CN103376322A - Application of apolipoprotein B 100 as marker of obesity-diabetes - Google Patents
Application of apolipoprotein B 100 as marker of obesity-diabetes Download PDFInfo
- Publication number
- CN103376322A CN103376322A CN2012101247337A CN201210124733A CN103376322A CN 103376322 A CN103376322 A CN 103376322A CN 2012101247337 A CN2012101247337 A CN 2012101247337A CN 201210124733 A CN201210124733 A CN 201210124733A CN 103376322 A CN103376322 A CN 103376322A
- Authority
- CN
- China
- Prior art keywords
- apolipoprotein
- obese diabetic
- reagent
- diabetes
- protein fragments
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Abstract
The invention relates to application of an apolipoprotein B 100 as a marker of obesity-diabetes and provides an apolipoprotein B 100 which is a useful marker for indicating the occurrence or development of obesity-diabetes. Since the content of the apolipoprotein B 100 in blood plasma of patients with obesity-diabetes is obviously higher than the content of the apolipoprotein B 100 in blood plasma of people without obesity-diabetes, the apolipoprotein B 100 can be used for developing diagnostic reagents or kits for diagnosing obesity-diabetes or risks of obesity-diabetes.
Description
Technical field
The invention belongs to biological technical field, specifically, the present invention relates to a kind of apolipoprotein B 100 (Apo B 100:Apolipoprotein B 100) as the application of the protein molecular marker thing that detects obese diabetic.
Background technology
Diabetes are diseases of a kind of serious threat human health, and world's incidence of disease is up to 2%, all present fast rise trend at the M ﹠ M of developing diabetes.In China, raising along with socioeconomic development and living standards of the people, diabetes prevalence day by day increases, China's diabetes total number of persons surpasses 4,000 ten thousand at present, become the severely afflicated area of diabetes, the annual medical expense that is used for treating diabetes greatly increases, this shows that diabetes have become serious harm China people life property safety's enemy, and be a key factor that affects socio-economic development, the fundamental research of therefore going into overdrive to carry out China's diabetes has strategic importance.
The diabetic is in the clinical diabetes diagnosis and is 12 years early stage, most of patient is just diagnosed after Diabetic Acute or chronic complicating diseases occurring and is treated, and in fact early detection, make a definite diagnosis to be only with early treatment and allow their restorative key in early days, thereby we need to search out suitable mark realizing the early detection of diabetes, thereby further realize making a definite diagnosis in early days and early treatment of diabetes; So, go to study and seek and can be of great significance as the protein molecular marker thing tool of prediction and diagnosing diabetes take early diagnosis and therapy as purpose.
Along with improving constantly of people's living standard, obese people is in continuous increase.Nationwide nutrition in 2002 and health survey show, nearly 22.8% overweight in greater than 18 years old Grown living in the China's Mainland, 7.1% reaches obese degree; Compare with 1992 investigation result, this two item number has increased respectively 40.7% and 97.2% according to during the decade.There are some researches show that obesity may directly cause various diseases, or greatly increase the ill risk of some disease.And along with the rising of body mass index (BMI), risk also progressively strengthens.The body mass index rising is an important risk factors (Must, the Spadano et al.1999 such as disease of cardiovascular system, type-II diabetes, respiratory disease chronic diseases; Kopelman2000; Kushner and Roth 2003; Kushner and Blatner 2005).In the middle of long-term fat crowd, the morbidity rate of diabetes is more than 5 times of general population.
Therefore, this area is necessary to find by all means the mark of early diagnosis obese diabetic or the ill risk of prediction obese diabetic very much, in the hope of providing foundation for clinical diagnosis.
Summary of the invention
The object of the present invention is to provide and carry fat egg B 100 (Apo B 100:Apolipoprotein B 100) as the application of the protein molecular marker thing that detects obese diabetic.
In a first aspect of the present invention, provide the purposes of a kind of apolipoprotein B 100 (Apolipoprotein B 100) as the obese diabetic mark.
In another preference, described apolipoprotein B 100 is as the protein molecular marker thing that detects obese diabetic or the generation of prediction obese diabetic.
In another preference, described mark is the mark of body fluid.
In another preference, described body fluid is blood plasma or serum.
In another preference, described apolipoprotein B 100 also comprises its protein fragments, and the amino acid sequence of this protein fragments is peculiar by apolipoprotein B 100.
In another preference, the amino acid sequence of the protein fragments of described apolipoprotein B 100 is such as SEQ ID NO:209 or wherein shown in the 2-21 amino acids.
In another aspect of this invention, provide a kind of polypeptide of separation, its amino acid sequence is such as SEQ ID NO:209 or wherein shown in the 2-21 amino acids.
In another aspect of this invention, provide a kind of polynucleotide of separation, its described polypeptide of encoding.
In another aspect of this invention, provide a kind of purposes of apolipoprotein B 100, for the preparation of the reagent that detects obese diabetic.
In another preference, described reagent is selected from: antibody, part.
In another preference, described antibody comprises monoclonal antibody and polyclonal antibody.
In another aspect of this invention, provide the purposes of the reagent of a kind of specific recognition apolipoprotein B 100 or its protein fragments, for the preparation of the kit that detects obese diabetic or differentiation obese diabetic people at highest risk.
In another preference, the reagent of described specific recognition apolipoprotein B 100 or its protein fragments is antibody.
In another aspect of this invention, provide the reagent of a kind of specific recognition apolipoprotein B 100 or its protein fragments, described reagent is polyclonal antibody.
In another aspect of this invention, provide a kind of kit that detects obese diabetic, described kit comprises container, and the described reagent that places described container.
In another aspect of this invention, provide a kind of test-strips (such as test paper or test strips), it comprises solid phase carrier, and is attached to the described reagent on the described solid phase carrier.
Other side of the present invention is because the disclosure of this paper is apparent to those skilled in the art.
Description of drawings
In Fig. 1, the mass spectrum multiple-reaction monitoring technology for detection of the MS/MS spectrogram of the peptide section VPSYTLILPSLELPVLHVPR of 100 concentration of apolipoprotein B in the blood plasma.
Branch's situation of the right R2 of this parent ion-daughter ion of 748.5_678.9 (y8) in Fig. 2,145 samples.Horizontal ordinate represents the R2 value, and ordinate represents the number of times that different R2 values occur.
Fig. 3, to the result of apolipoprotein B 100 parent ions-daughter ion to the mass spectrum multiple-reaction monitoring technology of (748.5_678.9); Wherein, normal is the normal health control group; Obese diabetic is obese diabetic patient group.Ordinate is light peptide divided by the ratio of heavy peptide, namely this section peptide VPSYTLILPSLELPVLHVPR in blood content than on the ratio of standard peptide.Do the t-test check for two groups, p value=0.0062, * * represents p＜0.01.
Embodiment
The inventor finally obtains a kind of generation for the indication obese diabetic or develops useful mark through studying widely and screening.Described mark is apolipoprotein B 100 (Apolipoprotein B 100) or its protein fragments, its content in obese diabetic patient body fluid is significantly higher than normal population, therefore can be used for determining obese diabetic or the ill risk of obese diabetic.
Apolipoprotein B 100
The Genebank accession number of aPoA po B100 is GI:105990532, and the login number of NCBI is: NP_000375, the Swissprot accession number is: P04114 is for IPI number: IPI00022229.Apo B100 mainly is distributed among blood plasma VLDL, IDL and the LDL, accounts for 25%, 60%, 95% of protein content in this three classes lipoprotein.APoA po-B100 participates in synthesizing, assemble and secretion of very low density lipoprotein (VLDL), being the necessary apolipoprotein of very low density lipoprotein (VLDL) that triglyceride is rich in the synthetic and secretion of liver, is mediation low-density lipoprotein and the requisite part of corresponding receptors bind.Known Apo B gene mutation can cause multiple plasma lipoprotein metabolic disorder.And approximately the diabetes B patient of 40%-50% is associated with dyslipidemia, and high triglyceride and low hdl cholesterol are the fat metabolic characteristics of diabetes B and insulin resistance, are again the hazards of diabetes B patient coronary heart disease.But, at present not with the report of aPoA poB100 as human obesity type diabetes molecular labeling.
Apolipoprotein B 100
Based on new discovery of the present invention, the peptide section that derives from apolipoprotein B 100 (polypeptide) is provided, described peptide section is that apolipoprotein B 100 albumen are peculiar, can be applied to well the molecular marked compound as obese diabetic.Preferably, described peptide section has the amino acid sequence shown in the SEQ ID NO:209, or has the amino acid sequence shown in the 2-21 position among the SEQ ID NO:1.The inventor has analyzed the peptide section that derives from a large number apolipoprotein B 100, and is best through identifying this peptide section intensity.The content that detects this peptide section just can directly reflect the content of apolipoprotein B 100 albumen, and is convenient, fast and accuracy is high.
The polynucleotide of peptide section of described apolipoprotein B 100 of encoding are also included within the present invention.Because described peptide section is that apolipoprotein B 100 albumen are peculiar, its corresponding polynucleotide sequence also is distinctive.The method of detection nucleic acid that can be by routine is known the content of the polynucleotide described in the testing sample.
The method that detects polypeptide or nucleic acid content is that those skilled in the art are known.For example, detecting polypeptide can be by means of mass spectrometer etc., maybe can be by methods such as Western Blot or ELISA.Detect nucleic acid and comprise the methods such as pcr amplification, Northern Blot.
The purposes of apolipoprotein B 100 or its protein fragments
The inventor identifies with ethanol precipitation classification-cation-exchange chromatography-reverse-phase chromatography Tandem Mass Spectrometry Analysis strategy normal health control group blood plasma and obese diabetic patient's plasma sample to protein wherein, success has all identified apolipoprotein B 100 in human normal plasma and obese diabetic human plasma, for follow-up MRM experiment provides sequence information.By mass spectrum multiple-reaction monitoring technology, confirm further and find that the expression that normal health control group and obese diabetic patient organize apolipoprotein B 100 in the blood plasma there are differences expression.
By detecting apolipoprotein B 100 expressions in normal health contrast and the obese diabetic patient blood plasma, the inventor find apolipoprotein B 100 normal health contrast and and obese diabetic patient blood plasma in there are differences expression, the expression of apolipoprotein B 100 in diabetic blood plasma is significantly higher than the expression in the normal health contrast blood plasma; Detect by MS/MS, search the storehouse, search protein, buildsummary analyzes, MRM detects, and quantitatively apolipoprotein B 100 confirms further and find that apolipoprotein B 100 expressions of normal health control group, obese diabetic patient group there are differences expression.
Therefore, first purpose of the present invention is that a kind of apolipoprotein B 100 or its protein fragments are provided, and obese diabetic occurs or the application of the protein molecular marker thing that the prediction obese diabetic occurs as detecting.
Therefore, the invention provides a kind of apolipoprotein B 100 or its protein fragments as the purposes of diabetes mark.It is a kind of multifunctional protein that is distributed in blood, ascites fluid, celiolymph and polytype cell surface that those skilled in the art understand apolipoprotein B 100 (apolipoprotein B 100), therefore, the apolipoprotein B 100 (apolipoprotein B 100) in the body fluid such as blood plasma, ascites fluid, celiolymph can be used as molecular marked compound.As optimal way of the present invention, apolipoprotein B 100 detects mark as the blood plasma of obese diabetic.Blood plasma detects and to draw materials conveniently, simple to operate, be convenient to check, the patient is easy to accept evaluate its prognosis, guiding treatment.
Based on new discovery of the present invention, can utilize apolipoprotein B 100 albumen: the antidiastole and/or the susceptibility analysis that (i) carry out obese diabetic; (ii) obese diabetic medicine, curative effect of medication, the prognosis of assessment correlated crowd, and select suitable methods for the treatment of; (iii) assess in early days the ill risk of correlated crowd obese diabetic, early monitoring early prevention.
It can also be for the preparation of the reagent of specific recognition apolipoprotein B 100, thereby for detection of the existence of apolipoprotein B100 whether and amount, as the basis for estimation of obese diabetic.
Reagent and the kit of identification apolipoprotein B 100
Based on new discovery of the present invention, the present invention also provides the reagent of specific recognition apolipoprotein B 100 or its protein fragments.Any reagent of identifying apolipoprotein B 100 or its protein fragments all comprises in the present invention, as the mark that detects obese diabetic.The reagent of described specific recognition apolipoprotein B 100 or its protein fragments for example is antibody or the part of specific binding apolipoprotein B 100.
The present invention also provides the purposes of the reagent of a kind of specific recognition apolipoprotein B 100 or its protein fragments, for detection of obese diabetic or obese diabetic people at highest risk.
As one embodiment of the present invention, described reagent is the antibody of anti-apolipoprotein B 100; It more particularly for example is polyclonal antibody.
Antibody of the present invention can be prepared by the known various technology of those skilled in that art.For example, the antigen of purifying can be applied to animal to induce the generation of polyclonal antibody, described animal such as rabbit, mouse, rat etc.Multiple adjuvant can be used for strengthening immune response, includes but not limited to Freunds adjuvant etc.
Antibody of the present invention also can be monoclonal antibody.This type of monoclonal antibody can utilize hybridoma technology to prepare that (see the people such as Kohler, Nature 256; 495,1975; The people such as Kohler, Eur.J.Immunol.6:511,1976; The people such as Kohler, Eur.J.Immunol.6:292,1976; The people such as Hammerling, In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981).
Described antibody can be used in the immunohistochemistry technology, detects apolipoprotein B 100 levels in the sample, thereby is used for the diagnosis obese diabetic or judges ill risk (neurological susceptibility).
Utilize described antibody, can detect the level of apolipoprotein B 100 in the body fluid, thereby can be used for detecting obese diabetic, can be used for predicting the generation of early stage obese diabetic, perhaps for the preparation of the preparation that detects obese diabetic or kit etc., for the preparation of the preparation of the generation of the early stage obese diabetic of prediction or kit etc.
The present invention also provides the kit that is used for the diagnosis obese diabetic, and this kit comprises: the reagent of specific recognition apolipoprotein B 100 or its protein fragments.Described reagent for example is: monoclonal antibody or polyclonal antibody.
Also can contain in the described kit: be used for the reagent of immunohistochemical analysis, described reagent such as second antibody, coloring agent, developer etc.In addition, also can comprise operation instructions etc. in the described kit.
More specifically, described kit can be a kind of kit based on enzyme linked immunoassay (ELISA) technology.For detection of obese diabetic or predict the generation of early stage obese diabetic.Elisa technique and be apparent for a person skilled in the art based on the detection reagent of this technology.
More specifically, the reagent of described specific recognition apolipoprotein B 100 or its protein fragments also can be fixed on the test paper, is prepared into immune colloid gold test paper or similar test material.
Major advantage of the present invention is:
(1) discloses first the mark that apolipoprotein B 100 or its protein fragments can be used as the diagnosis obese diabetic.
(2) prepared first the reagent of specific recognition apolipoprotein B 100 or its protein fragments, described reagent specificity is good, and detection sensitivity is high, and accuracy is high, has good clinical value.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: lab guide (New York:Cold Spring Harbor Laboratory Press, 2002) condition described in, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise number percent and umber calculate by weight.
Unless otherwise defined, employed all specialties are identical with the meaning that scientific words and one skilled in the art are familiar with in the literary composition.In addition, any method similar or impartial to described content and material all can be applicable among the present invention.The usefulness that better implementation method described in the literary composition and material only present a demonstration.
Embodiment 1, differential expression protein screening technique-ethanol precipitation classification-cation-exchange chromatography-reverse-phase chromatography tandem mass spectrum strategy
Experimental waters all in the present embodiment are all from Milli-Q (Millipore, Bedford, MA, USA) ultrapure water equipment; Dithiothreitol (DTT), SDS, urea, guanidine hydrochloride, 3-[(3-courage amido propyl)-diethyl] the-third sulfo group (CHAPS), Tris, ammonium bicarbonate (NH
4HCO
3) and iodoacetamide (IAA) buy from Bio-Rad company (Herc μ Les, CA, USA); Trypsase (Trypsin) is available from Promega company (Madison, WI, USA); Formic acid (formic acid, FA), trifluoroacetic acid (trifluoroacetic acid, TFA) and acetonitrile (Acetonitrile) are available from Aldrich company (MilWaukee, WI, USA); Phosphoric acid is available from Solution on Chemical Reagents in Shanghai company.Except acetonitrile was chromatographically pure, it is pure that remaining chemical reagent is analysis.
In 150 μ L plasma samples, add 750 μ L solution (pH 7.4 for 100mM NaCl, 10mM Tris), with 0.22 μ m filter membrane centrifugal removal lipid under the rotating speed of 4 ℃ of 10 000g; The plasma sample of getting after the 780 μ L grease removal adds 565 μ L cold alcohol solutions, places on 4 ℃ of vertical shaking tables and mixes 1h, then 4 ℃ of 16 centrifugal 45min of 000g.Supernatant is the high abundance component, and the precipitation part is low abundance components.With low abundance components with the cold ethanol rinse of 100% (v/v) a time, carry out low-temperature freeze-dry, adding 600 μ L 6M guanidine hydrochloride solutions dissolves, get 300 μ L, add 5 μ L 1M DTT, mixing, place 2.5h for 37 ℃, after being cooled to room temperature, add 25 μ L1M IAA, the room temperature lucifuge is placed 40min.Through the complete sex change of protein of above-mentioned processing, disulfide bond is opened, and sulfydryl is closed.Then add 1.6ml cryoproteins matter precipitated liquid (acetone: ethanol: acetic acid=50: 50: 0.1), behind-20 ℃ of placement 15h, 4 ℃ of centrifugal 45min of 15000g remove supernatant, add 1mL 100% (v/v) cold acetone, vibration 1min, 4 ℃ of centrifugal 45min of 15000g remove supernatant, add 1mL 70% cold ethanol, vibration 1min, 4 ℃ of centrifugal 45min of 15000g remove supernatant, freeze-drying, add 250 μ L 100mM ammonium bicarbonate solns, 50 μ g trypsase behind 37 ℃ of enzymolysis 4h, add 50 μ g trypsase again, 37 ℃ of enzymolysis are behind the 16h.The EP liquid in pipe is transferred in the Millipore 10K super filter tube, 4 ℃ of 10000g ultrafiltration 45min, remove enzyme and not by the protein of enzymolysis, collect filtrate, freeze-drying, every effective 0.1% aqueous formic acid redissolves, and gets former plasma proteins enzymolysis product (etc. quality) 50 μ g loading Mass Spectrometer Method.
Cation-exchange chromatography-reverse-phase chromatography tandem mass spectrum (SCX-RP-MS/MS) comprises an automatic sampler, two high pressure mixing pumps and is used for two-dimensional liquid chromatography cation-exchange chromatography separating column (SCX post) and C18 reversed phase chromatography separation post (RP post) and two the anti-phase trap posts of C18 that separate, a ten-way valve.High performance liquid chromatography solution comprises two kinds: one, cation-exchange chromatography separation solution comprises A:pH 2.5 solution; B:pH 8.5 solution; Two, reversed phase chromatography separation solution: A:0.1% formic acid; B:0.1% formic acid, 100% (v/v) acetonitrile.Through the elute soln flushing of different pH values from low to high, the peptide section that elutes under different pH gradients is passed through reversed phase chromatography separation to peptide section potpourri again, then carries out Mass Spectrometer Method first.Concrete experimentation is as follows.Peptide section potpourri behind the enzymolysis enters into the SCX post through automatic sampler with break-even pattern, under the flushing of the eluent of a certain pH value, the peptide section that does not keep is flushed on the C18 trap post of back, behind the 180min, ten-way valve switches, carry out the wash-out of next pH gradient on the SCX post, the peptide section that elutes is attached on No. two C18 trap posts, simultaneously, on C18 trap post of 100% acetonitrile solution flushing with 2-35% (v/v), and the peptide section that elutes on this trap carried out Re-isolation at C18 reversed phase chromatography separation post, Mass Spectrometer Method, behind the 180min, ten-way valve is cut valve again, at this moment, the peptide section that the pH gradient elution gets off is attached on C18 trap post, and washes the peptide section on the C18 trap post that is combined in No. two that a upper pH gradient elution gets off with 100% acetonitrile solution of 2-35%, and the peptide section that elutes on this trap is carried out Re-isolation at C18 reversed phase chromatography separation post, Mass Spectrometer Method is carried out so repeatedly.Used altogether 10 different pH gradients that the peptide section on the SCX pillar is carried out wash-out.Mass spectrum condition in whole process is: the scanning of the mass spectrum condition is set as full scan (full scan) back of a 400-2000M/z and carries out the MS/MS scanning at front 10 tops in the full scan, the setting of wherein dynamically getting rid of (dynamic exclusion) is: multiplicity (repeat count) is 2, repeating patient time (repeat duration) is 30s, and dynamically getting rid of the time (exclusion duration) is 120s.Mass Spectrometer Method repeats once.
Used 33 routine plasma samples to comprise in the experiment: 5 routine normal types and euglycemia sample (group 1), 5 routine normal types and hyperglycaemia sample (group 2), 5 routine normal types and diabetes sample (group 3), 6 routine obesities and euglycemia sample (group 4), 6 routine obesities and hyperglycaemia sample (group 5), 6 routine obese diabetic samples (group 6).Every group of sample takes out separately the equal-volume plasma sample and is mixed into 150 μ L blood plasma biased samples, is total to get six samples, carries out respectively above-mentioned experimental implementation.All sample clinical indices see Table 1.The clinical indices of measuring is respectively: fasting blood-glucose (FPG, mmol/l), insulin (Insulin, μ U/ml), triglyceride (TG, mmol/l), HDL-C (HDL_C, mmol/l), LDL-C (LDL_C, mmol/l), T-CHOL (TC, mmol/l)
Table 1,33 routine plasma sample clinical datas
Embodiment 2, the mass spectrum interpretation of result
The raw data that twice Mass Spectrometer Method in above-described embodiment obtained is with IPI human 3.51 checking storehouses, be buildsummary, peptide section screening strategy is the parameter of peptide FDR＜=1% and unipeptides＞=2, identify altogether 717 protein, identify 31 in the anti-storehouse, protein FDR is 4.32%
Table 2 shows be apolipoprotein B 100 in 6 groups of blood plasma (Apolipoprotein B 100) the peptide section (for Apolipoprotein B 100 distinctive) identify that number of times, the peptide section in the table all are enzymolysis apolipoprotein B 100 (Apolipoprotein B 100) and the peptide section sequence that obtains.
Fig. 1 has shown in the mass spectrum multiple-reaction monitoring technology MS/MS spectrogram for detection of the peculiar peptide section VPSYTLILPSLELPVLHVPR of apolipoprotein B in the blood plasma 100.Better through identifying this peptide section intensity, its detection of peptides hop count is higher, and it is moderate to comprise the amino acid number, is fit to be applied to many reaction monitorings of mass spectrum technology for detection checking (many reaction monitorings of mass spectrum technology can detect and comprise 7-22 amino acid whose peptide section usually).
The peptide hop count of apolipoprotein B 100 Mass Spectrometric Identifications in table 2, the 6 groups of blood plasma
Table 2, ". " represent tryptic restriction enzyme site; The trypsase specific recognition also cuts K and the peptide bond of the carboxyl of R participation formation.Enzyme is cut the peptide section between the rear acquisition two ". ".
Above form shows: apolipoprotein B 100 peptide section in obese diabetic patient blood plasma is identified number of times 5836 times, is higher than among the human normal plasma peptide section and identifies number of times 5406 times.This experiment provides follow-up MRM checking necessary sequence information simultaneously.
The dithiothreitol (DTT) that uses in the present embodiment (DTT) is available from Sigma company; Iodoacetamide (IAA), formic acid are available from Fluka company; Acetonitrile is available from Merck company; Capillary reversed-phase liquid chromatography, triple level Four bar mass spectrums are all available from Agilent company.The conventional method synthetic standards is heavily marked peptide section VPSYTLILPSLELPVLHVPR* (Heavy-Arg) (the heavy target amino acid of " * " expression).
The per 3 μ L stostes of plasma sample add 97 μ L 2D lysates (8M urea, 40mM Tris, 65mMDTT), place 1h for 4 ℃, add 1 μ L 1M DTT, place 2.5h in 37 ℃, to be cooled to room temperature, add 15 μ L 1M IAA, place 40min in the room temperature lucifuge, sample is transferred in the Millipore 10K super filter tube, 4 ℃, the centrifugal 45min of 10000g adds 100 μ L 50mM ammonium bicarbonate solutions, 4 ℃, then the centrifugal 45min of 10000g adds 12 μ g trypsase, 76 μ L 50mM ammonium bicarbonate solutions, 37 ℃ of enzymolysis 16h, then filter membrane is transferred in another centrifuge tube, 4 ℃, the centrifugal 45min of 10000g, add 100 μ L 50mM ammonium bicarbonate solutions, 4 ℃, the centrifugal 45min of 10000g collects centrifugal gained liquid twice, add 3 μ L formic acid, freeze-drying.Peptide section sample after the freeze-drying dissolves with 100 μ L 0.1% (v/v) FA solution.
Get the plasma proteins enzymolysis product (equal-volume) and 20pmol standard weight mark peptide section of the former blood plasma of corresponding 0.3 μ L, mix joining on the reversed-phase column with break-even pattern by automatic sampler.The solvent that is used for reversed-phase column is 0.1% (v/v) aqueous formic acid (A liquid), 0.1% (v/v) formic acid, 90% (v/v) acetonitrile solution (B liquid).With the flow velocity of 1.5 μ L/min, to reversed-phase column at 0-20min with 5% B liquid loading, 20-120min carries out gradient elution with the B liquid of 5-35% (volume ratio).The sample of wash-out enters QQQ 6410 (Agilent) and detects by receiving esi ion source from the reversed-phase column.Choose 748.5_465.8 (y8), 748.5_508.3 (y4), 748.5_621.4 (y5), 748.5_678.9 (y12), 748.5_735.5 (y13), 748.5_817.5 (y7) totally 6 parent ion-daughter ions to detecting, get the positive signal of signal at co-elute place and its peak area is carried out integration, finally get parent ion-daughter ion to the quantitative information of 748.5_678.9 (y8) quantitative information as this peptide section, carry out the follow-up data analysis, this parent ion-daughter ion is seen Fig. 2 to the light peptide of 748.5_678.9 (y8) in elution time and the correlativity of the ratio of the signal intensity of heavy peptide in all samples.Table 3 has shown apolipoprotein B 100 MRM mass spectrum verification msgs in the 36 routine normal health contrast blood plasma, table 4 has shown apolipoprotein B 100 MRM mass spectrum verification msgs in the 109 routine obese diabetic blood plasma, ratio is that light peptide is divided by the ratio of heavy peptide in the form, namely this section peptide in blood content than on the value of standard peptide, R2 is the correlativity of the ratio of light peptide and the signal intensity that weighs peptide, wherein 97.2% data R2＞=0.75.
Table 3
Table 4
Get parent ion-daughter ion to the quantitative information of (748.5_678.9) quantitative information as this peptide section (VPSYTLILPSLELPVLHVPR), carry out the follow-up data analysis.Normal person and this parent ion-daughter ion of obese diabetic people are done the t-test check to (748.5_678.9) gained ratio, p value=0.0062, less than 0.01, verified that apolipoprotein B 100 protein content in the obese diabetic human plasma is significantly higher than the normal person, sees Fig. 3.
In sum, apolipoprotein B 100 significantly raises in endomorphy type glycosuria patient blood plasma, obviously and the genesis close relation of endomorphy type glycosuria, so this albumen detects the prediction that can be used for the diabetes genesis as a molecular marked compound to its expression.
Analyze through ROC, when cutoff is 0.5649, accuracy 62.7%, sensitivity 65.1, specificity 55.6%, area under curve 0.636.
Embodiment 4, antibody preparation
The inventor is injected into total length apolipoprotein B 100 albumen (GenBank accession number GI:105990532) in the rabbit body, obtains immune serum.Concrete preparation method is as follows:
Immunoblot experiment shows: the rabbit polyclonal antibody of acquisition is only identified apolipoprotein B 100 albumen, and basic other albumen of nonrecognition can be used as the detection reagent that detects obese diabetic or the ill risk of obese diabetic.
The polyclonal antibody, two anti-(routines), the chromogenic reagent that obtain are placed respectively different containers, container is placed box with operation instructions, obtain detection kit.
All quote in this application as a reference at all documents that the present invention mentions, just as each piece document is quoted separately as a reference.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned instruction content of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (10)
1. an apolipoprotein B 100 is as the purposes of obese diabetic mark.
2. purposes as claimed in claim 1 is characterized in that, described apolipoprotein B 100 also comprises its protein fragments, and the amino acid sequence of this protein fragments is peculiar by apolipoprotein B 100.
3. purposes as claimed in claim 2 is characterized in that, the amino acid sequence of the protein fragments of described apolipoprotein B 100 is such as SEQ ID NO:209 or wherein shown in the 2-21 amino acids.
4. the polypeptide of a separation is characterized in that, its amino acid sequence is such as SEQ ID NO:209 or wherein shown in the 2-21 amino acids.
5. the polynucleotide of a separation is characterized in that, its polypeptide claimed in claim 4 of encoding.
6. the purposes of an apolipoprotein B 100 is for the preparation of the reagent that detects obese diabetic.
7. the purposes of the reagent of a specific recognition apolipoprotein B 100 or its protein fragments is for the preparation of detecting obese diabetic or distinguishing obese diabetic people at highest risk's kit.
8. the reagent of a specific recognition apolipoprotein B 100 or its protein fragments, described reagent is polyclonal antibody.
9. kit that detects obese diabetic, described kit comprises container, and the reagent claimed in claim 8 that places described container.
10. test-strips, it comprises solid phase carrier, and is attached to the reagent claimed in claim 8 on the described solid phase carrier.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012101247337A CN103376322A (en) | 2012-04-25 | 2012-04-25 | Application of apolipoprotein B 100 as marker of obesity-diabetes |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012101247337A CN103376322A (en) | 2012-04-25 | 2012-04-25 | Application of apolipoprotein B 100 as marker of obesity-diabetes |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103376322A true CN103376322A (en) | 2013-10-30 |
Family
ID=49461759
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2012101247337A Pending CN103376322A (en) | 2012-04-25 | 2012-04-25 | Application of apolipoprotein B 100 as marker of obesity-diabetes |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103376322A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2018516363A (en) * | 2015-04-22 | 2018-06-21 | ネステク ソシエテ アノニム | Biomarkers for predicting weight loss in female subjects |
| TWI702292B (en) * | 2018-12-28 | 2020-08-21 | 薩摩亞商康多富國際有限公司 | Method for determining a set of personalized metabolic disease healthy foods and non-transitory computer readable storage medium |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004085996A2 (en) * | 2003-03-20 | 2004-10-07 | Albert Einstein College Of Medicine Of Yeshiva University | Biomarkers for longevity and disease and uses thereof |
| CN1603825A (en) * | 2003-09-29 | 2005-04-06 | 杜凤鸣 | Quantitative enzyme linked immunosorbent assay kit for Apo B100 protein in human urine and preparation method thereof |
| WO2006083853A2 (en) * | 2005-01-31 | 2006-08-10 | Insilicos, Llc | Methods of identification of biomarkers with mass spectrometry techniques |
| CN101646942A (en) * | 2006-03-23 | 2010-02-10 | 埃梅利塔·德古兹曼·布雷耶 | Apolipoprotein fingerprinting technique |
| US20110124022A1 (en) * | 2008-07-23 | 2011-05-26 | Diabetomics, Llc | Methods for detecting pre-diabetes and diabetes |
-
2012
- 2012-04-25 CN CN2012101247337A patent/CN103376322A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004085996A2 (en) * | 2003-03-20 | 2004-10-07 | Albert Einstein College Of Medicine Of Yeshiva University | Biomarkers for longevity and disease and uses thereof |
| CN1603825A (en) * | 2003-09-29 | 2005-04-06 | 杜凤鸣 | Quantitative enzyme linked immunosorbent assay kit for Apo B100 protein in human urine and preparation method thereof |
| WO2006083853A2 (en) * | 2005-01-31 | 2006-08-10 | Insilicos, Llc | Methods of identification of biomarkers with mass spectrometry techniques |
| CN101646942A (en) * | 2006-03-23 | 2010-02-10 | 埃梅利塔·德古兹曼·布雷耶 | Apolipoprotein fingerprinting technique |
| US20110124022A1 (en) * | 2008-07-23 | 2011-05-26 | Diabetomics, Llc | Methods for detecting pre-diabetes and diabetes |
Non-Patent Citations (1)
| Title |
|---|
| KING C. CHAN ET AL: "Analysis of the human serum proteome", 《CLINICAL PROTEOMICS》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2018516363A (en) * | 2015-04-22 | 2018-06-21 | ネステク ソシエテ アノニム | Biomarkers for predicting weight loss in female subjects |
| TWI702292B (en) * | 2018-12-28 | 2020-08-21 | 薩摩亞商康多富國際有限公司 | Method for determining a set of personalized metabolic disease healthy foods and non-transitory computer readable storage medium |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Jacobs et al. | Utilizing human blood plasma for proteomic biomarker discovery | |
| Papafilippou et al. | Protein corona fingerprinting to differentiate sepsis from non-infectious systemic inflammation | |
| CN106796239B (en) | Composition for diagnosis of pancreatic cancer and the method using its diagnosis of pancreatic cancer | |
| CN106405104B (en) | A kind of new cirrhosis or hepatic fibrosis markers | |
| WO2010141469A2 (en) | Protein biomarkers and therapeutic targets for autoimmune and alloimmune diseases | |
| CN113176411B (en) | Biomarker for detecting novel coronavirus infection by saliva and application thereof | |
| Woo et al. | Characterization and modulation of surface charges to enhance extracellular vesicle isolation in plasma | |
| CN102687011A (en) | Cancer biomarker and the use thereof | |
| KR20150061816A (en) | Polypeptide markers for cancer diagnosis derived from blood sample and methods for the diagnosis of cancers using the same | |
| Carlsson et al. | Different fractions of human serum glycoproteins bind galectin-1 or galectin-8, and their ratio may provide a refined biomarker for pathophysiological conditions in cancer and inflammatory disease | |
| CN102539779A (en) | Application of Vitamin D binding protein serving as marker of diabetes | |
| KR20120125157A (en) | A method for the diagnosis using lectin | |
| CN104749380A (en) | Application Of Hce1 And Antibody Thereof In Preparation Of Composition Used For Diagnosing Main Body Liver Cancer | |
| CN107045062A (en) | Detect colloidal gold immuno-chromatography test paper strip, kit of human neutrophil genatinase associated lipocalin and preparation method thereof | |
| CN103376323A (en) | Application of apolipoprotein C-III as marker of obesity-diabetes | |
| CN105849562B (en) | The measuring method of soluble g PC3 protein | |
| KR20150062915A (en) | Serological markers for cancer diagnosis using blood sample | |
| Okada et al. | Serum complement C3 and α2-macroglobulin are potentially useful biomarkers for inflammatory bowel disease patients | |
| Azevedo et al. | Extracellular vesicles and their relationship with the heart–kidney axis, uremia and peritoneal dialysis | |
| CN103376324A (en) | Application of albumin as obesity-diabetes marker | |
| CN103376322A (en) | Application of apolipoprotein B 100 as marker of obesity-diabetes | |
| Miękus et al. | Gel electrophoretic separation of proteins from cultured neuroendocrine tumor cell lines | |
| CN107290552B (en) | The biomarker of high coagulation and its application | |
| Imai et al. | Comprehensive analysis and comparison of proteins in salivary exosomes of climacteric and adolescent females | |
| CN103370620A (en) | Diagnostic drug and diagnostic method for alzheimer's disease |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C05 | Deemed withdrawal (patent law before 1993) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20131030 |