CN107779503A

CN107779503A - The related difference expression gene of Alzheimer and its application

Info

Publication number: CN107779503A
Application number: CN201711272650.1A
Authority: CN
Inventors: 汪冰怡; 向常娟; 肖枫
Original assignee: Beijing Medintell Bioinformatic Technology Co Ltd
Current assignee: Beijing Medintell Bioinformatic Technology Co Ltd
Priority date: 2017-12-06
Filing date: 2017-12-06
Publication date: 2018-03-09

Abstract

The present invention relates to the related difference expression gene of Alzheimer and its application, is specifically related to C16ORF7 genes and its application in diagnosing and treating Alzheimer Disease.To solve the problems, such as that current Alzheimer molecular marked compound is rare, inventor carries out high-flux sequence to Alzheimer patient and Healthy People control peripheral blood sample, genescreen is carried out using bioinformatics method, pick out the closely related gene C 16ORF7 of Ahl tribulus sea silent sickness, available for Alzheimer disease assisting in diagnosis and treatment preparation is prepared, there is important clinical value.

Description

The related difference expression gene of Alzheimer and its application

Technical field

The present invention relates to biomedicine field, and in particular to the related difference expression gene of Alzheimer and its application, More particularly relate to C16ORF7 genes and its application in diagnosing and treating Alzheimer Disease.

Background technology

Alzheimer disease (AD) is that one kind is with age height correlation, with progressive cognitive disorder and memory infringement Main central nervous system degenerative disease.Due to highlighting for China human mortality Aging Problem, increasing for elderly population quantity makes One of an important factor for this kind of disease turns into influence population of China quality of life is obtained, Alzheimer disease disease does not still have at present Effective treatment method, it can only find by diagnosing and treating ahead of time to delay disease process, therefore and diagnose and predict in advance The molecular marker of Alzheimer disease is particularly important.

Diagnosis of Alzheimer disease is entered by AD biological markers in cerebrospinal fluid ((A beta molecules, Protein tau etc.)) at present OK, the shortcomings that accuracy rate of diagnosis is preferable, but presence 2 is obvious：Expense is too high, and related biological tissue's acquisition process is more difficult； It is larger to obtain cerebrospinal fluid pain to caused by patient, it is also possible to leave sequelae.By contrast, peripheral blood is a kind of compares The biological tissue easily obtained, study the related biology changes of AD in peripheral blood just has realistic meaning, existing patent very much In have revealed that the part molecular marker related to Alzheimer disease, the CRTAP bases as disclosed in ZL2015104634693 Cause, the EAPP genes that ZL2015104635287 is disclosed, 10 Ahl tribulus sea silent sickness correlations that CN2016104739651 is disclosed Disease-causing gene, still, said gene need further to verify.

To solve the problems, such as that current Alzheimer molecular marked compound is rare, inventor is to Alzheimer patient and is good for Health people compares peripheral blood sample and carries out high-flux sequence, carries out genescreen using bioinformatics method, picks out candidate's base Because of C16ORF7.Further, the present invention has carried out RT-PCR method and has confirmed that C16ORF7 Ahl tribulus sea silent sickness has well Correlation, available for Alzheimer assisting in diagnosis and treatment preparation is prepared, there is important clinical value.

The content of the invention

It is an object of the invention to provide the reagent of a kind of detection C16ORF7 genes and/or albumen to prepare alzheimer ' Application in silent diagnostic preparation.

To achieve the above object, the present invention screens candidate by high-flux sequence combination bioinformatics method first Gene C 16ORF7, further C16ORF7 and Alzheimer relation by molecular cytobiology method validation： C16ORF7 has good correlation with Alzheimer, and Alzheimer preparation and/or A Erci are treated available for preparing The silent diagnostic preparation in sea, has important clinical value.

Further, the diagnostic preparation of described Alzheimer include with fluorescence quantifying PCR method, method for gene chip, The expression of C16ORF7 genes in sequence measurement detection Alzheimer peripheral blood.

Fluorescence quantitative PCR method is the specific probe by fluorescent dye or fluorescence labeling, enters rower to PCR primer Note tracking, real time and on line monitoring course of reaction, can be analyzed product with reference to corresponding software, calculate testing sample mould The initial concentration of plate.The appearance of quantitative fluorescent PCR, greatly simplifies the process of quantitative detection, and is truly realized definitely It is quantitative.The appearance of a variety of detecting systems, make the selectivity of experiment stronger.Automation mechanized operation improves operating efficiency, and reaction is fast Fast, reproducible, high sensitivity, 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 Nucleic acid probe or cDNA fragments on polymer matrix film (nylon membrane, nitrocellulose membrane etc.) surface, generally with isotope marks Target gene is hybrid with it, and is detected by radiography technology.2) DNA probe battle array on a glass is fixed with point sample method Row, by being detected with the hybridization of the target gene of fluorescence labeling.3) oligonucleotides directly synthesized on the hard surfaces such as glass Probe array, the target gene hybridization with fluorescence labeling are detected.Genetic chip is as a kind of advanced, extensive, high flux Detection technique, applied to the diagnosis of disease, its advantage has the following aspects：First, the sensitivity and accuracy of height；Second, It is fast and convenient；Third, a variety of diseases can be detected simultaneously.

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 lost The solution reading rate of information is passed, to obtain all mRNA sequence information, decryption mRNA collection of illustrative plates provides guarantee.High flux simultaneously Sequencing makes it possible the analysis of the careful overall picture of transcript profile and genome progress to species, so the depth that is otherwise known as Degree sequencing.The representative of high-flux sequence platform is 454 sequenators (the Roch GSFLX of Roche Holding Ag (Roche) Sequencer), the Solexa genome analysises instrument (Illumina Genome Analyzer) of Illumina companies and ABI SOLiD sequenators (ABI SOLiD sequencer).

The described product for being used for C16ORF7 genes in fluorescence quantifying PCR method detection Alzheimer contains a pair of spies The primer of specific amplification C16ORF7 genes；Described genetic chip includes the spy with the nucleic acid array hybridizing of C16ORF7 genes Pin.

Further, the diagnostic preparation of described Alzheimer includes the table that C16ORF7 albumen is detected with immunization method Reach.It is preferred that in the immunologic detection method detection Alzheimer C16ORF7 protein expressions for western blot and/or ELISA and/collaurum 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).

Conventional 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 antigen or antibody point is added into sample to be checked on film after closing, resisting with colloid gold label after washing Corresponding antigen or antibody are surveyed in physical examination.(4) specific antigen or antibody are fixed on by colloidal gold immunity chromatography with ribbon On film, colloid gold label reagent (antibody or monoclonal antibody) is adsorbed on pad, when sample to be checked is added to test strips one end Sample pad on after, moved forward by capillarity, dissolve pad on colloid gold label reagent after react to each other, when When being moved to the region of fixed antigen or antibody, the conjugate of thing and gold marked reagent to be checked is specifically bound therewith again And be trapped, it is gathered in detection band, colour developing result can be observed by the naked eye.The method has developed into diagnosis test paper, Using very convenient.

Further, the ELISA method of the detection C16ORF7 albumen is to use ELISA detection kit.The kit In antibody can use commercially available C16ORF7 monoclonal antibodies.Further, described kit includes：It is mono- to be coated with C16ORF7 The solid phase carrier of clonal antibody, ELIAS secondary antibody, the substrate of enzyme, protein standard substance, negative controls, dilution, cleaning solution, enzyme Reaction terminating liquid etc..

Further, the colloidal gold method of the detection C16ORF7 albumen is that can be adopted using detection kit, described antibody With commercially available C16ORF7 monoclonal antibodies.Further, described gold-immunochromatographyreagent reagent for assay box uses colloidal gold immunochromatographimethod skill Art or collaurum percolation.Further, detection zone (T) specking on described gold-immunochromatographyreagent reagent for assay box nitrocellulose filter There are anti-C16ORF7 monoclonal antibodies, quality control region (C) specking to have Immunoglobulin IgG.

It is an object of the invention to provide a kind of PCR kit for fluorescence quantitative for detecting Alzheimer, its feature exists In the kit detects gene C 16ORF7, using special sense primer and anti-sense primer, upstream primer sequence SEQ ID NO.1, downstream primer sequence are SEQ ID NO.2.

Further, the PCR kit is suitable for presently, there are all types fluorescence quantitative gene extender of in the market, High sensitivity, 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.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.

Described kit also includes RNA extraction agents.It is preferred thatReagent carries out sample rna extraction.

The present invention also have detected this kit sensitivity, and it is 10 as a result to show this kit detection range⁶-10²copies/ μ l, minimum concentrations are 100copies/ μ l.

It is an object of the present invention to provide a kind of Alzheimer detection kit, the detection of described detection kit C16ORF7 albumen.Further, described kit also includes other detection reagents.

It is an object of the present invention to provide it is a kind of detect Alzheimer genetic chip, described genetic chip include with The probe of the nucleic acid array hybridizing of C16ORF7 genes.

It is an object of the invention to provide C16ORF7 genes and/or albumen in Alzheimer treatment preparation is prepared Using.

Further, the Alzheimer treatment preparation refers to the preparation for suppressing the expression of C16ORF7 genes.This area The expression of the known suppressor of personnel can generally use one kind in following methods and/or several：Include but is not limited to pass through The suppressor of activating genes of interest, the inhibition of gene expression of activating genes of interest albumen, using RNA perturbation techniques suppress Destination gene expression, activation promote the microRNA of target gene mRNA degradeds, import the encoding proteins degraded of promotion target gene Molecule, suppress promote destination gene expression the factor and albumen expression.Suppression base i.e. by activating C16ORF7 genes Cause, activate the albumen for suppressing C16ORF7 gene expressions, the siRNA for importing suppression C16ORF7 gene expressions, activate promotion The microRNA of C16ORF7 mRNA degradeds, import the molecule for promoting C16ORF7 protein degradations, suppression promotion C16ORF7 genes The factor of expression and the expression of albumen.

RNA interference (RNAi) refers to that exogenous and endogenous double-stranded RNA induces the mRNA of homologous target gene in vivo Selective degradation, cause the phenomenon of PTGS, be it is a kind of efficiently, specifically blocked using small double-stranded RNA it is internal The expression of certain specific gene, promotes mRNA to degrade, and cells show is gone out the technology of specific gene missing phenotype.SiRNA is designed After the completion of can use direct synthesis technique or structure SiRNA expression vector, the siRNA prepared can be total to by calcium phosphate Mechanical Method, the cation lipid such as the precipitation method, electroporation, DEAE- glucans and polybrene methods, microinjection or particle gun The approach transfectional cells such as body reagent method.

Further, one kind in one sequence of the siRNA sequence of the suppression C16ORF7 gene expressions and/or several Kind：SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.5、SEQ ID NO.6.

It is preferred that siRNA sequence is SEQ ID NO.1 and SEQ ID NO.2.

It is an object of the invention to provide a kind of preparation for treating Alzheimer, the system of the treatment Alzheimer Agent suppresses the expression of C16ORF7 genes in Alzheimer.Further, suppression is contained in the preparation of the treatment Alzheimer The siRNA of C16ORF7 gene expressions processed.

The preferable siRNA sequence for suppressing C16ORF7 gene expressions is SEQ ID NO.1 and SEQ ID NO.2.

It is an object of the invention to provide one kind to treat Alzheimer preparation, the treatment Alzheimer preparation suppression The expression of C16ORF7 genes processed.Further, suppression C16ORF7 gene expressions are contained in described treatment Alzheimer preparation Carrier.

Brief description of the drawings

Fig. 1 C16ORF7 genes relative expression's spirogram in Alzheimer peripheral blood and healthy human peripheral blood

Fig. 2 is C16ORF gene relative expression's spirograms after RNAi suppresses

Embodiment

With reference to specific embodiment, the present invention is expanded on further, is only used for explaining the present invention, and it is not intended that right The limitation of the present invention.It will be understood by those skilled in the art that：In the case where not departing from the principle and objective of the present invention These embodiments can be carried out with a variety of change, modification, replacement and modification, the scope of the present invention is by claim and its is equal Thing limits.The experimental method of unreceipted actual conditions in the following example, generally according to normal condition or according to proposed by manufacturer Condition examinations.

The high-flux sequence of embodiment 1 and analysis

Samples sources obtain subjects informed consent in BJ Union Hospital.15 Alzheimers are collected respectively Patient peripheral's blood sample and 9 Healthy People control peripheral blood samples, progress RNA extractions, agarose gel electrophoresis after RNA extractions, Whether up-to-standard the RNA sample that can be extracted from electrophoresis result with preliminary judgement is, if can be used for further transcribing component Analysis.And then the extraction situation of RNA samples is detected by NanoDrop1000 spectrophotometers, the sample of RNA-seq sequencings will Ask：OD260/OD280 is 1.8-2.2.

Microarray dataset is the high-flux sequence platforms of HiSeq 2500 of Illumina companies, and it is deep to carry out high flux transcript profile Degree 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, quality The position distribution of value, G/C content, PCR duplication contents, kmer frequency etc..In differential genes expression analysis When, according to obtained FPKM values, differential screening is carried out using internationally recognized algorithm EBSeq.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 to difference expression gene Onlogy and signal path analysis, and functional annotation and protein interaction network analysis, mirror are carried out to difference expression gene In the result of data above analysis, with reference to document, we have screened up-regulation difference expression gene C16ORF7.

The Alzheimer peripheral blood in patients of embodiment 2 and the material of healthy human peripheral blood C16ORF7 expression conditions one and Method

1st, material

95 Alzheimer peripheral blood in patients and 31 healthy human peripheral bloods are collected, it is grouped and numbered.

2nd, method

The extraction of 2.1 Alzheimer peripheral blood in patients and healthy human peripheral blood total serum IgE

UsingReagent carries out sample rna extraction, and experimental implementation is carried out by product description, concrete operations See 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 28S, 18S band；70 DEG C of water-baths be incubated 1 hour after electrophoresis pattern 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) enter 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, the μ g of template ribonucleic acid 1, aqua sterilisa is added to the μ l of total system 25.42 DEG C are incubated 1 Hour, 72 DEG C 10 minutes, of short duration centrifugation.It is standby that -20 DEG C of refrigerators are put in cDNA preservations.

2.3Real-Time PCR

2.3.1 instrument and analysis method

With the type quantitative real time PCR Instruments of ABI 7500, the relative quantitative assay of data is carried out using 2- △ △ CT methods.

2.3.2 design of primers

C16ORF7 sequences are that NM_004890.2 uses online primer-design software, by invitrogen after design of primers Company synthesizes.Specific primer sequence is as follows：

The primer sequence of table 1

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, 60 DEG C 55sec) × 35 circulations.

Table 2RealTime reaction systems

(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 mould after dilution Plate, expanded respectively with target gene primer and reference gene primer, while melt curve analysis analysis, root are carried out at 60-95 DEG C According to amplification efficiency is high primer screening is carried out with the unimodal principle of solubility curve.

(3) sample RealTimePCR is detected

After 10 times of dilutions of each sample cDNA 2 μ l will be taken to make template, respectively with target gene primer and reference gene primer Expanded.Simultaneously 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 of each reaction tube Similar efficiency, the limit is flat and present without raising up, and exponent phase slope is larger, illustrates that amplification efficiency is higher；Sample amplified production Solubility curve is all unimodal, illustrates that amplified production only has one, is specific amplification；It is public according to qRT-PCR relative quantification Formula：2- Δ Ct × 100%, compare expression water of the C16ORF7 genes in Alzheimer peripheral blood and healthy human peripheral blood It is flat.As a result (being specifically shown in Fig. 1) is shown：QRT-PCR stable amplification results, wherein C16ORF7 are in Alzheimer peripheral blood in patients In expression be higher than healthy human peripheral blood, about the 7 of control group times, result above demonstrates the expression of high flux transcript profile The result of the confluence analysis C16ORF7 of data high expression in Alzheimer peripheral blood.

Embodiment 3RNAi suppresses C16ORF gene expressions and its influence to Alzheimer model cell

SiRNA is built and synthesis：According to (the http of Photographing On-line software siDirect version 2.0:// Design.rnai.jp/), according to C16ORF7 genes in GenBank (NCBI Reference Sequence:NM_ 004890.2) the corresponding siRNA of sequences Design in.Synesis Company's synthesis is sent to after design, siRNA controls are provided by company.

Cell line：PC-12 cell lines are purchased from Chinese Academy of Sciences's cell bank；Aβ_25-35, purchased from Wuhan doctor's moral bioengineering Co., Ltd.

Alzheimer model cell：The cell in growth period of taking the logarithm is inoculated in blake bottle, is placed and is trained in incubator Support, it is 37 DEG C to control incubator temperature, CO₂Cell attachment after content is 5%, 24h, discards old nutrient solution, and cell is complete in DMEM After full nutrient solution culture 4h, appropriate A β are added_25-35, control A β_25-35Final concentration of 20 μm of ol/L, continue to cultivate, prepare to be used for Cell transfecting.

Cell is grouped：C groups：Transfect liposome group；C1 groups：Transfect nonspecific siRNA groups；S1, S2, S3 group：Transfection Specific siRNA groups.

Transfection：The step of being provided according to LipofectamineTM2000Transfection Reagent is carried out.

1. 24h before transfection, the cell pancreatin in growth period of taking the logarithm digest and counted, cell is adjusted with DMEM culture mediums Concentration is 1 × 10⁵/ ml, take 2m1 to be inoculated in six orifice plates, be positioned over 37 DEG C, 5%CO₂Cultivated in incubator, in cell up to 80% It is used to transfect during fusion.The DMEM medium cultures 3-4h without serum is used before transfection.

2. prepare transfection liquid：

A liquid：250u1 serum free mediums dilute 4.0ugDNA, gentle to mix；

B liquid：250u1 serum free mediums dilute 10u1Lipofectamine, gentle to mix, and room temperature places 5min；

3. transfect：A liquid mixes with B liquid, incubation at room temperature 20min, directly compound is added in every hole, shaken Dynamic culture plate, is gently mixed.In CO₂Liquid is changed after 37 DEG C of insulations 24-48h, 6h in incubator, adds the culture medium containing serum.

Using the change of C16ORF7 gene expressions before and after the detection transfection of Real-time PCR methods：Extract each group cell RNA, determine RNA concentration and purity, carry out reverse transcription reaction, every group of DNA profiling carries out C16ORF7's and actin simultaneously Real-time PCR react, and in triplicate, specific steps are with reference to embodiment 2 for experiment.

Mtt assay surveys cell propagation：It is difficult that succinate dehydrogenase in living cells mitochondria can reduce exogenous MTT The bluish violet crystal of dissolubility is simultaneously deposited in cell, and dead cell is without this function.Dimethyl sulfoxide (DMSO) (DMSO) can dissolve cell In purple crystal thing, its absorbance is determined at 490nm with ultraviolet specrophotometer, can indirect reaction living cells quantity. In the range of certain cell number, the amount that MTT crystals are formed is directly proportional to cell number.

(1) MTT solution：250mg MTT are weighed, are put into small beaker, add 50mI PBS (0.0lmol/L, pH7.4), 30min is stirred on electromagnetic force mixer, packing degerming with 0.22um miillpore filter, 4 DEG C of preservations, in two weeks effectively.

(2) cell is grouped：Experiment is divided into negative control group and experimental group.Negative control group transfects for non-specific siRNA 48h after Alzheimer model cell；Experimental group is 48h after siRNA transfection Alzheimer model cell；Every group If 3 repetitions.

(3) inoculating cell：With 0.25% Trypsin Induced single-layer culturing cell, trained with containing 10% hyclone DMEM Nutrient solution is made into individual cells suspension, and individual cell is inoculated in 96 well culture plates with every hole 1, per pore volume 100ul.

(4) cell is cultivated：Culture plate is put into CO₂Incubator, at 37 DEG C, 5%CO₂And cultivated under the conditions of saturated humidity.

(5) colour generation：0h, 24h, 48h, 72h, 96h after culture, addition MTT solution (5mg/ml) 20u1 per hole, 37 DEG C Continue to be incubated 4h, terminate culture, carefully discard in hole culture supernatant night.Supernatant is sucked, dimethyl sulfoxide (DMSO) 150ul is added, shakes Swinging 10min is completely dissolved crystallization.

(6) colorimetric：490nm wavelength is selected, each hole absorbance is determined on ultraviolet specrophotometer.If put down with test hole Row is not added with the blank control wells that cell only adds nutrient solution.It is repeated 3 times, records result, using the time as transverse axis, absorbance (A490) Cell growth curve is drawn for the longitudinal axis.

Experimental result：

Real-time PCR detect transfection efficiency.Using Real-time PCR methods structure C16ORF7's and actin Standard curve, coefficient correlation are respectively 0.988,0.991, and linear relationship is good, meets the requirements.With the method for double standard curves Compare the expression of each group C16ORF7 genes.Liposome transfection group, the expression of nonspecific transfection group gene are substantially similar, difference It is not statistically significant.C16ORF7-siRNA1, C16ORF7-siRNA2, C16ORF7-siRNA3, which have, suppresses C16ORF7 genes The effect of expression, C16ORF7-siRNA1 effect become apparent from, and suppress efficiency up to 78%, and C16ORF7-siRNA2 and C16ORF7-siRNA3 inhibitory action is respectively 28% and 61%, compared with liposome transfection group, nonspecific transfection group, Difference is statistically significant, P<0.05 (see Fig. 2).

Cell growth inhibition assay (MTT).Alzheimer model cell is blank control group, negative control group is elected as The Alzheimer model cell of nonspecific transfection, experimental group are the Alzheimer mould of transfection C16ORF7-siRNA1 groups Type cell.The blue colorimetric method of tetramethyl azo (MTT) experiment display, under identical primary condition, each group cell proliferation rate It is close, no difference of science of statistics (P<0.05).After 24h, specific siRNA transfection group cell proliferation rate slows down, blank control group Cell, negative control group cell proliferation rate are close, and siRNA transfection group cell proliferation rate slows down, and difference has statistics Learn meaning (P<0.05).As observing time extends, compare between preceding two groups of groups, growth rate is similar, no significant difference (P>0.05)；Compared with first two groups, growth rate substantially slows down siRNA transfection group, there is significant difference (P< 0.05).MTT test result indicates that, the cell growth of siRNA transfection group is substantially suppressed.

The present invention filters out Alzheimer pathogenic related gene C16ORF7, binding molecule biology using high-flux sequence Learn experimental verification, it was confirmed that C16ORF7 has the function that important in Alzheimer Disease.The present invention is Alzheimer Clinic diagnosis provide new target, have good potential applicability in clinical practice.

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Claims

A kind of 1. application of the reagent of detection C16ORF7 genes and/or albumen in Alzheimer diagnostic preparation is prepared.
2. application according to claim 1, it is characterised in that Alzheimer diagnostic preparation includes using quantitative fluorescent PCR The expression of method, method for gene chip, sequence measurement detection C16ORF7 genes.
3. application according to claim 2, it is characterised in that for fluorescence quantifying PCR method detection C16ORF7 genes Product contains the primer of a pair of specific amplification C16ORF7 genes；Genetic chip includes miscellaneous with the nucleotide sequence of C16ORF7 genes The probe of friendship.
4. application according to claim 1, it is characterised in that Alzheimer diagnostic preparation includes being detected with immunization method The expression of C16ORF7 albumen.
5. application according to claim 4, it is characterised in that immunization method detection C16ORF7 protein expressions for ELISA Detection kit and/gold-immunochromatographyreagent reagent for assay box.
6. a kind of PCR kit for fluorescence quantitative for detecting Alzheimer, it is characterised in that the kit detects gene C16ORF7, 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 inhibitor of 7.C16ORF7 genes and/or albumen in Alzheimer treatment preparation is prepared.
8. application according to claim 7, it is characterised in that the Alzheimer treatment preparation can use following sides The expression of one kind and/or several promotion C16ORF7 genes in method：Suppressor, activation suppression including activating C16ORF7 genes The albumen of C16ORF7 gene expressions processed, import the siRNA for suppressing C16ORF7 gene expressions, activation promotion C16ORF7mRNA drops The microRNA of solution, import the molecule for promoting C16ORF7 protein degradations, the factor and egg for suppressing promotion C16ORF7 gene expressions White expression.
9. application according to claim 8, it is characterised in that one kind in one sequence of siRNA sequence and/or several Kind：SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.5、SEQ ID NO.6.
10. one kind treats Alzheimer preparation, the treatment Alzheimer preparation suppresses the expression of C16ORF7 genes, its It is characterised by, the siRNA for suppressing C16ORF7 gene expressions is contained in preparation.