CN108034707A - Application of the SPAG7 genes in diagnosis of dementia preparation is prepared - Google Patents
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Abstract
The present invention relates to application of the SPAG7 genes in diagnosis of dementia preparation is prepared, and is specifically related to the application of SPAG7 genes and its expression product in diagnose and treat senile dementia disease.To solve the problems, such as that current senile dementia molecular marked compound is rare, inventor carries out high-flux sequence to old dementia patients and Healthy People control peripheral blood sample, pick out candidate gene SPAG7, and confirm that SPAG7 genes have good correlation with senile dementia by RT PCR methods, diagnosing senile dementia for clinical gene lays the foundation.
Description
Technical field
The present invention relates to biomedicine field, and in particular to SPAG7 genes answering in diagnosis of dementia preparation is prepared
With more particularly relating to the application of SPAG7 genes and its expression product in diagnose and treat senile dementia disease.
Background technology
Senile dementia also known as alzheimer (AD), the incidence of China's senile dementia are annual every thousand people of every 8 people, 60 years old
The later sick incidence and illness rate are all with the increase exponential increase at age.Due to showing for China human mortality Aging Problem
Existing, elderly population quantity increases so that this kind of disease becomes one of influence China's elderly population Health and Living key factor,
And the still no effective treatment method of senile dementia, doing sth. in advance diagnose and treat helps to delay disease process, therefore, finds
Senile dementia diagnoses and predicts relevant biomarker into seeming extremely important.
Existing disease process diagnostic method includes the MMSE marking tests based on questionnaire, and for A amyloid betas
Neuron image checking, also have some invasive methods such as by analyzing (A β points of the associated biomolecules in cerebrospinal fluid (CSF)
Son, Protein tau etc.) help to carry out the diagnosis of AD, study and thought, pass through AD biological markers in cerebrospinal fluid and carry out AD's
Accuracy rate of diagnosis is preferable, but there are 2 it is obvious the shortcomings that：Expense is excessive, and compared to peripheral blood, relevant biological tissue obtained
Journey is more difficult；It is larger to obtain cerebrospinal fluid pain caused by patient, it is also possible to leave sequelae.In contrast, peripheral blood is one
Kind is easier the biological tissue obtained, and study the relevant biology changes of AD in peripheral blood just has realistic meaning very much, existing
Patent in have revealed that part and the relevant molecular marker of senile dementia, the YAP1 bases as disclosed in ZL2015104636167
Cause, the EAPP genes that ZL2015104635287 is disclosed, 10 and the relevant cause of senile dementia that CN2016104739651 is disclosed
Ospc gene, still, said gene need further to verify.
To solve the problems, such as that current senile dementia molecular marked compound is rare, inventor is to old dementia patients and Healthy People pair
High-flux sequence is carried out according to peripheral blood sample, genescreen is carried out using bioinformatics method, picks out candidate gene
SPAG7.Existing literature research shows that the diseases such as SPAG7 genes and oral cavity property stomatitis, pharyngitis and adenopathy are related, can be used as above-mentioned disease
The diagnosis molecular marker of disease.Further, the present invention has carried out RT-PCR method and has confirmed that SPAG7 has very well with senile dementia
Correlation, for clinical gene diagnose senile dementia lay the foundation.
The content of the invention
Examined it is an object of the invention to provide the reagent of a kind of detection SPAG7 genes and/or albumen preparing senile dementia
Application in disconnected preparation.
To achieve the above object, the present invention screens candidate's base by high-flux sequence combination bioinformatics method first
Because of SPAG7, the further relation of SPAG7 and senile dementia by molecular cytobiology method validation：SPAG7 is crazy about with old age
Stay with good correlation, available for treatment senile dementia preparation and/or diagnosis of dementia preparation is prepared, have important
Clinical value.
Further, the diagnostic preparation of the senile dementia is included with fluorescence quantifying PCR method, method for gene chip, survey
The expression of SPAG7 genes in sequence method detection senile dementia peripheral blood.
Fluorescence quantitative PCR method is the specific probe by fluorescent dye or fluorescent marker, and PCR product is marked
Tracking, real time and on line monitoring reaction process, can analyze product with reference to corresponding software, calculate sample to be tested template
Initial concentration.The appearance of quantitative fluorescent PCR, greatly simplifies the process of quantitative detection, and is truly realized absolute quantitation.
The appearance of a variety of detecting systems, makes the selectivity of experiment stronger.Automation mechanized operation improves work efficiency, rapid reaction, repetition
The good, high sensitivity of property, high specificity, result are clear.
Genetic chip is also known as DNA microarray (DNA microarray), can be divided into three kinds of main Types：1) it is fixed on poly-
Nucleic acid probe or cDNA fragments on compound substrate (nylon membrane, nitrocellulose membrane etc.) surface, usually use the target of isotope marks
Gene is hybrid with it, and is detected by radiography technology.2) DNA probe array on a glass is fixed with point sample method,
By being detected with the hybridization of the target gene of fluorescent marker.3) oligonucleotide probe directly synthesized on the hard surfaces such as glass
Array, the target gene hybridization with fluorescent marker are detected.Genetic chip is as a kind of advanced, extensive, high throughput detection
Technology, applied to the diagnosis of disease, its advantage has the following aspects：First, the sensitivity and accuracy of height；It is second, quick
It is easy；Third, a variety of diseases can be detected at the same time.
High-flux sequence (High-throughput sequencing) is also known as sequencing technologies (next of future generation
Generation sequencing) it is the change for tradition being sequenced revolution, once to hundreds of thousands to millions of DNA
Molecule carries out sequencing, greatly improves sequencing efficiency.This kind of large scale sequencing technology greatly improves multiple species and loses
The solution reading rate of communication breath, to obtain the sequence information of all mRNA, decryption mRNA collection of illustrative plates provides guarantee.High pass measures at the same time
Sequence to carry out the analysis of careful overall picture to the transcript profile and genome of species, so the depth survey that is otherwise known as
Sequence.The representative of high-flux sequence platform is 454 sequenators (Roch GSFLX sequencer) of Roche Holding Ag (Roche),
The Solexa genome analysises instrument (Illumina Genome Analyzer) of Illumina companies and the SOLiD sequenators of ABI
(ABI SOLiD sequencer)。
The product for being used for SPAG7 genes in fluorescence quantifying PCR method detection senile dementia contains a pair of of specificity
Expand the primer of SPAG7 genes；The genetic chip includes the probe with the nucleic acid array hybridizing of SPAG7 genes.
Further, the diagnostic preparation of the senile dementia includes the expression with immunization method detection SPAG7 albumen.It is preferred that
SPAG7 protein expressions for western blot and/or ELISA and/colloid in immunologic detection method detection senile dementia
Golden detection method.
Enzyme-linked immunosorbent assay (ELISA) will known antigen or antibody absorption in surface of solid phase carriers, 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 detection macromolecular antigen and specific antibody
Deng, have the advantages that quick, sensitive, easy, carrier be easy to standardization.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 peripheral blood
Section, can be dyed with the antibody of colloid gold label, can also be strengthened with silver-colored developer solution and marked on the basis of colloid gold label
Note, the silver atoms for making to be reduced are deposited on marked gold grain surface, can be remarkably reinforced the sensitiveness of colloid gold label.(2)
Immune colloid gold electronic speculum decoration method can with the antibody of colloid gold label or antiantibody with negative staining Virus Sample or peripheral blood are ultra-thin cuts
Piece combines, and then carries out negative staining.Observation and viral diagnosis available for morphology of virus.(3) dot immunogold filtration assay is using micro-
Membrane as carrier is filtered in hole, first adds sample to be checked after closing, the antibody of colloid gold label is used after washing antigen or antibody point on film
Detect corresponding antigen or antibody.(4) specific antigen or antibody are fixed on film by colloidal gold immunity chromatography with ribbon
On, colloid gold label reagent (antibody or monoclonal antibody) is adsorbed on bonding pad, when sample to be checked is added to test strips one end
After in sample pad, move forward through capillary action, react to each other after dissolving the colloid gold label reagent on bonding pad, work as movement
To fixed antigen or antibody region when, the conjugate of thing and gold marked reagent to be checked occurs specific binding therewith again and is cut
Stay, be gathered in detection and take, colour developing result can be observed by the naked eye.The method has developed into diagnosis test paper, uses ten
It is convenient to divide.
Further, the ELISA method of the detection SPAG7 albumen is to use ELISA detection kit.In the kit
Antibody can use commercially available SPAG7 monoclonal antibodies.Further, the kit includes：It is coated with SPAG7 monoclonal antibodies
Solid phase carrier, ELIAS secondary antibody, the substrate of enzyme, protein standard substance, negative controls, dilution, cleaning solution, enzyme reaction terminate liquid
Deng.
Further, the colloidal gold method of the detection SPAG7 albumen is that can use city using detection kit, the antibody
The SPAG7 monoclonal antibodies sold.Further, the gold-immunochromatographyreagent reagent for assay box uses colloidal gold immunochromatographimethod technology or colloid
Golden percolation.Further, detection zone (T) specking on the gold-immunochromatographyreagent reagent for assay box nitrocellulose filter has anti-SPAG7
Monoclonal antibody, quality control region (C) specking have Immunoglobulin IgG.
It is an object of the invention to provide a kind of PCR kit for fluorescence quantitative for detecting senile dementia, it is characterised in that institute
Kit detection gene SPAG7 is stated, using special sense primer and anti-sense primer, upstream primer sequence is SEQ ID NO.1,
Downstream primer sequence is SEQ ID NO.2.
Further, which is suitable for presently, there are all types fluorescence quantitative gene extender of in the market, spirit
Sensitivity is high, it is quantitative quick and precisely, stability it is good, have a good application prospect.
Further, above-mentioned PCR kit for fluorescence quantitative component includes：Specific primer, internal control primer, quantitative fluorescent PCR
Reaction solution.The wherein described specific primer includes sense primer and anti-sense primer, and upstream primer sequence is SEQ ID NO.1,
Downstream primer sequence is SEQ ID NO.2.The internal control primer is β-actin internal control primers, and upstream primer sequence is SEQ ID
NO.3, downstream primer sequence are SEQ ID NO.4.
The kit also includes RNA extraction agents.It is preferred thatReagent carries out sample rna extraction.
The present invention also have detected this kit sensitivity, this kit of the results show detection range is 106-102copies/μ
L, minimum concentrations are 100copies/ μ l.
It is an object of the present invention to provide a kind of senile dementia detection kit, detection kit detection SPAG7 eggs
In vain.Further, the kit further includes other detection reagents.
It is an object of the present invention to provide it is a kind of detect senile dementia genetic chip, the genetic chip include with
The probe of the nucleic acid array hybridizing of SPAG7 genes.
It is an object of the invention to provide the application of SPAG7 genes and/or albumen in senile dementia treatment preparation is prepared.
Further, the senile dementia treatment preparation refers to the preparation that can promote the expression of SPAG7 genes.People from this area
Member is known to promote the expression of gene usually to use one kind in following methods and/or several：Regulated and controled by DNA level
SPAG7 genes：Including but not limited to increase the copy number of SPAG7 genes, transfect the over-express vector of the gene containing SPAG7；Pass through
Transcriptional level control SPAG7 genes：Including but not limited to activate the expression of SPAG7 genes, activation regulates and controls SPAG7 gene expressions
Promoter, the transcription factor for suppressing negative regulation SPAG7 gene expressions, using RNA perturbation techniques to suppressing SPAG7 gene expressions
Repressor is disturbed；SPAG7 genes are regulated and controled by post-transcriptional level：Including but not limited to suppress to promote SPAG7 gene mRNAs
The microRNA transcriptional expressions of degraded, import the microRNA for promoting SPAG7 gene expressions；Pass through level modulation after translation
SPAG7 genes：Including but not limited to import and promote the molecule of SPAG7 gene coded proteins, suppress negative regulation SPAG7 gene expressions
Albumen, promote SPAG7 gene expressions the factor and albumen expression.
It is an object of the invention to provide one kind to treat senile dementia preparation, and the anti-senile dementia preparation promotes old silly
The expression of SPAG7 genes in slow-witted patient.Further, promotion SPAG7 gene expressions are contained in the treatment senile dementia preparation
Carrier.
Brief description of the drawings
Fig. 1 SPAG7 genes relative expression's spirogram in senile dementia peripheral blood and healthy human peripheral blood
Embodiment
With reference to specific embodiment, the present invention is further explained, is only used for explaining the present invention, and it is not intended that to this
The limitation of invention.It will be understood by those skilled in the art that：Can in the case where not departing from the principle of the present invention and objective
These embodiments are carried out with a variety of change, modification, replacement and modification, the scope of the present invention is limited by claim and its equivalent
It is fixed.The experimental method of actual conditions is not specified in the following example, usually according to normal condition or according to the bar proposed by manufacturer
Part examinations.
1 high-flux sequence of embodiment and analysis
Samples sources obtain subjects informed consent in BJ Union Hospital.15 senile dementias are collected respectively to suffer from
Person's peripheral blood sample and 9 Healthy People control peripheral blood samples, progress RNA extractions, agarose gel electrophoresis after RNA extractions, from
Whether the RNA sample that electrophoresis result can be extracted with preliminary judgement is up-to-standard, if can be used for further transcriptome analysis.
And then the extraction situation of RNA sample, the sample requirement of RNA-seq sequencings are detected by NanoDrop1000 spectrophotometers：
OD260/OD280 is 1.8-2.2.
Microarray dataset is the 2500 high-flux sequence platforms of HiSeq of Illumina companies, carries out high throughput transcript profile depth
Sequencing, we use Fast-QC (http after sequencing://www.bioinformatics.babraham.ac.uk/projects/
Fastqc/) software carries out total evaluation to the quality of sequencing data, includes the quality Distribution value of base, the position point of mass value
Cloth, G/C content, PCR duplication contents, frequency of kmer etc..In differential genes expression analysis, according to obtaining
FPKM values, using internationally recognized algorithm EBSeq carry out differential screening.Wherein, during screening, LOG2FC>1 or<-1,FDR<
0.05.In order to be better understood from the function of difference expression gene, we have carried out Gene Onlogy and letter to difference expression gene
Number path analysis, and functional annotation and protein interaction network analysis are carried out to difference expression gene, in view of data above
Analysis as a result, we have screened downward difference expression gene SPAG7 with reference to document.
2 old dementia patients peripheral blood of embodiment and healthy human peripheral blood SPAG7 expression conditions
One material and method
1st, material
95 old dementia patients peripheral bloods and 31 healthy human peripheral bloods are collected, it is grouped and is numbered.2nd, side
Method
The extraction of 2.1 old dementia patients peripheral bloods and healthy human peripheral blood total serum IgE
UsingReagent carries out sample rna extraction, and experimental implementation is carried out by product description, and concrete operations are shown in
Specification.
RNA quality judging standards：The OD260/OD280 values of RNA samples are between 1.7-2.2；Total serum IgE electrophoresis pattern has clearly
Clear 28S, 18S band；Electrophoresis pattern after when 70 DEG C of water-bath insulations 1 are small and the collection of illustrative plates no significant difference before water-bath insulation.
2.2 reverse transcriptions synthesize cDNA
UsingIII Reverse Transcriptase (invitrogen, article No. 18080-044) into
Row cDNA reverse transcriptions, experimental implementation are carried out by product description, and concrete operations are as follows：
Using Reverse Transcriptase kit, converse record synthesis cDNA is carried out to l μ g total serum IgEs with RT Buffer.Using 25 μ l
Reaction system, each sample take 1 μ g total serum IgEs to be separately added into following components in PCR pipe as template ribonucleic acid：
5 × RT Buffer, 5 μ l, 10mmol/l dNTP, 1.25 μ l, 0.1mmol/l DTT 2.5 μ l, 30 μm of mol/l
2 μ l, 200U/ μ l MMLV of OligodT 1.25 μ l, 1 μ g of template ribonucleic acid, add aqua sterilisa to 25 μ l of total system.42 DEG C are incubated 1
Hour, 72 DEG C 10 minutes, of short duration centrifugation.It is spare that -20 DEG C of refrigerators are put in cDNA preservations.
2.3Real-Time PCR
2.3.1 instrument and analysis method
With 7500 type fluorescence quantitative PCR instruments of ABI, the relative quantitative assay of data is carried out using 2- △ △ CT methods.2.3.2
Design of primers
SPAG7 sequence NM_004890.2, using online primer-design software, by invitrogen companies after design of primers
Synthesis.Specific primer sequence is as follows：
1 primer sequence of table
Operating process is as follows：
(1) reaction system：Use PowerGreen PCR Master Mix (invitrogen, article No.
4367659) expanded, experimental implementation is carried out by product description.Amplification program is：95 ° of 10min, (95 DEG C of 15sec, 55 DEG C
60sec) × 35 circulation.
2 RealTime reaction systems of table
| Component | Addition |
| 2×mix | 10μl |
| Sense primer (10uM) | 0.5μl |
| Anti-sense primer (10uM) | 0.5μl |
| Template | 2μl |
| Add sterile purified water | 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,
Expanded respectively with target gene primer and reference gene primer, while melt curve analysis analysis is carried out at 60-95 DEG C, according to expansion
Increasing Efficiency is high and the unimodal principle of solubility curve carries out primer screening.
(3) sample RealTimePCR is detected
After 10 times of each sample cDNA dilution 2 μ l will be taken to make template, respectively with target gene primer and reference gene primer into
Row amplification.At the same time solubility curve analysis is carried out at 60-95 DEG C.
Two experimental results
Real-time quantitative PCR amplification curve flex point understands that amplification curve entirety collimation is good, shows the amplification effect of each reaction tube
Rate is close, and the limit is put down without raising up now, and exponent phase slope is larger, illustrates that amplification efficiency is higher；Sample amplified production is molten
Solution curve is all unimodal, illustrates that amplified production only has one, is specific amplification；According to the relative quantification formula of qRT-PCR：2-
Δ Ct × 100%, compares expression of the SPAG7 genes in senile dementia peripheral blood and healthy human peripheral blood.The results show
(being specifically shown in Fig. 1)：The expression of qRT-PCR stable amplification results, wherein SPAG7 in old dementia patients peripheral blood is less than
/ 6th of healthy human peripheral blood, about control group, result above demonstrate the integration point of high throughput transcript profile expression data
Analyse the result of SPAG7 low expressions in senile dementia peripheral blood.
The present invention filters out senile dementia pathogenic related gene SPAG7 using high-flux sequence, and binding molecule biology is real
Verification, it was confirmed that SPAG7 has the function that important in senile dementia disease.The present invention provides for senile dementia clinic diagnosis
New target, has a good potential applicability in clinical practice.
Sequence table
<110>Beijing Yang Shen biology information technologies Co., Ltd
<120>Application of the SPAG7 genes in diagnosis of dementia preparation is prepared
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> DNA
<213> Homo sapiens
<400> 1
gagaggagca tactacat 18
<210> 2
<211> 18
<212> DNA
<213> Homo sapiens
<400> 2
atagcgacag tcatcatc 18
<210> 3
<211> 22
<212> DNA
<213> Homo sapiens
<400> 3
agttgcgtta caccctttct tg 22
<210> 4
<211> 21
<212> DNA
<213> Homo sapiens
<400> 4
tcaccttcac cgttccagtt t 21
Claims (10)
- A kind of 1. application of the reagent of detection SPAG7 genes and/or albumen in diagnosis of dementia preparation is prepared.
- 2. application according to claim 1, it is characterised in that diagnosis of dementia preparation is included with quantitative fluorescent PCR side The expression of method, method for gene chip, sequencing approach detection SPAG7 genes.
- 3. application according to claim 2, it is characterised in that the production for fluorescence quantifying PCR method detection SPAG7 genes Product contain the primer of a pair of of specific amplification SPAG7 genes；Genetic chip includes the spy with the nucleic acid array hybridizing of SPAG7 genes Pin.
- 4. application according to claim 1, it is characterised in that diagnosis of dementia preparation includes being detected with immunization method The expression of SPAG7 albumen.
- 5. application according to claim 4, it is characterised in that immunization method detection SPAG7 protein expressions are examined for ELISA Test agent box and/gold-immunochromatographyreagent reagent for assay box.
- A kind of 6. PCR kit for fluorescence quantitative for detecting senile dementia, it is characterised in that the kit detects gene SPAG7, Using special sense primer and anti-sense primer, upstream primer sequence is SEQ ID NO.1, and downstream primer sequence is SEQ ID NO.2。
- Application of the accelerating agent of 7.SPAG7 genes and/or albumen in senile dementia treatment preparation is prepared.
- 8. application according to claim 7, it is characterised in that the senile dementia treatment preparation can use following methods In one kind and/or several promotion SPAG7 genes expression：Copy number, transfection base containing SPAG7 including increasing SPAG7 genes The over-express vector of cause；Activate the promoter of expression, the activation regulation and control SPAG7 gene expressions of SPAG7 genes, suppress negative regulation The transcription factor of SPAG7 gene expressions, using RNA perturbation techniques to suppress SPAG7 gene expressions repressor disturb；Suppression System promotes the microRNA transcriptional expressions of SPAG7 gene mRNAs degraded, imports the microRNA for promoting SPAG7 gene expressions；Lead Enter to promote the molecule of target gene encoding proteins, the albumen of suppression negative regulation SPAG7 gene expressions, promotion SPAG7 gene expressions The factor and albumen expression.
- 9. one kind treats senile dementia preparation, it is characterised in that the treatment senile dementia preparation promotes the table of SPAG7 genes Reach.
- 10. treatment senile dementia preparation according to claim 9, it is characterised in that in the anti-senile dementia preparation Contain the carrier for promoting SPAG7 gene expressions.
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| CN201711272540.5A CN108034707B (en) | 2017-12-06 | 2017-12-06 | SPAG7 gene is preparing the application in diagnosis of dementia preparation |
| US16/770,478 US11497817B2 (en) | 2017-12-06 | 2018-12-05 | Senile dementia treatment formulation and application thereof |
| PCT/CN2018/119432 WO2019109962A1 (en) | 2017-12-06 | 2018-12-05 | Senile dementia treatment formulation and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107779503A (en) * | 2017-12-06 | 2018-03-09 | 北京泱深生物信息技术有限公司 | The related difference expression gene of Alzheimer and its application |
| WO2019109962A1 (en) * | 2017-12-06 | 2019-06-13 | 北京泱深生物信息技术有限公司 | Senile dementia treatment formulation and application thereof |
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| CN108034707B (en) | 2019-01-08 |
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