CN105510592A - Protein marker of obstructive nephropathy - Google Patents
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Abstract
The invention relates to a protein marker of obstructive nephropathy, specifically to an application of human obstructive nephropathy urine differential protein obtained by unilateral ureteral ligation model in rats and mass spectrometry as a potential marker. A simpler, faster and noninvasive method and selection can be provided for detection in early diagnosis and therapeutic process of obstructive nephropathy and prognosis evaluation. Specifically, the invention relates to an application of a combination part capable of being selectively combined to one or more types of proteins in the preparation of a reagent for diagnosis of human obstructive nephropathy.
Description
Technical field
The present invention relates to the protein marker that people's obstructive nephropathy is relevant.Specifically, the present invention relates to utilization but the purposes of people's obstructive nephropathy of obtaining of side Unilateral Ureteral Obstruction Nephrotic Syndrome in Rats and mass spectrophotometry protein marker relevant to disease severity.
Background technology
The meaning of Urinary proteomics in kidney trouble research:
Urine is the important sources of biomarker, more easily change relative to blood urine, and these changes can be comparatively sensitive the change of reflection physical function, that is, see YouheG.Canurinebethegoldmineforbiomarkerdiscovery? SciChinaLifeSci2013; 43.Urine as the principal output of kidney, and can be easy to obtain in large quantities, non-invasively, is often called as external Renal biospy, has important diagnostic significance clinically.The changes of function concealment of urinary system, not easily discover, current diagnosis method depends on biopsy, make early diagnosis, treatment and cure rate raising very difficult.Urine often can in the function to a certain degree showing urinary system, and utilize urine variation diagnostic kidney trouble also have relatively convenient, simple, without wound, the advantage such as quick.Urinary proteome forms jointly primarily of the plasma proteins of glomerular filtration and the protein of renal tubule, concetrated pipe and the urinary tract secretion, so the change of kidney and urinary tract function can reflect by urinary proteome, its hypersensitivity also makes it the important method becoming the biomarker finding urinary injuries.
Obstructive nephropathy is chronic renal disease one of the main reasons, and be cause the most important reason Trnka of the whole renal damage in latter stage of children, P., etal., Urinarybiomarkersinobstructivenephropathy.ClinJAmSocNeph rol, 2012.7 (10): p.1567-75.In kidney maturation, ureteral obstruction can cause kidney development bad, and a series of Renal tissues damage may be caused at ripe kidney ureteral obstruction, main pathological manifestations is renal tubule atrophy, kidney region fibrosis and interstitial cell infiltration Bascands, J.L.andJ.P.Schanstra, Obstructivenephropathy:insightsfromgeneticallyengineered animals.KidneyInt, 2005.68 (3): p.925-37.This end-stage disease can cause the serious kidney region fibrosis of degree to have a strong impact on renal function, even causes end stagerenaldisease to threaten the health and lives of people.
Unilateral ostruction (UUO) rat model can simulate the pathogenetic process of obstructive nephropathy disease, and can well Control release time and the injury of kidney development order of severity.This model causes injury of proximal cells mainly through ligation Induced by Unilateral Ureteral Obstruction, induction Epithelial and stromal becomes, and then cause renal interstitial cell infiltration, tubular ectasia and apoptosis, the increasing of fibroblast and myofibroblast, extrtacellular matrix deposition, finally cause renal tubule atrophy Klahr, S.andJ.Morrissey, Obstructivenephropathyandrenalfibrosis.AmJPhysiolRenalPh ysiol, 2002.283 (5): p.F861-75.The pathological lesion that this model can cause these different fast, and there is good repeatability.Utilize this animal model to simulate the process of obstructive nephropathy, observing kidney from normal, Minimal change to there is the overall variation that serious pathological damages, having important directive significance to obstructive nephropathy early diagnosis clinically, treatment and prognosis.
Summary of the invention
Therefore, technical purpose of the present invention is the Urine proteins mark utilizing Rats Undergoing Unilateral Urethral Ligation nephropathy model relevant with people's obstructive nephropathy state of an illness that mass spectrophotometry obtains, and detects experimenter's health status, the monitoring obstructive nephropathy order of severity and treatment process to utilize it and/or assesses this kidney trouble prognosis.
In other words, this research conjugated protein omics technology and animal model, the biomarker that the research obstructive nephropathy state of an illness is relevant, for the early detection of obstructive nephropathy, the monitoring in therapeutic process, and prognostic evaluation.Present invention finds a series of protein biomarker for detection and prognosis evaluation in obstructive nephropathy early diagnosis, therapeutic process.
The present invention is in order to search out the relevant protein marker of the obstructive coincident with severity degree of condition of people, utilize unilateral ostruction animal model, animal used as test urine is obtained in progression of disease process, utilize mass-spectrometric technique to identify the Urine proteins group in disease generating process, obtain and obstructive nephropathy coincident with severity degree of condition associated protein.To obtained proteome data through strict bioinformatic analysis, acquisition can reflect the different lesions stage of obstructive nephropathy, the protein biomarker for detection and prognosis evaluation in this disease early diagnosis, therapeutic process.
The Urine proteins mark that discovery of the present invention is relevant to people's obstructive nephropathy coincident with severity degree of condition, totally 73, the albumen be not wherein in the news.On the one hand, the invention provides the purposes of bound fraction in the reagent for the preparation of diagnosis people obstructive nephropathy that alternative is bonded to one or more albumen, wherein, one or more albumen described are selected from the unilateral ostruction rat model people source protein corresponding with the differential protein that rats in sham-operated group urine protein analysis of spectrum produces." bound fraction " is meant to molecule or the entity of the mRNA that can be bonded to above-mentioned albumen or code for said proteins.
On the other hand, the invention provides the bound fraction binding selectively to one or more albumen, purposes in the reagent of preparation diagnosis people obstructive nephropathy, one or more albumen wherein said are selected from myosin-B, antithrombase 2, superoxide dismutase [Cu-Zn], ubiquitin carboxy terminal hydrolytic enzyme isodynamic enzyme, Induced by Retinoic Acid serine carboxypeptidase, nucleoside diphosphokinase, seralbumin, Galectin-3 associated proteins, ec-sod [Cu-Zn], type i collagen α 1 side chain, α actinin 1, basement membrane gathers candy, bladder chalone C, carboxypeptidase Q, isoprenoid synthetase structure domain albumen, deoxyribonuclease 1, fibrinogen β chain, γ paddy Acyl-hydrolase, 14-3-3 albumen ζ/Δ, cortisol binding globulin, alpha1 Antichymotrypsin, alpha1 Antichymotrypsin, 14-3-3 albumen beta/alpha, calgranulin B, calgranulin A, calcyclin, Cytochrome P450 2E1, dipeptidyl peptidase 2, L lactic dehydrogenase, Cathepsin H, people's epididymal proteins 4, coagulation factors 12, IST1 homolog, retinaldehyde binding protein 4, PGH2-D isomerase, secreting type urine adjusts element, Solute Carrier family 12 member 7, dipeptidyl peptidase 1, inhibitors of neutrophil elastase, β2-microglobulin, angiostatin, Annexin A1, hemopexin, transaldolase, table colloid albumen, apo E, ezrin, proteinase cathepsin L2, the nuclear translocation factor 2, gelsolin, matrix reconstruction associated protein 8, cadherins 1, α actinin 4, alpha2 Macroglobulin, kallikrein 1, haemoglobin alpha hypotype, keratin (I type cytoskeleton 10), gamma globulin 1 heavy chain, gamma globulin 2 heavy chain, gamma globulin 4 heavy chain, serotransferrin, aminopeptidase N, β hexokinase type β hypotype, G-protein coupled receptor C family member 5C, flesh type phosphorylase, flagellum transport protein 172 homolog, plasminogen activator inhibitor, vimentin, lectin, complement component C9, lysozyme C 1, moesin, glycan-phosphatidylinositol specific phospholipase D.
Further, described bound fraction comprises polypeptide or is made up of polypeptide.
Further, described bound fraction comprises antibody or its Fab or variant, or is made up of antibody or its Fab or variant.
We comprise the bound fraction being bonded to described albumen relative to other polypeptide more strongly at definition " selective binding "; Strong at least 10 times are combined into preferably, preferably strong at least 50 times and even more preferably strong at least 100 times.Preferred combination part is only bonded to described albumen.
Term used herein " polypeptide " is meant to several amino acids and is linked together by peptide bond.Term " peptide " can exchange with term " polypeptide " and use, but peptide can be made up of two or more polypeptide.
Further, described diagnostic reagent is for detecting experimenter's health status, the monitoring obstructive nephropathy order of severity and treatment process and/or assess disease prognosis.
Particularly, by distinguishing renal damage or the interstitial fibrosis monitoring obstructive nephropathy order of severity.
On the other hand, the invention provides a kind of method, it comprises
A): the urine obtaining experimenter,
B): be separated Urine proteins,
C): the level determining one or more albumen described in described any one of claim 1 to 5 in normal person and patient urine albumen,
D): the expression of wherein said albumen is the indication of the obstructive nephropathy order of severity, treatment process and/or prognosis evaluation.
Further, wherein step (b) uses quantitative mass spectral method, ELISA method, and Western method detects.
On the other hand, the invention provides the purposes of bound fraction in the reagent for the preparation of diagnosis obstructive nephropathy renal damage that alternative is bonded to one or more albumen, wherein, one or more albumen described are selected from antithrombase 2, ubiquitin carboxy terminal hydrolytic enzyme isodynamic enzyme, Induced by Retinoic Acid serine carboxypeptidase, seralbumin, Galectin-3 associated proteins, bladder chalone C, carboxypeptidase Q, deoxyribonuclease 1, fibrinogen β chain, 14-3-3 albumen ζ/Δ, cortisol binding globulin, alpha1 Antichymotrypsin, 14-3-3 albumen beta/alpha, calgranulin B, calcyclin, Cytochrome P450 2E1, dipeptidyl peptidase 2, L lactic dehydrogenase, people's epididymal proteins 4, coagulation factors 12, IST1 homolog, retinaldehyde binding protein 4, PGH2-D isomerase, secreting type urine adjusts element, Solute Carrier family 12 member 7, dipeptidyl peptidase 1, inhibitors of neutrophil elastase, β2-microglobulin, angiostatin, hemopexin, table colloid albumen, apo E, ezrin, proteinase cathepsin L2, the nuclear translocation factor 2, gelsolin, matrix reconstruction associated protein 8, cadherins 1, alpha2 Macroglobulin, kallikrein 1, keratin (I type cytoskeleton 10), serotransferrin, aminopeptidase N, β hexokinase type β hypotype, G-protein coupled receptor C family member 5C, flesh type phosphorylase, flagellum transport protein 172 homolog, plasminogen activator inhibitor, glycan-phosphatidylinositol specific phospholipase D.
On the other hand, the invention provides the purposes of bound fraction in the reagent for the preparation of diagnosis obstructive nephropathy kidney region fibrosis that alternative is bonded to one or more albumen, wherein, one or more albumen described are selected from myosin-B, superoxide dismutase [Cu-Zn], ubiquitin carboxy terminal hydrolytic enzyme isodynamic enzyme, Induced by Retinoic Acid serine carboxypeptidase, nucleoside diphosphokinase, seralbumin, ec-sod [Cu-Zn], type i collagen α 1 side chain, α actinin 1, basement membrane gathers candy, bladder chalone C, carboxypeptidase Q, isoprenoid synthetase structure domain albumen, deoxyribonuclease 1, fibrinogen β chain, γ paddy Acyl-hydrolase, alpha1 Antichymotrypsin, calgranulin B, calgranulin A, dipeptidyl peptidase 2, L lactic dehydrogenase, people's epididymal proteins 4, coagulation factors 12, retinaldehyde binding protein 4, PGH2-D isomerase, secreting type urine adjusts element, Solute Carrier family 12 member 7, inhibitors of neutrophil elastase, angiostatin, Annexin A1, hemopexin, transaldolase, table colloid albumen, ezrin, the nuclear translocation factor 2, gelsolin, matrix reconstruction associated protein 8, cadherins 1, α actinin 4, alpha2 Macroglobulin, kallikrein 1, haemoglobin alpha hypotype, keratin (I type cytoskeleton 10), gamma globulin 1 heavy chain, gamma globulin 2 heavy chain, gamma globulin 4 heavy chain, serotransferrin, flesh type phosphorylase, vimentin, lectin, complement component C9, lysozyme C 1, moesin, the change of glycan-phosphatidylinositol specific phospholipase D expression is the indication of obstructive nephropathy severe renal interstitial fibrosis.
Accompanying drawing explanation
Fig. 1: sham-operation 1,3 weeks groups and unilateral ostruction model 1,3 weeks group renal tissues of rats section HE coloration result, ligation 1 week group occurs that tubular ectasia, fiberization are not obvious, and 3 weeks groups occur that renal tubule atrophy, fibrosis are serious.
Fig. 2 A-Fig. 2 B shows serum creatinine and raises gradually to unilateral ostruction 1 week group, 3 weeks groups from sham-operation group, and Urine proteins/creatinine ratio significantly raises unilateral ostruction 3 weeks group rats.
Fig. 3: in urine, Westernblot verifies mass spectral results, shows: Annexin A1, vimentin increase in ligation for 3 weeks, α-actinine, moesin and lectin increases in ligation for 1 week, within 3 weeks, significantly increases
Fig. 4: in tissue, Westernblot verifies mass spectral results: Annexin A1, vimentin and moesin increase in ligation for 3 weeks, and α-actinine and lectin increase in ligation, significantly increase for 3 weeks for 1 week.
Fig. 5 A-Fig. 5 D: immunohistochemistry the result, display: in tissue, Annexin A1 (Fig. 5 A), vimentin (Fig. 5 B), α-actinine (Fig. 5 C) and moesin (Fig. 5 D) increase in ligation for 1 week, significantly increase for 3 weeks.
Embodiment
To further illustrate the present invention by following non-limiting example below, as well known to those skilled in the art, without departing from the spirit of the invention, can make many amendments to the present invention, such amendment also falls into scope of the present invention.
Following experimental technique if no special instructions, is conventional method, and the experiment material used if no special instructions, all can easily obtain from commercial company.
Embodiment
Embodiment 1
Material and reagent
1) instrument: rat urine collection tube: purchased from BD company.TripleTOF5600 mass spectrometer: purchased from ABI company; Agilent1200 high performance liquid chromatograph: purchased from Agilent company; MillliQRG ultrapure water system: purchased from Millipore company; C18 inverse analysis post (RP post, 0.1 × 150mm, 3 μm,
): purchased from MichromBioresources company.
2) main agents: deionized water derives from MillliQRG ultrapure water system; Chromatographic grade acetonitrile, formic acid and methyl alcohol are that Fisher company produces; Iodacetyl ammonium (IAA), ammonium bicarbonate, dithiothreitol (DTT) (DTT) are bought from Merck company; Order-checking level pancreatin is bought from Sigma company; Annexin A1, vimentin and moesin antibody are all from Abcam company, and α actinin and lectin antibody are all from ABGENT company.
3) animal: male SD rat (body weight 180 ~ 200g), purchased from Beijing medical experiment Institute of Botany, is raised in feeding standard environment.
Experimental technique
1) Rats Undergoing Unilateral Urethral Ligation model is set up and sample collection.
A) rats in sham-operated group model establishment step: after belly sterilization, median abdominal incision, is separated left side ureter and is left intact, close abdomen, give injection of antibiotic.
B) Rats Undergoing Unilateral Urethral Ligation model establishment step: after belly sterilization, median abdominal incision, is separated left side ureter and carries out binode bundle at its nearly bladder end, and cut in the middle of ligation, close abdomen, give injection of antibiotic.
C) sample collection flow process: 1 week after modeling and collect urine and the left kidney of sham-operation group and ligation group rat for three weeks respectively, the collection method of urine is that the kidney end ureter residual in left side carries out intra-ureteral cannula, after collecting intubate, 0.5 hr urine discards, then after collecting, the urine of 2.5 hours carries out follow-up study.After collecting left kidney, kidney is divided into halves by crown cutting, and a liquid nitrogen is freezing is placed on-80 DEG C of Refrigerator stores, and second half is placed in formalin and is fixed.
2) sample preparation:
A) Urine proteins extracts and preserves: sham-operation 1,3 weeks groups and ligation organize urine for 1,3 weeks in centrifugal 20 minutes of 4 DEG C of 2000g, get supernatant, are placed in new EP pipe, continue 12000g4 DEG C centrifugal 20 minutes; Get supernatant ,-80 DEG C of preservations.
B) Urine proteins carries out enzyme on film and cuts, see WisniewskiJR, ZougmanA, NagarajN, MannM.Universalsamplepreparationmethodforproteomeanalysi s.Naturemethods2009; 6:359-62.Reverse-phase chromatographic column is separated, Mass Spectrometric Identification.
3) reversed-phase high-performance liquid chromatography separate series Mass Spectrometric Identification:
A) chromatographic process: enzyme cuts rear peptide section adds Agilent company anti-phase Trap post through Agilent1200 high performance liquid chromatography autopipette, is changed by six-way valve, carry out RP chromatographic column (0.1 × 150mm, MagicC18,5 μm,
michromBioresources, Auburn, the U.S.) be separated.Elution time 120 minutes, column flow rate is 0.5 μ l/min.RP post gradient is that (mobile phase A is 5 ~ 40% Mobile phase B: 0.1% formic acid+2% acetonitrile+97.9% water; Mobile phase B is: 0.1% formic acid+89.9% acetonitrile+10% water).
B) mass spectrometry method: the polypeptide application TripleTOF5600 mass spectrum eluted from reversed-phase column is identified.
4) database retrieval:
All mass spectral results mascot software carries out database retrieval.Database used is Swissprot_2012_07.Search condition is: pancreatin enzyme is cut; 2 leakages have been allowed to cut site; The fixing modification of halfcystine+57Da; Variable modification: deamidating (NQ), Oxidation (M).Tripletof5600 data mass spectrometric data retrieval allowable error is: parent ion 0.05Da, daughter ion 0.05Da.
5) relative quantitative assay result:
All mass spectral results carry out relative quantification interpretation of result by elementary LC-MS/MS software.Software operation uses according to method shown in document, see HauckSM, DietterJ, KramerRL, etal.Decipheringmembrane-associatedmolecularprocessesint argettissueofautoimmuneuveitisbylabel-freequantitativema ssspectrometry.Molecular & cellularproteomics:MCP2010; 9:2292-305.
6) Westernblot checking
By sham-operation 1,3 weeks groups and ligation 1,3 weeks group rat urine albumen 30ug, histone 40ug, 10%SDS-PAGE is separated, at transferring film liquid (10%methanol, 25mMTrisbase, 192mMglycine, PH8.0) in by albumen transferring film on pvdf membrane, pvdf membrane room temperature in 5% skimmed milk power closes 1h, primary antibodie overnight incubation, primary antibodie is respectively Annexin A1 (1:1000), vimentin (1:1000), α actinin 1 (1:1000), moesin (1:1000), lectin (1:1000); Develop the color with two anti-(peroxidase-conjugated IgG, 1:10000) incubated at room 2h, ECL after washing film 30min.
7) SABC checking
Cut into slices after the nephridial tissue that sham-operation 1,3 weeks groups and ligation 1,3 weeks group rat fix is carried out paraffin embedding, dewaxing is to water, carry out Triton after PBS cleaning to punch 5 minutes, 5%BSA closes 20 minutes, dropping primary antibodie is spent the night, drip two after PBS cleaning to resist, DAB colour developing and haematoxylin are redyed, and brown area is positive colour developing.
Experimental result
1) derive from sham-operation group, ligation 1,3 weeks group the urine of three rats through Mass Spectrometric Identification, carrying out 3 technology respectively and repeat, is 501 based on the albumen more than two peptides jointly identified.Compared with sham-operation group, the rat differential protein in ligation 1 week group urine is 95, ligation three weeks differential proteins organized in urine are 96.Through ANOVA statistical analysis and the screening of change multiple, reliable differential protein does the homology comparison of people's albumen, present invention obtains 75 people's unilateral ostruction rat model urine albumen.
2) unilateral ostruction model and sham-operation rat model Urine proteins are compared, through ANOVA statistical analysis and the screening of change multiple, reliable differential protein people homology have 73, be respectively: myosin-B, antithrombase 2, superoxide dismutase [Cu-Zn], ubiquitin carboxy terminal hydrolytic enzyme isodynamic enzyme, Induced by Retinoic Acid serine carboxypeptidase, nucleoside diphosphokinase, seralbumin, Galectin-3 associated proteins, ec-sod [Cu-Zn], type i collagen α 1 side chain, α actinin 1, basement membrane gathers candy, bladder chalone C, carboxypeptidase Q, isoprenoid synthetase structure domain albumen, deoxyribonuclease 1, fibrinogen β chain, γ paddy Acyl-hydrolase, 14-3-3 albumen ζ/Δ, cortisol binding globulin, alpha1 Antichymotrypsin, alpha1 Antichymotrypsin, 14-3-3 albumen beta/alpha, calgranulin B, calgranulin A, calcyclin, Cytochrome P450 2E1, dipeptidyl peptidase 2, L lactic dehydrogenase, Cathepsin H, people's epididymal proteins 4, coagulation factors 12, IST1 homolog, retinaldehyde binding protein 4, PGH2-D isomerase, secreting type urine adjusts element, Solute Carrier family 12 member 7, dipeptidyl peptidase 1, inhibitors of neutrophil elastase, β2-microglobulin, angiostatin, Annexin A1, hemopexin, transaldolase, table colloid albumen, apo E, ezrin, proteinase cathepsin L2, the nuclear translocation factor 2, gelsolin, matrix reconstruction associated protein 8, cadherins 1, α actinin 4, alpha2 Macroglobulin, kallikrein 1, haemoglobin alpha hypotype, keratin (I type cytoskeleton 10), gamma globulin 1 heavy chain, gamma globulin 2 heavy chain, gamma globulin 4 heavy chain, serotransferrin, aminopeptidase N, β hexokinase type β hypotype, G-protein coupled receptor C family member 5C, flesh type phosphorylase, flagellum transport protein 172 homolog, plasminogen activator inhibitor, vimentin, lectin, complement component C9, lysozyme C 1, moesin, glycan-phosphatidylinositol specific phospholipase D.
3) Urine proteins/UCr ratio and serum creatinine value in the urine of common sham-operation 1,3 weeks groups and each 5 rats of ligation 1,3 weeks group are analyzed, find the remarkable rising occurring Urine proteins/UCr ratio in ligation 3 weeks groups; Serum creatinine value, at sham-operation group indifference, raises in ligation 1 week group, and ligation 3 weeks groups raise further.
4) select in 6 animal urines and tissue, to carry out Westernblot checking to the Annexin A1 in above-mentioned protein marker, vimentin, moesin, α-actinine and lectin, and then immunohistochemical checking has been carried out to the nephridial tissue sample of each group of rat.These results have confirmed mass spectral results further, namely wherein there is up-regulated in ligation 1 week group in moesin, α-actinine and lectin, the early diagnosis of this disease from now on is likely used for as the urinary biomarkers thing that obstructive nephropathy is potential, in addition, these five albumen can as the advance stages of urinary biomarkers thing determination obstructive nephropathy, provides theoretical foundation for the diagnosis of disease, treatment and prognosis.
Claims (10)
1. alternative is bonded to the purposes of bound fraction in the reagent for the preparation of diagnosis people obstructive nephropathy of one or more albumen, wherein, one or more albumen described are selected from the unilateral ostruction rat model people source protein corresponding with the differential protein that rats in sham-operated group urine protein analysis of spectrum produces.
2. purposes according to claim 1, one or more albumen wherein said are selected from: myosin-B, antithrombase 2, superoxide dismutase [Cu-Zn], ubiquitin carboxy terminal hydrolytic enzyme isodynamic enzyme, Induced by Retinoic Acid serine carboxypeptidase, nucleoside diphosphokinase, seralbumin, Galectin-3 associated proteins, ec-sod [Cu-Zn], type i collagen α 1 side chain, α actinin 1, basement membrane gathers candy, bladder chalone C, carboxypeptidase Q, isoprenoid synthetase structure domain albumen, deoxyribonuclease 1, fibrinogen β chain, γ paddy Acyl-hydrolase, 14-3-3 albumen ζ/Δ, cortisol binding globulin, alpha1 Antichymotrypsin, alpha1 Antichymotrypsin, 14-3-3 albumen beta/alpha, calgranulin B, calgranulin A, calcyclin, Cytochrome P450 2E1, dipeptidyl peptidase 2, L lactic dehydrogenase, Cathepsin H, people's epididymal proteins 4, coagulation factors 12, IST1 homolog, retinaldehyde binding protein 4, PGH2-D isomerase, secreting type urine adjusts element, Solute Carrier family 12 member 7, dipeptidyl peptidase 1, inhibitors of neutrophil elastase, β2-microglobulin, angiostatin, Annexin A1, hemopexin, transaldolase, table colloid albumen, apo E, ezrin, proteinase cathepsin L2, the nuclear translocation factor 2, gelsolin, matrix reconstruction associated protein 8, cadherins 1, α actinin 4, alpha2 Macroglobulin, kallikrein 1, haemoglobin alpha hypotype, keratin, gamma globulin 1 heavy chain, gamma globulin 2 heavy chain, gamma globulin 4 heavy chain, serotransferrin, aminopeptidase N, β hexokinase type β hypotype, G-protein coupled receptor C family member 5C, flesh type phosphorylase, flagellum transport protein 172 homolog, plasminogen activator inhibitor, vimentin, lectin, complement component C9, lysozyme C 1, moesin, glycan-phosphatidylinositol specific phospholipase D.
3. purposes according to claim 1 and 2, wherein said bound fraction comprises polypeptide or is made up of polypeptide.
4. purposes according to claim 1 and 2, wherein said bound fraction comprises antibody or its Fab or variant, or is made up of antibody or its Fab or variant.
5. the purposes according to any one of Claims 1-4, is characterized in that described diagnostic reagent is for detecting experimenter's health status, the monitoring obstructive nephropathy order of severity and treatment process and/or assess disease prognosis.
6. purposes according to claim 5, wherein by distinguishing renal damage or the interstitial fibrosis monitoring obstructive nephropathy order of severity.
7. the purposes according to any one of claim 1 to 6, described purposes comprises
A): the urine obtaining experimenter,
B): be separated Urine proteins,
C): the level determining one or more albumen described in described any one of claim 1 to 6 in normal person and patient urine albumen,
D): the expression of wherein said albumen is the indication of the obstructive nephropathy order of severity, treatment process and/or prognosis evaluation.
8. purposes according to claim 7, wherein step b) use quantitative mass spectral method, ELISA method, Western method detects.
9. alternative is bonded to the purposes of bound fraction in the reagent for the preparation of diagnosis obstructive nephropathy renal damage of one or more albumen, wherein, one or more albumen described are selected from: antithrombase 2, ubiquitin carboxy terminal hydrolytic enzyme isodynamic enzyme, Induced by Retinoic Acid serine carboxypeptidase, seralbumin, Galectin-3 associated proteins, bladder chalone C, carboxypeptidase Q, deoxyribonuclease 1, fibrinogen β chain, 14-3-3 albumen ζ/Δ, cortisol binding globulin, alpha1 Antichymotrypsin, 14-3-3 albumen beta/alpha, calgranulin B, calcyclin, Cytochrome P450 2E1, dipeptidyl peptidase 2, L lactic dehydrogenase, people's epididymal proteins 4, coagulation factors 12, IST1 homolog, retinaldehyde binding protein 4, PGH2-D isomerase, secreting type urine adjusts element, Solute Carrier family 12 member 7, dipeptidyl peptidase 1, inhibitors of neutrophil elastase, β2-microglobulin, angiostatin, hemopexin, table colloid albumen, apo E, ezrin, proteinase cathepsin L2, the nuclear translocation factor 2, gelsolin, matrix reconstruction associated protein 8, cadherins 1, alpha2 Macroglobulin, kallikrein 1, keratin, serotransferrin, aminopeptidase N, β hexokinase type β hypotype, G-protein coupled receptor C family member 5C, flesh type phosphorylase, flagellum transport protein 172 homolog, plasminogen activator inhibitor, glycan-phosphatidylinositol specific phospholipase D.
10. alternative is bonded to the purposes of bound fraction in the reagent for the preparation of diagnosis obstructive nephropathy kidney region fibrosis of one or more albumen, wherein, one or more albumen described are selected from: myosin-B, superoxide dismutase [Cu-Zn], ubiquitin carboxy terminal hydrolytic enzyme isodynamic enzyme, Induced by Retinoic Acid serine carboxypeptidase, nucleoside diphosphokinase, seralbumin, ec-sod [Cu-Zn], type i collagen α 1 side chain, α actinin 1, basement membrane gathers candy, bladder chalone C, carboxypeptidase Q, isoprenoid synthetase structure domain albumen, deoxyribonuclease 1, fibrinogen β chain, γ paddy Acyl-hydrolase, alpha1 Antichymotrypsin, calgranulin B, calgranulin A, dipeptidyl peptidase 2, L lactic dehydrogenase, people's epididymal proteins 4, coagulation factors 12, retinaldehyde binding protein 4, PGH2-D isomerase, secreting type urine adjusts element, Solute Carrier family 12 member 7, inhibitors of neutrophil elastase, angiostatin, Annexin A1, hemopexin, transaldolase, table colloid albumen, ezrin, the nuclear translocation factor 2, gelsolin, matrix reconstruction associated protein 8, cadherins 1, α actinin 4, alpha2 Macroglobulin, kallikrein 1, haemoglobin alpha hypotype, keratin (I type cytoskeleton 10), gamma globulin 1 heavy chain, gamma globulin 2 heavy chain, gamma globulin 4 heavy chain, serotransferrin, flesh type phosphorylase, vimentin, lectin, complement component C9, lysozyme C 1, moesin, glycan-phosphatidylinositol specific phospholipase D.
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CN108254568B (en) * | 2017-12-19 | 2020-06-05 | 李继承 | Kit for detecting serum specific protein in constitution with yin deficiency and excessive internal heat and application of kit |
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