CN108410978A - Major depressive disorder related gene and its application in diagnosis - Google Patents
Major depressive disorder related gene and its application in diagnosis Download PDFInfo
- Publication number
- CN108410978A CN108410978A CN201810498803.2A CN201810498803A CN108410978A CN 108410978 A CN108410978 A CN 108410978A CN 201810498803 A CN201810498803 A CN 201810498803A CN 108410978 A CN108410978 A CN 108410978A
- Authority
- CN
- China
- Prior art keywords
- slc35e1
- genes
- depressive disorder
- major depressive
- reagent
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/30—Psychoses; Psychiatry
- G01N2800/304—Mood disorders, e.g. bipolar, depression
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Microbiology (AREA)
- Pathology (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Hematology (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Cell Biology (AREA)
- General Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Application the invention discloses major depressive disorder related gene and its in diagnosis, the gene is SLC35E1, the present invention has found SLC35E1 genes up-regulated expression in major depressive disorder patient by high-flux sequence, it confirms that the albumen of SLC35E1 genes and its coding is implicitly present in differential expression in major depressive disorder patient by further verifying, the albumen of SLC35E1 mRNA and its coding is prompted to can be used as the clinical diagnosis that detection marker is applied to major depressive disorder.
Description
Technical field
The present invention relates to biomedical sectors, are related to major depressive disorder related gene and its application in diagnosis, specifically
Involved gene marker be SLC35E1.
Background technology
Depression refers to a kind of serious harm human physical and mental health by many factors as caused by heredity, wound, pressure etc.
Chronic recurrent psychotic disorder, based on mood, cognition and behavioral function disorder, complicated clinical manifestation, such as lasting feelings
Thread is low, and automatic nervous system function is disorderly (such as appetite, sleep disturbance), cognitive disorder (such as valueless sense and inappropriate crime
Sense) and hyperpraxia (apparent intense and sluggish) etc. even will appear suicidal idea or suicide.
With the becoming faster of social rhythm, the aggravation of life stress, depression has become increasingly common.It is reported that depression
Global population's lifetime prevalence be up to 20%, the about 50% patients with depression effect after anti depressant therapy is not notable, and
There is the patient of about 50%-60% to will appear recurrence social to patient, occupation or other functional bands and carry out serious damage, separately grinds
Study carefully and shows that 60% felo-de-se is and probably to have 15%-17% eventually to commit suicide and die in patients with depression because of depression.
This high illness rate of depression, high relapse rate, height are disabled lethality, and the feature that complicated clinical manifestation is various, and patient home is given
And society brings huge financial burden.According to the statistics of the World Health Organization, the whole world is used to treat the economic expense of depression every year
With up to 60,000,000,000 dollars, when to the year two thousand thirty, financial burden caused by depression will likely will be as in all diseases
One.Therefore, the pathogenesis for illustrating MDD, for the prevention of MDD, the design of related drugs and reasonable effective clinical treatment
It is of great significance, has become biomedical sector major issue urgently to be resolved hurrily.
With the development of high throughput sequencing technologies, research of the gene in disease is in widespread attention.Modern molecular
Science of heredity thinks that the factors such as many social environments or itself experience can cause the variation of gene expression, leads to certain specific genes
Epigenetic occurs to change, these variations cause the plasticity and dysfunction of neuron, the severe depression based on gene level
The research of disease pathogenesis and biomarker gradually causes the concern of scholars.Searching is answered with the relevant gene of major depressive disorder
For the clinical diagnosis of major depressive disorder, the pathogenesis of major depressive disorder is disclosed, improves the life quality of patient with important
Meaning.
Invention content
In order to make up for the deficiencies of the prior art, the purpose of the present invention is to provide relevant with major depressive disorder occurrence and development
Gene marker is diagnosed the illness using gene marker with promptness, specificity and sensitivity, and patient is in depression early stage
It can know risk, for risk height, corresponding prevention and treatment measure be taken, to improve the quality of living.
To achieve the goals above, the present invention adopts the following technical scheme that：
The present invention provides the reagents of detection SLC35E1 levels to prepare the application in diagnosing major depressive disorder product.
Further, the product includes by sequencing technologies, nucleic acid hybridization technique, nucleic acid amplification technologies, protein immunization skill
Art detects the reagent of SLC35E1 levels.
Further, the reagent includes：
The probe of specific recognition SLC35E1 genes；Or
The primer of specific amplification SLC35E1 genes；Or
Specifically bind the antibody or ligand of the albumen of SLC35E1 codings.
Further, the primer sequence of the specific amplification SLC35E1 genes is to such as SEQ IDNO.1 and SEQ IDNO.2
It is shown.
The present invention provides a kind of product of SLC35E1 expressions in vitro detection sample, the product include preparation,
Chip or kit；Wherein, the product passes through sequencing technologies, nucleic acid hybridization technique, nucleic acid amplification technologies, protein immunization technology
Method detection SLC35E1 genes or albumen expression.
The chip includes genetic chip, protein chip, and the genetic chip includes for detecting SLC35E1 genetic transcriptions
The horizontal oligonucleotide probe for SLC35E1 genes, the protein chip include SLC35E1 albumen specific antibody or
Ligand；The kit includes gene detecting kit, protein detection kit, and the gene detecting kit includes for examining
The reagent or chip of SLC35E1 gene transcription levels are surveyed, the protein detection kit includes for detecting SLC35E1 albumen
The reagent or chip of expression.
" sample " is the substance that can therefrom obtain nucleic acid, polypeptide or other analytes comprising cell or cellular material.It is excellent
Choosing, the sample is blood.
The present invention provides products recited above to prepare the application in diagnosing major depressive disorder tool.
The present invention provides a kind of kits of diagnosis major depressive disorder, which is characterized in that the kit includes：
Detect one or more reagents of SLC35E1 genes or protein expression level；With
One or more substances selected from the group below：Container, operation instructions, positive control, negative control object, buffering
Agent, auxiliary agent or solvent.
Further, the kit include by RT-PCR methods, qRT-PCR methods, biochip test method, southern blotting technique method,
The reagent of hybridization in situ, Western blot detection SLC35E1 genes or protein expression level.
Further, the reagent that SLC35E1 gene expression doses are detected with qRT-PCR methods includes such as sequence SEQ ID
The primer of specific amplification SLC35E1 genes shown in NO.1~2.
Further, the reagent that SLC35E1 gene expression doses are detected with qRT-PCR methods also gathers comprising SYBRGreen
Polymerase chain reaction system, the primer pair for expanding house-keeping gene；The SYBR Green PCRs system packet
Contain：PCR buffer solutions, dNTPs, SYBR Green fluorescent dyes.
Further, the amplification house-keeping gene is GAPDH, expand the primer sequence of GAPDH to such as SEQ ID NO.3 with
Shown in SEQ ID NO.4.
Description of the drawings
Fig. 1 is the expression figure in major depressive disorder patient using QPCR detection SLC35E1 genes；
Fig. 2 is the expression figure in major depressive disorder patient using protein immunization detection SLC35E1 albumen.
Specific embodiment
The present invention after extensive and in-depth study, by the method for high-flux sequence, detection major depressive disorder patient and
Difference expression gene in normal person inquires into its relationship between the generation of major depressive disorder, to the early stage for depression
Detection provides marker.It is carried by screening present invention firstly discovers that SLC35E1 conspicuousnesses raise in major depressive disorder patient
Show that SLC35E1 can be used as the clinical diagnosis that Testing index is applied to major depressive disorder.
SLC35E1 genes and albumen
SLC35E1 is located at No. 19 1st area of the short arm of a chromosome, 3 bands of people, in the context of the present invention, " SLC35E1 genes " packet
Include the polynucleotides of any functional equivalent of people SLC35E1 genes and people's SLC35E1 genes.It is a kind of representative
SLC35E1 genes (NM_ in the nucleotide sequence of SLC35E1 genes such as at present international public nucleic acid database GeneBank
024881.4) shown in.
It would be recognized by those skilled in the art that the practicability of the present invention is not limited to appointing to the target gene of the present invention
The gene expression of what specific variants is quantified.If when nucleic acid or its segment and other nucleic acid (or its complementary strand) optimal comparison
When (have nucleotides inserted appropriate or missing), at least about 60% nucleotide base, usually at least about 70%, more
Usually at least about 80%, it is preferably at least about 90% and there are nucleosides more preferably at least about in 95-98% nucleotide bases
The acid sequence phase same sex, then the two sequences are " substantially homologous " (or substantially similar).
The SLC35E1 genes of the present invention can be natural or artificial synthesized, or use can express SLC35E1
DNA fragmentation carrier transfectional cell obtain.Carrier described in the carrier includes viral vectors, carrier for expression of eukaryon.
Viral vectors can be any carrier appropriate, including but not limited to retroviral vector, adenovirus vector, gland
Viral related viral vectors, herpesviral (such as herpes simplex virus, vaccinia virus and Epstein-Barr virus) carrier, alphavirus vectors.
Carrier for expression of eukaryon can be any expression vector appropriate, including but not limited to pCMV-Myc expression vectors,
PcDNA3.0 expression vectors, pcDNA3.1 expression vectors, pEGFP expression vectors, pEF Bos expression vectors, pTet expression vectors,
PTRE expression vectors or modified carrier on the basis of known expression vector, such as pBin438, pCAMBIA1301
Deng.
In the context of the present invention, SLC35E1 gene expression products include people SLC35E1 albumen and people SLC35E1
The partial peptide of albumen.The partial peptide of the SLC35E1 albumen contains and the relevant functional domain of major depressive disorder.
" SLC35E1 albumen " includes any functional equivalent of SLC35E1 albumen and SLC35E1 albumen.The function
Equivalent includes SLC35E1 albumen conservative variation protein or its active fragment or its reactive derivative, allelic variant,
Natural mutation, induced mutants, can be encoded with the DNA of the DNA hybridization of people SLC35E1 under high or low stringent condition
Protein.
Detection technique
The SLC35E1 of the present invention is examined using multiple nucleic acids known to persons of ordinary skill in the art and protein techniques
It surveys, these technologies include but not limited to：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).
The PCR of commonly referred to as PCR uses denaturation, annealing and the primer extend of primer pair and opposite strand
Multiple cycles, exponentially increase target nucleic acid sequence copy number；The amplification of the transcriptive intermediate of TMA is (in substantial constant
Temperature, multiple copies of target nucleic acid sequence are autocatalytically synthesized under conditions of ionic strength and pH, wherein target sequence is more
A RNA copies autocatalytically generate other copy；The ligase chain reaction of LCR uses miscellaneous with the adjacent area of target nucleic acid
The two groups of complementary DNA oligonucleotides handed over；Other amplification methods include for example：The expansion based on nucleic acid sequence of commonly referred to as NASBA
Increase；Use the amplification of rna replicon enzyme (commonly referred to as Q β replicase) amplification probe molecule itself；Amplification method based on transcription；
And the sequence amplification of self―sustaining.
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
Chip includes genetic chip, protein chip in the present invention；The genetic chip includes solid phase carrier；And orderly
The oligonucleotide probe being fixed on the solid phase carrier, the oligonucleotide probe specifically correspond to SLC35E1 institutes
Some or all of show sequence.The protein chip includes solid phase carrier, and the SLC35E1 codings being fixed on solid phase carrier
Albumen specific antibody or ligand.
The solid phase carrier includes inorganic carrier and organic carrier, the inorganic carrier include but not limited to have silicon carrier,
Glass carrier, ceramic monolith etc.；The organic carrier includes polypropylene film, nylon membrane etc..
" 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 preciseness 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.
Exemplary probe in the present invention includes PCR primer and gene specific DNA oligonucleotide probe, such as fixed
In micro probe array, the quantitative nucleic acid enzyme protection on microarray substrate examine probe, the probe that is connect with molecular barcode and
The probe being fixed on pearl.
These probes have 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 pass through PNA (Polyamide nucleic in part of it or whole nucleotides
Acid, peptide nucleic acid), LNA (registered trademark, lockednucleic acid, Bridged Nucleic Acid, Cross-linked core
Acid), ENA (registered trademark, 2 '-O, 4 '-C-Ethylene-bridged nucleic acids), GNA (Glycerol
Nucleic acid, glycerine nucleic acid), the artificial replacement nucleic acid such as TNA (Threose nucleic acid, threose nucleic acid) obtains
Polynucleotides.
The ligand of the present invention may include that the peptide of SLC35E1, antibody or its segment or aptamers or widow can be specifically bound
Nucleotide.Antibody can be monoclonal antibody, polyclonal antibody or its segment that can specifically bind the SLC35E1.
The specific antibody of heretofore described SLC35E1 albumen includes monoclonal antibody, polyclonal antibody.It is described
The specific antibody of SLC35E1 albumen includes complete antibody molecule, any segment of antibody or modification, for example, chimeric antibody,
scFv、Fab、F(ab’)2, Fv etc..As long as the segment can retain the binding ability with SLC35E1 albumen.For examining
The preparation for surveying the antibody of protein level is well known to those skilled in the art, and the present invention may use any method to make
The standby antibody, segment as mentioned can be synthesized by chemical method de novo formation or using recombinant DNA technology.
The present invention provides a kind of kit, the kit can be used for detecting the expression water of SLC35E1 genes or albumen
It is flat, including for SLC35E1 detections and/or the ligand, and/or chip of the quantitative present invention.The optional explanation with kit
Book is together.
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.
Kit or chip can be used for detecting multiple genes including SLC35E1 genes (for example, with again in the present invention
Spend depression relevant multiple genes) expression, multiple markers of major depressive disorder are carried out at the same time detection, can be significantly
Improve the accuracy rate of major depressive disorder diagnosis.
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.
In the present invention, " sample " " is the nucleic acid of can therefrom obtain comprising cell or cellular material, polypeptide or other points
Analyse the sample of object.The example of biological sample includes in non-limiting manner：Urine, blood, serum, blood plasma, celiolymph, liquor pleurae, branch
Lavage of trachea, 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.In a specific embodiment of the present invention, the sample
This is blood.
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:ColdSpring 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 major depressive disorder
1, sample collection
5 normal human bloods and major depressive disorder blood samples of patients sample are collected respectively, and the acquirement of above-mentioned all samples is logical
Cross the agreement of Ethics Committee.
The inclusion criteria of major depressive disorder patient：Americanism medical diagnosis on disease and statistic handbook fourth edition (DSM- IV) depression
The diagnostic criteria of disease, Hamilton depressive scale>34 points；Without head trauma history, central nervous system infection or organic mental
Obstacle history.
Exclusion criteria：There is abuse or relies on psychoactive drug substance mental disease medical history and meet DSM-IV diagnostic criteria；Quilt
It is diagnosed as the patient of mental retardation；Other tract diseases such as nervous system or the serious heart, liver, kidney are suffered from present
Person；In addition to depression, presently, there are or previously be once diagnosed with other psychiatric conditions.
2, the preparation and quality analysis of RNA sample
The preparation of 2.1RNA samples
(1) blood is taken, 3 times of volume erythrocyte cracked liquids are added, 10min, 10,000rpm centrifugations are placed at room temperature for after mixing
Supernatant is abandoned in 1min, suction, collects leukocyte cell pellet；
(2) TRIzol is added, is placed at room temperature for 5min, 4 DEG C of 12,000rpm centrifuge 10min, take supernatant；
(3) 200 μ l chloroforms are added per 1ml Trizol, are placed at room temperature for 3-5min after shaking vigorously and mix well, 4 DEG C 12,
000rpm centrifuges 10-15min, water phase being transferred in new pipe carefully；
(4) isometric ice-cold isopropanol is added in supernatant, is placed at room temperature for 10-20min, 4 DEG C of 12,000rpm centrifugations
10min abandons supernatant；
(5) 75% ethyl alcohol of 1ml (being prepared with RNase-free water) is added in RNA precipitate, mildly vibrates centrifuge tube, suspends
Precipitation；
(6) 4 DEG C of 8,000rpm centrifuge 1-2min, abandon supernatant；Of short duration rapid centrifugation is carefully inhaled with pipettor and abandons supernatant, room
Temperature places 2min and dries precipitation；
(7) 50 μ l RNase-free water are added in precipitating, flick tube wall, fully to dissolve RNA, -70 DEG C of preservations.
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
Sequencing result shows that expression quantity of the SLC35E1 genes in major depressive disorder blood samples of patients dramatically increases, difference tool
Statistically significant (P<0.05).
The differential expression of 2 QPCR sequence verification SLC35E1 genes of embodiment
1, SLC35E1 genes are selected to carry out large sample QPCR verifications according to the testing result of high-flux sequence.According to implementation
Sample collection mode in example 1 selects major depressive disorder blood samples of patients and each 60 of normal human blood.
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：
It takes 2 μ g of total serum IgE to carry out reverse transcription, Oligo (dT) 2 μ l is added, mix well；70 DEG C of water-baths；Ice immediately after 5min
Bathe 2-3min；5 × RT Buffer, 55 μ l, RNasin 40U/ μ l, M-MLV 200U/ μ l of μ l, dNTP (2.5mM) are added, mend
Nuclease free water is to 25 μ l；
After 42 DEG C of water-bath 60min, to inactivate M-MLV, -20 DEG C store for future use 95 DEG C of water-bath 5min.
4, QPCR is expanded
(1) design of primers
QPCR amplifications are designed according to the coded sequence of SLC35E1 genes in Genebank and house-keeping gene GAPDH genes to draw
Object is synthesized by raw work.
The primer pair sequence of SLC35E1 genes is as follows：
Forward primer sequence is 5 '-AACATCTTAACAGACCACTT-3 ' (SEQ ID NO.1)；
Reverse primer sequences are 5 '-CACATCATAGCGGTTCAA-3 ' (SEQ ID NO.2).
The primer sequence of GAPDH genes is to as follows：
Forward primer sequence is 5 '-CTCTGGTAAAGTGGATATTGT-3 ' (SEQ ID NO.3)；
Reverse primer sequences are 5 '-GGTGGAATCATATTGGAACA-3 ' (SEQ ID NO.4).
(2) PCR reaction systems：Forward primer and each 1 μ l, SYBR Green PCR systems of reverse primer
12.5 μ l, 2 μ l of template, add deionized water to complement to 25 μ l.Wherein, SYBR Green PCRs system is purchased from
Invitrogen companies.
(3) PCR reaction conditions：95 DEG C of 10min, (95 DEG C of 15s, 60 DEG C of 60s) × 35 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, statistical method
Experiment is tested using 3 repetitions, and result data is indicated in a manner of mean+SD, is used
SPSS18.0 statistical softwares are next for statistical analysis, and difference between the two is examined using t, it is believed that works as P<There is system when 0.05
Meter learns meaning.
6, result
The results are shown in Figure 1, compared with normal human blood, table of the SLC35E1 genes in major depressive disorder blood samples of patients
Up to up-regulation, difference has statistical significance (P<0.05), consistent with high-flux sequence result, wherein in major depressive disorder patient
Up-regulated expression has 55, and that expresses downward has 1, and no significant difference has 4, and positive rate is 55/60 × 100%=
91.7%, it prompts SLC35E1 genes to can be used as the early diagnosis that gene marker is applied to major depressive disorder, is found to early,
It is early to intervene.
3 protein immunoblot experiment of embodiment detects the differential expression of SLC35E1 albumen
The differential expression that SLC35E1 albumen is detected using Western blot, using GAPDH as the internal reference of protein quantification,
Wherein SLC35E1 polyclonal antibodies are purchased from abcam companies.
1, blood is taken, 3 times of volume erythrocyte cracked liquids are added, 10min, 10,000rpm centrifugations are placed at room temperature for after mixing
Supernatant is abandoned in 1min, suction, collects leukocyte cell pellet；
2, leucocyte is cracked using the protein lysate that lysate (RIPA) and PMSF are configured to；
3, albumen concentration, BCA protein quantification kit of the concrete operation step with reference to CWBIO companies are measured using BCA methods
On illustrate carry out；
4, SDS-PAGE carries out gel electrophoresis
5, statistical analysis
According to destination protein SLC35E1 bands in gel images and the gray value of internal reference GAPDH bands, ratio is calculated,
The relative expression quantity of destination protein is obtained, in triplicate.
6, result
The results are shown in Figure 2, compared with normal person, SLC35E1 albumen up-regulated expression, difference in major depressive disorder patient
With statistical significance (P<0.05), expression is similar with the expression of mRNA, and up-regulated expression has 55, prompts
SLC35E1 albumen can be used as the clinical diagnosis that protein marker is applied to major depressive disorder.
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 table
<110>Beijing Yang Shen biology information technologies Co., Ltd
<120>Major depressive disorder related gene and its application in diagnosis
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
aacatcttaa cagaccactt 20
<210> 2
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
cacatcatag cggttcaa 18
<210> 3
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
ctctggtaaa gtggatattg t 21
<210> 4
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
ggtggaatca tattggaaca 20
Claims (10)
1. the reagent for detecting SLC35E1 levels is preparing the application in diagnosing major depressive disorder product.
2. reagent according to claim 1, which is characterized in that the product includes hybridizing skill by sequencing technologies, nucleic acid
The reagent of art, nucleic acid amplification technologies, protein immunization technology detection SLC35E1 levels.
3. application according to claim 1 or 2, which is characterized in that the reagent includes：
The probe of specific recognition SLC35E1 genes；Or
The primer of specific amplification SLC35E1 genes；Or
Specifically bind the antibody or ligand of the albumen of SLC35E1 codings.
4. application according to claim 3, which is characterized in that the primer sequence of the specific amplification SLC35E1 genes
To as shown in SEQ IDNO.1 and SEQ IDNO.2.
5. the product of SLC35E1 expressions in a kind of vitro detection sample, which is characterized in that the product includes preparation, core
Piece or kit.
6. product according to claim 5, which is characterized in that the sample is blood.
7. product described in claim 5 or 6 is preparing the application in diagnosing major depressive disorder tool.
8. a kind of kit of diagnosis major depressive disorder, which is characterized in that the kit includes：
Detect one or more reagents of SLC35E1 genes or protein expression level；With
One or more substances selected from the group below：Container, positive control, negative control object, buffer, helps operation instructions
Agent or solvent.
9. kit according to claim 8, which is characterized in that the kit includes by RT-PCR methods, qRT-PCR
Method, biochip test method, southern blotting technique method, hybridization in situ, Western blot detection SLC35E1 genes or protein expression water
Flat reagent.
10. kit according to claim 9, which is characterized in that described to detect SLC35E1 gene tables with qRT-PCR methods
Include the primer of the specific amplification SLC35E1 genes as shown in NO.1~2 sequence SEQ ID up to horizontal reagent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810498803.2A CN108410978A (en) | 2018-05-23 | 2018-05-23 | Major depressive disorder related gene and its application in diagnosis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810498803.2A CN108410978A (en) | 2018-05-23 | 2018-05-23 | Major depressive disorder related gene and its application in diagnosis |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108410978A true CN108410978A (en) | 2018-08-17 |
Family
ID=63140335
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810498803.2A Pending CN108410978A (en) | 2018-05-23 | 2018-05-23 | Major depressive disorder related gene and its application in diagnosis |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108410978A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109797215A (en) * | 2019-02-25 | 2019-05-24 | 天津市第三中心医院 | Marker is used in major depressive disorder diagnosis |
| CN112980944A (en) * | 2021-04-26 | 2021-06-18 | 武汉儿童医院 | Major depression detection reagent, system and application |
| CN113073137A (en) * | 2021-04-02 | 2021-07-06 | 武汉儿童医院 | Postpartum depression detection reagent, system and application |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016201299A1 (en) * | 2015-06-12 | 2016-12-15 | Indiana University Research & Technology Corporation | Predicting suicidality using a combined genomic and clinical risk assessment |
| CN107022637A (en) * | 2017-06-02 | 2017-08-08 | 邳州东大医院 | A kind of gene marker related to major depressive disorder |
| WO2017147720A1 (en) * | 2016-03-04 | 2017-09-08 | Exerkine Corporation | Method for treating a central nervous system disorder |
| CN107164536A (en) * | 2017-07-07 | 2017-09-15 | 邳州东大医院 | Application of the MRNIP genes in major depressive disorder diagnosis |
-
2018
- 2018-05-23 CN CN201810498803.2A patent/CN108410978A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016201299A1 (en) * | 2015-06-12 | 2016-12-15 | Indiana University Research & Technology Corporation | Predicting suicidality using a combined genomic and clinical risk assessment |
| WO2017147720A1 (en) * | 2016-03-04 | 2017-09-08 | Exerkine Corporation | Method for treating a central nervous system disorder |
| CN107022637A (en) * | 2017-06-02 | 2017-08-08 | 邳州东大医院 | A kind of gene marker related to major depressive disorder |
| CN107164536A (en) * | 2017-07-07 | 2017-09-15 | 邳州东大医院 | Application of the MRNIP genes in major depressive disorder diagnosis |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109797215A (en) * | 2019-02-25 | 2019-05-24 | 天津市第三中心医院 | Marker is used in major depressive disorder diagnosis |
| CN113073137A (en) * | 2021-04-02 | 2021-07-06 | 武汉儿童医院 | Postpartum depression detection reagent, system and application |
| CN112980944A (en) * | 2021-04-26 | 2021-06-18 | 武汉儿童医院 | Major depression detection reagent, system and application |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107022637B (en) | A kind of gene marker relevant to major depressive disorder | |
| CN107164536B (en) | Application of the MRNIP gene in major depressive disorder diagnosis | |
| CN107267649B (en) | Applications of the STRADB in asthma early diagnosis | |
| EP3644062B1 (en) | Method for predicting esophagus cancer response to anti-erbb3 antibody treatment | |
| CN108410978A (en) | Major depressive disorder related gene and its application in diagnosis | |
| CN112567051A (en) | Liver cancer specific biomarkers | |
| CN110331209A (en) | Application of the biomarker in adenocarcinoma of lung diagnosis | |
| CN109633156B (en) | Application of biomarker in evaluating oral squamous carcinoma risk degree | |
| CN110331201B (en) | Cervical squamous carcinoma related biomarker and application thereof | |
| CN107630085B (en) | Application of molecular marker in male osteoporosis | |
| EP2369016A1 (en) | Methods for diagnosing and treating graft rejection and inflammatory conditions | |
| CN113846164A (en) | Marker molecule for predicting sensitivity of patient to preoperative radiotherapy and chemotherapy combined total rectal resection and derivative product thereof | |
| CN113817825A (en) | Molecular marker for predicting sensitivity of rectal cancer patient to preoperative chemoradiotherapy combined total rectal resection treatment | |
| CN110656169B (en) | Diagnostic markers for atrial fibrillation | |
| KR101657051B1 (en) | Marker composition for diagnosis of chronic obstructive pulmonary disease | |
| JP6949315B2 (en) | Differentiation evaluation method for squamous cell lung cancer and adenocarcinoma of the lung | |
| CN108034707B (en) | SPAG7 gene is preparing the application in diagnosis of dementia preparation | |
| CN113403382B (en) | Application of UBE2F in diagnosis and treatment of femoral head necrosis | |
| KR101745297B1 (en) | Composition for diagnosis of obesity and uses thereof | |
| CN112941179B (en) | Application of gene in evaluating oral squamous cell carcinoma | |
| CN109576361A (en) | A kind of biomarker relevant to ischemic cardiomyopathy occurrence and development | |
| CN109797215A (en) | Marker is used in major depressive disorder diagnosis | |
| CN113862356A (en) | Product for predicting sensitivity of rectal cancer to preoperative chemoradiotherapy combined total rectal resection treatment scheme through marker | |
| CN113862357A (en) | Product for predicting sensitivity of rectal cancer to preoperative chemoradiotherapy combined total rectal resection based on biomarkers and application of product | |
| CN109486939A (en) | Application of the gene marker in ischemic cardiomyopathy diagnosis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB02 | Change of applicant information |
Address after: Room 1210, Building 3, Ronghua Xintai Building, 10 Ronghua South Road, Yizhuang Economic and Technological Development Zone, Daxing District, Beijing Applicant after: BEIJING MEDINTELL BIOMED Co.,Ltd. Address before: 100080 Beijing city Haidian District Shanyuan Street No. 1 cubic court building room 3103 Applicant before: BEIJING MEDINTELL BIOMED Co.,Ltd. |
|
| CB02 | Change of applicant information | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20200401 Address after: Room 2503, Qianshan building, building D2, Qingdao International Innovation Park Phase II, No. 1, Keyuan Weiyi Road, Laoshan District, Qingdao, Shandong Province Applicant after: Qingdao Yangshen biomedical Co.,Ltd. Address before: Room 1210, Building 3, Ronghua Xintai Building, 10 Ronghua South Road, Yizhuang Economic and Technological Development Zone, Daxing District, Beijing Applicant before: BEIJING MEDINTELL BIOMED Co.,Ltd. |
|
| TA01 | Transfer of patent application right | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180817 |
|
| RJ01 | Rejection of invention patent application after publication |