CN104984363A - Application of ZMYM1 in preparation of Parkinson's disease diagnosis and treatment reagents - Google Patents
Application of ZMYM1 in preparation of Parkinson's disease diagnosis and treatment reagents Download PDFInfo
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Abstract
The invention relates to an application of ZMYM1 in the preparation of Parkinson's disease diagnosis and treatment reagents. Candidate gene ZMYM1 is chosen after gene screening through adopting bioinformatics method analysis based on a high-flux sequencing result, and molecular biology experiments confirm that the ZMYM1 gene has very good correlation with the Parkinson's disease, so the ZMYM1 gene can be used in the preparation of auxiliary diagnosis and treatment preparations of the Parkinson's disease, and has important clinic application values.
Description
Technical field
The present invention relates to biomedicine field, be specifically related to the application of ZMYM1 in preparation parkinson diagnosis and treatment reagent.
Background technology
Parkinson disease (Parkinson's disease, PD) be a kind of Chronic Progressive disease, cannot cure at present, first it described in 1817 by British Parkinson, along with the progress to PD, we there has also been darker understanding to this disease, and it shows as mobility's symptom clinically if static tremor, bradykinesia, myotonia and posture gait disorder and non-athletic symptom are as constipation, sleep disorder, mental act exception etc.The pathogenesis of current PD is not clear, may be relevant with inherited genetic factors, mitochondrial function defect, oxidative stress, dysimmunity, apoptosis, environmental factors etc.In PD, the patient of about 10% is familial form PD patient, has obvious inherited character, and remaining 90% is sporadic PD patient.Present much research is thought, PD is the coefficient result of inherited genetic factors and environmental factors, and is the large focus at present to PD study of incident mechanism to the research of PD Disease-causing gene.Up to now, oneself is through finding that the gene relevant to heritability PD has SNCA, LRRK2, DJ-1, PINK-1, Parkin etc., and these Disease-causing genes also need further research, and can not meet clinical demand far away.For PD patient, find the therapy formulating personalized therapy program and the most applicable patient targetedly in advance in advance, thus improve the treatment situation of levodopa class medicine and improve the life quality of patient to greatest extent, alleviate the misery of patient, reduce the financial burden of family and society, this just needs to provide more molecular marker can diagnose out parkinson disease early.
For solving the rare problem of current parkinson molecular marked compound, inventor carries out high-flux sequence to 15 routine disturbances in patients with Parkinson disease peripheral blood samples and 9 routine Healthy People contrast peripheral blood samples, carry out genescreen in conjunction with bioinformatics method, pick out candidate gene zmym1.Further, invention has been molecular cytobiology method and confirm that zmym1 and Parkinsonian relation: zmym1 and parkinson disease have good dependency, can be used for preparation parkinson assisting in diagnosis and treatment preparation, there is important clinical value.
Summary of the invention
ZMYM1 gene and/or albumen is the object of the present invention is to provide to prepare the application in Parkinson treatment preparation.
For achieving the above object, first the present invention screens candidate gene ZMYM1 by high-flux sequence in conjunction with bioinformatics method, the relation of ZMYM1 and parkinson: ZMYM1 and parkinson have good dependency further by molecular cytobiology method validation, can be used for preparation treatment parkinson preparation and/or parkinson diagnostic preparation, there is important clinical value.
Further, described Parkinson treatment preparation refers to the preparation of the expression that can promote ZMYM1 gene.Those skilled in the art know and promote that the expression of gene can adopt the one in following method and/or several usually: by DNA level regulation and control ZMYM1 gene: include but not limited to increase the copy number of ZMYM1 gene, transfection containing the over-express vector of ZMYM1 gene; By transcriptional level control ZMYM1 gene: include but not limited to the expression of activation ZMYM1 gene, activate the promoter of regulation and control ZMYM1 gene expression, suppress the transcription factor of negative regulation ZMYM1 gene expression, adopt suppression of RNA perturbation technique to suppression ZMYM1 gene expression to disturb; By post-transcriptional level regulation and control ZMYM1 gene: include but not limited to suppress the microRNA transcriptional expression of promotion ZMYM1 gene mRNA degraded, import the microRNA promoting ZMYM1 gene expression; By translating rear level modulation ZMYM1 gene: include but not limited to import the molecule promoting ZMYM1 gene coded protein, the albumen suppressing negative regulation ZMYM1 gene expression, promote the factor of ZMYM1 gene expression and the expression of albumen.
RNA interference (RNAi) refers to that exogenous and endogenous double-stranded RNA induces the mRNA selective degradation of homology target gene in vivo, cause the phenomenon of PTGS, it is a kind of expression using little double-stranded RNA to block body certain specific gene interior efficiently, specifically, impel mRNA to degrade, make cells show go out the technology of specific gene disappearance phenotype.Can adopt direct synthesis technique after siRNA has designed or build SiRNA expression vector, the siRNA prepared can by approach transfectional cells such as Mechanical Method, lipofectamine reagent method such as calcium phosphate precipitation, electroporation, DEAE-glucosan and polybrene method, microinjection or particle guns.
The object of the present invention is to provide one to treat parkinson preparation, described anti-Parkinson preparation promotes the expression of ZMYM1 gene in disturbances in patients with Parkinson disease.Further, containing the carrier promoting ZMYM1 gene expression in described treatment parkinson preparation.
The object of the present invention is to provide ZMYM1 gene and/or the application of albumen in preparation parkinson diagnostic preparation.
Further, the diagnostic preparation of described parkinson comprises the expression detecting ZMYM1 gene in parkinson peripheral blood with fluorescence quantifying PCR method, method for gene chip.
Fluorescence quantitative PCR method is by fluorescent dye or fluorescently-labeled specific probe, carries out labelling tracking, real time and on line monitoring course of reaction, can analyze in conjunction with corresponding software to product PCR primer, calculates the initial concentration of testing sample template.The appearance of quantitative fluorescent PCR, greatly simplifies the process of detection by quantitative, and really achieves absolute quantitation.The appearance of multiple detection system, makes the selectivity of experiment stronger.Automation mechanized operation improves work efficiency, reacts quick, reproducible, highly sensitive, high specificity, result be clear.
Gene chip is also called DNA microarray (DNA microarray), three kinds of main Types can be divided into: 1) be fixed on polymer matrix film (nylon membrane, nitrocellulose membrane etc.) nucleic probe on surface or cDNA fragment, usually be hybrid with it with isotope-labeled target gene, detected by radiography technology.2) fixing DNA probe array on a glass by point sample method, detecting by hybridizing with fluorescently-labeled target gene.3) oligonucleotide probe array of directly synthesis on the hard surfaces such as glass, hybridizes with fluorescently-labeled target gene and detects.Gene chip is as a kind of advanced person, extensive, high throughput testing technology, and be applied to the diagnosis of disease, its advantage has the following aspects: one is susceptiveness highly and accuracy; Two is fast and convenient; Three is can detect various diseases simultaneously.
Described contains the primer of a pair specific amplification ZMYM1 gene for the product of ZMYM1 gene in fluorescence quantifying PCR method detection parkinson; Described gene chip comprises the probe with the nucleic acid array hybridizing of ZMYM1 gene.
Further, the diagnostic preparation of described parkinson comprises the expression detecting ZMYM1 albumen with immunization method.In preferred described immunologic detection method detection parkinson, ZMYM1 protein expression is western blot and/or ELISA and/gold colloidal detection method.
Enzyme-linked immunosorbent assay (ELISA) is adsorbed on surface of solid phase carriers by known antigen or antibody, makes the technology that the antigen antibody reaction of enzyme labelling is carried out at solid phase surface.This technology can be used for detecting macromole antigen and specific antibody etc., has quick, sensitive, easy, carrier and is easy to the advantages such as standardization.ELISA detection kit can be divided into indirect method, double-antibody method, competition law, dibit point one-step method, prize law to survey the ELISA of IgM antibody, application Avidin and biotin according to testing goal and operating procedure.In ELISA detection kit, chromogenic substrate can select horseradish peroxidase (HRP) or alkali phosphatase (AP).
Conventional immune colloid gold detection technique: (1) immune colloid gold light microscopic staining cell suspension smear or peripheral blood section, the antibody of available colloid gold label dyes, also can on the basis of colloid gold label, labelling is strengthened with silver-colored developer solution, make the silver atoms be reduced be deposited on the gold grain surface of labelling, obviously can strengthen the sensitivity of colloid gold label.(2) immune colloid gold staining method for electron microscopy can be combined with negative staining Virus Sample or peripheral blood ultrathin section with the antibody of colloid gold label or anti antibody, then carries out negative staining.Can be used for observation and the Viral diagnosis of morphology of virus.(3) dot immunogold filtration assay application microporous filter membrane makes carrier, first by antigen or antibody point on film, add sample to be checked after closing, wash the corresponding antigen of antibody test or the antibody of rear colloid gold label.(4) specific antigen or antibody are fixed on film with ribbon by colloidal gold immunity chromatography, colloid gold label reagent (antibody or monoclonal antibody) is adsorbed on pad, after in the sample pad that sample to be checked is added to test strips one end, moved forward by capillarity, react to each other after dissolving the colloid gold label reagent on pad, when moving to the region of fixing antigen or antibody, there is specific binding with it again and be trapped in the conjugate of thing to be checked and gold marked reagent, be gathered in detection zone, by being observed visually colour developing result.This method now develops into diagnosis test paper, uses very convenient.
Further, the ELISA method of described detection ZMYM1 albumen is for using ELISA detection kit.Antibody in described test kit can adopt commercially available ZMYM1 monoclonal antibody.Further, described test kit comprises: wrap by the solid phase carrier of ZMYM1 monoclonal antibody, ELIAS secondary antibody, the substrate of enzyme, protein standard substance, negative controls, diluent, cleaning mixture, enzyme reaction stop buffer etc.
Further, the colloidal gold method of described detection ZMYM1 albumen is for using detection kit, and described antibody can adopt commercially available ZMYM1 monoclonal antibody.Further, described gold-immunochromatographyreagent reagent for assay box adopts colloidal gold immunochromatographimethod technology or gold colloidal percolation.Further, detection zone (T) specking on described gold-immunochromatographyreagent reagent for assay box nitrocellulose filter has anti-ZMYM1 monoclonal antibody, quality control region (C) specking has immunoglobulin IgG.
The object of the present invention is to provide a kind of PCR kit for fluorescence quantitative detecting parkinson, it is characterized in that, described test kit detects gene ZMYM1, adopts special forward primer and downstream primer, forward primer sequence is SEQ ID NO.1, and downstream primer sequence is SEQ ID NO.2.
Further, this PCR kit is suitable for all types fluorescence quantitative gene extender deposited at present commercially, highly sensitive, quantitatively quick and precisely, good stability, has a good application prospect.
Further, above-mentioned PCR kit for fluorescence quantitative component comprises: Auele Specific Primer, internal reference primer, fluorescence quantitative PCR reaction solution.Wherein said Auele Specific Primer comprises forward primer and downstream primer, and forward primer sequence is SEQ ID NO.1, and downstream primer sequence is SEQ ID NO.2.Described internal reference primer is β-actin internal reference primer, and forward primer sequence is SEQ ID NO.3, and downstream primer sequence is SEQ ID NO.4.
Described test kit also comprises RNA extraction agent.Preferably
reagent carries out sample rna extraction.
The present invention also have detected this test kit susceptiveness, and it is 10 that result shows this test kit detection range
6-10
2copies/ μ l, minimum concentrations is 100copies/ μ l.
The object of the invention there is provided a kind of parkinson detection kit, and described detection kit detects ZMYM1 albumen.Further, described test kit also comprises other detectable.
The object of the invention there is provided a kind of gene chip detecting parkinson, and described gene chip comprises the probe with the nucleic acid array hybridizing of ZMYM1 gene.
Accompanying drawing explanation
Fig. 1 ZMYM1 gene relative expression's spirogram in parkinson peripheral blood and healthy human peripheral blood
Detailed description of the invention
Below in conjunction with specific embodiment, setting forth the present invention further, only for explaining the present invention, and can not limitation of the present invention be interpreted as.Those having ordinary skill in the art will appreciate that: can carry out multiple change, amendment, replacement and modification to these embodiments when not departing from principle of the present invention and aim, scope of the present invention is by claim and equivalents thereof.The experimental technique of unreceipted actual conditions in the following example, the usually conveniently conditioned disjunction condition examinations of advising according to manufacturer.
Embodiment 1 high-flux sequence and analysis
Samples sources, in BJ Union Hospital, all obtains subjects informed consent.Collect 15 routine disturbances in patients with Parkinson disease peripheral blood samples and 9 routine Healthy People contrast peripheral blood samples respectively, carry out RNA extraction, RNA extract after agarose gel electrophoresis, from electrophoresis result can preliminary judgement extract RNA sample whether up-to-standard, whether may be used for further transcriptome analysis.And then detected the extraction situation of RNA sample by NanoDrop1000 spectrophotometer, the sample requirement of RNA-seq order-checking: OD260/OD280 is 1.8-2.2.
Order-checking platform is the HiSeq 2500 high-flux sequence platform of Illumina company, carry out the order-checking of the high flux transcript profile degree of depth, after order-checking, we use Fast-QC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) software to carry out total evaluation to the quality of sequencing data, comprise the mass value distribution of base, the position distribution of mass value, GC content, PCR duplication content, the frequency etc. of kmer.When differential genes expression analysis, according to the FPKM value obtained, internationally recognized algorithm EBSeq is adopted to carry out differential screening.Wherein, during screening, LOG2FC>1 or <-1, FDR<0.05.In order to better understand the function of difference expression gene, we have carried out Gene Onlogy and signal path analysis to difference expression gene, and functional annotation and protein interaction analysis of network are carried out to difference expression gene, in view of the result of above data analysis, in conjunction with document, we have screened difference expression gene ZMYM1.
Embodiment 2 disturbances in patients with Parkinson disease peripheral blood and healthy human peripheral blood ZMYM1 expression conditions
One materials and methods
1, material
Collect 135 routine disturbances in patients with Parkinson disease peripheral bloods and 33 routine healthy human peripheral bloods, it is divided into groups and numbers.
2, method
The extraction of 2.1 disturbances in patients with Parkinson disease peripheral bloods and healthy human peripheral blood total serum IgE
Adopt
reagent carries out sample rna extraction, and experimental implementation is undertaken by product description, and description is shown in concrete operations.
RNA quality judging standard: the OD260/OD280 value of RNA sample is between 1.7-2.2; Total serum IgE electrophoresis pattern has 28S, 18S band clearly; The electrophoresis pattern of 70 DEG C of water bath heat preservations after 1 hour and the collection of illustrative plates no significant difference before water bath heat preservation.
2.2 reverse transcription synthesis cDNA
Adopt
iII Reverse Transcriptase (invitrogen, article No. 18080-044) carries out cDNA reverse transcription, and experimental implementation is undertaken by product description, and concrete operations are as follows:
Use Reverse Transcriptase kit, with RT Buffer, converse record synthesis cDNA is carried out to l μ g total serum IgE.Adopt 25 μ l reaction systems, each sample gets 1 μ g total serum IgE as template ribonucleic acid, in PCR pipe, add following component respectively:
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/lOligodT 2 μ l, 200U/ μ l MMLV 1.25 μ l, template ribonucleic acid 1 μ g, add aquesterilisa to total system 25 μ l.Hatch 1 hour for 42 DEG C, 72 DEG C 10 minutes, of short duration centrifugal.It is for subsequent use that-20 DEG C of refrigerators are put in cDNA preservation.
2.3 Real-Time PCR
2.3.1 instrument and analytical method
With ABI 7500 type quantitative real time PCR Instrument, 2-△ △ CT method is adopted to carry out the relative quantitative assay of data.
2.3.2 design of primers
Adopt online primer-design software, synthesized by invitrogen company after design of primers.Concrete primer sequence is as follows:
Table 1 primer sequence
Operating process is as follows:
(1) reaction system: use Power
green PCR Master Mix (invitrogen, article No. 4367659) increases, and experimental implementation is undertaken by product description.Amplification program is: 95 ° of 10min, (95 DEG C of 15sec, 60 DEG C of 60sec) × 45 circulations.
Table 2 RealTime reaction system
| Component | Addition |
| 2×mix | 10μl |
| Forward primer (10uM) | 0.5μl |
| Downstream 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, after dilution, sample is respectively got 2 μ l and is made template, increase with genes of interest primer and reference gene primer respectively, carry out melt curve analysis analysis at 60-95 DEG C simultaneously, carry out primer screening according to amplification efficiency height and the unimodal principle of solubility curve.
(3) sample RealTimePCR detects
Get 2 μ l after being diluted by each sample cDNA 10 times and make template, increase with genes of interest primer and reference gene primer respectively.Carry out solubility curve analysis at 60-95 DEG C simultaneously.
Two experimental results
Real-time quantitative PCR amplification curve flex point is clear, and the overall collimation of amplification curve is good, shows that the amplification efficiency of each reaction tube is close, and the limit is flat and without raising up now, exponent phase slope is comparatively large, illustrates that amplification efficiency is higher; Sample amplified production solubility curve is all unimodal, illustrates that amplified production only has one, is specific amplification; Relative quantification formula according to qRT-PCR: 2-Δ Ct × 100%, compares the expression of ZMYM1 gene in parkinson peripheral blood and healthy human peripheral blood.Result display (specifically seeing Fig. 1): qRT-PCR stable amplification result, wherein the expression of ZMYM1 in disturbances in patients with Parkinson disease peripheral blood is lower than healthy human peripheral blood, the result of confluence analysis ZMYM1 low expression in parkinson peripheral blood of above result verification high flux transcript profile expression data.
The present invention adopts high-flux sequence to filter out parkinson pathogenic related gene ZMYM1, and binding molecule biological experiment is verified, confirms that ZMYM1 has important effect in Parkinson disease.The present invention is that parkinson clinic diagnosis provides new target, has good potential applicability in clinical practice.
Claims (10)
1.ZMYM1 gene and/or albumen are preparing the application in Parkinson treatment preparation.
2. application according to claim 1, it is characterized in that, described Parkinson treatment preparation can adopt the expression of a kind of and/or several promotion ZMYM1 gene in following method: comprise increase ZMYM1 gene copy number, transfection is containing the over-express vector of ZMYM1 gene; Activating the expression of ZMYM1 gene, activate the promoter of regulation and control ZMYM1 gene expression, suppress the transcription factor of negative regulation ZMYM1 gene expression, adopting RNA perturbation technique to disturb suppressing suppression of ZMYM1 gene expression; Suppress to promote the microRNA transcriptional expression of ZMYM1 gene mRNA degraded, import the microRNA promoting ZMYM1 gene expression; Import the molecule promoting genes of interest encoding proteins, the albumen suppressing negative regulation ZMYM1 gene expression, promote the factor of ZMYM1 gene expression and the expression of albumen.
3. treat a parkinson preparation, it is characterized in that, described treatment parkinson preparation promotes the expression of ZMYM1 gene.
4. treatment parkinson preparation according to claim 3, is characterized in that, containing the carrier promoting ZMYM1 gene expression in described anti-Parkinson preparation.
5.ZMYM1 gene and/or the application of albumen in preparation parkinson diagnostic preparation.
6. application according to claim 5, is characterized in that, the diagnostic preparation of described parkinson comprises the expression detecting ZMYM1 gene with fluorescence quantifying PCR method, method for gene chip.
7. application according to claim 6, is characterized in that, the described product for fluorescence quantifying PCR method detection ZMYM1 gene contains the primer of a pair specific amplification ZMYM1 gene; Described gene chip comprises the probe with the nucleic acid array hybridizing of ZMYM1 gene.
8. application according to claim 5, is characterized in that, the diagnostic preparation of described parkinson comprises the expression detecting ZMYM1 albumen with immunization method.
9. application according to claim 8, is characterized in that, what described immunization method detected ZMYM1 protein expression is ELISA detection kit and/gold-immunochromatographyreagent reagent for assay box.
10. detect a PCR kit for fluorescence quantitative for parkinson, it is characterized in that, described test kit detects gene ZMYM1, and adopt special forward primer and downstream primer, forward primer sequence is SEQ ID NO.1, and downstream primer sequence is SEQ ID NO.2.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105385774A (en) * | 2015-12-24 | 2016-03-09 | 周丽丽 | Application of TMF1 as Parkinsonism diagnosis marker |
| CN105506171A (en) * | 2016-03-01 | 2016-04-20 | 北京泱深生物信息技术有限公司 | LYRM2 (LYR motif-containing 2) gene and new use of expression product thereof |
Citations (1)
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| US20130217028A1 (en) * | 2010-10-26 | 2013-08-22 | Silva A. Mandel | Peripheral blood gene markers for early diagnosis of parkinson's disease |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130217028A1 (en) * | 2010-10-26 | 2013-08-22 | Silva A. Mandel | Peripheral blood gene markers for early diagnosis of parkinson's disease |
Non-Patent Citations (2)
| Title |
|---|
| JOSE A. SANTIAGO 等: ""Network-based metaanalysis identifies HNF4A and PTBP1 as longitudinally dynamic biomarkers for Parkinson’s disease"", 《PNAS》 * |
| LEONID MOLOCHNIKOV 等: ""A molecular signature in blood identifies early Parkinson’s disease"", 《MOLECULAR NEURODEGENERATION》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105385774A (en) * | 2015-12-24 | 2016-03-09 | 周丽丽 | Application of TMF1 as Parkinsonism diagnosis marker |
| CN105385774B (en) * | 2015-12-24 | 2018-09-25 | 周丽丽 | Purposes of the TMF1 as parkinsonism diagnosis marker |
| CN105506171A (en) * | 2016-03-01 | 2016-04-20 | 北京泱深生物信息技术有限公司 | LYRM2 (LYR motif-containing 2) gene and new use of expression product thereof |
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