CN109557198A - A kind of protein biomarkers and its application for diagnosis of osteoporosis - Google Patents

Abstract

The invention discloses a kind of protein biomarkers for diagnosis of osteoporosis and its applications, belong to technical field of biological.Biomarker of the present invention using 1 protein of Abl Interactor as diagnosis of osteoporosis.Protein biomarkers provided by the present invention can be used for preparing diagnostic kit, and it has excellent diagnostic value when being applied to blood sample Diagnosis of osteoporosis.Protein of the invention is individually used, diagnosis AUC can reach 0.76.Using 0.376ng/ml as section, sensitivity and specificity are respectively 73.3% and 64.3%.Joint CTX and P1NP, diagnosis AUC can reach 0.78.The protein can be used as the biomarker of the osteoporosis blood sample diagnosis of people, facilitate the diagnosis of osteoporosis, the identification and its prevention and treatment of osteoporotic fracture people at highest risk.

Description

A kind of protein biomarkers and its application for diagnosis of osteoporosis

Technical field

The present invention relates to a kind of protein biomarkers for diagnosis of osteoporosis and its applications, belong to biological inspection Survey technology field.

Background technique

Osteoporosis (Osteoporosis, OP) is a kind of common chronic disease, and the clinical statures that show as become short, waist more Back is ached, Yi Fasheng low-wound fractures (also known as osteoporotic fracture, OF).OF disability rate is high, and lethality is up to 31%.Currently, the illness rate of Chinese residents osteoporosis is up to 8.8 ‰.The new biomarkers of screening OP, disclose its point Sub- pathogenic mechanism establishes OP early diagnosis prediction model, research and development target the particularly important strategy that intervening measure is control OP and arrange It applies.

OP is a kind of complex disease regulated and controled jointly by environmental factor and inherent cause, and wherein inherent cause is OP morbidity Important factor in order, OP morbidity in proportion be about 60-80%.Existing genome-wide association study has found largely OP tumor susceptibility gene, but the molecular mechanism of related OP morbidity not yet studies maturation at present.The science of heredity susceptibility loci joint having found Get up to explain OP heritability very little, still there is a large amount of gene to remain to be discovered, and the function of known disease gene is also big It is mostly indefinite.Product of the protein as gene expression is the main executive of gene function, can be by influencing cytoskeleton Structure adjusts the activity of cellular gene expression, cell etc. to play a role.

Peripheral blood mononuclear cells is the precursor of osteoclast, and can migrate to bone tissue Surface Differentiation is osteoclast, Bone resorption is participated in, it is closely related with bone loss and morbidity.Contain protein abundant in blood plasma, is reached by blood circulation complete Body respectively organizes (including bone), participates in life process.Unconventionality expression is filtered out from the peripheral blood mononuclear cells of patient OP and blood plasma Protein as biomarker, and develop corresponding auxiliary diagnostic box, it will help the early stage of osteoporosis examines Disconnected and prevention and treatment.

Summary of the invention

In order to solve the above technical problems, providing a kind of protein biomarkers for diagnosis of osteoporosis, simultaneously There is provided it is a kind of can be used in course of disease early detection and when detecting have higher sensitivity and the osteoporosis compared with high specific Detection kit and its application.

The first purpose of the invention is to provide a kind of biomarker for diagnosis of osteoporosis, the biology mark Will object is a-protein bl Interactor 1, also referred to as Abelson interactor 1 or Abelson-binding protein 4.Since the albumen can be by the ABI1 coded by said gene of people, hereinafter referred to as ABI1 protein.

In one embodiment of the invention, the amino acid sequence of the ABI1 protein such as SEQ ID NO.1 institute Show.

In one embodiment of the invention, the ABI1 protein source is in peripheral blood mononuclear cells.

In one embodiment of the invention, the ABI1 protein source is in blood plasma.

A second object of the present invention is to provide a kind of antibody for diagnosis of osteoporosis, the antibody can specificity It identifies and combines ABI1 protein.

Third object of the present invention is to provide a kind of kits for diagnosis of osteoporosis, comprising to plasma A BI1 The antibody that protein content is measured.

In one embodiment of the invention, the antibody can specific recognition ABI1 protein.

It in one embodiment of the invention, further include the component of ELISA detection, the component includes detection plate, delays Fliud flushing and ABI1 protein standard specimen.

Fourth object of the present invention is to provide the protein biomarkers in preparing bone health screening product Application.

Protein biomarkers of the invention are evaluated in bone health, including in application, including: in diagnosis of osteoporosis The plasma concentration of subject's biomarker is measured to obtain measured value, the protein biomarkers are ABI1 protein； By the measured value compared with reference value, if measured value is lower than reference value, it is determined that subject suffers from osteoporosis.

The application specifically includes: collecting limosis vein blood, the separated plasma of subject；It provides and the biomarker Corresponding protein antibody kit；The measured value is measured by ELISA detection method.

The beneficial effects of the present invention are:

Protein biomarkers provided by the present invention can be used for preparing diagnostic kit, and it is being applied to blood plasma sample When product Diagnosis of osteoporosis, with excellent sensitivity and specificity.Protein of the invention is individually used, diagnosis AUC can Reach 0.76.Using 0.376ng/ml as section, sensitivity and specificity are respectively 73.3% and 64.3%.Joint CTX and P1NP, diagnosis AUC can reach 0.78.The protein can be used as the biomarker of the osteoporosis plasma sample diagnosis of people, Facilitate the diagnosis of osteoporosis, the identification and its prevention and treatment of osteoporotic fracture people at highest risk.

Detailed description of the invention

Fig. 1 is the Mass Spectrometric Identification figure of ABI1 protein in peripheral blood mononuclear cells；Wherein, A is osteoporotic fracture trouble Person；B is normal healthy controls individual；

Fig. 2 is that ABI1 protein expresses significant imbalance in peripheral blood mononuclear cells；Wherein, left figure is osteoporotic bone It rolls over patient (N=36, FC=0.73, P < 0.05), right figure is sufferers of osteoporosis face (N=18, FC=0.82, P < 0.05)；

Fig. 3 is patients with osteoporotic bone fracture (OF), sufferers of osteoporosis face (OP), bone density normal individual (CTRL) The expression of ABI1 protein in peripheral blood mononuclear cells；Wherein, A is mass spectrum label-free result (LFQ signal strength Value)；B is Western Blotting result；

Fig. 4 is that ABI1 protein compares in individual blood plasma in 20 patients with osteoporotic bone fracture and 64 without History of Bone Fracture Concentration difference (N=84；FC=0.39；P<0.05)；

Fig. 5 is concentration difference (N=75 of the ABI1 protein in 38 sufferers of osteoporosis face and 37 human normal plasmas； FC=0.70；P<0.05)；

Fig. 6 is concentration difference (N=64 of the ABI1 protein in 32 sufferers of osteoporosis face and 32 human normal plasmas； FC=0.32；P<0.05)；

Fig. 7 is Receiver Operating Characteristics (ROC) curve of sufferers of osteoporosis face in ABI1 protein identification crowd；Wherein, A-B is that 2 known Bone markers are used alone, and C is that ABI1 protein marker is used alone in the invention, and D is the invention The evaluation result that middle ABI1 protein marker and 2 known Bone markers are used in combination.

Specific embodiment

The present invention will be further explained below with reference to the attached drawings and specific examples, so that those skilled in the art can be with It more fully understands the present invention and can be practiced, but illustrated embodiment is not as a limitation of the invention.

In embodiment do not specify actual conditions experiment, usually according to normal condition as manufacturers instruction, experiment guide or The condition of textbook content.

Before being described to example, it is necessary to remarks explanation: will cause experiment knot using the reagent of different manufacturers, different batches The difference of fruit, belongs to normal phenomenon.

Embodiment 1: the screening (9:9) for protein of lacking of proper care in sufferers of osteoporosis face peripheral blood mononuclear cells

1, after obtaining informed consent, 9 patients with osteoporosis and 9 bone health individuals are had collected as normal right According to acquisition empty stomach peripheral blood sample is placed in sodium citrate anticoagulant tube.

2, peripheral blood mononuclear cells obtained by density-gradient centrifugation method and MACS positive method for separating, carry out cell protein The identification and quantification of matter group.Specific step is as follows:

(1) peripheral blood mononuclear cells separates

1. 15ml density gradient separation liquid is added in leucosep pipe, centrifugation, 100g, 1min.

2. the fresh whole blood being collected into is poured into the centrifuge tube of 50ml, equivalent PBS is added and mixes, then moves to leucosep Guan Zhong, centrifugation, 310g, 20min.

It is discarded 3. the remaining supernatant on leucocyte group upper layer in leucosep pipe is sucked out, leucocyte group is moved into new 50ml Centrifuge tube adds PBS to 15ml, centrifugation, 400g, 10min.

After 4.50ml centrifuge tube is centrifuged leucocyte, supernatant is discarded, the cell precipitation of tube bottom is only stayed.

(2) magnetic mark

1. 500 μ l BSA buffer are added, piping and druming is mixed.

2. 50 μ l CD14 magnetic beads are added, piping and druming is mixed.

3. being incubated for 15min (2-8 DEG C) (incubation time is unsuitable too long, non-specific binding otherwise easily occurs) in refrigerator.

4. 10ml BSA buffer is added, it is centrifuged 310g, 10min, supernatant is discarded after centrifugation.

5. 500 μ lBSA buffer are added, piping and druming precipitating is mixed.

(3) peripheral blood mononuclear cells separates

1. installing LS/MS pipe in magnetic field.Rinse: 500 μ l BSA buffer are added in pipe and rinse MS pipe.

2. collecting unlabelled cell with 15ml centrifuge tube: the cell liquid that piping and druming is completed being added in MS pipe, every time with 500 μ l PBS rinses MS pipe, rinses 3 times.Liquid is allowed to drip naturally in the process.

3. MS pipe is removed magnetic field, CD14+ is collected with 15ml centrifuge tube and marks cell: 1ml PBS being added in MS, will live Plug is pushed into rapidly the bottom of LS/MS pipe.

4. target cell 1 manages (1ml) centrifugation, 2000rpm/min, 10min.

5. discarded supernatant drains Liquid Residue with toilet paper.CD14+ target cell saves in -80 DEG C, is spare.

(4) extraction, pretreatment of peripheral blood mononuclear cells total protein and quantitative

1. 300 μ l SDT buffer (4%SDS, 1.0mM DTT, 100mM are added in every pipe CD14+ target cell sample Tris-HCl, PH=7.6) lytic cell, 15min is incubated in boiling water bath.

2. high speed centrifugation, 14000g, 40min collect supernatant, peripheral blood mononuclear cells total protein is obtained.

3. 9 samples of case group and 9 samples of control group are mixed respectively in group with any three for a small group It closes, obtains the peripheral blood mononuclear cells albumen mixing sample of 3 case groups and 3 control groups.

4. taking 6 albumen sample boiling water bath 5min after merging, ultrasound cracking.

5. centrifugation, 13400rpm/min, 30min take supernatant.

6.BCA method carries out protein quantification.

(5) the SDS-PAGE experiment of protein

Every group of sample take 20 μ g protein sample 5:1 (v/v) be added 5 times of concentration sample-loading buffers, boiling water bath 5min, 14000g centrifugation 10min takes supernatant, carries out 12.5%SDS-PAGE electrophoresis.Deposition condition: constant current: 15mA, electrophoresis time 60min.Coomassie brilliant blue staining.

(6) the FASP enzymatic hydrolysis of protein

Every part of sample takes 200 μ g respectively, is separately added into DTT to final concentration of 100mM, and boiling water bath 5min is cooled to room temperature. 200 μ L UA buffer (8M Urea, 150mM Tris-HCl pH8.0) mixing is added, is transferred to 30kd ultra-filtration centrifuge tube, is centrifuged 14000g 15min.200 μ L UA buffer centrifugation 14000g 15min is added, abandons filtrate.100 μ L IAA (50mM are added IAA in UA), 600rpm vibrates 1min, is protected from light room temperature 30min, is centrifuged 14000g 10min.100 μ L UA buffer are added, Centrifugation 14000g 10min is repeated 2 times.100 μ L NH4HCO3buffer are added, centrifugation 14000g 10min is repeated 2 times.It is added 40 μ L Trypsin buffer (2 μ g Trypsin in, 40 μ L NH4HCO3buffer), 600rpm vibrate 1min, 37 DEG C 16-18h.Renew collecting pipe, be centrifuged 14000g 10min, collects filtrate, liquor C 18-SD Extraction Disk Cartridge desalting processing, OD280 are quantitative.

(7) the LC-MS/MS analysis of enzymolysis product

According to quantitative result, each sample respectively takes 2 μ g enzymolysis products to carry out LC-MS/MS analysis.Using a nanoliter flow velocity HPLC Liquid phase systems EASY-nLC1000 is separated.Liquid phase A liquid is 0.1% formic acid acetonitrile solution (acetonitrile 2%), and B liquid is 0.1% formic acid acetonitrile solution (acetonitrile 84%).150 μm of * 100mm of chromatographic column Thermo EASY column SC200 (RP-C18) it is balanced with 100% A liquid.Sample is loaded to Thermo EASY column SC001traps by autosampler 150 μm of * 20mm (RP-C18) (Thermo), then through chromatography post separation, flow velocity 400nL/min.Related fluid phase gradient is as follows: 0 Minute --- 100 minutes, B linear gradient was from 0% to 45%；100 minutes --- 108 minutes, B linear gradient from 45% to 100%；108 minutes --- -120 minutes, B liquid maintained 100%.Enzymolysis product is used after capillary high performance liquid chromatography separates Q-Exactive mass spectrograph (Thermo Finnigan) is analyzed by mass spectrometry.Analyze duration: 120min, detection mode: just from Son, precursor scans range: the mass-charge ratio of the fragment of 300-1800m/z, polypeptide and polypeptide acquires in following manner: every Secondary full scan (full scan) acquires 20 fragment patterns storeds (MS2scan, HCD) afterwards.MS1 resolution ratio in M/Z 200 is 70, 000, MS2 in M/Z 200 resolution ratio be 17,500.

(8) the label-free analysis of Maxquant

The LC-MS/MS original document of 6 samples imports Maxquant software (version number 1.3.0.5) and carries out checking storehouse, carries out The nonstandard quantitative analysis of LFQ.Database be uniprot_human_151619_20160229.fasta (accession sequence 151619, Under be loaded in 2016-02-29).Major parameter is as follows:

Main search ppm:6Missed cleavage:2

MS/MS tolerance ppm:20De-Isotopic:TRUE

Enzyme:Trypsin

Database:uniprot_human_151619_20160229.fasta

Fixed modification:Carbamidomethyl (C)

Variable modification:Oxidation (M), Acetyl (Protein N-term)

Decoy database pattern:reverse

LFQ:TRUE

LFQ min.ratio count:1

Match between runs:2min

Peptide FDR:0.01

Protein FDR:0.01

(9) statistical analysis of Perseus

Maxquant treated data are for statistical analysis using Perseus 1.3.0.4 software, by comparing sclerotin Proteomic expression profile in osteoporosis patient group and Normal group peripheral blood mononuclear cells is filtered out to express in patient group and be lost The protein of tune.Group difference analysis the result shows that: ABI1 protein expression level exists significant between patient group and normal group Difference (P < 0.05).

Figure 1A shows the second order ms qualification figure of ABI1 protein in peripheral blood mononuclear cells.

Fig. 2 (left figure) shows that ABI1 protein expression is significantly lacked of proper care in sufferers of osteoporosis face peripheral blood mononuclear cells.By scheming As it can be seen that the ABI1 protein expression level of sufferers of osteoporosis face group is remarkably decreased (Fold Change compared with Normal group (FC)=0.82；P<0.05).

The screening (18:18) of embodiment 2:OF patient peripheral's blood monocyte imbalance protein

The present embodiment includes 18 OF patients and 18 controls without History of Bone Fracture.The age and gender of patient and control Match.Acquire peripheral blood, separation blood mononuclear cell, extract gross protein, digestion, identification and quantification, case-control variance analysis, Identify differential expression protein.

Specific experiment and analysis method process, same as Example 1, details are not described herein again.Analysis the result shows that: sclerotin There are significant difference (P < 0.05) for ABI1 protein expression level between osteoporotic fractures group (OF) and control group (NF).Fig. 2 is (right Figure) show ABI1 protein expression imbalance in patients with osteoporotic bone fracture peripheral blood mononuclear cells.As seen from the figure, with control group It compares, the ABI1 protein expression level of osteoporotic fracture group is remarkably decreased (FC=0.73；P<0.05).

Embodiment 3: the verifying of patients with osteoporosis peripheral blood mononuclear cells protein markers

One, the identification of osteoporosis protein markers

This embodiment contains 27 subjects altogether.Wherein, 9 be bone density normal individual (CTRL), 9 be osteoporosis suffer from Person (OP), 9 be patients with osteoporotic bone fracture (OF).It acquires peripheral blood, separation blood mononuclear cell, extract total protein of cell Matter is mixed using any 3 samples in organizing as a small group, measures concentration.Specific experiment and analysis method process, with implementation Example 1 is identical, and details are not described herein again.

Fig. 3 shows bone density normal individual (CTRL), sufferers of osteoporosis face (OP), patients with osteoporotic bone fracture (OF) There are significant difference (P=0.0053) for ABI1 protein expression level in peripheral blood mononuclear cells.As seen from the figure, just with bone density Normal group of individuals is compared, under the ABI1 protein expression level of sufferers of osteoporosis face group and patients with osteoporotic bone fracture group is significant Drop；Group difference has statistical significance.

Fig. 3 (A) is LFQ quantitative result.

Two, the verifying of osteoporosis protein markers

ABI1 protein in peripheral blood mononuclear cells is quantified using Western blotting.Specific step is as follows.

1.SDS-PAGE gel is prepared

The PAGE gel reagent preparation box (the green skies, P0012A) produced using the green skies, mentioning to specifications Show the separation gel and spacer gel (concentration glue) of successively preparation 8%.

2. protein sample is handled

5 × SDS-PAGE albumen the sample-loading buffer (the green skies, P0015) being concentrated in right amount is added in protein sample, 100 DEG C of white 3min of boiled egg.

3. loading and electrophoresis

After pretreated protein sample is cooled to room temperature, protein sample is directly loaded to SDS-PAGE glue well It is interior, for the ease of observation electrophoretic effects and transferring film effect, and judge molecular weight of albumen size, using pre- dsred protein (CST, Cat No.#14208) molecular weight standard as reference.

It is used when preparing electrophoresis liquid (the green skies, P0014B) electrophoresis to specifications and uses low-voltage constant pressure in upper layer glue Electrophoresis (80V, 20min), and high voltage (100V, 80min) is used when bromophenol blue enters lower layer's glue.Usual bromophenol blue reaches The bottom end of glue nearby can stop electrophoresis, or according to the electrophoresis situation of pre-dyed standard protein, prompt destination protein by It fits when disengaged, electrophoresis can be stopped.

Glue after electrophoresis is cut according to the size of albumen sample separated region, the glue after having cut is spare.

4. transferring film

It cuts with the sizable pvdf membrane of glue surface product, the film after cutting first impregnates 3-5min with methanol, to specifications It prepares transferring film liquid (the green skies, P0021B), it can multi-functional electrophoresis apparatus progress transferring film operation (330mA, 60min) using day.

Serious fever phenomenon can be generated in During migration, carry out transferring film so transferring film slot is placed in ice water.Transferring film Effect can be found in the standard protein of pre-dyed, the usual maximum 1-2 band of molecular weight is more difficult to be transferred completely on film, can also To be dyed with Coomassie brilliant blue rapid dye liquor to the SDS-PAGE for completing transferring film, the residual condition of albumen is observed.

5. closing

After transferring film, protein film is placed into preprepared 5% skimmed milk power (TBST preparation) immediately, is being shaken It is slowly shaken on bed, room temperature closes 60min, and the moisturizing of film is paid attention to during whole operation, avoids the drying of film, otherwise easily Generate higher background.

6. primary antibody is incubated for

Film is cut off into two parts according to the position of pre-dyed standard protein, a part includes molecules of interest amount (55kDa) The ABI1 target protein of size, another part internal reference albumen of GAPDH containing house-keeping gene.With reference to ABI1 (CST, Cat No.# 39444), GAPDH (CST, Cat No.#5174) primary antibody specification, the dilution ratio prompted to specifications, with 5% degreasing Milk powder dilutes primary antibody.Confining liquid is exhausted, primary antibody is added immediately respectively, room temperature is incubated for 1h on shaking table.

7. washing

Primary antibody liquid is exhausted, Western cleaning solution (TBST) is added immediately, washing 10min is shaken on shaking table, exhausts washing After liquid, cleaning solution washing 10min is added, is washed altogether three times.

8. secondary antibody is incubated for

ABI1 and GAPDH primary antibody are rabbit source, and secondary antibody selects HRP to mark goat anti-rabbit IgG antibody (good fortune Mace, FMS- RB01), the dilution ratio for recommending 1:2000 to specifications, with 5% skimmed milk power dilution secondary antibody, spare.Exhaust cleaning solution Afterwards, the secondary antibody diluted is added immediately, room temperature is slowly incubated for 1h on shaking table.

9. washing

Washing step such as step 7 10. is developed

Using the super quick ECL chemical luminescence reagent kit (the green skies, P0018) of BeyoECL Plus, exposure development detects albumen Expression.As a result as shown in Fig. 3 (B).

Fig. 3 B is Western blotting result-protein development figure.The signal strength of ABI1 protein band in figure Display: bone density normal individual (CTRL), sufferers of osteoporosis face (OP), patients with osteoporotic bone fracture (OF) peripheral blood mononuclear are thin It is visible group difference that ABI1 protein expression level, which has naked eyes, in born of the same parents.Image quantitative analysis and comparison among groups show: with Bone density normal individual group is compared, the ABI1 protein expression level of sufferers of osteoporosis face group and patients with osteoporotic bone fracture group It is remarkably decreased；Group difference has statistical significance (P < 0.05).

The identification of embodiment 4:OF patients blood plasma's protein markers

The embodiment includes 84 elderly men samples, wherein 20 are patients with osteoporotic bone fracture, 64 are bone-free Roll over the control individual of history.Plasma A BI1 protein detection step is as follows:

One, blood specimen collection, blood plasma separation

1. extracting empty stomach volunteer about 10ml venous blood with sodium citrate anticoagulant tube.

2. under the revolving speed of 500g, high speed centrifugation 10min upper plasma is carefully sucked out spare.

Two, ELISA quantifies plasma A BI1 protein concentration

Restore 1. ABI1 protein ELISA kit and test plasma sample are taken out to place to room temperature.

2. the standard sample of 50 μ l or the test plasma sample of 50 μ l, blank tune are added in the hole of ELISA detection plate The Sample dilution zeroing of 50 μ l is added in zero hole.

3. the enzyme solutions of 100 μ l and mixing are added in each hole.

1h is incubated for 4. will test plate and be placed in 37 DEG C of incubator.

5. take out detection plate, by board-washing machine, with 1 × cleaning solution board-washing four times.

6. each Kong Zhongxian is added 50 μ l substrate A and adds 50 μ l substrate B, 37 DEG C are protected from light incubation 15min.

7. 50 μ l terminate liquids are added in every hole and mix, color can be turned yellow by blue at this time.

8. detecting the absorbance value in every hole with microplate reader under the conditions of wavelength 450nm.

9. data are analyzed: drawing standard curve according to the method for ELISA kit prompt, acquire corresponding to each sample Plasma A BI1 concentration.

Fig. 4 shows ABI1 protein in the control individual blood plasma of 20 patients with osteoporotic bone fracture and 64 without History of Bone Fracture In concentration difference (N=84；FC=0.39；P<0.05).

The verifying of embodiment 5:OP patients blood plasma's protein markers

The embodiment includes 75 elderly men samples, wherein 38 are sufferers of osteoporosis face, 37 are normal healthy controls.Hip Whether bone bone density is measured using DEXA and is diagnosed with osteoporosis.Plasma A BI1 determination of protein concentration step and reality It is identical to apply example 4, repeats no more.

Fig. 5 shows concentration difference schematic diagram of the ABI1 protein in 38 sufferers of osteoporosis face and 37 human normal plasmas (N=75；FC=0.70；P<0.05).

The verifying of embodiment 6:OP patients blood plasma's protein markers

The embodiment includes 64 elderly men samples, wherein 32 are sufferers of osteoporosis face, 32 are normal healthy controls.Hip Whether bone bone density is measured using DEXA and is diagnosed with osteoporosis.Plasma A BI1 determination of protein concentration step and reality It is identical to apply example 4, repeats no more.

Fig. 6 shows concentration difference (N=of the ABI1 protein in 32 sufferers of osteoporosis face and 32 human normal plasmas 64；FC=0.32；P<0.05).

Embodiment 7: the performance evaluation of plasma A BI1 Diagnosis of osteoporosis: ROC analysis

The embodiment includes 64 elderly men samples, wherein 32 are sufferers of osteoporosis face, 32 are normal healthy controls.Hip Whether bone bone density is measured using DEXA and is diagnosed with osteoporosis.Plasma A BI1 determination of protein concentration step and reality It is identical to apply example 4, repeats no more.As reference, commercial kits (I type is respectively adopted in P1NP the and beta-CTX content in blood plasma Collagenous amino end extends peptide detection kit and beta- Collagen Degradation Products detection kit) measurement.ROC analysis uses SPSS 20.0 softwares are completed, as a result as shown in Figure 7.

Fig. 7 shows the ROC curve of plasma A BI1 protein.Wherein A-B is that 2 known Bone markers are used alone, C It is used alone for the protein markers ABI1 of the invention, D is ABI1 protein and 2 known bone metabolic indicators in the invention Object (P1NP, beta-CTX) is used in combination.As can be seen from Figure: protein biomarkers of the invention are individually used, under curve Area (AUC) is 0.76 (95% confidence interval: 0.63-0.88), is had diagnostic value (P=0.001)；And it is substantially better than Beta-CTX (AUC=0.65) and P1NP (AUC=0.61).After joint beta-CTX and P1NP, diagnosis performance is slightly improved (AUC=0.78,95% confidence interval: 0.599-0.965, P=0.016).Using 0.376ng/ml as section, plasma proteins It is respectively 73.3% and 64.3% that ABI1, which individually distinguishes case and the sensitivity and specificity of control,.

In conclusion protein biomarkers ABI1 provided by the present invention can be used for making by above-mentioned technical proposal Standby diagnostic kit, and it has the performance better than markers with known when being applied to the diagnosis of osteoporosis of plasma sample, Its application helps to improve the early diagnosis and and prevention and treatment of China's osteoporosis.

Embodiment described above is only to absolutely prove preferred embodiment that is of the invention and being lifted, protection model of the invention It encloses without being limited thereto.Those skilled in the art's made equivalent substitute or transformation on the basis of the present invention, in the present invention Protection scope within.Protection scope of the present invention is subject to claims.

Sequence table

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Claims (9)

1. a kind of biomarker for diagnosis of osteoporosis, which is characterized in that the biomarker is Abl 1 protein of Interactor.

2. biomarker according to claim 1, which is characterized in that the Abl Interactor1 protein Amino acid sequence is as shown in SEQ ID NO.1.

3. biomarker according to claim 1, which is characterized in that 1 protein of Abl Interactor comes Derived from peripheral blood mononuclear cells.

4. biomarker according to claim 1, which is characterized in that 1 protein of Abl Interactor comes Derived from blood plasma.

5. a kind of antibody for diagnosis of osteoporosis, which is characterized in that the antibody can specific recognition and combine Abl 1 protein of Interactor.

6. a kind of kit for diagnosis of osteoporosis, which is characterized in that include the antibody described in claim 5.

7. kit according to claim 6, which is characterized in that further include the component of ELISA detection, the component includes 1 protein standard specimen of detection plate, buffer and Abl Interactor.

8. biomarker described in claim 1 is preparing the application in bone health screening product.

9. biomarker described in claim 1 is preparing the application in diagnosing osteoporosis product.