CN107267649B - Applications of the STRADB in asthma early diagnosis - Google Patents

Abstract

The invention discloses applications of the STRADB in asthma early diagnosis.The present invention prompts STRADB to can be used as the molecular indexes that asthma clinical early diagnoses experiments prove that the differential expression of conspicuousness is presented in asthmatic patient and normal person in albumen of STRADB genes and its coding.

Description

Applications of the STRADB in asthma early diagnosis

Technical field

The invention belongs to biomedicine fields, are related to applications of the STRADB in asthma early diagnosis.

Background technology

Bronchial asthma abbreviation asthma is respiratory disease type relatively common in clinic diagnosis, is a kind of multiple Miscellaneous syndrome, the chronic inflammation in following faint breath road are main feature, and it is narrow to be usually accompanied by pulmonary airways, Airway Remodeling, The cough of airway hyperreactivity and recurrent exerbation, wheezing is uncomfortable in chest, out of breath.According to the frequency of these these diseases appearance and sternly Weight degree, people are divided into asthma：Slightly, moderate, three kinds of different grades of severe.The recurrent exerbation of asthma can also be lured further Complication is sent out, such as：Pulmonary emphysema, chronic obstructive pulmonary disease, pulmonary heart disease, respiratory failure, heart failure etc..Currently, the whole world has greatly About 300,000,000 populations suffer from asthma and illness rate also has increased trend year by year.Asthma all can to the daily life and work of patient Generation seriously affects, meanwhile, asthma also gives personal and society to bring huge financial burden.There are two most important two for asthma A feature is shrinkage enhancing and the excitability response of air flue of airway smooth muscle cells.Since the pathogenesis of asthma is multiple Miscellaneous, there are many factor for inducing and participating in morbidity, not yet get across so far to its mechanism.

Asthma is coefficient by multiple factors such as immune, h and Es as a result, having during its occurrence and development more The participations such as kind cell, inflammatory mediator, and easily prolonged and repeated breaking-out.The research of the past focuses mostly on the acidophil granules mediated in Th2 Cellular inflammatory.In recent years, with the continuous development of science of heredity and molecular biology, asthma genetic teiology becomes the heat of research Point.Asthma genetic mechanism and its related gene research are gradually goed deep into, the change of gene is during the occurrence and development of asthma It plays an important role.

The traditional treatment of asthma mainly uses glucocorticoid, bronchodilator, but the state of an illness of some patientss obtains not To alleviation, therefore improves diagnosis and monitoring to asthma and implement early prevention treatment with important meaning to filter out susceptible individual Justice.Traditional diagnostic techniques is measured dependent on clinical manifestation, pulmonary function test or peak flow velocity, but above-mentioned diagnostic method is not easy to examine Go out the unconspicuous asthma of early symptom or easy mistaken diagnosis.With the development of biotechnology, the analysis of biomarker is asthma Early diagnosis provide a kind of new way.

Invention content

An object of the present invention provides a kind of biomarker of asthma, is applied to the early diagnosis of asthma, to reality The prevention of existing asthma.

The second object of the present invention, provides a kind of product of diagnosis asthma, and the product can realize the spy of asthmatic patient Specific diagnosis.

The present invention provides application of the STRADB genes in the product for preparing diagnosis asthma.Wherein, STRADB genes exist It expresses and lowers in asthmatic patient.

Further, the product includes the reagent for detecting STRADB expressions in sample.

Further, the reagent includes：It is examined by RT-PCR, real-time quantitative PCR, immune detection, in situ hybridization or chip Survey the reagent of STRADB expressions.

Further, the reagent that STRADB expressions are detected by RT-PCR includes at least a pair of of specific amplified The primer of STRADB genes；The reagent that STRADB expressions are detected by real-time quantitative PCR includes at least a pair of special The primer of amplification of STR ADB genes；The reagent by immune detection STRADB expressions includes special with STRADB albumen Property combine antibody and/or ligand；It is described by situ hybridization detect STRADB expressions reagent include and STRADB bases The probe of the nucleic acid array hybridizing of cause；It is described by chip detect STRADB expressions reagent include and STRADB gene cores The probe of acid sequence hybridization or the antibody and/or ligand combined with STRADB protein-specifics.

In the present invention, " sample " is can therefrom to obtain nucleic acid, polypeptide or other analyses comprising cell or cellular material The sample of object.The example of biological sample includes in non-limiting manner：Urine, blood, serum, blood plasma, celiolymph, liquor pleurae, branch gas Pipe lavation, phlegm, peritoneal fluid, bladder irrigation, secretion (for example, mammal gland secretion), dentilave, swab are (for example, oral cavity is wiped Son), separation cell, tissue samples, touching prepare object and fine needle puncture object.Preferably, the sample is human peripheral.

The present invention provides a kind of products of diagnosis asthma, including preparation, chip or kit, wherein the preparation, core Piece or kit include detecting the reagent of STRADB expressions.

Further, the reagent for STRADB expressions being detected in the chip includes the spy of specific recognition STRADB genes Needle, or specifically bind the antibody or ligand of the albumen of STRADB codings.The chip can be used for detecting including STRADB Multiple genes or albumen (for example, with the relevant multiple genes of asthma or albumen) expression.

Further, the reagent for STRADB expressions being detected in the kit includes drawing for specific amplification STRADB genes Object；Or the probe of specific recognition STRADB genes；Or the antibody or ligand of the albumen of specific binding STRADB codings.

In a specific embodiment of the present invention, the primer sequence of the specific amplification STRADB genes such as SEQ SEQ ID Shown in NO.3 and SEQ ID NO.4.

Further, the reagent further includes SYBR Green PCRs system, for expanding house-keeping gene Primer pair；The SYBR Green PCR systems include：PCR buffer solutions, dNTPs, SYBR Green fluorescence dye Material.

Further, the detection kit further includes operation instructions or label.

In the present invention, the kit can be used for detecting multiple genes including STRADB or albumen (for example, with The relevant multiple genes of asthma or albumen) expression.

Description of the drawings

Fig. 1 is the expression figure in asthmatic patient using QPCR detection STRADB genes；

Fig. 2 is the figure for the differential expression for detecting STRADB genes on protein level using immunoblotting.

Specific implementation mode

The present invention after extensive and in-depth study, it is aobvious to be found that STRADB is presented in asthmatic patient and normal person for the first time Sex differernce expression is write, is that the early detection of asthma finds better approaches and methods.

Biomarker

" biomarker " is also referred to as " molecular marker ", is the expression in tissue or cell and normal or health The expression of cell or tissue compares any gene or albumen to change.

It would be recognized by those skilled in the art that the practicability of the present invention is not limited to the marker gene of the present invention The gene expression of any specific variants is quantified.As unrestricted example, STRADB includes comprising SEQ ID NO.2 Polypeptide and other STRADB natural sequence polypeptides, such as naturally occurring variant and natural sequence polypeptide, by SEQ ID NO.1 institutes That shows is nucleotide sequence coded.

Molecular marker of the present invention includes gene and albumen.Such marker includes containing coding molecule marker Nucleic acid sequence or this sequence complementary series complete or partial sequence DNA.Molecular marker nucleic acid further includes containing Complete or partial sequence the RNA of any nucleic acid sequence of concern.Molecular marker albumen is by the DNA molecular mark of the present invention The albumen of object coding or corresponding to the present invention DNA molecular marker.Molecular marker albumen includes any molecular marker The complete or partial amino-acid series of albumen or polypeptide.The segment and variant of molecular marker gene and albumen are also included within this hair In bright range.

Detection technique

The present invention can use multiple nucleic acids known to persons of ordinary skill in the art and protein techniques to be detected, this Technologies include but not limited to a bit：Nucleic acid sequencing, nucleic acid hybridization, nucleic acid amplification technologies, protein immunization technology.

The present invention simultaneously can expand nucleic acid before detection or with detection.The exemplary non-limit of nucleic acid amplification technologies Property example processed includes but not limited to：PCR (PCR), reverse transcriptase polymerase chain reaction (RT-PCR), transcription are situated between Amplification (TMA), ligase chain reaction (LCR), strand displacement amplification (SDA) and the amplification (NASBA) based on nucleic acid sequence led. Those skilled in the art it will be recognized that certain amplification techniques (for example, PCR) need before amplification by RNA reverse transcriptions at DNA (for example, RT-PCR), and other amplification techniques then direct cloning RNA (for example, TMA and NASBA).

In general, PCR uses denaturation, primer pair and the annealing of opposite strand and multiple cycles of primer extend, with index side Formula increases the copy number of target nucleic acid sequence；RT-PCR then is used for reverse transcriptase (RT) to prepare complementary DNA (cDNA) from mRNA, Then cDNA is generated to multiple copies of DNA by PCR amplification；TMA is in the temperature of substantial constant, ionic strength and pH Under the conditions of autocatalytically synthesize multiple copies of target nucleic acid sequence, multiple RNA copies of wherein target sequence are autocatalytically given birth to At other copy, TMA is optionally included using blocking, part, terminate part and other modified parts, to improve TMA processes Sensitivity and accuracy；LCR uses the two groups of complementary DNA oligonucleotides hybridized with the adjacent area of target nucleic acid.DNA few nucleosides Acid is covalently attached in thermal denaturation, hybridization and the multiple cycles of the repetition of connection by DNA ligase, to generate detectable double-strand Connect oligonucleotide product；SDA uses multiple cycles of following steps：Primer sequence pair and the opposite strand of target sequence move back Fire carries out primer extend to generate (hemiphosphorothioated) of half thiophosphorylation of double-strand under there are dNTP α S Primer extension product, the nicking that the endonuclease that semi-modified restriction enzyme enzyme recognition site carries out mediates, and from cutting The polymerase-mediated object drawn that 3 ' end of mouth carries out, which extends, to be set with replacing existing chain and generating for next round primer annealing, nicking and chain The chain changed expands so as to cause the geometry of product.

The nucleic acid of non-amplification or amplification can be detected by any conventional means in the present invention.

Nucleic acid hybridization technique in the present invention include but not limited in situ hybridization (ISH), microarray and Southern or Northern traces.In situ hybridization (ISH) be it is a kind of use label complementary DNA or RNA chains as probe with position tissue one Part or slice (original position) or if organize it is sufficiently small if for entirely organize (full organization embedding ISH) in specific DNA or The hybridization of RNA sequence.DNA ISH can be used for determining the structure of chromosome.RNA ISH are for measuring with position tissue slice or entirely MRNA in organization embedding and other transcripts (for example, ncRNA).Usually sample cell and tissue are handled in situ solid Targeting transcript, and increase the entrance of probe.Probe hybridizes with target sequence at high temperature, then washes off extra probe.Point Not Shi Yong autoradiograph, fluorescence microscopy or immunohistochemistry, in tissue with radiation, fluorescence or antigenic mark base The probe of label is positioned and is quantified.ISH can also be used two or more to pass through radioactivity or other nonradioactive labelings The probe of substance markers, to detect two or more transcripts simultaneously.

Southern and Northern traces are respectively used to detection specific DNA or RNA sequence.Make to extract from sample DNA or RNA fracture, it is separated by electrophoresis on matrix gel, be then transferred on molecular filter.Make filter combine DNA or RNA with and the label probe of sequence of interest complementation hybridize.Detection is attached to the hybridization probe of filter.A kind of change of the program Change form is reverse northern trace, wherein the substrate nucleic acid fixed to film is the set of the DNA fragmentation of separation, and probe is From tissue extraction and the RNA that is marked.

Protein immunization technology includes sandwich immunoassay, such as sandwich ELISA, wherein using identifying on biomarker not The detection of the biomarker is carried out with two kinds of antibody of epitope；Radiommunoassay (RIA), direct, indirect or comparison are enzyme-linked Immunosorbent assay (ELISA), enzyme immunoassay (EIA) (EIA), fluorescence immunoassay (FIA), immunoblotting, immuno-precipitation With the immunoassays based on any particle (as used gold particle, Argent grain or latex particle, magnetic-particle or quantum dot).It can example Such as implement immunization in the form of microtiter plate or item.

Immunization according to the present invention can be based on, for example, any one of following methods.

Immuno-precipitation is simplest method of immunity；This method measures the amount of sediment, reagent antibodies With sample be incubated with and with its present in target antigen react and form the precipitation after insoluble aggregate to be formed.It is immune Precipitation reaction can be qualitative or quantitative.

In particle immunoassays, Multiple Antibodies are connect with the particle, and the particle can be in combination with many antigens Molecule.This has greatly speeded up the speed of visible reaction.This allows the quick and sensitive detection of biomarker.

In immunoturbidimetry (immunonephelometry), the phase interaction of the target antigen on antibody and biomarker With the formation for causing immune complex, the immune complex is too small and cannot precipitate.But these compounds will scatter incidence Light, nephometer can be used to measure in this.The concentration of antigen (i.e. biomarker) can be measured within a few minutes of reaction.

Radiommunoassay (RIA) method comes labelled antigen or antibody using radioactive isotope such as I125.It is used Isotope emits gamma-rays, usually measures the ray after removing uncombined (free) radioactive label.With it is other Immunoassays compare, the main advantage of RIA be higher sensitivity, easy signal detection and confirmation, quickly It measures.Main disadvantage be health and safety risk caused by the use by radiation and with safeguard license radiation safety and Processing routine relevant time and expense.For this reason, routine clinical laboratory practice in, RIA largely by Enzyme immunoassay (EIA) is replaced.

Enzyme immunoassay (EIA) (EIA) develops into the substitute of radiommunoassay (RIA).These methods are marked anti-using enzyme Body or target antigen.The sensitivity of EIA is close to the sensitivity of RIA, and there is no dangerous caused by radioactive isotope.For examining One of the most widely used EIA methods surveyed are enzyme linked immunosorbent assay (ELISA) (ELISA).Two kinds of antibody can be used in ELISA method, One is specific for target antigen, and another and enzyme is coupled, and the addition of zymolyte causes chemiluminescence signal or fluorescence to be believed Number generation.

Fluorescence immunoassay (FIA) refers to the immunoassays using fluorescent marker or enzyme label, the fluorescent marker or enzyme mark Be denoted as on substrate to form fluorescence-causing substance.Fluorescence measurement is inherently sensitiveer than colorimetric (spectrophotometry) measurement. Therefore, FIA methods have the higher sensitivity for analysis of EIA methods that Billy is measured with (optical density) is absorbed.

Chemiluminescence immunoassay uses chemiluminescent labeling, and light is generated when it is excited by chemical energy；Use light detection Device measures transmitting.

Therefore, method carries out immunization according to the present invention known to can be used.In the inspection of the biomarker of the present invention Any direct (as used sensor chip) or round-about way can be used in survey.

Chip, kit

Term " chip " is also referred to as " array ", refers to the solid support of the nucleic acid comprising connection or peptide probes.Array is usual Including being connected to a variety of different nucleic acid or peptide probes of substrate surface according to different known locations.These arrays, also referred to as " microarray " usually can generate these arrays using mechanical synthesis methods or light guiding synthetic method, and the light guiding is closed The combination of photolithography method and solid phase synthesis process is incorporated at method.Array can include flat surface, or can be pearl Son, gel, polymer surfaces, the fiber of such as optical fiber, glass or the nucleic acid in any other suitable substrate or peptide.It can be with Certain mode carrys out array of packages, to allow the manipulation of diagnosis for carrying out global function device or other means.

" microarray " is that hybridised arrays original paper is ordered in matrix, and the hybridised arrays original paper such as polynucleotide is visited Needle (such as oligonucleotides) or bonding agent (such as antibody).The matrix can be solid matrix, for example, glass or silica Slide, pearl, fibre optics binder or semi-solid matrix, such as nitrocellulose filter.Nucleotide sequence can be DNA, RNA or Any arrangement therein.

Various probe arrays have been described in the literature and can be used in the context of the invention detecting may be with this paper The relevant marker of phenotype.For example, DNA probe array chip or larger DNA probe array chip (otherwise, Ke Yitong Cross and interrupt chip and obtain each individual chip) it is used for one embodiment of the invention.DNA probe array chip generally comprises glass Glass chip placed high-density DNA probe (short dna segment) array thereon.These chips can respectively keep for example, about 6000 The DNA of ten thousand longer sample DNA sequences (for example, from individual or group, for example, including marker of interest) for identification Probe.It is carried out by DNA hybridization with the DNA probe group identification sample DNA on chip glass.When DNA sample and DNA probe array When hybridization, sample is incorporated into those of sample DNA sequence complementation probe.It is more steady by evaluating individual sample DNA and those probes Admittedly hybridize, it is possible to determine that known nucleic acid sequence whether there is in sample, thereby determine that the marker found in nucleic acid It whether there is.It can also be using this means by controlling hybridization conditions to allow to distinguish single nucleotide, for example, being used for SNP The sample gene parting with one or more SNP is identified to carry out ASH.It is multiple that array provides a kind of (or series winding) simultaneously detection The convenience embodiment of polymorphic marker.

In the present invention, chip includes genetic chip, protein chip；The genetic chip includes solid phase carrier；And have Sequence is fixed on the oligonucleotide probe on the solid phase carrier, and the oligonucleotide probe specifically corresponds to STRADB institutes Some or all of show sequence.The protein chip includes solid phase carrier, and the STRADB codings being fixed on solid phase carrier Albumen specific antibody or ligand.

" probe " refers to the molecule that can be combined with the particular sequence of another molecule or subsequence or other parts.Unless otherwise finger Go out, term " probe " is often referred to match by complementary base and (often referred to as " target polynucleotide ") be combined with another polynucleotides Polynucleotide probes.According to the stringency of hybridization conditions, probe energy and the target for lacking sufficient sequence complementarity with the probe are more Nucleotide combines.Probe can make direct or indirect label, and range includes primer.Crossing system includes, but are not limited to：It is molten Liquid phase, solid phase, mixed phase or in situ hybridization measuring method.

Above-mentioned probe has the base sequence with the specific base sequence complementary of target gene.Here, so-called " complementation ", As long as hybridization, can not be complete complementary.These polynucleotides have usually relative to the specific base sequence 80% or more, preferably 90% or more, more preferable 95% or more, particularly preferred 100% homology.These probes can be DNA, Can also be RNA, furthermore it is possible to be artificial by PNA, LNA, ENA, GNA, TNA etc. in part of it or whole nucleotides The polynucleotides that replacement nucleic acid obtains.

The ligand of the present invention may include that the peptide of STRADB, antibody or its segment or aptamers or widow can be specifically bound Nucleotide.The antibody for above-mentioned STRADB protein used in the present invention is used with broadest, and is specifically covered for example Monoclonal antibody, polyclonal antibody, the antibody with multi-epitope specificity, multi-specificity antibody and antibody fragment.Such antibody Can be it is chimeric, humanization, people's and synthesis.

" monoclonal antibody " refers to the antibody obtained from the antibody of a group substantially homogeneity as used herein, that is, constitutes group Each antibody it is identical and/or combine same epitope, in addition to production monoclonal antibody during issuable possible variant Outside, such variant is generally with indivisible presence.Such monoclonal antibody is typically include comprising the polypeptide sequence in conjunction with target Antibody, wherein target combination polypeptide sequence are by including selecting single target combination polypeptide sequence to exist in comforming the more peptide sequence What interior process obtained.For example, selection course can be comform polyclonal such as hybridoma clone, phage clone or recombination Unique clones are selected in the set of DNA clone.It should be appreciated that selected target binding sequence can further change, such as in order to Improve to the affinity of target, by target binding sequence humanization, improve its yield in cell culture, reduce it in body Interior immunogenicity creates multi-specificity antibody etc., and include the antibody of target binding sequence after changing is also the present invention Monoclonal antibody.From the typical polyclonal antibody preparations comprising the different antibodies for different determinants (epitope) not Together, each monoclonal antibody of monoclonal antibody preparations is for the single determinant on antigen.Other than their specificity, The advantage of monoclonal antibody preparations is that they are generally not affected by the pollution of other immunoglobulins.Modifier " monoclonal " refers to Show antibody basically homogeneity antibody population obtain feature, should not be construed as require generated by any ad hoc approach it is anti- Body.For example, need to be generated by multiple technologies according to the monoclonal antibody that the present invention uses, including：Hybridoma, recombination DNA methods, display technique of bacteriophage and for from having part or whole human immunoglobulin gene's seat or encoding human that ball is immunized The animal of the gene of protein sequence generates the technology of people or human-like antibodies.

Monoclonal antibody clearly includes " chimeric " antibody (immunoglobulin), wherein heavy chain and/or light chain herein A part be derived from particular species or belong to corresponding sequence in the antibody of specific antibodies classification or subclass it is identical or homologous, and The remainder of chain with derived from another species or belong to corresponding sequence in the antibody of another antibody isotype or subclass it is identical or The segment of homologous and such antibody, as long as they show desired biological activity.

It includes the sequence derived from non-human immunoglobulin that " humanization " form of inhuman (such as mouse) antibody, which refers to bottom line, The gomphosis immunoglobulins of row, immunoglobulin chain or its segment such as Fv, Fab, Fab ', F (ab ')²Or antibody is other anti- Original combines subsequence.

" antibody fragment " includes a part for full length antibody, usually its antigen binding domain or variable region.Antibody fragment Example includes Fab, Fab ', F (ab ')²With Fv segments；Double antibody；Linear antibodies；Single-chain antibody molecules；And by antibody fragment shape At multi-specificity antibody.

" Fv " is the minimum antibody fragment for including intact antigen identification and binding site.The segment is by close, non-covalent knot The dimer composition of the heavy chain variable domain and a light-chain variable domain that close.It is distributed from the foldable structure of the two structural domains Go out six hypervariable loops (heavy chain and each 3 rings of light chain), facilitates the amino acid residue in conjunction with antigen and assign antibody with antigen binding Specificity.Even knowing however, single variable domain (or only including half of Fv of three CDR to antigen-specific) can also have Ability that is other and combining antigen, only affinity is less than entire binding site.

" functional fragment " of the antibody of the present invention refers to those and retains with they derivative complete full chain molecule with substantially Identical affinity combination polypeptide and it is active at least one measuring method (such as inhibit TH2 induction asthma approach, such as In mouse model, or in vitro inhibit antibody fragment combine antigen biological activity) segment.

In the present invention, kit can be used for detecting the expression of STRADB genes or albumen, including being examined for STRADB The ligand, and/or chip of survey and/or the quantitative present invention.Together with the optional specification with kit.

Kit includes one or more sterile chambers, such container can be box, ampoule, bottle, phial, pipe, Bag, pouch, blister package or other suitable vessel forms known in the art.Such container can by plastics, glass, Laminated paper, metal foil or the other materials suitable for container drug.

In the present invention, term " comprising " is for referring to phrase " including but not limited to ", and with phrase " including but not limited to " It may be used interchangeably.

Statistical method

In the present invention, experiment at least uses 3 repetitions to test, and result data is all in a manner of mean+SD It indicates, come using SPSS18.0 statistical softwares for statistical analysis, difference between the two is using t inspections, it is believed that works as P< There is statistical significance when 0.05.

Embodiment

The present invention is described in further detail with reference to the accompanying drawings and examples.Following embodiment is merely to illustrate this It invents rather than limits the scope of the invention.Test method without specific conditions in embodiment, usually according to conventional strip Part, such as Sambrook et al., molecular cloning：Laboratory manual (New York:Cold Spring HarborLaboratory Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.

Embodiment 1 is screened and the relevant gene marker of asthma

1, sample collection

According to the diagnostic criteria of bronchial asthma diagnosis and treatment guide, 10 normal human bloods and asthmatic patient are respectively collected The acquirement of blood peripheric venous blood 3ml, the equal informed consent of patient, above-mentioned all samples pass through the agreement of Ethics Committee.

2, the preparation and quality analysis of RNA sample

The preparation of 2.1 RNA samples

Total serum IgE is extracted using the RNA extracts kits of Promega companies.It is as follows：

1) it takes 1ml to collect the whole blood in heparin or the processed test tubes of EDTA, puts into sterile centrifugation tube；

2) 3000rpm (400g) centrifuges 5min, carefully siphons away supernatant at the top of sample；

3) plus 1ml haemocyte lysates, careful inhale are put 4-5 times, sediment are resuspended, 3000rpm centrifuges 5min；

4) repeatedly step 3 (is total to three times) twice；

5) cell precipitate is avoided, nearly all supernatant is carefully siphoned away, only retains 100 μ l supernatants；Check BME Through being added in RNA lysates, then plus in 175 μ l RNA lysates to cell, resuspension and lytic cell are put in suction；

6) add 350 μ l RNA dilutions, overturn mixing 3-4 times, 13000g centrifuges 10min at room temperature, by limpid lysate It is transferred in a sterile centrifugation tube；

7) 200 μ l, 95% ethyl alcohol is added into cleared lysate, 3-4 times is put with mixing with liquid-transfering gun suction；By this mixture It is transferred in centrifugal column assembly, 13000g centrifuges 1min；

8) centrifugal column is taken down from centrifugal column assembly, discards the liquid in collecting pipe, centrifugal column is installed onto collecting pipe On, add 600 μ l RNA cleaning solutions in centrifugal column assembly, 13000g centrifuges 1min；

9) liquid in collecting pipe is discarded, centrifugal column is installed onto on collecting pipe, the freshly prepared DNase of 50 μ l are incubated Mixture is applied directly on the film in centrifugal column；

10) it is incubated 15min at room temperature, 200 μ l DNA enzymatics stop buffers are added into centrifugal column, and (second has been added in confirmation Alcohol), 13000g centrifuges 1min；

11) 600 μ l RNA cleaning solutions (ethyl alcohol has been added) are added, 13000g centrifuges 1min；

12) collecting pipe is emptied, 250 μ l RNA cleaning solutions (ethyl alcohol has been added), high speed centrifugation 2min are added into centrifugal column；

13) centrifugal column is transferred on elution pipe from collecting pipe, 100 μ l nuclease-free waters is added on film, by centrifugal column Assembly puts centrifuge into and makes the lid facing outwards of elution pipe, and 13000g centrifuges 1min, abandons centrifugal column, covers and fill RNA's Elution pipe, is stored in -70 DEG C.

The quality analysis of 2.2 RNA samples

The RNA concentration and purity extracted are detected using Nanodrop2000, agarose gel electrophoresis detects RNA Integrality, Agilent2100 measure RIN values.Concentration >=200ng/ μ l, OD260/280 is between 1.8~2.2.

3, rRNA is removed

The rRNA in total serum IgE is removed using Ribo-Zero kits.

4, construction cDNA library

It is specific to grasp using the structure for carrying out cDNA library using the Truseq RNA sample Prep Kit of Illumina Make by specification progress.

5, upper machine sequencing

CDNA library is sequenced using Hiseq4000 microarray datasets, concrete operations by specification carries out.

6, high-throughput transcript profile sequencing data analysis

Bioinformatic analysis is carried out to sequencing result, RNA-seq read positioning is carried out using TopHat v1.3.1, leads to It crosses Cufflinks v1.0.3 and RNA-seq segment numbers is standardized to the relative abundance for calculating transcript, utilize Cuffdiff detects differential expression, works as FDR<When 0.05, it is believed that mRNA significant differences are expressed.

7, result

RNA-seq is the results show that expression quantity of the STRADB genes in blood of asthmatic patients is substantially less than the table of normal person Up to amount.

The differential expression of 2 QPCR sequence verification STRADB genes of embodiment

1, STRADB genes are selected to carry out large sample QPCR verifications according to the testing result of high-flux sequence.According to embodiment Method in 1 collects selection blood of asthmatic patients and each 80 of normal human blood sample.

2, RNA extraction steps are the same as embodiment 1.

3, reverse transcription：It is operated using the reverse transcription reagent box of TAKARA companies.It is as follows：

(1) it takes 2 μ g of total serum IgE to carry out reverse transcription, Oligo (dT) 2 μ l is added, mix well；After 70 DEG C of water-bath 5min immediately Ice bath 2-3min；

(2) 25 μ l reaction systems are built, including 5 × RT Buffer 5 μ l, dNTP (2.5mM) 5 μ l, RNasin 40U/ μ l, M-MLV 200U/ μ l mend nuclease free water to 25 μ l；

After (3) 42 DEG C of water-bath 60min, 95 DEG C of water-bath 5min are to inactivate M-MLV；

(4) -20 DEG C store for future use.

4, QPCR is expanded

(1) design of primers

QPCR amplimers are designed according to the coded sequence of STRADB genes and GAPDH genes in Genebank, by Shanghai Sheng Gong biotechnologies Services Co., Ltd synthesizes.Wherein, the primer sequence of amplification of STR ADB genes is to such as SEQ ID NO.3 Shown in~4, the primer sequence of amplification reference gene GAPDH is to as shown in NO.5~6 SEQ ID.

(2) 25 μ l PCR reaction systems are prepared：

Deionized water 8.5 μ l, 12.5 μ l of SYBR Green PCRs system, just are added into sterile EP tube (anti-) is uniformly mixed to 1 μ l of primer, 12.5 μ l of SYBR Green PCRs system, 2 μ l of template；Wherein, SYBR Green PCR systems are purchased from Invitrogen companies.

(3) PCR reaction conditions：95 DEG C of 10min, (95 DEG C of 15s, 60 DEG C of 60s) × 45 cycles.Using SYBR Green as Fluorescent marker carries out PCR reactions on Light Cycler fluorescence quantitative PCR instruments, true by melt curve analysis analysis and electrophoresis Determine purpose band, Δ Δ CT methods carry out relative quantification.

5, result

The results are shown in Figure 1, and compared with normal human blood, expression of the STRADB genes in blood of asthmatic patients is lowered, Difference has statistical significance (P<0.05), consistent with RNA-sep results.

3 protein level of embodiment verifies the differential expression of STRADB

1, each histone is extracted according to RIPA protein lysate kit specifications, uses BCA determination of protein concentration reagents Box detects albumen concentration in sample.

2, with the detection STRADB albumen variation of conventional Western-blot methods, each group experiment is repeated 3 times, with β- Actin is internal reference, does STRADB protein band absorbance quantitative analyses, expression quantity is with STRADB albumen/β-actin absorbances Ratio represents.

3, result

The results are shown in Figure 2, and compared with normal person, the protein level of STRADB is significantly lowered in asthmatic patient, difference tool Statistically significant (P<0.05).

The explanation of above-described embodiment is only intended to understand the method and its core concept of the present invention.It should be pointed out that for this For the those of ordinary skill in field, without departing from the principle of the present invention, several improvement can also be carried out to the present invention And modification, these improvement and modification will also be fallen into the protection domain of the claims in the present invention.

SEQUENCE LISTING

<110>The second the People's Hospital of Changzhou

<120>Applications of the STRADB in asthma early diagnosis

<160> 6

<170> PatentIn version 3.5

<210> 1

<211> 1134

<212> DNA

<213>People source

<400> 1

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cgtccatcca ctagagccag tgaagtacta tgttccacca acgtttctca ctatgagctc 180

caagtagaaa taggaagagg atttgacaac ttgacttctg tccatcttgc acggcatact 240

cccacaggaa cactggtaac tataaaaatt acaaatctgg aaaactgcaa tgaagaacgc 300

ctgaaagctt tacagaaagc cgtgattcta tcccactttt tccggcatcc caatattaca 360

acttattgga cagttttcac tgttggcagc tggctttggg ttatttctcc atttatggcc 420

tatggttcag caagtcaact cttgaggacc tattttcctg aaggaatgag tgaaacttta 480

ataagaaaca ttctctttgg agccgtgaga gggttgaact atctgcacca aaatggctgt 540

attcacagga gtattaaagc cagccatatc ctcatttctg gtgatggcct agtgaccctc 600

tctggcctgt cccatctgca tagtttggtt aagcatggac agaggcatag ggctgtgtat 660

gatttcccac agttcagcac atcagtgcag ccgtggctga gtccagaact actgagacag 720

gatttacatg ggtataatgt gaagtcagat atttacagtg ttgggattac agcatgtgaa 780

ttagccagtg ggcaggtgcc tttccaggac atgcatagaa ctcagatgct gttacagaaa 840

ctgaaaggtc ctccttatag cccattggat atcagtattt tccctcaatc agaatccaga 900

atgaaaaatt cccagtcagg tgtagactct gggattggag aaagtgtgct tgtctccagt 960

ggaactcaca cagtaaatag tgaccgatta cacacaccat cctcaaaaac tttctctcct 1020

gccttcttta gcttggtaca gctctgtttg caacaagatc ctgagaaaag gccatcagca 1080

agcagtttat tgtcccatgt tttcttcaaa cagccttatt ttgagtttct ttaa 1134

<210> 2

<211> 377

<212> PRT

<213>People source

<400> 2

Met Ser Leu Leu Asp Cys Phe Cys Thr Ser Arg Thr Gln Val Glu Ser

1 5 10 15

Leu Arg Pro Glu Lys Gln Ser Glu Thr Ser Ile His Gln Tyr Leu Val

20 25 30

Asp Glu Pro Thr Leu Ser Trp Ser Arg Pro Ser Thr Arg Ala Ser Glu

35 40 45

Val Leu Cys Ser Thr Asn Val Ser His Tyr Glu Leu Gln Val Glu Ile

50 55 60

Gly Arg Gly Phe Asp Asn Leu Thr Ser Val His Leu Ala Arg His Thr

65 70 75 80

Pro Thr Gly Thr Leu Val Thr Ile Lys Ile Thr Asn Leu Glu Asn Cys

85 90 95

Asn Glu Glu Arg Leu Lys Ala Leu Gln Lys Ala Val Ile Leu Ser His

100 105 110

Phe Phe Arg His Pro Asn Ile Thr Thr Tyr Trp Thr Val Phe Thr Val

115 120 125

Gly Ser Trp Leu Trp Val Ile Ser Pro Phe Met Ala Tyr Gly Ser Ala

130 135 140

Ser Gln Leu Leu Arg Thr Tyr Phe Pro Glu Gly Met Ser Glu Thr Leu

145 150 155 160

Ile Arg Asn Ile Leu Phe Gly Ala Val Arg Gly Leu Asn Tyr Leu His

165 170 175

Gln Asn Gly Cys Ile His Arg Ser Ile Lys Ala Ser His Ile Leu Ile

180 185 190

Ser Gly Asp Gly Leu Val Thr Leu Ser Gly Leu Ser His Leu His Ser

195 200 205

Leu Val Lys His Gly Gln Arg His Arg Ala Val Tyr Asp Phe Pro Gln

210 215 220

Phe Ser Thr Ser Val Gln Pro Trp Leu Ser Pro Glu Leu Leu Arg Gln

225 230 235 240

Asp Leu His Gly Tyr Asn Val Lys Ser Asp Ile Tyr Ser Val Gly Ile

245 250 255

Thr Ala Cys Glu Leu Ala Ser Gly Gln Val Pro Phe Gln Asp Met His

260 265 270

Arg Thr Gln Met Leu Leu Gln Lys Leu Lys Gly Pro Pro Tyr Ser Pro

275 280 285

Leu Asp Ile Ser Ile Phe Pro Gln Ser Glu Ser Arg Met Lys Asn Ser

290 295 300

Gln Ser Gly Val Asp Ser Gly Ile Gly Glu Ser Val Leu Val Ser Ser

305 310 315 320

Gly Thr His Thr Val Asn Ser Asp Arg Leu His Thr Pro Ser Ser Lys

325 330 335

Thr Phe Ser Pro Ala Phe Phe Ser Leu Val Gln Leu Cys Leu Gln Gln

340 345 350

Asp Pro Glu Lys Arg Pro Ser Ala Ser Ser Leu Leu Ser His Val Phe

355 360 365

Phe Lys Gln Pro Tyr Phe Glu Phe Leu

370 375

<210> 3

<211> 19

<212> DNA

<213>Artificial sequence

<400> 3

acttgacttc tgtccatct 19

<210> 4

<211> 18

<212> DNA

<213>Artificial sequence

<400> 4

ttcaggcgtt cttcattg 18

<210> 5

<211> 21

<212> DNA

<213>Artificial sequence

<400> 5

ctctggtaaa gtggatattg t 21

<210> 6

<211> 20

<212> DNA

<213>Artificial sequence

<400> 6

ggtggaatca tattggaaca 20

Claims (10)

1. Application of the detection reagent of 1.STRADB genes in the product for preparing diagnosis asthma.
2. 2. application according to claim 1, which is characterized in that the product includes STRADB expressions in detection sample Reagent.
3. 3. application according to claim 2, which is characterized in that the reagent includes：By RT-PCR, real-time quantitative PCR, The reagent of immune detection, in situ hybridization or chip detection STRADB expressions.
4. 4. application according to claim 3, which is characterized in that the examination for detecting STRADB expressions by RT-PCR Agent includes at least the primer of a pair of of specific amplified STRADB genes；It is described that STRADB expressions are detected by real-time quantitative PCR Reagent include at least the primers of a pair of of specific amplified STRADB genes；The examination by immune detection STRADB expressions Agent includes the antibody and/or ligand combined with STRADB protein-specifics；It is described that STRADB expression water is detected by situ hybridization Flat reagent includes the probe with the nucleic acid array hybridizing of STRADB genes；It is described that STRADB expressions are detected by chip Reagent includes the probe hybridized with STRADB gene nucleic acid sequences or the antibody combined with STRADB protein-specifics and/or matches Body.
5. 5. according to claim 2-4 any one of them applications, which is characterized in that the sample is selected from human peripheral.
6. 6. application according to claim 2, which is characterized in that the product includes preparation, chip or kit, wherein The preparation, chip or kit include detecting the reagent of STRADB expressions.
7. 7. application according to claim 6, which is characterized in that detect the reagent packet of STRADB expressions in the chip Include the probe of specific recognition STRADB genes, or the antibody or ligand of the albumen of specific binding STRADB codings.
8. 8. application according to claim 6, which is characterized in that detect the reagent of STRADB expressions in the kit Include the primer of specific amplification STRADB genes；Or the probe of specific recognition STRADB genes；Or specific binding STRADB The antibody or ligand of the albumen of coding.
9. 9. application according to claim 8, which is characterized in that the primer sequence of the specific amplification STRADB genes is such as Shown in SEQ SEQ ID NO.3 and SEQ ID NO.4.
10. 10. application according to claim 9, which is characterized in that the reagent further includes SYBR Green polymerase chains Reaction system, the primer pair for expanding house-keeping gene；The SYBR Green PCR systems include：PCR is slow Fliud flushing, dNTPs, SYBR Green fluorescent dyes.