CN109061192A - One kind urine albumen relevant to osteoarthritis and its application - Google Patents

Abstract

The invention provides the application of ADIRF protein in the preparation of products for detection, auxiliary diagnosis or treatment of osteoarthritis, the expression of the ADIRF protein is up-regulated in the urine of patients with osteoarthritis; further, the invention also provides a method for detecting osteoarthritis An ELISA kit for inflammatory diseases, the test sample of the kit is a urine sample, which has the advantages of non-invasiveness, large-scale repeated sampling, and convenient storage. The osteoarthritis-related urine protein provided by the present invention can be used as an early biomarker of osteoarthritis, and provides a new direction for further studying the pathological mechanism of osteoarthritis and exploring drug targets for early prevention and treatment of osteoarthritis.

Description

One kind urine albumen relevant to osteoarthritis and its application

Technical field

The present invention relates to technical field of biomedical detection, and in particular to a kind of urine albumen relevant to osteoarthritis And its application.

Background technique

Osteoarthritis (osteoarthritis, OA), which is that one kind is multifactor, leads to morbidity, in mid-aged population very Common chronic degenerative joint disease, extensive in distribution on global, women is more than male, is common in knee joint, finger little Guan Section, hip joint, also seen in shoulder joint, wrist joint, ankle-joint etc..Osteoarthritis is with articular cartilage abrasion, regression and joint Periphery osteoproliferation is characterized, and acute stage is also often accompanied by synovitis.According to investigations, osteoarthritis illness in 60 years old or more crowd Rate is up to 50% or more, and prevalence is up to 80% within 75 years old or more, and the disability rate of the disease is up to 53%.

Knee joint osteoarthritis causes gonalgia, swelling, stiff, knee articulation degree limited and dysfunction etc., The quality of life of patient is seriously affected, finally also influences its life expectancy indirectly.Currently, since the teiology to OA still lacks The understanding of essence, medical field, which eradicates it, still lacks effective pharmaceutical means, and joint replacement surgery often becomes trouble The therapeutic modality that person must finally select.In order to walk out the predicament of OA treatment aspect, the teiology mechanism of OA is illustrated and in its base The effective scheme that treatment OA is explored on plinth, becomes the research direction that medical field needs to be broken through.

The definition of protein group refers to all eggs expressed by a genome, one or more cells or certain tissues No matter white matter, be small enough to a cell or greatly to an organism, is all that go research to participate in life from an integral level living Dynamic all proteins.These all proteins are not a unalterable set, but vary whenever and wherever possible, Because as the progress of vital movement and the adjusting of stable state always have different protein being expressed or modifying.Protein is raw The component part of the movable undertaker and most of organism structures are ordered, the formation of protein will pass through a series of transcription After modify, therefore, from genome and transcript profile can not direct derivation go out protein group the case where.

The sample of proteomics research can be tissue, cell, blood, joint fluid, urine etc., in one's early years to Urine proteins The research of matter group is often confined to disease in the urological system, gradually expand to later it is systemic with metabolic disorder be performance Disease.Due to the influence of high-abundance proteins few in urine, for blood and joint fluid, Urinary proteomics more have Conducive to the biomarker of searching disease, while may be found on pathogenic mechanism.

Orthopaedics joint field is that sample carries out the research of Urinary proteomics and few with urine at present, with iTRAQ The Urinary proteomics research of the emergence and development of the high throughput proteins omics technology such as technology, this field shows gradually The trend increased.As mentioned in the introduction, more and more evidences show that the occurrence and development of osteoarthritis are related with metabolic factor, At the same time, many scholars also have no resolution in the biomarker for attempting to look for osteoarthritis for many years, therefore, are well suited for urine The method of proteomics come explore osteoarthritis biomarker and study its related pathologies mechanism.

Summary of the invention

To solve the above-mentioned problems, the purpose of the present invention is to provide it is a kind of for osteoarthritis disorders early diagnosis Urine proteins group biomarker ADIRF, and it is provided and is applied in preparation detection osteoarthritis product.

Present invention firstly provides ADIRF albumen answering in preparation detection, auxiliary diagnosis or treatment osteoarthritis product With.

Preferably, the ADIRF albumen expresses up-regulation in osteoarthritis sample.

Preferably, the osteoarthritis sample is urine specimen.

Preferably, the product includes reagent, kit, chip or drug.

Preferably, the product can be detected whether by the expression of ADIRF albumen in detection Samples subjects Risk with osteoarthritis.

Preferably, described detect includes:

A) urine of subject and normal control are obtained,

B) optionally, Urine proteins are separated,

C) expression of ADIRF albumen in the urine of the subject and normal control is determined,

D) expression of albumen in the urine of the subject and normal control is compared,

E) according to the comparison result of step d), detect whether the subject suffers from osteoarthritis disorders.

Preferably, using method selected from the following carry out step c) in the determination: mass spectrometry method, ELISA method, Western method or combinations thereof.

Further, the present invention provides a kind of for detecting the ELISA kit of osteoarthritis disorders, it includes The detection reagent of ADIRF albumen.

Preferably, the detection reagent of the ADIRF albumen includes the specific antibody reagent of ADIRF albumen.

Preferably, the specific antibody of the ADIRF albumen is monoclonal antibody and/or polyclonal antibody.

The coated antibody that the ELISA kit that the present invention detects osteoarthritis disorders uses is anti-for anti-ADIRF monoclonal Body, the effect of coated antibody are the ADIRF captured in sample to be tested (for example, urine sample), and capture principle is to pass through antigen The specific binding of antibody.Requirement of the present invention to coated antibody is that the antigen that can be captured on antigen molecule ADIRF determines Cluster, thus specifically bind the two can effectively, it therefore meets the antibody that can be specifically bound with antigen molecule, including The anti-ADIRF monoclonal antibody of the kinds such as mouse, rabbit, chicken, dog or monkey source property can be used as coated antibody.Wherein, it is preferable to use The anti-human ADIRF monoclonal antibody of mouse, main cause are that the specificity of murine antibody identification single antigenic determinat is high, favorably In from sample combine antigen to be measured.

The detection antibody that the ELISA kit that the present invention detects osteoarthritis disorders uses is anti-ADIRF Anti-TNF-α Body, the effect for detecting antibody is combined with the antigen molecule ADIRF on coated antibody, to detect in sample to be tested ADIRF.Requirement of the present invention to detection antibody is to be able to detect to combine on the antigenic determinant on antigen molecule ADIRF , it therefore meets the antibody that can be specifically bound with antigen molecule, anti-including the kinds such as mouse, rabbit, chicken, dog, monkey source property ADIRF polyclonal antibody can be used to detect antibody.Wherein, it is preferable to use rabbit-anti people coated antibody can pass through commercially available way Diameter obtains, and can also voluntarily prepare, the preparation method is known to technical field personnel.

It should be strongly noted that although coated antibody and detection antibody are all anti-ADIRF antibody, but cannot use The anti-ADIRF antibody of same kind.Reason is: the anti-ADIRF antibody using different genera is to enable to detect antibody It is integrated on the antigen site different from coated antibody, to detect different epitopes, and when using same kind When antibody, the antibody with chromogenic reaction enzyme can be combined with capture and detection antibody simultaneously, and chromogenic reaction is caused not have spy The opposite sex can not determine the amount of determined antigen.Therefore, it when selecting coated antibody and detection antibody, needs to choose from difference The anti-ADIRF antibody of kind.

Preferably, the kit further includes anti-more grams of ADIRF of the coated ELISA Plate of anti-ADIRF monoclonal antibody, label Grand antibody, protein standard substance ADIRF, tmb substrate solution, dilution, washing buffer, stop bath.

Further, the present invention provides the kit in the kit of preparation detection or auxiliary diagnosis osteoarthritis Application.

Further, the present invention provides the specific antibody of ADIRF albumen in preparation treatment osteoarthritis drugs Using.

Preferably, the specific antibody of the ADIRF albumen include complete polyclonal antibody, monoclonal antibody, its What antigen-binding fragment (i.e. " antigen-binding portion thereof ") or single-stranded.

Fall into the range of the antibody of present disclosure is polyclonal antibody, monoclonal antibody and recombinant antibodies.Use this Technology known to field can easily prepare these antibody.In addition, the antibody that can be used for present disclosure can be by two entirely The function fragment of complete antibody or complete antibody molecule that long light chain and two total length heavy chains form.The antibody molecule " function fragment " means the segment for retaining antibody binding function, such as Fab, F (ab'), F (ab') 2 and Fv.

Further, the present invention also provides a kind of in Human Osteoarthritis urine specimen screens the side of differentially expressed protein Method, comprising the following steps:

(1) the urine of Human Osteoarthritis and control group is obtained；

(2) Urine proteins are separated；

(3) the albumen sample described in two groups carries out reductive alkylation processing respectively, obtains two histone sample solutions；

(4) trypsase is added into two histone sample solutions respectively and carries out enzymolysis processing；

(5) different labelled reagents is used, the processing of iTRAQ label is carried out to two groups of samples after enzymolysis processing respectively, and Label treated Human Osteoarthritis group is mixed with the sample solution of control group；

(6) liquid chromatogram separation is carried out to mixed sample, then the peptide fragment of liquid chromatogram separation is subjected to tandem mass spectrum Analysis；

(8) protein identification is carried out to mass spectrometry results using protein identification software, it is horizontal according to protein abundance, Screening analysis obtains the differentially expressed protein in the sample of Human Osteoarthritis and control group；

(9) differential protein is identified by bioinformatic analysis and illustrate the biological function of differential protein；

(10) ADIRF albumen relevant to osteoarthritis is finally screened.

Preferably, (9) middle bioinformatic analysis includes that differential protein GO enrichment analysis and KEGG signal are logical to the step Road enrichment analysis.

Beneficial effect

The present invention is confirmed compared with the control group, on ADIRF albumen is expressed in Human Osteoarthritis urine by research Adjust, and study its osteoarthritis early detection, the reagent of auxiliary diagnosis or kit and or treatment drug in application before Scape；The further present invention provides a kind of for detecting the ELISA kit of osteoarthritis disorders, the kit detection sample For urine specimen, acquisition is noninvasive, can repeat to sample on a large scale, save convenient advantage.It is provided by the invention with osteoarthritis phase The urine albumen of pass can be used as the early stage biomarker of osteoarthritis, for the pathomechanism of further research osteoarthritis, be The drug target for exploring osteoarthritis early prevention and treatment provides new direction.

Detailed description of the invention

Expression of the Fig. 1 using Western blot detection ADIRF albumen in urine specimen.

Specific embodiment

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, implementing The conventional means that technological means used in example is well known to those skilled in the art.

Test method without specific conditions in embodiment, usually conventional method in that art.

The present inventor carries out iTRAQ experiment to 4 osteoarthritis samples and 4 check samples, believes in conjunction with biology Breath method is screened, and picks out candidate albumen ADIRF albumen, and there is no ADIRF albumen as marker in existing research Report relevant to osteoarthritis, further, inventor also use western blot to verify above-mentioned albumen in osteoarthritis trouble Expression in the urine specimen of person and control group, it was confirmed that the ADIRF expresses up-regulation in osteoarthritis urine specimen, Related product can be used for diagnosing, treat osteoarthritis.

ADIRF albumen of the invention is known albumen before making the present invention, and essential information is as follows:

ADIRF NCBI Reference Sequence:NP_006820.1；

ADIRF (adipogenesis regulatory factor, fat generate regulatory factor) in the present invention be by The protein of ADIRF gene coding, now studying disease relevant to ADIRF includes sarcoma of vagina and vagina smooth muscle sarcoma.Its Related pathways are peroxisome proliferator-activated receptor alpha (PPARalpha) and Developmental Biology regulating lipid metabolism.

Fat generates regulatory factor and plays crucial effect in fat cell development: promoting Adipocyte Differentiation and preceding Adipocyte Differentiation early stage stimulation primary fat generates the transcription initiation of the factor such as PPARG and CEBPA.It, which is over-expressed, assigns The drug resistance of anticancer chemotherapeutic agent cis-platinum.

Present invention application iTRAQ combines mass spectrographic method and identifies protein marker in Human Osteoarthritis urine.Together Position element label is opposite and absolute quantitation (isobaric tags for relative and absolute quantitation, ITRAQ a kind of) the opposite and absolute quantitation technology for external isotope labelling of the same race that technology is researched and developed by AB SCIEX company.It should Technology utilizes a variety of isotope reagent labelled protein polypeptide N-terminals or lysine side-chain group, through the series connection point of high-precision mass spectrograph Analysis, can compare the expressing quantity between up to 8 kinds of samples simultaneously, be that quantitative proteomics are commonly high-throughput in recent years Screening technique.ITRAQ quantitative proteomics are to form polypeptide after proteolytic cleavage, with iTRAQ isotope reagent labeling polypeptide N End or lysine side-chain group.Peptide fragment after label passes through liquid phase separation, carries out first mass spectrometric and second mass analysis, Before second order ms, the same peptide fragment in labeled different samples shows as identical mass-to-charge ratio and other physicochemical properties.And In second order ms, signal ion shows as the peak of different mass-to-charge ratioes (114~121), can be with according to the height and area of wave crest It identifies protein and analyzes the quantitative information of same protein different disposal.

ITRAQ reagent includes three parts: report section, peptide reactive moieties, balance portion.(1) report section has eight kinds: 113-121 (without 120), therefore iTRAQ reagent can 8 groups of samples of label simultaneously.(2) peptide reactive moieties: can with peptide fragment N-terminal and rely Propylhomoserin side-chain amino group occurs to be covalently attached and mark upper peptide fragment.(3) balance portion: guarantee the matter lotus of labeled same peptide fragment Than identical.Compared with traditional dielectrophoresis quantitative analysis, iTRAQ has following technological service advantage: (1) high sensitivity, inspection It is low to survey limit, can detect that low-abundance protein；(2) separating capacity is strong, and analyst coverage is wide, and iTRAQ can be to any kind of egg White matter carries out separation identification, including high molecular weight protein, acidic protein and basic protein, memebrane protein and insoluble protein； (3) high-throughput: while 8 samples are analyzed, experiment flux is improved, it can be simultaneously to multiple time points or different disposal Protein analyzed；(4) result is reliable: quantification and qualification result is relatively reliable；(5) high degree of automation: liquid phase It is used in conjunction with mass spectrum, automatic operation, analysis speed is fast, good separating effect.

Term " sample " used herein includes the sample or culture obtained from any source.Sample can be obtained from Blood (including any blood product, such as whole blood, blood plasma, serum or certain types of haemocyte), urine, saliva.Sample is also Including tissue sample.In one embodiment, sample comes from urine.

Terms used herein " expression " refer to by methods known to those skilled in the art measure given nucleic acid or Protein can measure.

Terms used herein " control ", which refer to, not to be shown any OA symptom and has not been diagnosed as osteoarthritis or the individual of OA Or group of individuals.Preferably, the drug for influencing OA is not used in the control individual, and has not been diagnosed as with any other disease. It is highly preferred that control individual has similar gender, age and Body Mass Index (BMI) compared with test sample.

Terminology used in the present invention " enzyme linked immunosorbent assay (ELISA) (enzyme-linked immunosorbent assay letter Claim ELISA) " refer to it is the characteristics of capable of specifically binding with antigen molecule using antibody molecule, by free foreign protein and to be incorporated into The destination protein of solid phase carrier separates, and using special marker to a kind of detection method of its qualitative or quantitative analysis. ELISA process includes: Antigen adsorption on solid phase carrier, this process is known as being coated with, and adds test antibodies, then plus corresponding enzyme mark Remember antibody, generates the compound of antigen -- test antibodies -- enzymic-labelled antibody, then generate coloured production with the substrate reactions of the enzyme Object.By the amount of the light absorption calculating antibody of spectrophotometer.The amount of test antibodies is directly proportional to color products.Similarly may be used Coated antibody measures antigenic content.The most common four kinds of methods of ELISA: Direct Determination antigen；Indirect Determination antibody；It is double Antibody sandwich measures antigen；Competition law measures antigen.

The collection of 1 sample of embodiment

Case-control study is method used by this research.The research object of experimental group is to assist in Beijing in 2015 years With hospital orthopedics be hospitalized suffer from severe osteoarthritis but also be not treated surgically patient 4, the research object of control group For relative healths person 4 for not suffering from osteoarthritis, experimental group meets Chinese Medical Association's osteology branch osteoarthritis diagnosis mark It is quasi-.The age of experimental group is 58.5 ± 2.0 years old (56-61 years old), and the age of control group is 57.6 ± 2.1 years old (55-61 years old).Root " adult body major punishment the is fixed " standard formulated for 2013 according to People's Republic of China's national health and Family Planning Committee, two groups Choose super severe one of the BMI between 24-28.Experimental program is agreed to through Ethics Committee of BJ Union Hospital, and obtains every The written informed consent of position subject.Experimental group and control group basic document are shown in Table 1.

Collect second of midstream urine from the morning of experimental group and control group, first with sterile large container splendid attire, after be transferred to Centrifuge tube, 4,000g centrifugations 10min (4 DEG C), supernatant are divided into 50mL sterile tube, and -80 DEG C save backup.

The basic document of 1 sample of table

OA case group is included in standard:

There are complete clinical data and imaging data (double knee positive sides position adds patella axial X-ray film), according to medical history and examines Disconnected standard diagnostics are the overweight female patient of severe osteoarthritis (K-L is classified 4 grades), and sign informed consent form.

The exclusion criteria of OA case group:

Knee trauma surgery history, gnotobiotic mice, adult knee joint deformity, metabolic bone disease, leg length discrepancy, knee close Save tumour history, osteoporosis, liver and kidney disease, hyperlipidemia, hypertension, diabetes, hyperthyroidism, parathyroid gland Hyperfunction medical history, takes estrogen, progestogen therapy person in the recent period, and other joint diseases such as gout, rheumatoid arthritis connect It has received and has changed state of an illness antirheumatic drug or immunosuppressive therapy, be inscribed within nearly 6 months and received intra-articular injection drug therapy.BMI is less than 24 or be greater than 28.

Control group is included in standard:

Control group choose be not suffer from osteoarthritis overweight women patient, gender, age and the BMI of control group with OA case group matches, and signs informed consent form.

The exclusion criteria of control group:

Knee trauma surgery history, gnotobiotic mice, adult knee joint deformity, metabolic bone disease, leg length discrepancy, knee close Save tumour history, osteoporosis, liver and kidney disease, hyperlipidemia, hypertension, diabetes, hyperthyroidism, parathyroid gland Hyperfunction medical history, takes estrogen, progestogen therapy person in the recent period, and other joint diseases such as gout, rheumatoid arthritis connect It has received and has changed state of an illness antirheumatic drug or immunosuppressive therapy, be inscribed within nearly 6 months and received intra-articular injection drug therapy.BMI is less than 24 or be greater than 28.

Embodiment 2iTRAQ experiment screening differential protein

One, instrument and reagent

Turbula shaker (its woods Bell's instrument manufacturing Co., Ltd of Haimen City, model: QL-901)

Centrifuge (Thermo, model: PICO17)

Ultrasonic cell disruption instrument (Nanjing Xian Ou instrument manufacturing Co., Ltd, model: XO)

Microplate reader (Thermo, model: M μ Ltiskan MK3)

Constant temperature incubates bath (Pudong, Shanghai Rong Feng scientific instrument Co., Ltd, model: HH.S4)

Vacuum freeze drier (Thermo, model: SPD2010-230)

RIGOL L-3000 highly effective liquid phase chromatographic system (BeiJing PuYuanJing power Science Co., Ltd), mobile phase A: 98% ddH₂O, 2% acetonitrile (pH 10)；Mobile phase B: 98% acetonitrile, 2%ddH₂O (pH 10) high performance liquid chromatograph: (Thermo Scientic EASY-nLC 1000System (Nano HPLC)), mobile phase A: 100% ultrapure water, 0.1% formic acid；Flowing Phase B:100% acetonitrile, 0.1% formic acid mass spectrometer system (Thermo, model: Q-Exactive)

Urea (Bio-Rad, article No.: 161-0731, the U.S.)

Sulphur urine (Sigma-Aldrich, article No.: T7875, the U.S.)

CHAPS (Bio-Rad, article No.: 161-0460, the U.S.)

Protease Inhibitor Cocktail (Roche, article No.: 04693116001, the U.S.)

Protein quantification dye liquor (Thermo Scientific, article No.: 23238, the U.S.)

Bovine serum albumin(BSA) (Bovine Serum Albumin, BSA) (Sigma-Aldrich, article No.: A2058, beauty State)

DTT (Bio-Rad, article No.: 161-0611, the U.S.)

Iodoacetamide (Bio-Rad, article No.: 163-2109, the U.S.)

Dissolution Buffer (AB Sciex, PN:4381664) in kit

Pancreatin (Promega, article No.: V5111, the U.S.)

10K super filter tube (milipore, PN:UFC5010BK)

8 marksKit (AB Sciex, PN:4390812, PN:4381664)

Ziptip (Millipore, PN:ZTC18M096 (2 μ g))

Chromatographic column: Durashell-C18,4.6mm × 250mm, 5 μm,(Agela, article No.: DC952505-0)

Acetonitrile (Merck, article No.: 100030, Germany)

Ammonium hydroxide (Sigma-Aldrich, article No.: 17837, the U.S.)

Pre-column (100 μm of AcclaimPepMap100column, 2cm x, C18,5 μm)

Chromatographic column (75 μm, C18,3 μm of EASY-Spray column, 12cmx)

Sample injection bottle (Thermo, 11190533)

Bottle cap (Thermo, 11150635)

Nozzle needle (Thermo, PN:ES542)

Two, iTRAQ quantitative experiment process

1. sample protein extracts

1) by sample according to 1:10 (W/V) be added lysis buffer (7M urea, 2M thiocarbamide, 0.1%CHAPS, piece/ 50mL Protease Inhibitor Cocktail), it is vortexed and mixes.

2) ultrasound 60s (0.2s on, 2s off), amplitude 22%.

3) room temperature is extracted 30 minutes.

4) 15,000g, 4 DEG C of centrifugation 20min carefully take out supernatant, freeze after packing in -80 DEG C.

2. protein quantification (Bradford method)

1) using Bradford method [Marion M.Bradford.AnalyticalBiochemistry, 1976,72: 248-254] measurement sample extraction protein concentration.First mix the sample with lysis buffer (7M urea, 2M thiocarbamide, 0.1% CHAPS) carrying out certain multiple dilution falls in its final concentration in the bent range of mark, and the sample and standard items diluted (is used BSA Lysis buffer is dissolved into the standard protein of series of concentrations) respectively take 10 μ L to be protected from light respectively with 300 μ L protein quantification dyestuffs 20min measures the light absorption value of standard items and sample at 595nm simultaneously with microplate reader, according to the every pipe light absorption value of standard items and dense The relationship of degree draws standard curve (curve equation: y=1.4337 × x2+0.0406 × x+0.03728, R²=0.98906).

2) each sample protein concentration is calculated according to curve equation.

3. proteolysis (FilterAided Sample Preparation, FASP)

1) 200 μ g protein solutions is taken to be placed in centrifuge tube after protein quantification；

2) final concentration 25mM DTT is added, 60 degree are reacted 1 hour；

3) addition final concentration 50mM iodoacetamide, room temperature 10 minutes；

4) protein solution after reductive alkylation is added in the super filter tube of 10K, 12,000 turns are centrifuged 20 minutes, discard Collecting pipe bottom solution；

5) the 100 μ L of Dissolution Buffer in iTRAQ kit is added, 12,000 turns are centrifuged 20 minutes, abandon Fall collecting pipe bottom solution, is repeated 3 times；

6) trypsase, 4 μ g of total amount (with albumen quality ratio 1:50), body is added in the collecting pipe more renewed in super filter tube 50 μ L of product, 37 DEG C of reactions are overnight；

7) next day, 12,000 turns are centrifuged 20 minutes, and the peptide fragment solution centrifugation after enzymolysis, digestion is in collection bottom of the tube；

8) 50 μ L DissolutionBuffer are added in super filter tube, 12,000 turns are centrifuged 20 minutes again, with upper step Merge, collects the sample that bottom of the tube is obtained after 100 μ L enzymatic hydrolysis.

4.iTRAQ label

1) iTRAQ reagent is taken out from refrigerator, equilibrates to room temperature, it willReagent is centrifuged to tube bottom.

2) to every pipe150 μ L isopropanols, vortex oscillation, centrifugation to tube bottom are added in reagent.

3) 50 μ L samples (100 μ g enzymolysis product) is taken to be transferred in new centrifuge tube.

4) iTRAQ reagent is dosed in sample, vortex oscillation, centrifugation to tube bottom reacts at room temperature 2 hours.

5) 100 μ L water are added and terminate reaction.

6) in order to detect labeling effciency and dosing accuracy, 1 μ L mixing is respectively taken out from 4 groups of samples, with Ziptip desalination MALDI-TOF-TOF (AB SCIEX 4800Plus) identification is carried out afterwards, and confirmation flag reaction is good；

7) sample after mixed mark, vortex oscillation, centrifugation to tube bottom.

8) vacuum refrigeration centrifugal drying.

9) the sample freezen protective after draining is stand-by.

5. the offline pre-separation of peptide hydrolysis and LC-MS/MS mass spectral analysis

Reversed phase chromatography separation under the conditions of 5.1 high pH

1) sample after mixed mark is dissolved with 100 μ L mobile phase As, and 14000g is centrifuged 20min, takes supernatant stand-by.

2) (45 DEG C of column temperature, Detection wavelength 214nm), detection system situation are separated using the BSA that 400 μ g have been digested.

3) the ready sample loading of 100 μ L, separation, flow velocity 0.7mL/min are taken.

5.2 nanoliter level reverse-phase chromatography-Q Exactive carry out protein analysis

1. experimental procedure

1) component for obtaining high pH reverse phase separation 20 μ L, 2% methanol, 0.1% formic acid redissolve.

2) 12,000rpm is centrifuged 10 minutes, draws supernatant loading.

3) 10 μ L of loading volume, takes sandwich method loading.

4) Loading Pump flow velocity 350nl/min, 15 minutes.

5) flow velocity 300nl/min is separated, gradient such as the following table 2 is separated:

2 nanoliter level reversed phase chromatography separation gradient of table

2. mass spectrometry parameters are arranged

A) source parameters:

Spray voltage:2.3kv；

Capillary Temperature:320 DEG C；

Ion Source:EASY-Spray source；

DP:100；

B) F μ Ll MS:

Resolution:70000FWHM；

F μ Ll Scan AGC target:3e6；

F μ Ll Scan Max.IT:20ms；

Scan range:300-1800m/z；

C) dd-MS2:

Resolution:17500FWHM；

AGC target:1e5；

Maximum IT:120ms；

Intensity threshold:8.30E+03；

Fragmentation Methods:HCD；

NCE:32%；

Top N:20；

6. MASS SPECTRAL DATA ANALYSIS

6.1 database

Selecting for database is using required species, database annotation completeness and sequence reliability as reference frame.? The database selected in this experiment is from UniProt (http://www.uniprot.org/), this version of database are as follows: UniProt.Rat.201509.fasta。

6.2 retrieval softwares

The mass spectral analysis of iTRAQ is completed by Thermo Q-Exactive type mass spectrum, the mass spectrum original document * of generation .RAW library processing is searched using Mascot 2.5.1 software, Quality Control is carried out to library result is searched using scaffold software.

7. bioinformatic analysis

7.1 differential protein quantitative informations statistics

The two groups of samples to be compared are counted using Perseus 1.3.0.4. (www.maxquant.org) software Value≤0.05 P, and the differential protein of albumen ratio >=1.25 or ratio≤0.75 in two groups of samples are screened in analysis.

7.2 differential protein functional annotations

Comprehensive biological function is carried out to all protein using UniProt (http://www.uniprot.org/) Annotation obtains and all relevant functional informations of these protein.Believe including the annotations such as Gene Ontology (GO) and access Breath.

The enrichment analysis of 7.3 differential protein functions

Function enrichment analysis is carried out to different group differences albumen using MetaCore software.Using MetaCore to each Group differential expression protein has carried out the biological process (biologicalprocess) based on Gene Ontology (GO), The enrichment of cellular component (cell μ Lar component) and molecular function (molec μ Lar function) is analyzed.The analysis is Finger with high throughput annotates each protein using functional annotation tool, obtains experimental identification protein in different kind organism Distribution situation in process or molecular function, and distribution distribution corresponding to overall protein matter is compared, thus really The protein of experimental identification significant enrichment (i.e. p value is less than 0.05) in which quasi-biology process or molecular function is recognized, from whole The degree of agreement between the protein of identification and expectation function distribution is held on body, can also be obtained and certain specific function phases The protein molecule of pass.It is analyzed by MetaCore, obtains biological process, the cellular component, molecular function of differential protein It is enriched with result.

The enrichment analysis of 7.4 differential protein accesses

Path analysis is carried out to differential expression protein using MetaCore software, is obtained and differential expression protein institute There is the access of relevant path information and enrichment.

8. result

Based on iTRAQ technology, the present invention has identified 1413 kinds of protein altogether from experimental group and the urine of control group, Wherein differential protein a total of 394 between two groups, 170 protein raise in OA experimental group, and 224 protein are in OA It is lowered in experimental group.According to GO analyze: differential protein be predominantly located at extracellular vesica, cell exocrine corpusculum, extracellularly Base portion and cell membrane；Differential protein is related with the adjusting of peptase and endopeptidase etc. and inhibiting effect, and also and cell The molecular functions such as adhesion molecule and the combination of protein receptor are related；Differential protein is primarily involved in cell adherence, injury repair The bioprocess such as adjusting, stress reaction.

Inventor is analyzed by Gene Onlogy and signal path, and has been screened in conjunction with document inventor in Bones and joints The differential protein ADIRF of up-regulation is expressed in the urine of scorching patient.ADIRF albumen can be used as the urine biology mark of osteoarthritis Will object provides theoretical foundation for the diagnosis, treatment and prognosis of disease.

3 western blot of embodiment detects expression of the ADIRF albumen in Human Osteoarthritis

Further verify above-mentioned 4 Human Osteoarthritis and 4 collator's urine specimen ADIRF protein expression situations.Ginseng Albumen in sample is proposed according to embodiment 2, uses health for century micro BCA protein quantification kit (article No.: CW2011), specifically Step is shown in its specification.Then, SDS- polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting are carried out, specifically Steps are as follows:

The SDS- polyacrylamide gel electrophoresis:

1, protein example is denaturalized:

A) according to BCA determination of protein concentration as a result, the total protein extraction of phase homogenous quantities is added in each gel well Object.The ratio of 0.25 microlitre of albumen sample-loading buffer is added according to every 1 microlitre of protein sample, on mixed protein sample and albumen Sample buffer (5x).

B) 100 DEG C or boiling water bath heating 3-5 minutes, with abundant albuminate.

C) it after being cooled to room temperature, is directly loaded in SDS-PAGE glue well.

2, prepared by offset plate:

The gel of 0.75mm thickness is prepared using the miniature vertical plate electrophoresis device of Bio-Rad company, book installs as directed After glass plate, the separation gel of 5mL 10% is first prepared in small beaker, is formulated as follows:

Table 3 separates glue formula

Component	Dosage
		30% acrylamide solution	1.7mL
Tris-HCL (1.5M, pH8.8)	1.3mL
		10%SDS	0.05mL
10%AP	0.05mL
		TEMED	0.002mL
Sterilize ddH2O	It is supplemented to 5mL

Then plus the covering of 1mL distilled water encapsulating immediately after mixing places about 30min after glue polymerization, with steaming at room temperature Distilled water is washed 2-3 times, then is blotted with filter paper.Then the concentration glue of 2mL 5% is prepared, is formulated as follows:

Glue formula is concentrated in table 4

Encapsulating, insertion sample comb avoid generating bubble immediately after mixing, after being gelled admittedly, take out sample comb, rear use is steamed Distilled water and 1x protein electrophoresis buffer successively rinse sample well.

3, loading and electrophoresis

By gel slab on electrophoretic apparatus, 1x protein electrophoresis buffer is filled it up in inside groove, 1x protein electrophoresis is slow in outer groove Fliud flushing should be more than platinum filament, in order loading.Protein quality standard protein gradient is added in the swimming lane of end.It is blue when electrophoresis The bottom end that dyestuff reaches glue can nearby stop electrophoresis.

The Western blotting:

1, NC film, filter paper, foam-rubber cushion are impregnated with transfer buffer in advance.Gel is taken out after SDS-PAGE, is removed dense Contracting glue rinses the several seconds in Tris/ glycine buffer, is subsequently placed in transfer buffer and impregnates 15-30min.Electricity is opened to turn One piece of dedicated foam rubber cushion impregnated with transfer buffer is padded in print folder, every side, then respectively puts the filter that one block of transfer liquid is impregnated with Paper, filter paper it is identical as foam rubber cushion size or with NC film, gel size is identical, gel is lain on cathode side filter paper, most NC film is lain on gel afterwards, removes bubble removing, clips electricity transfer folder.Electricity transfer liquid is filled it up in electrophoresis tank, is inserted into electricity transfer Electrophoresis tank is put into refrigerator and (to be put into pre-cooling in refrigerator before electricity transfer liquid), connects electrode, turn-on current turns by folder The anode of the NC film reply electrophoresis tank of print folder.

2, it closes: being rinsed with 1xTBS primary.The alipoidic milk power TBS Block buffer containing 5% is added, is placed in shaken cultivation It is closed in case；

3, primary antibody hybridizes: abandoning confining liquid, is added and is hybridized with the primary antibody of primary antibody (LSBio, LS-C140569) diluted Solution is placed in 4 DEG C of hybridized overnights, is hybridized in shaken cultivation case within second day；

4, primary antibody hybridization solution is recycled, is washed film 3 times with TBST；

5, TBST is abandoned, is added and uses the diluted secondary antibody of Block buffer (Goat Anti-Rabbit IgG, HRP Conjugated, CW0103) hybridization solution, it is placed in shaken cultivation case and is hybridized；

6, two corresponding anti-solution is abandoned, is washed film 3 times with TBST；

7, ECL chemiluminescence and Image Acquisition and analysis: according to highly sensitive chemical luminescence detection kit (Kang Weishi Discipline article No. CW0049B), specific steps are referring to specification.

8, data normalization is carried out using β-Actin as internal reference, using normal person as sample for reference, observes osteoarthritis The relative expression levels of ADIRF albumen in urine specimen.

Shown in result figure 1, compared with the control group, ADIRF albumen is in osteoarthritis group in Human Osteoarthritis urine Expression up-regulation.It is with uniformity with protein science experimental result.Prompting ADIRF may be pass relevant to osteoarthritis generation The key factor.

The assembling of 4 osteoarthritis correlation ADIRF protein detection kit of embodiment

The present inventor can pass through inspection using ADIRF albumen and/or the foundation of its antibody using the above results as foundation ADIRF protein expression level is surveyed to diagnose the ELISA kit of Human Osteoarthritis urine specimen, it include: solid phase carrier, Marker and the substrate that chromogenic reaction can occur with the marker；Marker is that enzyme marks ADIRF albumen, enzyme to mark ADIRF Protein antibodies, biotin labeling ADIRF albumen or biotin labeling ADIRF protein antibodies.Specific kit can have following examinations Agent composition:

1) ELISA Plate (solid phase carrier)；

2) people ADIRF standard protein；

3) marker；

4) Sample dilution；

5) antibody diluent；

6) washing lotion；

7) colour reagent；

8) terminate liquid.

It is known to those skilled in the art that the above component is only illustrative, the ELISA Plate can be what primary antibody was coated with (being used for double sandwich-ELISA method, competition law) or (for Salmonella method) not being coated with；People's ADIRF standard protein can Be enrichment or expression, synthesis holoprotein or comprising the peptide fragment of antigenic determinant；Marker can be horseradish peroxidase Label or biotin labeling；Colour reagent can be TMB, OPD or ABTS etc..ELISA method can be double sandwich methods, direct method Or competition law.

By the ELISA kit of following assembling detection osteoarthritis by taking the bis- sandwich methods of ELISA as an example:

Conventional reagent in 1.ELISA experiment:

It is coated with buffer (carbonate buffer solution of pH9.6): Na₂CO₃1.59g NaHCO₃2.93g adds distilled water to 1L.

Washing buffer (pH7.4): 8.0g NaCl；0.2g KH₂PO₄；2.9g Na₂HPO₄·12H₂O； 0.2g KCl； 0.05% Tween-20 of 0.5mL, adds ddH₂O to 1L.

Dilution: bovine serum albumin(BSA) (BSA) 0.1g adds washing buffer to 100mL；

The anti-human ADIRF albumen source of mouse monoclonal antibody that the present invention uses is purchased by Shanghai Yuan Mu Biotechnology Co., Ltd It buys, anti-ADIRF rabbit polyclonal antibody is purchased from abcam.

Protein standard substance ADIRF is that source of people recombinant protein is purchased from abcam company.

2. the coating of ELISA Plate:

The anti-coated ELISA Plate of ADIRF source of mouse monoclonal antibody is prepared via a method which: with the carbon of pH9.6 Hydrochlorate coating buffer anti-ADIRF monoclonal antibody will be diluted to 0.63 μ g/mL of purpose concentration after purification；It is anti-by what is diluted Liquid solution is added in micropore after mixing, 100 holes μ L/, and 4 DEG C overnight；Board-washing 3 times, 200 holes μ L/；3%BSA confining liquid is added, 300 holes μ L/, 4 DEG C overnight；Board-washing 3 times, 200 holes μ L/；- 20 DEG C of preservations.

3. the preparation of enzyme labelled antibody:

Anti- ADIRF albumen rabbit source polyclonal antibody is taken, is coupled respectively with HRP, obtains enzymic-labelled antibody.It takes certain The enzymic-labelled antibody of amount is added in dilution, is mixed well, make its final concentration of 2 μ g/mL (can according to specific condition and It is fixed), 2-8 DEG C is kept in dark place.

4. the assembling of kit:

The ELISA kit of detection scoliosis includes the component in table 5:

5 ELISA detection kit of table

The application method of the kit is as follows:

(1) taking ADIRF standard protein concentration respectively is respectively 900pg/mL, 600pg/mL, 300 pg/mL, 150pg/mL, The ELISA Plate that coated antibody is added in 50 μ L of 75pg/mL solution draws standard curve；

(2) blank well (sample and enzyme marking reagent is not added in blank control wells, remaining each step operation is identical), to be measured is set respectively Sample well first adds 40 μ L of sample diluting liquid on ELISA Plate in sample to be tested hole, then adding 10 μ L of sample to be tested again, (sample is most Whole dilution is 5 times).Sample is added on ELISA Plate hole bottom by sample-adding, is not touched hole wall as far as possible, is shaked gently mixing；

(3) it is incubated 30 minutes with 37 DEG C of sealing plate film sealing plate postposition；

(4) it carefully takes sealing plate film off, discards liquid, dry, cleaning solution is filled it up in every hole, is discarded after standing 30 seconds, so weight It is 5 times multiple, it pats dry；

(5) 50 μ L of enzyme marking reagent is added in every hole；

(6) it incubates, washing, step is same as above；

(7) 50 μ L of tmb substrate solution A is first added in every hole, adds 50 μ L of tmb substrate solution B, and gently concussion mixes, 37 DEG C are protected from light colour developing 15 minutes；

(8) every hole adds 50 μ L of terminate liquid, terminates reaction；

(9) it is returned to zero with blank well, 450nm wavelength sequentially measures the absorbance (OD value) in each hole, calculates in each serum sample Protein content.

ELISA kit of the invention can prepare the application in osteoarthritis disorders detection kit.

Kit performance urine specimen acquisition of the present invention is noninvasive, can repeat to sample on a large scale, save convenient advantage, benefit ADIRF albumen and its polypeptide fragment are detected with urine specimen.

Although above the present invention is described in detail with a general description of the specific embodiments, On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause This, these modifications or improvements, belong to claimed model without departing from theon the basis of the spirit of the present invention It encloses.

Claims (10)

1. The application of ADIRF protein in the preparation of products for detection, auxiliary diagnosis or treatment of osteoarthritis. 2. The application according to claim 1, characterized in that the expression of the ADIRF protein is up-regulated in osteoarthritis samples. 3. The application according to claim 2, wherein the osteoarthritis sample is a urine sample. 4. The application according to claim 1, characterized in that the product can detect whether the patient suffers from osteoarthritis by detecting the expression level of ADIRF protein in the subject sample. 5. The application according to any one of claims 1-4, wherein the product includes reagents, kits, chips or medicines. 6. An ELISA kit for detecting osteoarthritis disease, characterized in that the kit includes a specific antibody reagent for the ADIRF protein. 7. The kit according to claim 6, wherein the specific antibody for the ADIRF protein is a monoclonal antibody and/or a polyclonal antibody. 8. test kit according to claim 7, is characterized in that, described test kit also comprises the enzyme label plate that anti-ADIRF monoclonal antibody coats, mark anti-ADIRF polyclonal antibody, protein standard item ADIRF, TMB substrate solution , Diluent, Wash Buffer, Stop Solution. 9. The use of the kit according to any one of claims 6-8 in the preparation of a kit for detection or auxiliary diagnosis of osteoarthritis. 10. The application of the specific antibody of ADIRF protein in the preparation of medicine for treating osteoarthritis.