CN103376324A - Application of albumin as obesity-diabetes marker - Google Patents
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- CN103376324A CN103376324A CN2012101247572A CN201210124757A CN103376324A CN 103376324 A CN103376324 A CN 103376324A CN 2012101247572 A CN2012101247572 A CN 2012101247572A CN 201210124757 A CN201210124757 A CN 201210124757A CN 103376324 A CN103376324 A CN 103376324A
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Abstract
The invention relates to an application of albumin (ALB, Albumin) as an obesity-diabetes marker. The invention provides a marker-albumin that is useful for indicating occurrence or development of obesity-diabetes, of which the content in the plasma of an obesity-diabetes patient is remarkably higher than that of normal people, therefore the application of the albumin can be used for developing a diagnostic reagent or a kit for diagnosing obesity-diabetes or risks of obesity-diabetes patients.
Description
Technical field
The invention belongs to biological technical field, specifically, the present invention relates to a kind of albumin (ALB, Albumin) 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% (Chen 2008) 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.At present, obese diabetic is classified clinically as a class of diabetes.
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 albumin (ALB, Albumin) 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 albumin as the obese diabetic mark.
In another preference, described albumin 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 albumin also comprises its protein fragments, and the amino acid sequence of this protein fragments is peculiar by albumin.
In another preference, the amino acid sequence of described albuminous protein fragments is such as SEQ ID NO:1 or wherein shown in the 2-8 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:1 or wherein shown in the 2-8 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 albuminous purposes, 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 albumin 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 albumin or its protein fragments is antibody.
In another aspect of this invention, provide the reagent of a kind of specific recognition albumin 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 YLYEIAR of albumin (ALB, Albumin) concentration.
The right R of this parent ion-daughter ion of 464.25_651.3 (y5) in Fig. 2,145 samples
2Distribution situation.Horizontal ordinate represents R
2Value, ordinate represents different R
2The number of times that value occurs.
Fig. 3, to the result of albumin (ALB, Albumin) parent ion-daughter ion to the mass spectrum multiple-reaction monitoring technology of (464.25_651.3); Ordinate is light peptide divided by the ratio of heavy peptide (L/H), namely this section p277 LYEIAR in blood content than on the ratio of standard peptide.Do the t-test check for two groups, p value=0.0208, * represents p＜0.05.
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 albumin (ALB, ALBUMIN) or its protein fragments, and its content in obese diabetic patient body fluid is significantly higher than normal population, therefore can be used for diagnosing obese diabetic or the ill risk of obese diabetic.
Albumin (ALB, Albumin)
Albumin (ALB), the login number of its NCBI is: NP_00468, the Swissprot accession number is: P02768 is for IPI number: IPI00384697/IPI00745872.Albumin is the water-soluble very high globular protein that is present in the blood plasma, accounts for the 50-60% of protein content in the blood, and water, calcium ion, potassium ion, sodion, fatty acid, hormone, cholerythrin etc. are had good binding ability.Albumin is synthetic in liver, plays an important role to keeping blood plasma body osmotic pressure.Simultaneously, albumin can with the multiple ligands combination, play the transportation effect.Known in diabetic population, albumin can be carried out saccharification and modify.There is the document prompting can be with albumin as the mark (Diabetes Metab.2012 Feb 18.) of diabetes pathological state still, the SRM technology that is out of use is at present verified large quantities of crowds, proves that albumin can be used as human obesity type diabetes molecular labeling.
Based on new discovery of the present invention, provide to derive from albuminous peptide section (polypeptide), described peptide section is that albumin is 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:1, or has the amino acid sequence shown in the 2-8 position among the SEQ ID NO:1.The inventor has analyzed and derived from a large number albuminous peptide section, and is best through identifying this peptide section intensity.The content that detects this peptide section just can directly reflect albuminous content, and is convenient, fast and accuracy is high.
The polynucleotide of the described albuminous peptide section of encoding are also included within the present invention.Because described peptide section is that albumin is 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 albumin 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, all identified albumin in normal health contrast and the obese diabetic patient blood plasma, provide to be applied to the albuminous possibility sequence that later stage MRM detects.By mass spectrum multiple-reaction monitoring technology, confirm further and find that normal health control group and obese diabetic patient organize that albuminous expression there are differences expression in the blood plasma.
By detecting Expression of Albumin amount in normal health contrast and the obese diabetic patient blood plasma, the inventor find albumin normal health contrast and and obese diabetic patient blood plasma in there are differences expression, the expression of albumin in obese diabetic patient 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, and MRM detects, and quantitatively albumin confirms and find that the Expression of Albumin level existence difference of normal health control group, obese diabetic patient group is expressed.
Therefore, first purpose of the present invention is that a kind of albumin 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 albumin or its protein fragments as the purposes of diabetes mark.It is a kind of multifunctional protein that is distributed in blood, urine and polytype cell surface that those skilled in the art understand albumin, and therefore, the albumin in the body fluid such as blood, urine can be used as molecular marked compound.As optimal way of the present invention, albumin 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 albumin: 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 albuminous reagent of specific recognition, thereby for detection of albuminous existence whether and amount, as the basis for estimation of obese diabetic.
Identify albuminous reagent and kit
Based on new discovery of the present invention, the present invention also provides the reagent of specific recognition albumin or its protein fragments.Any reagent of identifying albumin or its protein fragments all comprises in the present invention, as the mark that detects obese diabetic.The reagent of described specific recognition albumin or its protein fragments for example is the albuminous antibody of specific binding or part.
The present invention also provides the purposes of the reagent of a kind of specific recognition albumin 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 antialbumin; 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 the albumin level in the sample, thereby is used for the diagnosis obese diabetic or judges ill risk (neurological susceptibility).
Utilize described antibody, can detect albuminous level 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 albumin 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 albumin 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 albumin or its protein fragments can be used as the diagnosis obese diabetic.
(2) prepared first the reagent of specific recognition albumin 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 (NH4HCO3) and iodoacetamide (IAA) be from (the Herc μ Les of Bio-Rad company, CA, USA) buy; 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 10000g; 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 centrifugal 45min of 16000g.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, 50ug trypsase behind 37 ℃ of enzymolysis 4h, adds 50ug 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) 50ug 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% (v/v) 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 C18trap 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 C18trap posts, simultaneously, on C18trap post of 100% acetonitrile solution flushing with 2-35%, 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 C18trap post, and washes the peptide section on the C18trap 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 obese diabetic 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
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 albumin (ALB, Albumin) in 6 groups of human plasmas the peptide section (for ALB distinctive) identify that number of times, the peptide section in the table all are albumin hydrolyzate 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 YLYEIAR of ALB in the blood plasma (Albumin).Higher through identifying this peptide section intensity, its detection of peptides hop count is higher, and comprise amino acid number moderate (comprising 10 amino acid), be 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 albumin (ALB, Albumin) Mass Spectrometric Identification in table 2, the 6 groups of blood plasma
In the table 2, ". " represents 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 ". "." * " represents on the methionine with oxidative modification.
This embodiment success has identified albumin (ALB, Albumin) in human normal plasma and obese diabetic patient blood plasma, the possible peptide section that is applied to follow-up MRM checking is provided.Found the important protein of this function of albumin (ALB, Albumin) through current embodiment, the back is verified with MRM.
Embodiment 3, the albumin differential expression the technical identification of mass spectrum multiple-reaction monitoring
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 YLYEIAR* (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, 65mM DTT), 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 12ug 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 464.25_175.1 (y1), 464.25_246.2 (y2), 464.25_359.2 (y3), 464.25_488.3 (y4), 464.25_651.3 (y5), 464.25_764.4 (y6) 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 464.25_651.3 (y5) 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 464.25_651.3 (y5) in elution time and the correlativity of the signal intensity of heavy peptide in all samples.Table 3 has shown albumin (ALB in the 36 routine normal health contrast blood plasma, Albumin) (ALB, Albumin) MRM mass spectrum verification msg, table 4 has shown albumin (ALB in the 109 routine obese diabetic blood plasma, Albumin) ratio is that light peptide (L) is divided by the ratio of heavy peptide (H) in (ALB, Albumin) MRM mass spectrum verification msg, form, namely this section peptide in blood content than on the value of standard peptide, R
2Be the correlativity of light peptide with the signal intensity of heavy peptide, wherein 100% data R
2＞=0.92.
Table 3
Table 4
Get parent ion-daughter ion to the quantitative information of (464.25_651.3) quantitative information as this peptide section (YLYEIAR), 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 (464.25_651.3) gained ratio, p value=0.0208, less than 0.05, verified albumin (ALB, Albumin) (ALB, Albumin) protein content is significantly higher than the normal person in the obese diabetic human plasma, sees Fig. 3.
Do the ROC curve with MRM result, area under curve: 0.6205, Albumin cut point=21.9, sensitivity=0.633, false positive rate=0.4444, specificity=0.5556.
In sum, albumin (ALB, Albumin) (ALB, Albumin) in endomorphy type glycosuria patient blood plasma, significantly rise, obviously and the genesis close relation of endomorphy type glycosuria, so this albumen detects the prediction that can be used for the obese diabetic genesis as a molecular marked compound to its expression.
Embodiment 4, antibody preparation
The inventor is total length ALB, and Albumin albumen (GenBank accession number GI:178344) is injected in the rabbit body, obtains immune serum.Concrete preparation method is as follows:
ALB (Albumin) albumen is expelled to rabbit with after Freunds adjuvant mixes with 1: 1 according to volume ratio, and immunity amount 0.5mg is every 2 week immunity 1 time, immunity 3 times.Behind the affinity purification of routine, obtain the rabbit polyclonal antibody for ALB (Albumin) albumen.
Immunoblot experiment shows: the rabbit polyclonal antibody of acquisition is only identified ALB (Albumin) 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 albumin is as the purposes of obese diabetic mark.
2. purposes as claimed in claim 1 is characterized in that, described albumin also comprises its protein fragments, and the amino acid sequence of this protein fragments is peculiar by albumin.
3. purposes as claimed in claim 2 is characterized in that, the amino acid sequence of described albuminous protein fragments is such as SEQ ID NO:1 or wherein shown in the 2-8 amino acids.
4. the polypeptide of a separation is characterized in that, its amino acid sequence is such as SEQ ID NO:1 or wherein shown in the 2-8 amino acids.
5. the polynucleotide of a separation is characterized in that, its polypeptide claimed in claim 4 of encoding.
6. albuminous purposes is for the preparation of the reagent that detects obese diabetic.
7. the purposes of the reagent of a specific recognition albumin 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 albumin 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.
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| CN106979982A (en) * | 2016-01-19 | 2017-07-25 | 上海市第六人民医院 | It is a kind of to be predicted for diabetes risk, treat the method evaluated and kit |
| CN111323473A (en) * | 2020-03-16 | 2020-06-23 | 上海中科新生命生物科技有限公司 | Method for analyzing exosome protein based on SP3 enzymolysis |
| CN113917152A (en) * | 2021-09-22 | 2022-01-11 | 北京松果天目健康管理有限公司 | Application of urine protein marker in preparation of kit for detecting diabetic nephropathy |
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| CN111323473A (en) * | 2020-03-16 | 2020-06-23 | 上海中科新生命生物科技有限公司 | Method for analyzing exosome protein based on SP3 enzymolysis |
| CN113917152A (en) * | 2021-09-22 | 2022-01-11 | 北京松果天目健康管理有限公司 | Application of urine protein marker in preparation of kit for detecting diabetic nephropathy |
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