CN105087822B

CN105087822B - Methylate CLSTN2 gene and its application

Info

Publication number: CN105087822B
Application number: CN201510629984.4A
Authority: CN
Inventors: 杨承刚; 任静
Original assignee: Beijing Medintell Bioinformatic Technology Co Ltd
Current assignee: Beijing Medintell Bioinformatic Technology Co Ltd
Priority date: 2015-09-29
Filing date: 2015-09-29
Publication date: 2019-02-15
Anticipated expiration: 2035-09-29
Also published as: CN105087822A

Abstract

The present invention relates to methylation CLSTN2 gene and its applications, more particularly relate to CLSTN2 gene, the DNA methylation assay of CLSTN2 gene and its application in diagnosis and treatment Osteoarthritis.Invention goes out the gene C LSTN2 of the low expression in osteoarthritis patient by the screening of transcript profile sequencing analysis, the sequencing analysis that methylates shows that CLSTN2 gene is in hyper-methylation in case group, further experimental verification gene C LSTN2 low expression in osteoarthritis patient's knee cartilage tissue, while gene methylation in patient's knee articular cartilage tissue is apparently higher than normal group.The present invention provides a kind of new Osteoarthritis diagnosis and treatment target spots, have important clinical value.

Description

Methylate CLSTN2 gene and its application

Technical field

The present invention relates to biomedicine technical fields, and in particular to methylation CLSTN2 gene and its application, more specifically It is related to CLSTN2 gene, the DNA methylation assay of CLSTN2 gene and its application in diagnosis and treatment Osteoarthritis.

Background technique

Osteoarthritis (Osteoarthritis, OA) is one kind with articular cartilage retrogression pathological changes and periarticular sclerotin Hyperplasia is the chronic progressive osteoarthropathy of major pathologic features.It is the most common joint disease in the elderly, and is caused The most common reason of middle-aged and the old's arthralgia.Chinese society steps into aging now, and the incidence of the disease is also in year by year Ascendant trend.

Osteoarthritis mainly encroaches on articular cartilage, bone and synovial tissue, leads to arthralgia, deformity and dysfunction.Mesh The arthritic cause of disease of prebone not yet illustrates, according to previous epidemiological study, academia generally believe OA be it is a kind of heredity because The complex disease that element is participated in environmental factor collective effect, multiple-factor.

Epigenetics is a big hot spot of current hereditary academia's research, is referred in the premise for not changing genome sequence Under, by the method for the modification of DNA and histone come controlling gene expression.The modification of this DNA and histone is with DNA first Baseization is most commonly seen.DNA methylation effect is a kind of gene modification effect generally existing in various organisms, is gene table Up to one of important regulating and controlling factor in the process.It has been reported and shows that the DNA methylation of OA generation, development process and cartilage cell have Certain correlation, but relevant research is also very deficient, it would be highly desirable to and more researchs disclose methylation relevant to Osteoarthritis Gene, and illustrate the methylation sites and the relationship of OA.

The present invention carries out high-throughput methyl by the knee cartilage tissue compareed to 5 Osteoarthritis patients and 2 Change sequencing analysis and transcript profile sequencing analysis, filter out it is several in case group in hyper-methylation and the candidate of down regulation of gene expression Gene and its methylation sites.Further molecular biosciences experiment results show candidate gene CLSTN2 in bone joint Low expression in scorching patient's knee articular cartilage tissue, while gene methylation in patient's knee articular cartilage tissue is obviously high In normal group.The present invention provides a kind of new Osteoarthritis diagnosis and treatment target spots, have important clinical value.

Summary of the invention

The purpose of the present invention is to provide a kind of Osteoarthritis diagnosis and treatment reagent, one of described diagnosis and treatment reagent component or Several island CpG methylations for being used to detect or reduce SEQ ID NO.3 in sequence table.

Further, one or more of described diagnosis and treatment reagent component is for detecting or reducing SEQ ID in sequence table The island CpG methylation at the 112bp of NO.3.

Further, methylation is detected using any one or the combination of several of them in following method: methylation Restriction enzyme enzyme method, the NaHSO of sensitivity₃Method, chip technology approach；The NaHSO₃Method includes BSP PCR sequencing PCR, joint NaHSO₃Restriction analysis method, methylation status of PTEN promoter, fluorescent quantitation；The chip technology approach includes that methylation is highly dense Spend chip, differential methylation hybridization, methylation-specific oligonucleotide chip.

The restriction enzyme enzyme method of methyl-sensitive is classical methylation analysis methods, mainly according to some restricted Restriction endonuclease cannot cut the DNA sequence dna of methylation.Since in eukaryotic DNA or mammalian DNA, only CG connected born of the same parents are phonetic Pyridine can be methylated, and therefore, the restriction enzyme in restriction enzyme site comprising CG sequence will encounter problems.This method Used two classical enzymes are to being HPa II-Msp I (CCGG) and SmaI-Xma I (CCCGGG).Due to second pair of restriction enzyme Identify that sequence is very rare, so general all use HPa II-Msp I (CCGG).Two enzymes all identify CCGG sequence, and ought be wherein Cytosine methylation when, HPaII can not be cut, using Hpa II-Msp work this attribute handle DNA, this just makes Southern can then be carried out or PCR amplification separates vertical object as the tool of rapid methylation analysis by obtaining HPa II-Msp I, Specify methylation state.

NaHSO₃The C of non-methylation can be converted to U, the latter becomes T through PCR amplification and generates T:A and match, but methylates C can then resist NaHSO₃Modification, the methylation information that such DNA includes can be converted into the difference of DNA sequence dna.Thus it sends out A variety of islands CpG methylation detecting method is opened up, such as BSP sequencing, PCR (COBRA, MSP, Methelight etc.), chip hybridization skill Art (microarray), such method are adapted to detect the methyl in several sites CpG on the one or more island CpG in own major gene Change, belongs to locus specificity DNA methylation detection technique.

The development of chip technology provides new method for high-throughput researching DNA methylation.Its Method And Principle substantially may be used Be divided into 3 kinds: (1) principle changed based on C/T after bisulf iotate-treated, (2) are based on methylation sensitive restriction endonuclease or first Method enriching methylate is obtained based on immune flutter of 5-methylcytosine antibody or MBD in the method for base dependence restriction endonuclease, (3) DNA fragmentation.

Preferably, it is detected using methylation of the BSP PCR sequencing PCR to SEQ ID NO.3, it is furthermore preferred that using drawing Object sequence is that sequence table SEQ ID NO.4 and SEQ ID NO.5 carries out BSP sequencing detection.

Preferably, methylation is detected using methylation status of PTEN promoter method, methylation status of PTEN promoter method packet Include a pair of of DNA methylation assay primer and a pair of non-DNA methylation assay primer.Preferably, methylation status of PTEN promoter method is in sequence table The island CpG methylation is detected at the 112bp of SEQ ID NO.3.

Preferably, primer sequence is used to carry out methylation-specific for sequence table SEQ ID NO.6 to SEQ ID NO.9 PCR method detects methylation.

The purpose of the present invention is to provide application of the above-mentioned diagnosis and treatment reagent in preparation Osteoarthritis clinic tool.

The purpose of the present invention is to provide a kind of reagents for detecting CLSTN2 gene in preparation diagnosis Osteoarthritis diagnosis Application in preparation.

Further, detection CLSTN2 gene expression amount and/or detection CLSTN2 gene methyl are contained in the diagnostic preparation The reagent of change degree.

Preferably, CLSTN2 gene expression amount: quantitative fluorescent PCR reagent is detected using one or more of following manner Box, genetic chip, ELISA method and/or colloidal gold method, it is furthermore preferred that use primer sequence for sequence table SEQ ID NO.1 and SEQ ID NO.2 carries out the detection of CLSTN2 gene expression amount.

Genetic chip is also known as DNA microarray (DNA microarray), can be divided into three kinds of main Types: 1) be fixed on poly- The nucleic acid probe or cDNA segment on object substrate (nylon membrane, nitrocellulose membrane etc.) surface are closed, the target of isotope labelling is usually used Gene is hybrid with it, and is detected by radiography technology.2) DNA probe array on a glass is fixed with point sample method, Hybridized by the target gene with fluorescent marker and is detected.3) oligonucleotide probe directly synthesized on the hard surfaces such as glass Array hybridizes with the target gene of fluorescent marker and is detected.Genetic chip is as a kind of advanced, extensive, high-throughput detection Technology, applied to the diagnosis of disease, advantage has the following aspects: first is that the sensitivity and accuracy of height；Second is that quickly It is easy；Third is that a variety of diseases can be detected simultaneously.

Known antigen or antibody are adsorbed on surface of solid phase carriers by enzyme-linked immunosorbent assay (ELISA), make enzyme mark The technology that the antigen-antibody reaction of note is carried out in solid phase surface.The technology can be used for detecting macromolecular antigen and specific antibody Deng having many advantages, such as that quick, sensitive, easy, carrier is easy to standardize.ELISA detection kit is according to testing goal and operation Step can be divided into indirect method, double-antibody method, competition law, double site one-step method, prize law survey IgM antibody, using Avidin and The ELISA of biotin.Horseradish peroxidase (HRP) or alkaline phosphatase may be selected in chromogenic substrate in ELISA detection kit Enzyme (AP).

Common immune colloid gold detection technique: (1) immune colloid gold light microscopic decoration method cell suspension smear or tissue are cut Piece can be dyed with the antibody of colloid gold label, can also be enhanced with silver-colored developer solution and be marked on the basis of colloid gold label, So that the silver atoms being reduced is deposited on marked gold particle surface, the sensibility of colloid gold label can be remarkably reinforced.(2) it is immunized Colloidal gold staining method for electron microscopy with the antibody of colloid gold label or antiantibody and negative staining Virus Sample or can be organized in conjunction with ultra-thin section, Then negative staining is carried out.It can be used for observation and the viral diagnosis of morphology of virus.(3) dot immunogold filtration assay application miillpore filter is made Antigen or antibody point are first added sample to be examined on film by carrier after closing, corresponding with the antibody test of colloid gold label after washing Antigen or antibody.(4) antigen of specificity or antibody are fixed on film by colloidal gold immunity chromatography with ribbon, colloidal gold Labelled reagent (antibody or monoclonal antibody) is adsorbed on bonding pad, when sample to be examined is added in the sample pad of test strips one end Afterwards, it moves forward, reacts to each other after dissolving the colloid gold label reagent on bonding pad through capillary action, it is fixed when being moved to When the region of antigen or antibody, the conjugate of object and gold marked reagent to be checked occurs specific binding therewith again and is trapped, and assembles It is taken in detection, colour developing result can be observed by the naked eye.The method has developed into diagnosis test paper, and use is very convenient.

The purpose of the present invention is to provide a kind of preparation for treating Osteoarthritis, contain promotion CLSTN2 in the preparation The reagent or compound of transcription or the expression of gene.

Those skilled in the art are known to promote the expression of gene and its expression product usually can be using one of following methods And/or several: the promoter of activation CLSTN2 gene, the albumen for activating CLSTN2 gene expression or the factor import promotion The carrier of CLSTN2 genetic transcription or expression.

The purpose of the present invention is to provide a kind of gene detecting kit for detecting Osteoarthritis, the kit detection Gene C LSTN2.

It is an object of the present invention to provide a kind of Osteoarthritis protein detection kit, the detection kit detection CLSTN2 albumen.Further, the kit further includes other detection reagents.

It is an object of the present invention to provide it is a kind of detect Osteoarthritis genetic chip, the genetic chip include with The probe of the nucleic acid array hybridizing of CLSTN2 gene.

Detailed description of the invention

CLSTN2 gene methylation situation in Fig. 1 Osteoarthritis group and control group

Fig. 2 osteoarthritis patient organizes interior CLSTN2 protein expression and gene methylation correlation analysis figure

Specific embodiment

Present invention will be further explained below with reference to specific examples, for explaining only the invention, and should not be understood as to this The limitation of invention.It will be understood by those skilled in the art that: without departing from the principle and spirit of the present invention may be used To carry out a variety of change, modification, replacement and modification to these embodiments, the scope of the present invention is limited by claim and its equivalent It is fixed.In the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition or according to item proposed by manufacturer Part examinations.

The acquisition and sequencing of 1 sample of embodiment

Control group: collect 2012-2014 because traffic accident hospital progress meniscus injury and cruciate ligament into The patient of row arthrocsopic surgery treatment, takes knee cartilage tissue speed to be stored in liquid nitrogen container.

Osteoarthritis patient's group: 2012-2014 is collected in hospital admission, according to nineteen ninety-five U.S.'s rheumatism association bone Arthritic diagnostic criteria is diagnosed as severe Osteoarthritis, there is the patient cases of knee prosthesis indication.

Wherein, clinical criteria is according to the prepared standard of nineteen ninety-five American Rheumatism Association:

Most of time has goinyalgia in (1) 1 month；

(2) sound when joint motion；

(3) morning stiffness is not more than 30 minutes；

(4) age is not less than 40 years old；

(5) swelling of knee joint bone is with snap；

(6) swelling of knee joint bone is not accompanied by snap.

It is minimum to there is ((1), (2), (3), (4) or (1), (2), (3), (5) or (1), (6) i.e. diagnosable OA.

After sample acquisition, selects 5 patient groups, 2 control groups to be sent to Mei Ji biotech firm and carry out transcript profile sequencing and full base Because of a group methylation sequencing, and provide preliminary analysis result.Sequencing result shows that candidate gene CLSTN2 suffers from Osteoarthritis Low expression in person's knee cartilage tissue, while gene methylation in patient's knee articular cartilage tissue is apparently higher than just Normal group.

2 osteoarthritis patient of embodiment and control synovial tissue CLSTN2 expression conditions

1. experimental material

The synovial tissue for choosing 37 osteoarthritis patient synovial tissues and 9 controls, is grouped it and numbers. Case group meets knee joint OA diagnostic criteria, carries out replacement knee in arthroplasty patient；Control group is meniscus injury and intersection The patient of ligament injury progress arthrocsopic surgery treatment.

2. experimental method

The extraction of synovial tissue's total serum IgE of 2.1 osteoarthritis patients and control

UsingReagent (invitrogen, article No. 15596-018) carries out sample rna extraction, experimental implementation It is carried out by product description.

RNA quality judging standard: the OD260/OD280 value of RNA sample is between 1.7-2.2；Total serum IgE electrophorogram has clearly Clear 28S, 18S band；70 DEG C of water-baths keep the temperature 1 hour after electrophorogram and the map no significant difference before water-bath heat preservation.

2.2 reverse transcriptions synthesize cDNA

UsingIII Reverse Transcriptase (invitrogen, article No. 18080-044) into Row cDNA reverse transcription, experimental implementation are carried out by product description, and concrete operations are as follows:

Using Reverse Transcriptase kit, converse record is carried out to l μ g total serum IgE with RT Buffer and synthesizes cDNA.Using 25 μ l Reaction system, each sample take 1 μ g total serum IgE as template ribonucleic acid.It is spare that -20 DEG C of refrigerators are put in the cDNA preservation of acquisition.

2.3 Real-Time PCR

Design of primers

Using online primer-design software, gene order is referring to NCBI:NM_022131.2 (CLSTN2), interior participation in the election GAPDH is synthesized by invitrogen company after design of primers.Specific primer sequence is as follows:

1 Real-Time PCR primer sequence of table

Operating process is as follows:

(1) reaction system: Power is usedGreen PCR Master Mix (invitrogen, article No. 4367659) it is expanded, experimental implementation is carried out by product description.Amplification program are as follows: 95 DEG C of 5min pre-reactions carry out 40 Recycle the amplified reaction of (95 DEG C of 15s, 60 DEG C of 45s).

2 Real-Time PCR reaction system of table

Component	Additional amount
		2×mix	10μl
Upstream primer (10uM)	0.5μl
		Downstream primer (10uM)	0.5μl
Template	2μl

Sterile purified water is added

To 25 μ l

(2) primer screening

After each sample cDNA is mixed, 5 times of gradient dilutions are carried out as template, sample respectively takes 2 μ l to make template after dilution, It is expanded respectively with target gene primer and reference gene primer, while in 60-95 DEG C of progress melt curve analysis analysis, according to expansion Increasing Efficiency height and the unimodal principle of solubility curve carry out primer screening.

(3) sample Real-Time PCR is detected

2 μ l will be taken to make template after 10 times of each sample cDNA dilutions, respectively with target gene primer and reference gene primer into Row amplification.Simultaneously in 60-95 DEG C of progress solubility curve analysis.

3. experimental result

Real-time quantitative PCR amplification curve inflection point understands that amplification curve entirety collimation is good, shows the amplification effect of each reaction tube Rate is close, and the limit is flat and present without raising up, and exponent phase slope is larger, illustrates that amplification efficiency is higher；Sample amplified production is molten Solution curve be all it is unimodal, illustrate that amplified production only has one, be specific amplification；According to the relative quantification formula of qRT-PCR, than Compared with expression of the CLSTN2 gene in Osteoarthritis group and control group.As the result is shown: qRT-PCR stable amplification result, Its expression of CLSTN2 gene in Osteoarthritis group is only about 0.3 times of control tissue, and result above demonstrates height Flux transcript profile expresses the result of confluence analysis CLSTN2 gene low expression in osteoarthritis patient of data.

The extraction and methylation modification of 3 DNA of embodiment

1. phenol-chloroform extraction process handles DNA

Extracting genome DNA uses phenol-chloroform extraction process, and detailed step is as follows:

(1) it takes 0.1g cartilaginous tissue to be placed in grinding, liquid nitrogen is added and is quickly ground, when to be ground to powdered, Being transferred to is in centrifuge tube, and 600 μ l is added to digest buffer, then plus 4 μ l Proteinase Ks (1M), shake up and be placed on 55 DEG C of water-baths In it is clear to digesting；

(2) it with isometric steaming saturated phenol again, mixes, shaking table shakes at a slow speed 20min, and 10000g is centrifuged 10min, takes supernatant Liquid；

(3) chlorination imitates/isoamyl alcohol (24:1) phenol: extract=1:1:2, mixes, shaking table shakes at a slow speed 20min, after centrifugation Take its supernatant；

(4) chlorination it is imitative/isoamyl alcohol (24:1): extract==1:1, its supernatant of centrifuging and taking again after mixing；

(5) plus the dehydrated alcohol of 0.1 times of volume sodium acetate (3M, pH=5.2) and the frost of 2 times of volumes, mixing freezed Night；

(6) 4 DEG C, 12000g is centrifuged 15min and abandons supernatant；

(7) precipitating is sufficiently washed with 70% ethyl alcohol that 500 μ l are freezed, and 12000g is centrifuged 5min, abandons supernatant；

(8) step 7 is repeated, at room temperature naturally dry；

(9) sufficiently dissolve precipitating with 30-50 μ l distilled water, agarose electrophoresis detects the integrality of DNA, -20 DEG C freeze it is standby With.

2. the modification and purifying of DNA sample:

The modification of DNA methylation is using EZ DNA Methylation^TMKit (ZYMO RESEARCH, article No. D5002) It carries out, principle and the modification of classical sodium hydrogensulfite are substantially similar.After kit is handled, the cytimidine of methylation does not become Change, and the cytimidine (C) not methylated will all become uracil (U).Redesign is respectively for methylation and non-methylation The specific primer of sequence carries out the amplification of PCR, and the methylation shape of DNA is determined by the method for agarose gel electrophoresis analysis State variation.

Specific steps:

(1) preparation of DNA: the DNA double extracted before steaming water is diluted to after 25-100 μ g/ml and draws 20 μ l and is placed in PCR Guan Zhong.

(2) 900 μ l the preparation of CT conversion reagent (Conversion Reagent): are added in a CT conversion reagent pipe The M- of water, the M- dilution buffer of 300 μ l and 50 μ l dissolve buffer, dissolve at room temperature and shake 10min；

(3) 130 μ l CT conversion reagents, 20 μ l DNA samples are added in PCR pipe, if DNA sample volume is less than 20 μ L can supply residual quantity with deionized water, pass through liquid-transfering gun or the operations mixing sample such as flick test tube；

(4) sample is put into temperature cycling device, prior to 98 DEG C place 10 minutes, then at 64 DEG C place 2.5 hours after, horse On carry out step；

(5) the M- combination buffer of 600 μ l is added to Zymo-Spin IC^TMIn adsorption column, and adsorption column is put into receipts In collector；

(6) in the Zymo-Spin IC containing M- buffer^TMThe sample in step (4) is added in adsorption column, is mixed after capping Even sample；

(7) remove efflux after 30 seconds are centrifuged at full speed；

(8) 100 μ l M- wash buffers are added in adsorption column, and be centrifuged 30 seconds at full speed；

(9) by 200 μ l M- vulcanization buffer be added in adsorption column, at room temperature place 20 minutes, after culture at full speed from The heart 30 seconds；

(10) 200 μ l M- wash buffers are added in column, it is slow to add 200 μ l M- rinsing again for centrifugation at full speed after 30 seconds In fliud flushing to column, it is centrifuged 30 seconds at full speed again；

(11) directly l0 μ l M- elution buffer is added in column, adsorption column is placed in the EP pipe of 1.5m1, entirely Speed centrifugation eluted dna；

(12) step 11 is repeated, is the DNA for modifying and purifying in EP pipe；

(13) DNA is stored in spare in -70 DEG C of refrigerators.

The research in 4 site CLSTN2 gene methylation CpG of embodiment

DNA that DNA and 5 control group that 5 Osteoarthritis groups in selection example 3 are extracted extracts (into The modification and purifying of sample are gone, that is, the cytimidine (C) that the cytimidine to methylate does not change, and do not methylate all will Become uracil (U)).With corresponding BSP primer amplification SEQ ID NO.3 (the long 224bp of sequence), the primer is shown in Table 3, with solidifying Whether gel electrophoresis verifies design of primers and succeeds, and obtains purpose amplified fragments, size and expected size one as the result is shown It causes.

3 BSP primer of table

The PCR product of recovery purifying is connect with pUC18-T carrier to construct BSP-PCR product recombinant plasmid, so respectively Connection product conversion is carried out afterwards and prepares competent cell, selects the white colony grown on IPTG/X-gal plate.Use gel Electrophoresis carries out PCR identification to the bacterium colony that target fragment is cloned to exclude false positive clones, and the correct clone of screening carries out subsequent Sequencing analysis.

Be the validity improved to clone's methylation sequencing result, each experimental group choose 5 identifications correctly clone into Row sequencing analysis.It has been illustrated in MATERIALS METHODS, sequence location one to be measured shares 4 sites CpG.Utilize SeqMan software pair Sequence alignment is carried out without the sequencing result for significantly interfering with signal, obtains the methylation shape in 4 sites CpG to be detected in each clone State.Analysis meets sulphite transformation experiment requirement, shows the result shows that sulphite transformation efficiency reaches 98% or more It is reliable that BSP analyzes result.It is methylated as the result is shown in the site (112bp) of the 3rd CpG, control group does not methylate, says The methylation in the bright site may be CLSTN2 gene low expression and pathogenic related one of the reason of Osteoarthritis.In addition, Partial methylation has occurred in the site (79bp) of 2nd CpG.

5 methylation status of PTEN promoter of embodiment (MSP)

1. CLSTN2 gene methylation and the design of non-methylated primers and synthesis

The synthesis of design of primers Hou Song company.

4 MSP primer of table

2. MSP reaction system

5 MSP reaction system of table

Reagent	Volume (μ l)
		2×HS PCR Mix	12.5
M/U upstream primer (10 μM)	1
		M/U downstream primer (10 μM)	1
Template	1
		Aqua sterilisa	Filling-in is to 25

Reaction condition: pre-reaction: 94 DEG C of 5min；React 35 circulations (94 DEG C 30 seconds, 60 DEG C 30 seconds, 72 DEG C 30 seconds)；Prolong 72 DEG C of 5min are stretched, takes out carry out agarose gel electrophoresis analysis after reaction.

3. experimental result

The amplified production of non-Methylation-specific primer exists, and there is no be for the amplified production of Methylation-specific primer Non- methylation；The amplified production of non-Methylation-specific primer is not present, and the amplified production of Methylation-specific primer, which exists, is For methylation.Electrophoresis result is shown: in 37 osteoarthritis patient synovial tissues, methylation positive has 26, gene first Base rate be 70.27%, 9 control synovial tissues in methylation positive only 2, gene methylation rate be 22.22% (see Table 6, Fig. 1).

6 Osteoarthritis group of table and control group CLSTN2 gene methylation situation

6 WB method of embodiment detects the expression of CLSTN2 in Osteoarthritis synovial membrane

One, protein example is prepared and is quantified

1. RIPA lysate (Beyotime) carries out protein example preparation, operating procedure is as follows:

Synovial tissue is taken out from refrigerator, wipes structural PBS solution with filter paper, weighs weight, and by synovial tissue It is placed in mechanical tissue homogenizers, 1:10 tissue carries out even than the lysate that respective volume is added in the bulking value ratio of lysate Slurry, 10000-14000g are centrifuged 3-5 minutes, take supernatant, and sample buffer strength mixing is added by 1:1 and is placed on 100 degree Water bath heating water bath 3-5 minutes, 10000g was centrifuged 10 minutes, is taken supernatant, is transferred in the test tube of another cleaning.

2. it is quantitative to carry out total protein using BCA determination of protein concentration kit

Use health for century micro BCA protein quantification kit (article No.: CW2011), specific steps are shown in its specification.

Two, SDS- polyacrylamine gel electrophoresis (SDS-PAGE)

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, mixed protein sample and albumen loading are added according to every 1 microlitre of protein sample Buffer (5 ×).

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 7 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 lml distilled water encapsulating immediately after mixing places about 30min after glue polymerization, with distillation at room temperature Washing 2-3 times, then blotted with filter paper.Then the concentration glue of 2m15% is prepared, is formulated as follows:

Glue formula is concentrated in table 8

Component	Dosage
		30% acrylamide solution	0.33ml
Tris-HCl (1.0M, pH6.8)	0.25ml
		10%SDS	0.02ml
10%AP	0.02ml
		TEMED	0.002ml
Sterilize ddH2O	It is supplemented to 2ml

Encapsulating, insertion sample comb avoid generating bubble immediately after mixing, after being gelled admittedly, take out sample comb, rear to use distillation Water and 1 × protein electrophoresis buffer successively rinse sample well.

Three, loading and electrophoresis

By gel slab on electrophoretic apparatus, l × protein electrophoresis buffer, l × protein electrophoresis in outer groove are filled it up in inside groove Buffer 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.

Four, Western blotting

1. first carrying out PAGE gel according to the method described above is separated by electrophoresis albumen.

2. impregnating NC film, filter paper, foam rubber cushion 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 paper that one block of transfer liquid is impregnated with, Filter paper and sea pad size it is identical or with NC film, gel size is identical, and gel is lain on cathode side filter paper, finally will NC film is lain on gel, removes bubble removing, clips electricity transfer folder.Electricity transfer liquid is filled it up in electrophoresis tank, is inserted into electricity transfer folder, it will be electric Swimming slot is put into refrigerator and (to be put into pre-cooling in refrigerator before electricity transfer liquid), connects electrode, turn-on current transfers the NC film of folder Cope with the anode of electrophoresis tank.

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

4. primary antibody hybridizes: abandoning confining liquid, primary antibody (the Anti-CLSTN2 antibody for using primary antibody diluted is added (ab156205)) hybridization solution is placed in 4 DEG C of hybridized overnights, is hybridized in shaken cultivation case within second day；

5. recycling primary antibody hybridization solution, washed film 3 times with TBST；

6. abandoning TBST, it 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；

7. abandoning two corresponding anti-solution, washed film 3 times with TBST；

8. 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.

9. data normalization is carried out using β-Actin as internal reference, using CLSTN2 in control group synovial tissue as referring to sample This, the relative expression levels of CLSTN2 albumen in experiment with computing group.

Five, experimental result

The results show that there are 14 positives in 37 osteoarthritis patient's groups, there are 6 positives, CLSTN2 in 9 control groups Protein positive rate is 37.84% and 66.67% respectively, and two groups of differences have statistical significance.Further Osteoarthritis is suffered from Person organizes interior CLSTN2 protein expression and gene methylation state does correlation analysis, as a result sees Fig. 2,23 CLSTN2 protein expressions There are 17 methylation positive all occur in negative patient, shows that CLSTN2 protein expression is significant related in bearing to gene methylation Property.

9 osteoarthritis patient of table organizes interior CLSTN2 protein expression and gene methylation correlation analysis

Claims

1. a kind of Osteoarthritis diagnostic reagent, which is characterized in that one or more of described diagnostic reagent component is for examining Survey the island CpG methylation at the 112bp of SEQ ID NO.3.

2. diagnostic reagent according to claim 1, which is characterized in that using BSP PCR sequencing PCR to the of SEQ ID NO.3 The island CpG methylation is detected at 112bp.

3. diagnostic reagent according to claim 2, which is characterized in that use sequence such as SEQ ID NO.4 and SEQ ID Primer sequence shown in NO.5 carries out BSP sequencing detection.

4. diagnostic reagent according to claim 1, which is characterized in that using methylation status of PTEN promoter method to SEQ ID The island CpG methylation is detected at the 112bp of NO.3.

5. diagnostic reagent according to claim 4, which is characterized in that use sequence such as SEQ ID NO.6 to SEQ ID Primer sequence shown in NO.9 carries out methylation status of PTEN promoter method and detects to methylation.

6. diagnostic reagent according to claim 1, which is characterized in that using the group of any one or a few in following method Conjunction detects the island CpG methylation at the 112bp of SEQ ID NO.3: the restriction enzyme side of methyl-sensitive Method, NaHSO3 method, chip technology approach；The NaHSO3 method includes BSP PCR sequencing PCR, joint NaHSO3 restriction analysis method, first Base specific PCR, fluorescent quantitation；The chip technology approach include methylation superchip, differential methylation hybridization, Methylation-specific oligonucleotide chip.

7. application of the diagnostic reagent as claimed in any one of claims 1 to 6 in preparation Osteoarthritis diagnostic tool.

8. the reagent of the island CpG methylation exists at the 112bp of SEQ ID NO.3 in a kind of detection CLSTN2 gene order table Application in preparation diagnosis Osteoarthritis diagnostic preparation.

9. application according to claim 8, which is characterized in that contain detection CLSTN2 gene expression amount in diagnostic preparation And/or the reagent of detection CLSTN2 gene methylation degree.

10. application according to claim 8, which is characterized in that using the detection of one or more of following manner CLSTN2 gene expression amount: PCR kit for fluorescence quantitative, genetic chip, ELISA method, colloidal gold method.

11. application according to claim 10, which is characterized in that use sequence such as sequence table SEQ ID NO.1 and SEQ Primer sequence shown in ID NO.2 carries out the detection of CLSTN2 gene expression amount.