of apoC-II in sample.
With it, inventor have detected apoC-II content in 100 example normal health check-up random urine,
Rate of accuracy reached, to more than 97%, has good specificity.
Taking the random urine of two normal health check-ups respectively, utilize said method to carry out ELISA mensuration, every day surveys
Determining once, be repeated 10 times altogether, by the formula coefficient of variation (CV)=S/X × 100%, (S is standard deviation, and X is flat
Average) calculate batch between and variation within batch coefficient.Be respectively with interassay coefficient of variation in finally giving batch 2.81% and
3.26%, good stability is described.
Although the present invention discloses as above with preferred embodiment, so it is not limited to the present invention, Ren Hesuo
Belong to those skilled in the art, without departing from the spirit and scope of the present invention, when making a little change
With improvement, therefore protection scope of the present invention is when being as the criterion depending on as defined in claim.
Claims (5)
1. the expression in urine of apoC-II and polypeptide thereof and the application in urine content detects thereof.
Application the most according to claim 1, it is characterised in that described urine apoC-II's
Aminoacid sequence is as shown in SEQ ID NO:1.
Application the most according to claim 1, it is characterised in that described preparation is urine apolipoprotein
C-II or polypeptide detection kit.
Application the most according to claim 3, it is characterised in that described test kit is antigen antibody reaction.
Application the most according to claim 4, it is characterised in that described reaction is urine apolipoprotein
C-II or polypeptide and its antibody are coated or be marked at solid phase or liquid phase carrier.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510067730.8A CN105988007A (en) | 2015-02-10 | 2015-02-10 | Use of urea apolipoprotein C-II |
| PCT/CN2015/000625 WO2016127277A1 (en) | 2015-02-10 | 2015-09-01 | Application of urine apolipoprotein c-ii |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201510067730.8A CN105988007A (en) | 2015-02-10 | 2015-02-10 | Use of urea apolipoprotein C-II |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012122024A2 (en) * | 2011-03-04 | 2012-09-13 | Russell Medford | Screening method for identifying patients at risk of drug induced liver injury |
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| JP2008547028A (en) * | 2005-06-24 | 2008-12-25 | サイファージェン バイオシステムズ， インコーポレイテッド | Biomarkers for ovarian cancer |
| EP2721179A4 (en) * | 2011-06-16 | 2014-10-01 | Caris Life Sciences Luxembourg Holdings S A R L | Biomarker compositions and methods |
| US9494606B2 (en) * | 2012-05-09 | 2016-11-15 | President And Fellows Of Harvard College | Quantification of lipoproteins |
| CN103063848B (en) * | 2012-12-26 | 2015-06-03 | 潍坊三维生物工程集团有限公司 | Kit for determination of apolipoprotein C2 by using immunoturbidimetry |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012122024A2 (en) * | 2011-03-04 | 2012-09-13 | Russell Medford | Screening method for identifying patients at risk of drug induced liver injury |
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