CN109486942A

CN109486942A - A kind of biomarker and its application for diagnosis of rheumatoid arthritis

Info

Publication number: CN109486942A
Application number: CN201811599941.6A
Authority: CN
Inventors: 邓飞艳; 雷署丰; 何培
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2018-12-26
Filing date: 2018-12-26
Publication date: 2019-03-19
Anticipated expiration: 2038-12-26
Also published as: CN109486942B

Abstract

The invention discloses a kind of biomarker for diagnosis of rheumatoid arthritis and its applications, belong to technical field of biological.The present invention is using the mRNA of gene RPN2 and protein as the biomarker of diagnosis of rheumatoid arthritis.Corresponding primer sets provided by the present invention and probe and protein can be used for reagent preparation box, and it has excellent sensitivity and specificity when being applied to peripheral blood mononuclear cells and plasma sample diagnosis rheumatoid arthritis.The AUC value of RPN2 mRNA of the present invention can achieve 0.911, have excellent diagnostic value；It is section with 3.3, sensitivity and specificity are respectively 82.1% and 83.3%.The AUC value of RPN2 protein of the present invention is 0.672, and using 14.29ng/ml as section, sensitivity and specificity are respectively 65.0% and 67.5%.Therefore, RPN2 mRNA and protein shown in the present invention can be used as the biomarker of rheumatoid arthritis, and for the diagnosing and treating of rheumatoid arthritis, having can application value.

Description

A kind of biomarker and its application for diagnosis of rheumatoid arthritis

Technical field

The present invention relates to a kind of biomarker for diagnosis of rheumatoid arthritis and its applications, belong to biological detection Technical field.

Background technique

Rheumatoid arthritis (rheumatoid arthritis, RA) be it is a kind of with corrode joint be mainly show it is slow Property autoimmune disease.The disease, which usually passes through to corrode cartilage and destroy skeletal joint, leads to that muscle and bone be unsound, body Body hypofunction and induction symbiosis disease, advanced stage disability rate are higher.If do not treated or to treatment reactionless, inflammation and destruction of joint It will lead to body function forfeiture, seriously affect the quality of life of patient, and with heavy social economical burden.Therefore screening RA New biomarkers, disclose its molecule pathogenic mechanism, establish early stage diagnosing model and preventive means be control RA exempt from Later period skeletal injury or the particularly important Strategic Measure of deformity are prevented and slowed down to epidemic disease inflammation.

RA is a kind of complex disease regulated and controled jointly by environmental factor and inherent cause, and nosogenesis and mechanism are not yet bright Really.Inherent cause is the important factor in order of RA morbidity, and proportion is about 50-60% in RA morbidity.Previous full genome Group association study has found more than 100 tumor susceptibility gene relevant to RA, and wherein most related to being immunized.However it has been found that Science of heredity susceptibility loci joins together to explain RA heritability very little (15%), still has a large amount of gene to remain to be discovered.It is clinical Upper traditional serodiagnosis index includes the anti-citrulling protein antibodies (ACPA) of autoantibody and rheumatoid factor (RF).ACPA's Sensitivity about 67%, it is lower in early stage RA patient.The specificity of RF is 38%-85%.Two indices join together have than The single better diagnosis effect of index.However, some researches show that the ACPA and RF diagnostic models combined to be only capable of correctly identifying 54%- 57% RA patient.Therefore, there are also to be developed for the biomarker with superior function.

Containing a series of and immune-related cell in peripheral blood mononuclear cells, such as T cell, B cell, monocyte Deng, therefore the functional group researchs for RA most at present are with peripheral blood mononuclear cells for research target cell.Accumulate in blood plasma Contain protein abundant, is respectively organized by blood circulation arrival whole body, participates in life process.In peripheral blood mononuclear cells and The gene of the unconventionality expression in RA is filtered out in blood plasma as biomarker, and develops corresponding auxiliary diagnostic box, it will Strong early diagnosis, predicted treatment, the monitoring recurrence etc. for pushing China RA, has important clinical value.

Summary of the invention

In order to solve the above technical problems, providing a kind of peripheral blood mononuclear cells base for diagnosis of rheumatoid arthritis Cause and plasma proteins biomarker.There is provided one kind simultaneously can be used in early detection and has higher sensitivity when detecting With compared with the rheumatoid arthritis detection kit of high specific and its application.

It is described the first purpose of the invention is to provide a kind of biomarker for diagnosis of rheumatoid arthritis Biomarker be gene RPN2 (full name: ribophorin II) coding RPN2mRNA or RPN2 protein (protein title: dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 2；Not Name: dolichyl-diphosphooligosaccharide--protein glycosyltransferase 63kDa subunit；oligosaccharyltransferase complex subunit(non-catalytic)；ribophorin- 2)。

In one embodiment of the invention, the nucleotide sequence of the RPN2mRNA is as shown in SEQ ID NO.1.

In one embodiment of the invention, the amino acid sequence of the RPN2 protein is as shown in SEQ ID NO.2.

In one embodiment of the invention, RPN2mRNA the and RPN2 protein is respectively derived from peripheral blood list A nucleus and blood plasma.

A second object of the present invention is to provide a kind of RPN2mRNA primer sets for diagnosis of rheumatoid arthritis and The combination of probe.

Third object of the present invention is to provide a kind of antibody for diagnosis of rheumatoid arthritis, the antibody can be special The opposite sex identifies and combines RPN2 protein.

Fourth object of the present invention is to provide a kind of kit for diagnosis of rheumatoid arthritis, comprising to periphery The combination of RPN2mRNA expression quantity is detected in blood mononuclear cell mRNA primer sets and mRNA probe；Or comprising to blood plasma The antibody that RPN2 protein content is measured.

In one embodiment of the invention, the mRNA primer sets include the quantitative PCR reverse transcription of RPN2mRNA Forward primer and reverse primer.

In one embodiment of the invention, the quantitative PCR reverse transcription forward primer of the RPN2mRNA and reversely draw The nucleotide sequence of object is respectively as shown in SEQ ID NO.3 and SEQ ID NO.4, or such as SEQ ID NO.5 and SEQ ID NO.6 It is shown.

In one embodiment of the invention, the kit further includes the component of qPCR augmentation detection, described The component of qPCR augmentation detection includes reverse transcriptase, buffer, dNTPs, MgCl₂、dd H₂O, fluorescent dye, Taq enzyme, standard items And reference material.

In one embodiment of the invention, the antibody can specific recognition RPN2 protein.

In one embodiment of the invention, the kit further includes the component of ELISA detection, the component packet Include detection plate, buffer and RPN2 protein standard specimen.

Fifth object of the present invention is to provide a kind of chips for diagnosis of rheumatoid arthritis, including for pair The mRNA probe that RPN2mRNA is detected.

In one embodiment of the invention, the nucleotide sequence of the mRNA probe and the mRNA probe The overall length maturation mRNA complete complementary of test object.

Sixth object of the present invention is to provide the mRNA biomarkers to detect rheumatoid arthritis in preparation Product in application.

The application of mRNA biomarker of the invention in diagnosis of rheumatoid arthritis includes: measurement from tested To obtain measured value, the biomarker is the expression of the biomarker of the peripheral blood mononuclear cells of person RPN2mRNA；By the measured value compared with reference value, if RPN2mRNA measured value is higher than reference value, it is determined that subject suffers from There is rheumatoid arthritis.

In one embodiment of the invention, the application specifically includes: extracting the single core of peripheral blood of subject Biomarker in cell；Primer sets corresponding with the biomarker and probe are provided；And pass through the detection side PCR Method measures the measured value.Wherein, PCR detection is positive/negative to primer sequence such as SEQ ID NO.3 (forward direction)/SEQ ID NO.4 Shown in (reversed) or SEQ ID NO.5 (forward direction)/SEQ ID NO.6 (reversed).

In one embodiment of the invention, the application specifically includes: the single core of peripheral blood for extracting subject is thin MRNA biomarker in born of the same parents；There is provided detection chip, the detection chip be loaded with the overall length of the biomarker at The mRNA probe of ripe mRNA fully-complementary sequence；And the detected value is measured with the detection chip.

The beneficial effects of the present invention are:

RPN2 gene biological marker provided by the present invention and its corresponding primer sets and probe can be used for preparing diagnosis Kit, and it has excellent when being applied to the diagnosis of rheumatoid arthritis of peripheral blood mononuclear cells and plasma sample Sensitivity and specificity.The AUC value of RPN2mRNA of the present invention can achieve 0.911, be section with 3.3, sensitivity and special Property is respectively 82.1% and 83.3%；The AUC value of RPN2 protein of the present invention can achieve 0.672, be to cut with 14.29ng/ml Point, sensitivity and specificity are respectively 65.0% and 67.5%.The gene can be used as the rheumatoid arthritis periphery of people The biomarker of blood mononuclear cell and plasma sample diagnosis is conducive to that the early stage of China's rheumatoid arthritis is pushed to examine Disconnected, predicted treatment, monitoring recurrence development.

Detailed description of the invention

In Fig. 1, A is expression (N=of the RPN2mRNA of the present invention in rheumatoid arthritis patients and normal control 43, FC=2.09, P < 0.05)；B is that RPN2mRNA of the present invention is used to distinguish patient with rheumatoid arthritis and normal control ROC curve figure；

Fig. 2 is RPN2mRNA of the present invention in 35 rheumatoid arthritis and 35 healthy human peripheral blood mononuclearcell samples Expression in this changes schematic diagram (N=70, FC=1.14, P < 0.05)；

Fig. 3 is expression water of the RPN2mRNA of the present invention in 6 rheumatoid arthritis and 3 healthy human T-cell's samples Flat schematic diagram (N=9, FC=1.79, P < 0.05)；

Fig. 4 is RPN2 protein in peripheral blood mononuclear cells of the present invention in 18 rheumatoid arthritis and 10 health Expression schematic diagram (N=28, FC=1.29, P < 0.05) in people；

Fig. 5 is the Mass Spectrometric Identification figure of RPN2 protein in peripheral blood mononuclear cells of the present invention；Wherein, A is rheumatoid Arthritic；B is normal healthy controls individual；

In Fig. 6, A is expression water of the RPN2 protein in 40 rheumatoid arthritis patients and 40 normal control blood plasma Flat (N=80, FC=2.12, P < 0.05)；B is invention RPN2 protein for distinguishing patient with rheumatoid arthritis and normal right According to ROC curve figure；

In Fig. 7, A is expression water of the RPN2mRNA in the post-stimulatory Jurkat cell of PHA and the control cell not stimulated Flat (P < 0.05)；B is influence (P < 0.05) of the RPN2 overexpression to the cell cycle of Jurkat cell；C is RPN2 overexpression pair The influence (P < 0.05) of the Apoptosis of Jurkat cell；D is influence of the RPN2 overexpression to the cell activation of Jurkat cell (P<0.05)。

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.

When carrying out small scale experiments, to guarantee the repeatability between parallel laboratory test, it is proposed that after configuration reagent, mix well simultaneously Packing, to guarantee the homogeneity of each experiment reagent.

Embodiment 1: the cDNA microarray of differential expression mRNA in peripheral blood mononuclear cells

1, after obtaining patient's informed consent, 25 peripheral blood samples for being diagnosed as patient with rheumatoid arthritis are had collected And 18 Healthy Peoples as normal control peripheral blood sample in sodium citrate anticoagulant tube.

2, patient with rheumatoid arthritis and PBMC of healthy people, tool are obtained by density-gradient centrifugation method Steps are as follows for body:

(1) in leucosep separating pipe be added 15ml density gradient separation liquid, be centrifuged 100g, 1 minute.

(2) 15ml peripheral blood sample being centrifuged 2000rpm, the supernatant after layering in 2 minutes focuses on 15ml centrifuge tube, from Heart 2500g, 15 minutes.Then packing is stored in -80 DEG C of refrigerators to 2ml centrifuge tube.

(3) the supernatant amount being extracted is supplemented with the PBS of equivalent, then mixed liquor is concentrated in the centrifuge tube of 50ml.Then The PBS of equivalent mixed liquor is added.

(4) mixed liquor is moved in the leucosep pipe in (1), be centrifuged 500g, 20 minutes.

(5) supernatant abandoned in the separating pipe of top is inhaled.Middle layer leucocyte is extracted to new 50ml centrifuge tube, PBS is added extremely Final volume is 15ml, be centrifuged 400g, 10 minutes.

(6) supernatant abandoned in the centrifuge tube of top is inhaled.4ml PBS is added to mix, draws 10ul and carries out cell count, it is remaining thin Cytosol average mark is filled in 2ml cryovial, be centrifuged 2000rpm, 2 minutes.

(7) after draining centrifuge tube, wherein 3 are taken, is separately added into 1ml TRIzol to prevent RNA from degrading.After piping and druming mixes It is stored in -80 DEG C of refrigerators.

3, single from the peripheral blood of patient with rheumatoid arthritis and normal healthy controls added with TRIzol in discovery experiment After extracting Total RNA in nucleus, pass through full-length genome chip of expression spectrum (lncRNA+mRNA Human Gene Expression Microarray V4.0) detection mRNA expression.

Steps are as follows for major experimental:

After 3.1 extract the RNA in RA and PBMC of healthy people, further use RNA clean-up kit purifies Total RNA, then quantitative with spectrophotometer methods, uses agarose gel electrophoresis Method detects its integrality.

3.2 Total RNA synthesize cDNA

(1) reverse transcription synthesizes First Strand cDNA.Following examination is sequentially added in 0.2ml nuclease free centrifuge tube Agent:

a.5ul Total RNA(100-500ng)；

B. it is addedJoin (Cat.No.360030) 1ul outside gene expression profile, while respective volume is added according to table 1 Agilent spike-in.

Table 1

C. reverse transcription Master Mix is prepared on ice, is mixed gently, of short duration centrifugation is placed on ice bath, and Master Mix is anti- Answer system are as follows: the First Strand Enzyme of lncRNA+mRNA First Strand the Buffer Mix, 1ul of 4ul Mix。

D. 5ul reverse transcription Master Mix in c is taken out, is added in the centrifuge tube containing Total RNA sample, reverse transcription is most End reaction volume is 10ul.

E. soft pressure-vaccum mixes 2-3 times, and brief centrifugation is placed on ice.

F. reverse transcription centrifuge tube is placed in PCR instrument, 25 DEG C of reactions 1h, 42 DEG C of reactions 1h, 4 DEG C of holding 5min or more.It takes Reverse transcription centrifuge tube out, brief centrifugation are placed on ice, are ready for Second Strand cDNA synthetic reaction.

(2) Second Strand cDNA is synthesized.

A. Second Strand Master Mix is prepared on ice, is mixed gently, ice bath after of short duration centrifugation.Second Strand Master Mix reaction system are as follows: the Second Strand of the Nuclease-free Water, 12.5ul of 32.5ul The Second Strand Enzyme Mix of Buffer Mix, 5ul.

B. 50 μ L Second Strand Master Mix is taken to be added to the First Strand cDNA step of step synthesis In the reaction tube of middle f, mixed volume is 60 μ L；Pressure-vaccum mixes 2-3 times, and brief centrifugation is placed on ice.

C. the second chain synthesis centrifuge tube is placed in PCR instrument, 16 DEG C of reaction 1h (closing PCR instrument lid heating function), 65 DEG C Reaction 10 minutes, 4 DEG C are kept for 5 minutes or more.

D. reaction tube is placed in after reaction and continues synthetic reaction on ice, or frozen in -20 DEG C rapidly.

3.3 are transcribed in vitro synthesis cRNA

(1) cRNA is synthesized.

A. it prepares and Master Mix is transcribed in vitro, mix gently, solution is collected in tube bottom by of short duration centrifugation.Master Mix Reaction system are as follows: the T7Buffer Mix of 24ul；The T7Enzyme Mix of 6ul.

B. in the reaction tube in cDNA step for taking 30 μ L IVT Master Mix to synthesize to upper step, pressure-vaccum is mixed, wink When centrifugation place on ice.

C. synthesis centrifuge tube will be transcribed in vitro to be placed in PCR instrument, 40 DEG C of reaction 16h, 4 DEG C of holdings.

D. after reaction, brief centrifugation usesRNA clean-up kit (MN company, 740.948.250) product is purified, and cRNA product after purification is quantified using ultraviolet specrophotometer.

3.4cRNA reverse transcription generates cDNA

A. 10 μ g of cRNA purified product is taken, adjustment volume to 22 μ L is added in 0.2mL nuclease free centrifuge tube, and is added Enter 2 μ L Random Primer mixing, be placed in PCR instrument, 70 DEG C 5 minutes, 25 DEG C 5 minutes, 4 DEG C, 2 minutes, brief centrifugation receive Collect liquid to tube bottom, places on ice.

B. cRNA reverse transcription Master Mix is prepared, is mixed gently, solution is collected in tube bottom by of short duration centrifugation.Master Mix reaction system are as follows: the 2nd-Cycle Enzyme Mix of 2nd-Cycle the Buffer Mix, 8ul of 8ul.

C. in the centrifuge tube after taking 16 μ L reverse transcription Master Mix that 4.1.1 step reaction is added, total volume is 40 μ L, is blown Persorption is 2-3 times even, brief centrifugation.

D. cRNA reverse transcription centrifuge tube is placed in PCR instrument, 25 DEG C are reacted 10 minutes, 40 DEG C of reaction 1.5h, 70 DEG C of reactions 10 minutes, 4 DEG C were reacted 5 minutes, and centrifuge tube is placed on ice.

E. it operates on ice, 2 μ L RNase H mixing is added in cRNA reverse transcription centrifuge tube, brief centrifugation is placed in PCR instrument On, 37 DEG C are reacted 45 minutes, and 95 DEG C are reacted 5 minutes, and 4 DEG C maintain 5 minutes.

F. after reaction, it can be frozen at -20 DEG C overnight, or carry out purification process immediately.It uses Extract II (MN company, Cat.No.740609.250) kit carries out cDNA purifying, and uses ultraviolet specrophotometer pair CRNA product after purification is quantified.

3.5 fluorescent marker

(1) fluorochrome label reacts

A. the cDNA bulk product obtained by reverse transcription and after purification is concentrated into 14 μ L, and 4 μ L Random Primer are added It mixes, of short duration centrifugation is placed in PCR instrument, and 95 DEG C are denaturalized 3 minutes, ice bath 5 minutes.

B. the Cy5-dCTP (or Cy3-dCTP) of 5 × Klenow Buffer, 1ul of 5ul are sequentially added, 1.2ul's Klenow Fragmen。

C. it after of short duration centrifugation, is placed in PCR instrument, 37 DEG C are reacted 1.5 hours, and 70 DEG C are reacted 5 minutes.4 DEG C of holdings.

D. fluorochrome label after reaction, usesExtract II (MN company, Cat.No.740609.250) kit carries out cDNA purifying, and is produced using ultraviolet specrophotometer to fluorescent marker after purification Object carries out Fluorescent dye incorporation amount and nucleic acid quantification.

3.6 chip hybridization

(1) marked product hybridization prepares

A. it passes throughMarked product after Extract II kits, elution volume is in 30 μ L or so.

B. if carrying out single channel chip hybridization, single tube label (cy3-dCTP or cy5-dCTP) purifying eluted product is taken out Vacuum concentration or moisturizing volume are spare to 27.5 μ L.

C. if carrying out binary channels chip hybridization, 1 pipe cy3-dCTP label purified product and other 1 pipe cy3-dCTP are marked After remembering purified product mixing, total 60 μ L or so vacuumize that be concentrated into 27.5 μ L spare.

(2) preparation of hybridization system, hybridization reaction

A. by it is upper it is step by step rapid in ready marked product, 2 × GEx Hyb Buffer with 55ul, 27.5ul's The Sample of Formamide, 27.5ul are mixed.

B. 100 μ L hybridization solutions is taken to be loaded into hybridization cover plate fence, " Agilent " label gently covers fence down, After installing Agilent hybridizing box and screwing, hybridizing box can be gently horizontally rotated, check liquid in the hybridization cavity of each sub- battle array Whether flow.

C. hybridizing box is mounted on the rotor of hybrid heater, pays attention to being symmetrically installed, while addition is appropriate ultrapure in pallet After water, 45 DEG C of hybridized overnights.

The cleaning of 3.7 chips and scanning

A. after, taking-up chip is washed dry instrument in rich 8 chip of Austria Slide Washer and is cleaned, and cleaning procedure is as follows:

I:0.2%SDS, 2 × SSC, 42 DEG C of 120S of washing lotion are cleaned 2 times.

II:0.2%SDS, 2 × SSC, 42 DEG C of 80S of washing lotion are cleaned 3 times.

After the completion of cleaning procedure, centrifuge dripping is to be scanned

B. the chip after cleaning is scanned using Agilent chip scanner (G2565CA), obtains hybridization picture.

3.8 chip data analysis

The fluorescent scanning image of chip through AGCC software (CommandSoftware digital signal) is converted by picture signal, obtains the fluorescence signal intensity of each probe.Then fortune It is carried out with Robust Multi-array Average (RMA) module of Affimetrix Expression Console software Whether the pretreatment of data, normalization, probe signals including initial data are significantly higher than the differentiation of background signal, believe probe Number it is integrated into probe groups signal.Agilent GeneSpring software is reused data to be normalized and variance analysis.

MRNA Differential expression analysis.Analysis specifically include that by fold-change value (FC) method compare case group with it is right According to the mRNA differential expression of group；Utilize the difference p value between t checking computation case group and control group.Screening conditions are p < 0.05.

4, to the peripheral blood mononuclear cells gene expression of 25 patient with rheumatoid arthritis and 18 normal controls into Row Differential expression analysis, compared with normal control, RPN2mRNA expression up-regulation.The differential expression of RPN2mRNA has conspicuousness (Figure 1A, p < 0.05, Fold Change (FC)=2.09), and area (AUC) is 0.91 (p < 0.05 under the ROC curve of mRNA； 95% confidence interval: 0.83-0.99), there is diagnostic value.When section value is 3.3, sensitivity and specificity are respectively 82.1% With 83.3% (Figure 1B).

Embodiment 2: the qRT-PCR experiment of mRNA in peripheral blood mononuclear cells

1, according to chip results, qRT-PCR verifying is carried out to RPN2mRNA.To patient with rheumatoid arthritis and normal right According to peripheral blood mononuclear cells carry out the qRT-PCR detection of RPN2mRNA, implement strict quality control, each sample in entire research This continuous detection is at least three times.Forward and reverse primer used is as shown in SEQ ID NO.3 and SEQ ID NO.4.

2, after obtaining patient's informed consent, collect peripheral blood sample that 35 are diagnosed as patient with rheumatoid arthritis and 35 Healthy Peoples as normal control peripheral blood sample in sodium citrate anticoagulant tube.

3, it is obtained outside patient with rheumatoid arthritis and normal person using density-gradient centrifugation method in the same manner as in Example 1 All blood mononuclear cells.

4, the reverse transcription to extraction, mRNA added with TRIzol peripheral blood mononuclear cells progress sample total serum IgE and cDNA Quantitative PCR.Steps are as follows for specific experiment:

The extraction of 4.1 sample rnas

(1) mononuclearcell being immersed in TRIzol is taken out, thaw at RT from refrigerator, is mixed；

(2) chloroform is added in 200ul chloroform/1mL TRIzol ratio, mixes rapidly 15 seconds and (does not have to vortex instrument to mix To prevent genomic DNA fracture), it is placed at room temperature for 2-3 minutes；

12000g is centrifuged 15 minutes at (3) 4 DEG C, extracts upper strata aqueous phase；

(4) be added with the isometric isopropanol of water phase, be mixed by inversion, be placed at room temperature for after ten minutes, at 4 DEG C 12000rpm from The heart 10 minutes, make RNA precipitate in tube bottom.

(5) supernatant is sucked out, -20 DEG C of 75% alcohol being pre-chilled suspension precipitatings are added into the pipe for have precipitating, and (alcohol is spent RNA enzyme water configuration)；

7500rpm is centrifuged 5 minutes at (6) 4 DEG C, is discarded upper layer waste liquid, air drying 5-10 minutes, is removed RNA with 30ul The water of enzyme dissolves RNA precipitate.

The digestion of genomic DNA is remained in 4.2RNA

The RNA stoste 30ul for taking above-mentioned steps to extract carries out DNA digestion reaction.Response procedures are 37 DEG C, 30 minutes. Reaction system is following (50ul): 30ul RNA stoste；0.5ul Recombinant Rnase Inhibitor；5ul DNaseI buffer；13.5ul Rnase Free H₂O；1ul Dnase I(Rnase free).

500ul TRIzol is added into the RNA that reaction obtains, carries out RNA extraction according to the step of 3.1 in embodiment 1, Being eventually adding 30ul goes the water of RNA enzyme to dissolve RNA.

4.3 is anti-by operation manual progress reverse transcription using 1st-Strand cDNA Synthesis kit (border is as biology) It answers.

4.4 carry out qPCR reaction using SYBR Green qPCR Master Mix Kit (border is as biology), and instrument uses Roche real-time fluorescence quantitative PCR instrument Roche light cycler 480II (Roche company).

5, data are analyzed

PCR amplification result indicates with Ct value, i.e. recurring number when fluorescence signal reaches set threshold value in PCR reaction.Meter The △ Ct value for calculating the mRNA of differential expression, is standardized, with relative quantification as internal reference using B2MMethod calculates case The differential expression of mRNA between control group.Comparison among groups use t-test comparing difference, and P < 0.05 is thought with statistical difference.

6, data analysis result is shown, the differential expression of RPN2mRNA has conspicuousness (Fig. 2, FC=1.14, p < 0.05).

Embodiment 3: the qRT-PCR experiment of mRNA in human T-cell

1, the qRT-PCR for carrying out RPN2mRNA to the T cell of patient with rheumatoid arthritis and normal control is detected, whole Implement strict quality control in a research, each sample continuously detects at least three times.Forward and reverse primer used such as SEQ ID NO.5 With shown in SEQ ID NO.6.

2, after obtaining patient's informed consent, 6 peripheral blood samples and 3 for being diagnosed as patient with rheumatoid arthritis are collected Example Healthy People as normal control peripheral blood sample in sodium citrate anticoagulant tube.

3, human T-cell extracts

(1) peripheral blood mononuclear cells is extracted according to described in example 1.

(2) EasySep Human Whole Blood CD3 Positive Selection Kit (STEMCELL is used Technologies, Canada) kit extract peripheral blood mononuclear cells in T cell.

A. 1ml PBS is added, peripheral blood mononuclear cells is resuspended, be transferred in new circular non-opaque pipe.

B. EasySep Human CD3 Positive the Selection Cocktail and EasySep of 60ul is added Two kinds of reagents of magnetic nanoparticles mark CD3+ cell, stand 3 minutes.

C. it shakes transparent pipe 30 seconds, the Rapidsphere of 45ul is added in transparent pipe, stand 3 minutes.

D. above-mentioned solution is placed in magnet, stands 3 minutes.

F. it has been inverted magnet and transparent pipe simultaneously, suspension has been poured out.Transparent pipe is removed into magnet, CD3+ cell pastes at this time On tube wall.

G. PBS is added, cell is resuspended, be centrifuged, 300g, 5 minutes.Abandon supernatant.Tube bottom pellet is target cell.

4, appropriate TRIzol is added in the T cell of extraction, carries out the extraction of sample total serum IgE.Specific experiment step is the same as real The extraction of RNA in example 3.

5, the RNA of 2ug is taken, is usedFirst-Strand cDNA Synthesis kit (Promega, the U.S.) carries out reverse transcription reaction by operation manual.

6, it usesQPCR Master Mix Kit (Promega, the U.S.) carries out qPCR reaction, and instrument uses 6 Flex of QuantStudioTM (Life Technologies, the U.S.).

7, data are analyzed

PCR amplification result indicates with Ct value, i.e. recurring number when fluorescence signal reaches set threshold value in PCR reaction.Meter The △ Ct value for calculating the mRNA of differential expression, is standardized, with relative quantification as internal reference using GAPDHMethod calculates disease The differential expression of mRNA between example and control group.Comparison among groups use t-test comparing difference, and P < 0.05 thinks poor with statistics It is different.

8, data analysis result is shown, the differential expression of RPN2mRNA has conspicuousness (Fig. 3, FC=1.79, p < 0.05).

Embodiment 4: the screening of differentially expressed protein in peripheral blood mononuclear cells

1, after obtaining patient's informed consent, collect peripheral blood sample that 18 are diagnosed as patient with rheumatoid arthritis and 10 Healthy Peoples as normal control peripheral blood sample in sodium citrate anticoagulant tube.

2, it is obtained outside patient with rheumatoid arthritis and normal person using density-gradient centrifugation method in the same manner as in Example 1 All blood mononuclear cells.

3, the extraction, pretreatment of peripheral blood mononuclear cells total protein and quantitative

(1) be added in every pipe peripheral blood mononuclear cells sample 300 μ l SDT buffers (4%SDS, 1.0mM DTT, 100mM Tris-HCl, PH=7.6) lytic cell, it is incubated for 15 minutes in boiling water bath.

(2) high speed centrifugation, 14000g 40 minutes, collect supernatant, obtain peripheral blood mononuclear cells total protein.Boiling water bath 5 Minute, ultrasound cracking.

(3) albumen sample is centrifuged, 13400rpm/min, 30 minutes, takes supernatant.

(6) BCA method carries out protein quantification.

4, the SDS-PAGE experiment of protein

Each sample respectively take 20 μ g protein sample 5:1 (v/v) be added 5 times of concentration sample-loading buffers, boiling water bath 5 minutes, 14000g, which is centrifuged, takes supernatant for 10 minutes, carries out 12.5%SDS-PAGE electrophoresis.Deposition condition: constant current: 15mA, electrophoresis time 60 divide Clock.Coomassie brilliant blue staining.

5, every part of sample takes 200 μ g respectively, is separately added into DTT to final concentration of 100mM, boiling water bath 5 minutes, 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, Centrifugation 14000g 15 minutes.200 μ L UA buffer are added to be centrifuged 14000g 15 minutes, abandon filtrate.100 μ L IAA are added (50mM IAA in UA), 600rpm vibrate 1 minute, are protected from light room temperature 30 minutes, are centrifuged 14000g 10 minutes.100 μ L are added UA buffer, centrifugation are repeated 2 times for 14000g 10 minutes.100 μ L NH4HCO3buffer are added, are centrifuged 14000g 10 minutes It is repeated 2 times.It is added 40 μ L Trypsin buffer (2 μ g Trypsin in, 40 μ L NH4HCO3buffer), 600rpm oscillation 1 minute, 37 DEG C of 16-18h.Renew collecting pipe, be centrifuged 14000g 10 minutes, collects filtrate, liquor C 18-SD Extraction Disk Cartridge desalting processing, OD280 are quantitative.

6, 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 uses Q- after capillary high performance liquid chromatography separates Exactive mass spectrograph (Thermo Finnigan) is analyzed by mass spectrometry.Duration: 120min is analyzed, detection mode: cation, Precursor scans range: the mass-charge ratio of the fragment of 300-1800m/z, polypeptide and polypeptide acquires in following manner: every time 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/Z200 resolution ratio be 17,500.

7, 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:6 Missed cleavage:2

MS/MS tolerance ppm:20 De-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 class wind Proteomic expression profile in wet arthritis patient group and Normal group peripheral blood mononuclear cells, filters out in patient group Express the protein of imbalance.Group difference analysis the result shows that: RPN2 protein expression level is deposited between patient group and normal group In significant difference (P < 0.05).

Fig. 4 is shown, compared with Normal group, RPN2 protein table in rheumatoid arthritis peripheral blood mononuclear cells Up to horizontal significant up-regulation (FC=1.29；P<0.05).

Fig. 5 is the second order ms qualification figure of RPN2 protein in peripheral blood mononuclear cells.Wherein, A is rheumatoid joint Scorching patient；B is normal healthy controls individual.

Embodiment 5: the identification of plasma proteins marker

After obtaining patient's informed consent, 40 peripheral blood samples and 40 for being diagnosed as patient with rheumatoid arthritis are collected Example Healthy People as normal control peripheral blood sample in sodium citrate anticoagulant tube.Blood plasma RPN2 protein detection step is as follows:

1, blood specimen collection, blood plasma separation

(1) about 10ml venous blood is under the revolving speed of 500g, high speed centrifugation 10min, upper plasma is carefully sucked out spare.

2, blood plasma RPN2 is quantified with the ELISA kit (MyBioSource, Cat.No:MBS9312546) of RPN2 albumen Protein concentration.It is tested by operation manual.

Fig. 6 shows that RPN2 protein compares the concentration difference in individual blood plasma in 40 patient with rheumatoid arthritis and 40 Different (Fig. 6 A, N=80；FC=2.12；P<0.05).Area (AUC) is 0.672 (Fig. 6 B, p < 0.05 under ROC curve；95% confidence Section: 0.55-0.79).Using 14.29ng/ml as section, sensitivity and specificity are respectively 65.0% and 67.5%.

Embodiment 6: the effect that specific RPN2 gene grows cell and activate in Jurkat immunocyte

Above-described embodiment shows: RPN2mRNA detects rheumatoid arthritis peripheral blood mononuclear cells with higher Diagnostic value (highly sensitive and specificity).The present embodiment carries out its pass between rheumatoid arthritis around RPN2 gene Join Mechanism Study.Jurkat cell is common CD4+T lymphocytic series.The stimulation of research inflammatory factor is to Jurkat cell first The influence of middle RPN2mRNA expression, as shown in Figure 7 A, Jurkat cell RPN2mRNA expression under PHA stimulation are significant Up-regulation.RPN2 overexpression is constructed in Jurkat cell using PGMLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro carrier Stably transfected cell line (OE) and control cell (NC), RPN2 overexpression can influence the Jurkat cell period (Fig. 7 B), promote Cell Proliferation (Fig. 7 C) and the cell-stimulating (Fig. 7 D) of PHA induction.

In conclusion by above-mentioned technical proposal, RPN2 gene biological marker provided by the present invention and its accordingly draw Object group and probe can be used for preparing diagnostic kit, and it is closed in the rheumatoid for being applied to peripheral blood mononuclear cells sample When section inflammation diagnosis, with excellent sensitivity and specificity.Further, inventor is confirmed by research, RPN2mRNA AUC value can achieve 0.911, when section value be 3.3 when, sensitivity and specificity are respectively 82.1% and 83.3%.RPN2 The AUC value of protein can achieve 0.672, and using 14.29ng/ml as section, sensitivity and specificity are respectively 65.0% He 67.5%.The gene can be used as the biomarker of human rheumatoid arthritis's peripheral blood mononuclear cells sample diagnosis, have Conducive to the development for pushing the early diagnosis of China's rheumatoid arthritis, predicted treatment, monitoring recurrence.

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

<110>University Of Suzhou

<120>a kind of biomarker and its application for diagnosis of rheumatoid arthritis

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<170> SIPOSequenceListing 1.0

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<212> DNA

<213>(artificial synthesized)

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ttggccctga caatcatagc cagcacctgg gctctgacgc ccactcacta cctcaccaag 120

catgacgtgg agagactaaa agcctcgctg gatcgccctt tcacaaattt ggaatctgcc 180

ttctactcca tcgtgggact cagcagcctt ggtgctcagg tgccagatgc aaagaaagca 240

tgtacctaca tcagatctaa ccttgatccc agcaatgtgg attccctctt ctacgctgcc 300

caggccagcc aggccctctc aggatgtgag atctctattt caaatgagac caaagatctg 360

cttctggcag ctgtcagtga ggactcatct gttacccaga tctaccatgc agttgcagct 420

ctaagtggct ttggccttcc cttggcatcc caagaagcac tcagtgccct tactgctcgt 480

ctcagcaagg aggagactgt gctggcaaca gtccaggctc tgcagacagc atcccacctg 540

tcccagcagg ctgacctgag gagcatcgtg gaggagattg aggaccttgt tgctcgcctg 600

gatgaactcg ggggcgtgta tctccagttt gaagaaggac tggaaacaac agcgttattt 660

gtggctgcca cctacaagct catggatcat gtggggactg agccatccat taaggaggat 720

caggtcatcc agctgatgaa cgcgatcttc agcaagaaga actttgagtc cctctccgaa 780

gccttcagcg tggcctctgc agctgctgtg ctctcgcata atcgctacca cgtgccagtt 840

gtggttgtgc ctgagggctc tgcttccgac actcatgaac aggctatctt gcggttgcaa 900

gtcaccaatg ttctgtctca gcctctgact caggccactg ttaaactaga acatgctaaa 960

tctgttgctt ccagagccac tgtcctccag aagacatcct tcacccctgt aggggatgtt 1020

tttgaactaa atttcatgaa cgtcaaattt tccagtggtt attatgactt ccttgtcgaa 1080

gttgaaggtg acaaccggta tattgcaaat accgtagagc tcagagtcaa gatctccact 1140

gaagttggca tcacaaatgt tgatctttcc accgtggata aggatcagag cattgcaccc 1200

aaaactaccc gggtgacata cccagccaaa gccaagggca cattcatcgc agacagccac 1260

cagaacttcg ccttgttctt ccagctggta gatgtgaaca ctggtgctga actcactcct 1320

caccagacat ttgtccgact ccataaccag aagactggcc aggaagtggt gtttgttgcc 1380

gagccagaca acaagaacgt gtacaagttt gaactggata cctctgaaag aaagattgaa 1440

tttgactctg cctctggcac ctacactctc tacttaatca ttggagatgc cactttgaag 1500

aacccaatcc tctggaatgt ggctgatgtg gtcatcaagt tccctgagga agaagctccc 1560

tcgactgtct tgtcccagaa ccttttcact ccaaaacagg aaattcagca cctgttccgc 1620

gagcctgaga agaggccccc caccgtggtg tccaatacat tcactgccct gatcctctcg 1680

ccgttgcttc tgctcttcgc tctgtggatc cggattggtg ccaatgtctc caacttcact 1740

tttgctccta gcacgattat atttcacctg ggacatgctg ctatgctggg actcatgtat 1800

gtctactgga ctcagctcaa catgttccag accttgaagt acctggccat cctgggcagt 1860

gtgacgtttc tggctggcaa tcggatgctg gcccagcagg cagtcaagag aacagcacat 1920

tagttccaga agaaagatgg aaattctgaa aactgaatgt caagaaaagg agtcaagaac 1980

aattcacagt atgagaagaa aaatggaaaa aaaaaacttt atttaaaaaa gaaaaaagtc 2040

cagattgtag ttatactttt gcttgttttt cagtttcccc aacacacagc agatacctgg 2100

tgagctcaga tagtctcttt ctctgacact gtgtaagaag ctgtgaatat tcctaactta 2160

cccagatgtt gcttttgaaa agttgaaatg tgtaattgtt ttggaataaa gagggtaaca 2220

ataggaa 2227

<210> 2

<211> 631

<212> PRT

<213>(artificial synthesized)

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Met Ala Pro Pro Gly Ser Ser Thr Val Phe Leu Leu Ala Leu Thr Ile

1 5 10 15

Ile Ala Ser Thr Trp Ala Leu Thr Pro Thr His Tyr Leu Thr Lys His

20 25 30

Asp Val Glu Arg Leu Lys Ala Ser Leu Asp Arg Pro Phe Thr Asn Leu

35 40 45

Glu Ser Ala Phe Tyr Ser Ile Val Gly Leu Ser Ser Leu Gly Ala Gln

50 55 60

Val Pro Asp Ala Lys Lys Ala Cys Thr Tyr Ile Arg Ser Asn Leu Asp

65 70 75 80

Pro Ser Asn Val Asp Ser Leu Phe Tyr Ala Ala Gln Ala Ser Gln Ala

85 90 95

Leu Ser Gly Cys Glu Ile Ser Ile Ser Asn Glu Thr Lys Asp Leu Leu

100 105 110

Leu Ala Ala Val Ser Glu Asp Ser Ser Val Thr Gln Ile Tyr His Ala

115 120 125

Val Ala Ala Leu Ser Gly Phe Gly Leu Pro Leu Ala Ser Gln Glu Ala

130 135 140

Leu Ser Ala Leu Thr Ala Arg Leu Ser Lys Glu Glu Thr Val Leu Ala

145 150 155 160

Thr Val Gln Ala Leu Gln Thr Ala Ser His Leu Ser Gln Gln Ala Asp

165 170 175

Leu Arg Ser Ile Val Glu Glu Ile Glu Asp Leu Val Ala Arg Leu Asp

180 185 190

Glu Leu Gly Gly Val Tyr Leu Gln Phe Glu Glu Gly Leu Glu Thr Thr

195 200 205

Ala Leu Phe Val Ala Ala Thr Tyr Lys Leu Met Asp His Val Gly Thr

210 215 220

Glu Pro Ser Ile Lys Glu Asp Gln Val Ile Gln Leu Met Asn Ala Ile

225 230 235 240

Phe Ser Lys Lys Asn Phe Glu Ser Leu Ser Glu Ala Phe Ser Val Ala

245 250 255

Ser Ala Ala Ala Val Leu Ser His Asn Arg Tyr His Val Pro Val Val

260 265 270

Val Val Pro Glu Gly Ser Ala Ser Asp Thr His Glu Gln Ala Ile Leu

275 280 285

Arg Leu Gln Val Thr Asn Val Leu Ser Gln Pro Leu Thr Gln Ala Thr

290 295 300

Val Lys Leu Glu His Ala Lys Ser Val Ala Ser Arg Ala Thr Val Leu

305 310 315 320

Gln Lys Thr Ser Phe Thr Pro Val Gly Asp Val Phe Glu Leu Asn Phe

325 330 335

Met Asn Val Lys Phe Ser Ser Gly Tyr Tyr Asp Phe Leu Val Glu Val

340 345 350

Glu Gly Asp Asn Arg Tyr Ile Ala Asn Thr Val Glu Leu Arg Val Lys

355 360 365

Ile Ser Thr Glu Val Gly Ile Thr Asn Val Asp Leu Ser Thr Val Asp

370 375 380

Lys Asp Gln Ser Ile Ala Pro Lys Thr Thr Arg Val Thr Tyr Pro Ala

385 390 395 400

Lys Ala Lys Gly Thr Phe Ile Ala Asp Ser His Gln Asn Phe Ala Leu

405 410 415

Phe Phe Gln Leu Val Asp Val Asn Thr Gly Ala Glu Leu Thr Pro His

420 425 430

Gln Thr Phe Val Arg Leu His Asn Gln Lys Thr Gly Gln Glu Val Val

435 440 445

Phe Val Ala Glu Pro Asp Asn Lys Asn Val Tyr Lys Phe Glu Leu Asp

450 455 460

Thr Ser Glu Arg Lys Ile Glu Phe Asp Ser Ala Ser Gly Thr Tyr Thr

465 470 475 480

Leu Tyr Leu Ile Ile Gly Asp Ala Thr Leu Lys Asn Pro Ile Leu Trp

485 490 495

Asn Val Ala Asp Val Val Ile Lys Phe Pro Glu Glu Glu Ala Pro Ser

500 505 510

Thr Val Leu Ser Gln Asn Leu Phe Thr Pro Lys Gln Glu Ile Gln His

515 520 525

Leu Phe Arg Glu Pro Glu Lys Arg Pro Pro Thr Val Val Ser Asn Thr

530 535 540

Phe Thr Ala Leu Ile Leu Ser Pro Leu Leu Leu Leu Phe Ala Leu Trp

545 550 555 560

Ile Arg Ile Gly Ala Asn Val Ser Asn Phe Thr Phe Ala Pro Ser Thr

565 570 575

Ile Ile Phe His Leu Gly His Ala Ala Met Leu Gly Leu Met Tyr Val

580 585 590

Tyr Trp Thr Gln Leu Asn Met Phe Gln Thr Leu Lys Tyr Leu Ala Ile

595 600 605

Leu Gly Ser Val Thr Phe Leu Ala Gly Asn Arg Met Leu Ala Gln Gln

610 615 620

Ala Val Lys Arg Thr Ala His

625 630

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tgacttcctt gtcgaagttg aa 22

<210> 4

<211> 20

<212> DNA

<213>(artificial synthesized)

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gtgatgccaa cttcagtgga 20

<210> 5

<211> 28

<212> DNA

<213>(artificial synthesized)

<400> 5

ccggaattca cctgctcgga ggaatggc 28

<210> 6

<211> 36

<212> DNA

<213>(artificial synthesized)

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ccgctcgagc taatgtgctg ttctcttgac tgcctg 36

Claims

1. a kind of biomarker for diagnosis of rheumatoid arthritis, which is characterized in that the biomarker is base Because of RPN2 mRNA or the RPN2 protein of RPN2 coding.

2. biomarker according to claim 1, which is characterized in that the nucleotide sequence such as SEQ of the RPN2 mRNA Shown in ID NO.1.

3. biomarker according to claim 1, which is characterized in that the amino acid sequence of the RPN2 protein is such as Shown in SEQ ID NO.2.

4. a kind of for the mRNA primer sets of diagnosis of rheumatoid arthritis and the combination of probe, which is characterized in that described The combination of mRNA primer sets and probe can in quantitative detection peripheral blood mononuclear cells RPN2 mRNA expression quantity.

5. a kind of antibody for diagnosis of rheumatoid arthritis, which is characterized in that the antibody specific recognition and can combine RPN2 protein.

6. a kind of kit for diagnosis of rheumatoid arthritis, which is characterized in that draw comprising mRNA as claimed in claim 4 The combination of object group and probe；Or include the antibody described in claim 5.

7. kit according to claim 6, which is characterized in that the mRNA primer sets include determining for RPN2 mRNA Measure PCR reverse transcription forward primer and reverse primer, the quantitative PCR reverse transcription forward primer and reverse primer of the RPN2 mRNA Nucleotide sequence respectively as shown in SEQ ID NO.3 and SEQ ID NO.4, or such as SEQ ID NO.5 and SEQ ID NO.6 institute Show.

8. a kind of chip for diagnosis of rheumatoid arthritis, which is characterized in that including what is detected to RPN2 mRNA MRNA probe.

9. chip according to claim 8, which is characterized in that the nucleotide sequence and RPN2 of the mRNA probe MRNA nucleotide sequence complete complementary.

10. the described in any item mRNA biomarkers of claims 1 to 3 are in the product of preparation detection rheumatoid arthritis Application.