CN102539779A - Application of Vitamin D binding protein serving as marker of diabetes - Google Patents
Application of Vitamin D binding protein serving as marker of diabetes Download PDFInfo
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Abstract
The invention relates to an application of a vitamin D binding protein serving as a marker of diabetes. The invention provides the vitamin D binding protein serving as the marker which is useful for indicating the occurrence or development of the diabetes; and the content of the vitamin D binding protein in the serum of a diabetic patient is obviously lower than that of the normal crowd so that the vitamin D binding protein can be used for diagnosing the diabetes or disease risk of the diabetes.
Description
Technical field
The invention belongs to biological technical field, specifically, the present invention relates to the application of a kind of DBP (Vitamin D-binding protein:DBP) as the protein molecular marker thing that detects diabetes.
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 The development in society and economy and living standards of the people; Diabetes prevalence day by day increases, and China's diabetes total number of persons surpasses 4,000 ten thousand at present, has become the severely afflicated area of diabetes; The medical expense that is used for treating diabetes every year greatly increases; This shows that diabetes have become serious harm China people life property safety's enemy, and is a key factor that influences socio-economic development, and 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 was 12 years early stage; Most of patient is just diagnosed after the acute or chronic complicating diseases of diabetes occurring and is treated; And in fact early detection, make a definite diagnosis to be only and let their restorative key in early days with early treatment; Thereby we need 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, be that purpose is gone to study and seek and can be had crucial meaning as the protein molecular marker thing of prediction and diagnosing diabetes with the early diagnosis and therapy.
Therefore, this area is necessary to find by all means the mark of early diagnosis diabetes or the ill risk of prediction diabetes very much, in the hope of for clinical diagnosis foundation being provided.
Summary of the invention
The object of the present invention is to provide the application of DBP (Vitamin D-binding protein:DBP) as the protein molecular marker thing that detects diabetes.
In first aspect of the present invention, the purposes of a kind of DBP (DBP) as the diabetes mark is provided.
In another preference, described DBP is as detecting diabetes or the pathogenetic protein molecular marker thing of prediction glycosuria.
In another preference, described mark is the mark of body fluid.
In another preference, described body fluid is serum.
In another preference, described DBP also comprises its protein fragments, and the amino acid sequence of this protein fragments is that DBP institute is peculiar.
In another preference, the amino acid sequence of the protein fragments of described DBP is like SEQ ID NO:1 or wherein shown in the 2-15 amino acids.
In another aspect of this invention, a kind of separated polypeptide is provided, its amino acid sequence is like SEQ ID NO:1 or wherein shown in the 2-15 amino acids.
In another aspect of this invention, a kind of polynucleotide of separation are provided, its described polypeptide of encoding.
In another aspect of this invention, a kind of purposes of DBP is provided, is used to prepare the reagent that detects diabetes.
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, the purposes of the reagent of a kind of specific recognition DBP or its protein fragments is provided, is used to prepare the kit that detects diabetes or distinguish the diabetes people at highest risk.
In another preference, the reagent of described specific recognition DBP or its protein fragments is antibody.
In another aspect of this invention, the reagent of a kind of specific recognition DBP or its protein fragments is provided, said reagent is polyclonal antibody.
In another aspect of this invention, a kind of kit that detects diabetes is provided, said kit comprises container, and the described reagent that places said container.
In another aspect of this invention, a kind of test-strips (like test paper or test strips) is provided, it comprises solid phase carrier, and is attached to the described reagent on the said solid phase carrier.
Others of the present invention are because the disclosure of this paper is conspicuous to those skilled in the art.
Description of drawings
Fig. 1 has shown sample strip cutting situation.
Fig. 2 shows is the MS/MS spectrogram that is used for detecting the peptide section FPSGTFEQVSQLVK of serum DBP concentration in the mass spectrum multiple-reaction monitoring technology.
Fig. 3 has shown the right RSD situation of this parent ion-daughter ion of 783.9_1077.6 (y9).Horizontal ordinate is represented the RSD value, and ordinate is represented relative ratio.
Fig. 4 has shown the result of DBP (DBP) parent ion-daughter ion to the mass spectrum multiple-reaction monitoring technology of (783.9_1077.6); Wherein, SN is the normal health control group; DM is diabetes B patient group.Ordinate is light peptide divided by the ratio of heavy peptide, promptly this section peptide FPSGTFEQVSQLVK in blood content than on the ratio of standard peptide.Do the t-test check for two groups, the p value is 0.04952＜0.05, and * representes p＜0.05.
Fig. 5 is the MRM ROC curve map as a result of DBP.
Embodiment
The inventor is through extensive studies and screening, finally obtain a kind of for the indication diabetes generation or develop useful mark.Described mark is DBP (DBP) or its protein fragments, and its content in diabetic's body fluid significantly is lower than normal population, therefore can be used for diagnosing diabetes or the ill risk of diabetes.
DBP
The Genebank accession number of DBP is GI:32483410, and the login number of NCBI is: NP_000574, the Swissprot accession number is: P02774 is for IPI number: IPI00555812.4.DBP is one and is present in the body fluid and the multifunctional protein of a lot of cell surfaces that in blood plasma, DBP can combine the vitamin D sterol, can stop the polymerization of actin through combining the actin monomer.DBP also combines with the immunoglobulin (Ig) on bone-marrow-derived lymphocyte surface, and the IgG Fc acceptor of T cell surface combines, and points out it in immunity disease, to work.Have report DBP also with the diabetes disease in glucose tolerance, hypoinsulinemia/hyperinsulinemia, insulin resistance; GDM be correlated with (PATURI V.RAO, GUDIGEGIRISESH, GUNDUPALLE NEELIMA; Et al.Journal of DIABETES CARE; 30,629-637,2007).
The most important member of vitamin D family is ergocalciferol (calciferol) and Vitamin D3 (cholecalciferol); The latter can absorb in the small intestine in photosynthetic synthesizing perhaps of skin; But, and in liver, generate 25-hydroxycholecalciferol by a kind of mixed-function oxidase hydroxylation no matter the cholecalciferol which kind of form obtains all need be transported in the liver through DBP.In vivo; 25-hydroxycholecalciferol is main circulation form, and it is transported in the kidney with after DBP combines; Generate 1 by a kind of mitochondria mixed-function oxidase cytochrome p450 hydroxylation; The 25-dihydroxyvitamin D3, this is the main activity form of vitamin D, and has the 25-hydroxycholecalciferol more than 99% all to combine with DBP in the body.But, at present not with the report of DBP as the diabetes molecular labeling.
DBP
Based on new discovery of the present invention, the peptide section that derives from DBP (polypeptide) is provided, described peptide section is that DBP albumen is peculiar, can be applied to the molecular marked compound as diabetes well.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 2-15 position among the SEQ ID NO:1.The inventor has analyzed the peptide section that derives from DBP in a large number, and is best through identifying this peptide section intensity.The content that detects this peptide section just can directly reflect the content of DBP albumen, and is convenient, fast and accuracy is high.
The polynucleotide of peptide section of described DBP of encoding are also included within the present invention.Because described peptide section is that DBP albumen is peculiar, its corresponding polynucleotide sequence also is distinctive.The method of detection nucleic acid that can be through 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 know.For example, detecting polypeptide can be by means of mass spectrometer etc., maybe can be through methods such as Western Blot or ELISA.Detect nucleic acid and comprise methods such as pcr amplification, Northern Blot.
The purposes of DBP or its protein fragments
The inventor cuts glue-tandem mass spectrum (LTQ-Orbi) technology with tandem mass spectrum analysis of enzymolysis-a scapus and sample in ethanol precipitation classification serum proteins-solution respectively with normal health control group serum and diabetes B patient's blood serum sample (all obtaining from the Shanghai City Sixth People's Hospital diabetes study) and protein is wherein identified and has been compared wherein protein expression level, finds that DBP is lower than the expression in the normal health control serum in diabetic expression of serum level; Through mass spectrum multiple-reaction monitoring technology, confirm further and find that normal health control group and diabetes B patient organize that the expression of DBP there are differences expression in the serum.
Through detecting DBP expression among normal health contrast and the diabetes B patients serum; The inventor find DBP normal health contrast and with the diabetes B patients serum in there are differences expression, DBP significantly is lower than the expression in the normal health control serum in diabetic expression of serum level; Detect through MS/MS, search the storehouse, search protein, buildsummary analyzes, and MRM detects, and quantitatively DBP confirms further and find that the DBP expression of normal health control group, diabetes B patient group there are differences expression.
Therefore, first purpose of the present invention is that a kind of DBP or its protein fragments are provided, and diabetes take place or the application of the pathogenetic protein molecular marker thing of prediction glycosuria as detecting.
Therefore, the invention provides a kind of DBP or its protein fragments purposes as the 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 DBP (DBP), thereby it can combine to be transported to destination organization with vitamin D and relevant blood metabolin.Therefore, the DBP (DBP) in body fluid such as serum, ascites fluid, celiolymph can be used as molecular marked compound.As optimal way of the present invention, DBP detects mark as the serum of diabetes.Serum 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 DBP albumen: (i) carry out the antidiastole and/or the neurological susceptibility analysis of diabetes; The (ii) relevant crowd's of assessment Remedies for diabetes, curative effect of medication, prognosis, and select suitable methods of treatment; The sick risk of the relevant crowd's patient of diabetes of (iii) early stage assessment, the early monitoring early prevention.
It can also be used to prepare the reagent of specific recognition DBP, thereby whether and amount the existence that is used to detect DBP, as the basis for estimation of diabetes.
The reagent and the kit of identification DBP
Based on new discovery of the present invention, the present invention also provides the reagent of specific recognition DBP or its protein fragments.The reagent of any DBP of identification or its protein fragments all comprises in the present invention, as the mark that detects diabetes.The reagent of described specific recognition DBP or its protein fragments for example is antibody or the part that specificity combines DBP.
The present invention also provides the purposes of the reagent of a kind of specific recognition DBP or its protein fragments, is used to detect diabetes or diabetes people at highest risk.
As one embodiment of the present invention, described reagent is the antibody of anti-DBP; It more particularly for example is polyclonal antibody.
Antibody of the present invention can prepare through 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 the enhance immunity reaction, includes but not limited to Freund etc.
Antibody of the present invention also can be monoclonal antibody.This type of monoclonal antibody can utilize hybridoma technology to prepare that (see people such as Kohler, Nature 256; 495,1975; People such as Kohler, Eur.J.Immunol.6:511,1976; People such as Kohler, Eur.J.Immunol.6:292,1976; People such as Hammerling, InMonoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981).
Described antibody can be used in the immunohistochemistry technology, detects the DBP level in the sample, thereby is used for diagnosing diabetes or judges ill risk (neurological susceptibility).
Utilize described antibody; Can detect the level of DBP in the body fluid, thereby can be used for detecting diabetes, can be used for predicting the generation of early diabetes; Perhaps be used to prepare the preparation that detects diabetes or kit etc., be used to prepare preparation or kit of the generation of predicting early diabetes etc.
The present invention also provides the kit that is used for diagnosing diabetes, and this kit comprises: the reagent of specific recognition DBP 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 for example: SA, 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.Be used to detect the generation of diabetes or prediction early diabetes.Elisa technique and be conspicuous for a person skilled in the art based on this technological detectable.
More specifically, the reagent of described specific recognition DBP 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 the mark that DBP or its protein fragments can be used as diagnosing diabetes first.
(2) prepared the reagent of specific recognition DBP or its protein fragments first, said 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 to the present invention is described and be not used in the restriction scope of the present 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.
Only if definition separately, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.In addition, any with the institute similar content of putting down in writing or the equalization method and material all can be applicable among the present invention.The usefulness that preferable implementation method described in the literary composition and material only present a demonstration.
Experimental waters all in the present embodiment are all from Milli-Q (Millipore, Bedford, MA, USA) ultrapure water equipment; (Hercules, CA USA) buy from Bio-Rad company for dithiothreitol (DTT), SDS, urea, 3-[(3-courage amido propyl)-diethyl]-third sulfo group (CHAPS), Tris, ammonium bicarbonate (NH4HCO3) and iodoacetamide (IAA); Trypsase (Trypsin) available from Promega company (Madison, WI, USA); Formic acid (formic acid, FA), trifluoroacetic acid (trifluoroacetic acid, TFA) and acetonitrile (Acetonitrile) available from Aldrich company (MilWaukee, WI, USA); Phosphoric acid is available from Shanghai chemical reagents corporation.Except that acetonitrile was chromatographically pure, it is pure that remaining chemical reagent is analysis.
Normal health control serum and diabetes B patient serum sample are provided by Shanghai Communications University's attached Shanghai City Sixth People's Hospital diabetes study.
In 50 μ L blood serum samples, add 250 μ L solution (pH 7.4 for 100mM NaCl, 10mM HEPES), with 0.22 μ m filter membrane centrifugal removal lipid under the rotating speed of 4 ℃ of 10000g; The blood serum sample of getting after the 260 μ L grease removal adds 182 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 deposition part is low abundance components.Two components are all carried out low-temperature freeze-dry, respectively add 200 μ L 2D lysates then and dissolve.Get each 100 μ L of high abundance and low abundance components, add 1 μ L 1M DTT, mixing is placed 2.5h for 37 ℃, be cooled to room temperature after, add 5 μ L 1M 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.With the ultra filtration membrane of every pipe solution with 3K, the 12000g centrifugal ultrafiltration is removed salt and other micromolecular compounds in the solution then, and making solvent exchange is the ammonium bicarbonate soln of 100mM.Accomplish after the above-mentioned all operations; Add the Trypsin of 20 μ g respectively to two components, 37 ℃ of water enzyme digestion 24h use the ultrafiltration of Millipore 10K ultra filtration membrane again; Remove enzyme and not by the protein of enzymolysis; Collect filtrating, freeze-drying, every effective 60 μ L, 0.1% aqueous formic acid redissolves, and gets 1/3 sample and carries out mass spectrophotometry.
One scapus tandem mass spectrum (IMDL-MS/MS) comprises that an automatic sampler, a high pressure mixing pump are used for the Bi-phase integral post that two-dimensional liquid chromatography separates with one.The Bi-phase integral post is divided into two sections: preceding half section is strong cat ion exchange column, and the second half section is C 18 reversed-phase columns (300 μ m ID; 50mm length, Column Technology Inc, USA).High performance liquid chromatography solution comprises A:0.1% formic acid; B:0.1% formic acid, 100% acetonitrile.Peptide section potpourri behind the enzymolysis enters into a scapus through automatic sampler with break-even pattern; Peptide section potpourri elder generation is through the first half (being the strong cat ion exchange column part) of a scapus; Most of peptide section is bonded on the strong cat ion exchange column; Reserve part is not flushed on the C18 reversed-phase column of latter half, and the B solution with 2-40% washes 120min then, and carries out Mass Spectrometer Method.In automatic sampler; With different pH gradients and free of losses sample introduction pattern 100 μ L pH solution sample introductions in a scapus; Promptly be eluted to latter half (C18 reversed-phase column) to a part of peptide section potpourri from the first half (strong cat ion exchange column part) of a scapus; The B solution flushing 120min of 2-40%, and carry out Mass Spectrometer Method; Repeat said process totally 11 times with different pH solution.Mass spectrum condition in whole process is: the scanning of the mass spectrum condition enactment is the MS/MS scanning that preceding 10 tops in the full scan are carried out in full scan (full scan) back of a 400-2000M/z; 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 90s.
With method for preparing 10 routine normal health control serums and 17 routine diabetes B patient serum sample.Diabetes B patient's clinical indices sees table 1 (sex " 1 " the expression male sex for details; " 2 " expression women; As follows), the clinical indices of normal serum sees table 2 for details.The clinical indices of measuring is respectively: and triglyceride (TG, mmol/L), T-CHOL (TC, mmol/L); HDL (HDL, mmol/L), low-density lipoprotein (LDL, mmol/L); Fasting blood-glucose (FPG, mmol/L), 2 hours after the meal blood sugar (PG2H, mmol/L).
Table 1,17 routine diabetes B patients' clinical data
| Sex | Age | FPG | PG2H | TG | TC | HDL | | BMI | |
| DM1 | |||||||||
| 1 | 69 | 9.73 | 19.14 | 0.64 | 4.30 | 2.11 | 1.82 | 20.40 | |
| |
1 | 67 | 7 | 7.50 | 2.34 | 5.93 | 1.36 | 3.58 | 25.70 |
| |
2 | 69 | 7.2 | 16.8 | 4.52 | 8.03 | 1.76 | 5.15 | 24.77 |
| |
1 | 69 | 9.84 | 18.34 | 1.87 | 5.20 | 1.06 | 3.11 | 23.05 |
| |
1 | 65 | 14.71 | 16.78 | 1.71 | 6.20 | 1.58 | 4.12 | 28.20 |
| |
1 | 68 | 8.50 | 14.60 | 1.45 | 6.20 | 1.38 | 4.03 | 22.50 |
| |
1 | 68 | 7.1 | 16 | 2.32 | 5.7 | 1.59 | 4.14 | 25.1 |
| |
1 | 67 | 9.44 | 14.90 | 0.71 | 3.70 | 0.98 | 2.64 | |
| DM9 | |||||||||
| 1 | 66 | 8.09 | 9.10 | 1.10 | 4 | 1.01 | 2.81 | | |
| DM10 | |||||||||
| 1 | 67 | 9.70 | 17.80 | 1.54 | 3.72 | 1.24 | 2.56 | 25.20 | |
| |
1 | 67 | 9.5 | 9 | 1.69 | 4.13 | 1.34 | 2.77 | 25.6 |
| |
1 | 69 | 7.10 | 15.30 | 0.85 | 3.91 | 1.36 | 2.84 | 25.50 |
| |
1 | 67 | 7.40 | 17.50 | 1.53 | 4.86 | 1.34 | 3.55 | 25.70 |
| |
1 | 66 | 7.98 | 11.67 | 0.86 | 5.60 | 2.30 | 2.53 | 23.90 |
| |
1 | 68 | 6.7 | 13.6 | 1.51 | 4.29 | 1.58 | 2.98 | 33.95 |
| |
1 | 68 | 6.10 | 11.60 | 3.02 | 5.84 | 1.01 | 4.87 | 26.85 |
| |
1 | 65 | 5 | 11.40 | 4.98 | 5.27 | 1.02 | 3.30 | 26.08 |
The clinical data of table 2,10 routine normal health control serums
| Sex | Age | FPG | PG2H | TG | TC | HDL | | BMI | |
| SN1 | |||||||||
| 1 | 70 | 4.80 | 6 | 0.55 | 3.64 | 1.08 | 2.56 | 22.46 | |
| |
2 | 66 | 4.20 | 5.60 | 1.05 | 3.81 | 1.16 | 2.71 | 19.74 |
| |
2 | 66 | 4.60 | 5.10 | 1.22 | 4.66 | 1.51 | 3.09 | 22.06 |
| |
2 | 70 | 4.70 | 4 | 0.99 | 4.35 | 1.24 | 3.01 | 18.86 |
| |
1 | 69 | 4.40 | 7.70 | 0.96 | 4.01 | 1.26 | 2.77 | 22.92 |
| |
1 | 69 | 5.1 | 6 | 1 | 4.62 | 1.35 | 3.22 | 22.41 |
| |
1 | 65 | 4.9 | 2.5 | 1.02 | 3.1 | 1.09 | 1.91 | 20.83 |
| |
1 | 69 | 4.8 | 6.1 | 1.35 | 4.97 | 1.55 | 3.5 | 21.58 |
| |
1 | 68 | 4.5 | 5.4 | 1.53 | 5.4 | 1.56 | 4.17 | 22.6 |
| |
1 | 67 | 4.8 | 3.9 | 0.88 | 3.66 | 1.07 | 2.34 | 20.94 |
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; The kapillary reversed-phase liquid chromatography is available from Agilent company.
The people source L02 cell that uses in the present embodiment is available from Chinese Academy of Sciences's cell bank.
(FPG, mmol/L) (PG2H mmol/L) was divided into 4 groups with 2 hours after the meal blood sugar with sample evidence physical signs fasting blood-glucose among the embodiment 1.First group is that (FPG is mmol/L) with 2 hours after the meal blood sugar (PG2H, mmol/L) index all normal (FPG＜7, PG2H＜11) for fasting blood-glucose; Second group of FPG＞7, PG2H＞11; The 3rd group of FPG＞7, PG2H＜11; The 4th group of FPG＜7, PG2H＞11.
Table 3, packet
| |
SN6 | | SN8 |
| Group | |||
| 2 | DM10 | | DM13 |
| Group | |||
| 3 | DM2 | | DM11 |
| Group | |||
| 4 | DM15 | DM16 | DM17 |
Each one-tenth is obtained 2ul (about 200ug) in the group, blendes together an integral body (about 600ug), adds the 600ug secretory protein as one group.Secretory protein is from people source L02 cell, and the preparation method is: people source L02 cell under normal condition, i.e. 10%CO
2, the DMEM nutrient culture media, 10%FBS cultivates, and treats that cell grows to about 90% and expires, and the hungry cultivation of nutrient culture media of no FBS is used in the PBS flushing instead, stimulates and secretes.After 24 hours, microscopy is collected nutrient culture media, the 0.22um membrane filtration, and the nutrient culture media after the filtration adds PMSF, NaVO
4, NaF proteinase inhibitors of phosphatases, centrifugal removal cell fragment, the ultrafiltration and concentration sample adds cocktail (1:25) ,-80 ℃ of preservations obtain secreted albumen.Every group adds 1 μ L 1M DTT respectively, to be cooled to room temperature in 56 ℃ of placement 30min, adds 5 μ L 1M IAA, places 40min in the room temperature lucifuge.The heavily mark of secretory protein: the powder with K8R10 in the time of the configuration nutrient solution joins in the nutrient culture media; Make that the KR in the nutrient culture media is K8R10; Cell is grown in this nutrient culture media, cultivates (like 5 generations) after some generations through going down to posterity and thinks that promptly protein in the last cell all is (in the albumen on the K mark 8 of K8R10 marks
13C, on the R mark 10
13C), promptly K8R10 heavily marks (as confidential reference items).
The plasma sample of above-mentioned processing is separated, behind the coomassie brilliant blue staining, according to the stripe information on the glue, each sample is cut into 24 bands (glue is divided into 24 equal portions through one dimension gel electrophoresis (7.5-17.5% gradient glue); The cutting distribution situation is seen Fig. 1), after decolouring, the freeze-drying, (it is indefinite that enzyme is cut back peptide segment length to add trypsase; Can by Mass Spectrometer Method arrive usually at 8-25 amino acid); After 37 ℃ of enzymolysis spend the night, carry out extracting in the glue, with the peptide section that obtains; After cryogenic freezing becomes dry powder, be stored in-80 ℃ of refrigerators.
With the peptide section dry powder of above-mentioned acquisition with 0.1% formic acid dissolving after, identify through the LC-Orbi-MS/MS mass spectrum.The scanning of the mass spectrum condition enactment is the full scan (full scan) of a 400-2000; The MS/MS scanning at preceding 10 tops in the full scan is carried out in the back; 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 90s.
The raw data that mass spectrum among above-mentioned two embodiment is obtained is with IPI human 3.07 checking storehouses; Be buildsummary; Light peptide is used the non-marked Policy Filtering, and promptly the parameter of peptide FDR＜=0.5% and unipeptides＞=2 identifies 1010 protein altogether; Identify 54 in the anti-storehouse, protein FDR is 5.35%; What the heavy peptide of light peptide all identified screens with labelling strategies, i.e. peptide FDR＜=1% and hits＞2 (or unihits＞=2, hits=2) strategy; Identify 1715 protein altogether, identify 41 in the anti-storehouse, protein FDR is 2.39%; Both merge, and remove redundancy, identify 2074 protein altogether; Identify 94 in the anti-storehouse, protein FDR is 4.53%.
What table 4 and table 5 showed is peptide section (being that DBP is distinctive) the evaluation number of times of the DBP (DBP) of diabetes B patients serum and normal health control serum; Show that DBP (DBP) protein content in the diabetes B patients serum significantly is lower than the normal person, wherein the peptide section of two tables all is enzymolysis DBP (DBP) and the peptide section sequence that obtains.Table 6 has been showed the evaluation situation of DBP (DBP) in 4 groups that table 3 divided.
Fig. 2 has shown the MS/MS spectrogram of the peptide section FPSGTFEQVSQLVK that is used for detecting serum DBP concentration in the mass spectrum multiple-reaction monitoring technology.
The peptide hop count that DBP (DBP) mass spectrum is identified among table 4, the 17 routine diabetes B patients serums
The peptide hop count that DBP (DBP) mass spectrum is identified in table 5, the 10 routine normal health control serums
Table 6
More than among the table 4-6, ". " represented tryptic restriction enzyme site; The peptide bond that the carboxyl of the identification of trypsase selectivity and cutting K and R is participated in formation.Enzyme is cut the back and is obtained the peptide section between two ". ".
Above form shows: DBP (DBP) protein content in the diabetes B patients serum significantly is lower than the normal person, through T-test check P value=0.000239.This has the protein of significant difference to have found DBP (DBP) through current embodiment, and the back is verified with MRM.
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; The kapillary reversed-phase liquid chromatography is available from Agilent company.The conventional method synthetic standards is heavily marked peptide section FPSGTFEQVSQLV*K (5
13C-Val) (the heavy target amino acid of " * " expression).Plasma proteins sample preparation enzymolysis process is the same, and per 3 μ L stostes add 1 μ L 1M DTT, and is to be cooled to room temperature in 56 ℃ of placement 30min, adds 5 μ L 1M IAA, places 40min in the room temperature lucifuge.
Get the plasma proteins enzymolysis product (equal-volume) and 10ng standard weight mark peptide section of the former blood plasma of corresponding 0.2 μ L, mix after automatic sampler joins on the reversed-phase column with break-even pattern.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 acetonitrile solution (B liquid).To shunt preceding 130 μ L/min, shunt the flow velocity of back 400nl/min, at the B liquid of 0-15min with 2%-35%, 15-25min carries out gradient elution with the B liquid of 35-95% (volume ratio) to reversed-phase column.The sample of wash-out gets into TSQ VANTAGE (Thermo scientific) and detects through receiving esi ion source from the reversed-phase column.TSQ VANTAGE condition enactment is spray voltage 2000V; Collision gas argon gas, collision atmospheric pressure 1.5mTorr; Q2 impact energy 36V; Q1 half-peak breadth resolution 0.2Da, Q3 half-peak breadth resolution 0.7Da; Residence time 20ms.Choose 783.9_574.4 (y5); 783.9_673.4 (y6); 783.9_930.5 (y8), 783.9_1077.6 (y9), 783.9_1322.7 (y12) totally 5 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 (783.9_1077.6) quantitative information as this peptide section, carry out the follow-up data analysis, this parent ion-daughter ion distributes to the RSD of (783.9_1077.6) and sees Fig. 3 in all samples.Table 7 has shown DBP (DBP) MRM mass spectrum verification msg in 4 routine normal health control serums and the 14 routine diabetes B serum; Ratio-1, ratio-2 represent twice experiment repetition, and data are the ratio of light peptide divided by heavy peptide in the form; Promptly this section peptide in blood content than on the value of standard peptide; On average shown mean value, SD is a standard variance, and RSD is a relative standard deviation.Table 8 shown DBP (DBP) MRM mass spectrum in 4 routine normal health control serums and the 14 routine diabetes B serum verify different parent ion-daughter ions between relative variance, 100% data RSD＜0.15 wherein.
Table 7
Table 8
(FPSGTFEQVSQLV*K (5 as this peptide section to the quantitative information of (783.9_1077.6) to get parent ion-daughter ion
13C-Val)) quantitative information carries out the follow-up data analysis.Parent ion-daughter ion is to data＜0.05 of the RSD relative standard deviation 77.8% between (783.9_1077.6) two repetitions; 100% data＜0.15; Normal person and this parent ion-daughter ion of diabetes patient are done the t-test check to (783.9_1077.6) gained ratio, and the p value is 0.04952, less than 0.05; Verified that DBP (DBP) protein content in the diabetes B patients serum significantly is lower than the normal person, sees Fig. 4.
Do the ROC curve with MRM result, the result is as shown in Figure 5, according to this figure, and TG-AUC: 0.875, DBP cut point=0.029166, sensitivity=0.786, false positive rate=0.25, specificity=0.75.
In sum; DBP (DBP) significantly reduces in the diabetes B patients serum; Incidence and development obvious and diabetes B has substantial connection, so this albumen detects the prediction that can be used for the diabetes incidence and development as a molecular marked compound to its expression.
The inventor is injected into total length DBP albumen (GenBank accession number GI:32483410) in the rabbit body, obtains immune serum.Concrete preparation method is following:
DBP albumen is expelled to rabbit with after Freund mixes with 1: 1 according to volume ratio, and immunity amount 0.5mg is every at a distance from 2 week immunity 1 time, immune 3 times.Behind conventional affinity purification, obtain rabbit polyclonal antibody to DBP albumen.
Immunoblot experiment shows: the rabbit polyclonal antibody of acquisition is only discerned DBP albumen, and basic other albumen of nonrecognition can be used as the detectable that detects diabetes or the ill risk of diabetes.
The polyclonal antibody that is obtained, two anti-(routines), chromogenic reagent are placed different containers respectively, container is placed box with operation instructions, obtain detection kit.
All documents in that the present invention mentions are all quoted as a reference in this application, are just quoted such as a reference separately as each piece document.Should be understood that in addition after having read above-mentioned teachings of the present invention, those skilled in the art can do various changes or modification to the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (10)
1. a DBP is as the purposes of diabetes mark.
2. purposes as claimed in claim 1 is characterized in that described DBP also comprises its protein fragments, and the amino acid sequence of this protein fragments is that DBP institute is peculiar.
3. purposes as claimed in claim 2 is characterized in that, the amino acid sequence of the protein fragments of described DBP is like SEQ ID NO:1 or wherein shown in the 2-15 amino acids.
4. a separated polypeptide is characterized in that, its amino acid sequence is like SEQ ID NO:1 or wherein shown in the 2-15 amino acids.
5. the polynucleotide of a separation is characterized in that, the described polypeptide of its coding claim 4.
6. the purposes of a DBP is used to prepare the reagent that detects diabetes.
7. the purposes of the reagent of a specific recognition DBP or its protein fragments is used to prepare the kit that detects diabetes or distinguish the diabetes people at highest risk.
8. the reagent of a specific recognition DBP or its protein fragments, said reagent is polyclonal antibody.
9. kit that detects diabetes, said kit comprises container, and the described reagent of claim 8 that places said container.
10. test-strips, it comprises solid phase carrier, and is attached to the described reagent of claim 8 on the said solid phase carrier.
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| CN103376326A (en) * | 2012-04-25 | 2013-10-30 | 中国科学院上海生命科学研究院 | Application of vitamin D binding protein as obesity-diabetes marker |
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| WO2014121715A1 (en) * | 2013-02-05 | 2014-08-14 | 北京九强生物技术股份有限公司 | 25-hydroxyl vitamin d detection kit and preparation method therefor |
| CN105393117A (en) * | 2013-01-31 | 2016-03-09 | 凯普里昂蛋白质组学公司 | Type 2 diabetes biomarkers and uses thereof |
| CN106220710A (en) * | 2016-08-04 | 2016-12-14 | 广州军区广州总医院 | A kind of vitamin D binding peptide and application thereof |
| CN106243192A (en) * | 2016-08-04 | 2016-12-21 | 广州军区广州总医院 | Vitamin d binding peptide and application thereof |
| CN112773889A (en) * | 2020-12-15 | 2021-05-11 | 重庆医科大学附属永川医院 | VDBP/VSIG 4-based immune negative regulation multivalent vaccine and preparation method thereof |
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| CN103376326A (en) * | 2012-04-25 | 2013-10-30 | 中国科学院上海生命科学研究院 | Application of vitamin D binding protein as obesity-diabetes marker |
| CN103376324A (en) * | 2012-04-25 | 2013-10-30 | 中国科学院上海生命科学研究院 | Application of albumin as obesity-diabetes marker |
| CN105393117A (en) * | 2013-01-31 | 2016-03-09 | 凯普里昂蛋白质组学公司 | Type 2 diabetes biomarkers and uses thereof |
| WO2014121715A1 (en) * | 2013-02-05 | 2014-08-14 | 北京九强生物技术股份有限公司 | 25-hydroxyl vitamin d detection kit and preparation method therefor |
| CN106220710A (en) * | 2016-08-04 | 2016-12-14 | 广州军区广州总医院 | A kind of vitamin D binding peptide and application thereof |
| CN106243192A (en) * | 2016-08-04 | 2016-12-21 | 广州军区广州总医院 | Vitamin d binding peptide and application thereof |
| CN112773889A (en) * | 2020-12-15 | 2021-05-11 | 重庆医科大学附属永川医院 | VDBP/VSIG 4-based immune negative regulation multivalent vaccine and preparation method thereof |
| CN115060815A (en) * | 2022-05-30 | 2022-09-16 | 广西大学 | Solid phase extraction-liquid chromatography-ion trap/time-of-flight mass spectrometry combined detection method for fluoroamidone metabolites in human urine |
| CN115060815B (en) * | 2022-05-30 | 2023-06-16 | 广西大学 | Solid phase extraction-liquid chromatography-ion trap/time-of-flight mass spectrometry combined detection method for flumidone metabolites in human urine |
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