CN108085383B - Gene detection composition and application thereof - Google Patents
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Abstract
The invention relates to a gene detection composition and application thereof, in particular to application of a TMED2, CCDC72 and ERC1 gene composition in detection of postmenopausal osteoporosis and clinical medication of Liuwei Dihuang pills. The inventor carries out high-throughput sequencing on peripheral blood samples before and after taking Liuwei Dihuang pills for postmenopausal osteoporosis patients and healthy person control peripheral blood samples by a high-throughput sequencing technology, shows candidate genes TMED2, CCDC72 and ERC1 and postmenopausal osteoporosis, and obviously changes the 3 genes after taking Liuwei Dihuang pills for postmenopausal osteoporosis patients, and further verifies the high-throughput data analysis result by molecular biological experiments. The invention provides a genome composition for clinical accurate diagnosis of postmenopausal osteoporosis and accurate medication, and has good market application value.
Description
Technical Field
The invention relates to the field of biological medicines, in particular to a gene detection composition and application thereof, and more particularly relates to application of a TMED2, CCDC72 and ERC1 gene composition in detection of postmenopausal osteoporosis and clinical medication of Liuwei Dihuang pills.
Background
The current studies on the function of TMED2 are mostly focused on its involvement in the process of vesicular transport of proteins between the endoplasmic reticulum and golgi apparatus. In recent years, TMED2 has been shown to be a specific transport receptor for G protein-coupled receptors (GPCRs) and to control the transport processes in the pathway of GPCR biosynthesis, regulating GPCR signaling pathways in astrocytes. In addition, TMED2 has been reported in the literature to be expressed both in the placenta and in the chorionic carcinoma cell line throughout pregnancy in humans, and mutation of TMED2 in mice results in defects in embryo and placenta development, suggesting that it may play a role in placental development (histones deacetylaseinium leucocytes to TRAIL-induced apoptosis by c-myc-mediated depression of cflp. oncogene,2012Jan 23.).
CCDC72 was first in human CD34+Hematopoietic stem cell discovery for maintaining DPC agglutinative growth and DPCSOther properties may play an important role. The functional domain of the CCDC72 gene is found by Wangjun et al to be homologous with the T2FA gene, namely, the functional domain can play a role of a transcription initiation factor in the gene transcription process and play a role in cell division, nuclear gene replication, repair and recombination (the gene is differentially expressed by the coagulation growth of hair papilla cells)Screening and bioinformatics analysis of full-length HSPC 016. China journal of medicine 2004,84(18): 1513-; the CCDC72 gene is widely expressed in human tissues, and the tissues have the characteristics of high metabolism and vigorous cell division.
There are few studies on ERC1 gene, and a screening article about congenital diaphragmatic hernia-related gene in 2016 shows that ERC1 gene may be related to congenital diaphragmatic hernia, but the correlation is not confirmed (screening of congenital diaphragmatic hernia-related gene, shandong medicine, vol 56, No. 19 in 2016).
Our previous studies showed that TMED2 gene is associated with secondary disc lesions (marker for diagnosis and treatment of degenerative disc lesions, CN 2015109830657), CCDC72 gene is associated with myeloma (a molecular marker for diagnosis and treatment of multiple myeloma, CN 201610602127X), ERC1 is associated with hypopharyngeal cancer (application of ERC1 in preparation of a tool for diagnosis or treatment of hypopharyngeal cancer, CN 2016107521916).
In the invention, the inventor carries out high-throughput sequencing on the peripheral blood samples before and after taking the Liuwei Dihuang pill for the postmenopausal osteoporosis patient and the healthy person control peripheral blood sample by a high-throughput sequencing technology, shows that the candidate genes TMED2, CCDC72 and ERC1 and the postmenopausal osteoporosis are generated, and the 3 genes are obviously changed after taking the Liuwei Dihuang pill for the postmenopausal osteoporosis patient, and verifies the analysis result of high-throughput data through molecular biological experiments, and the verification result is consistent with the sequencing result. The invention provides a genome composition for clinical accurate diagnosis of postmenopausal osteoporosis and accurate medication, and has good market application value.
Disclosure of Invention
The invention aims to provide a gene composition, which comprises one or more of the following genes: TMED2, CCDC72, and ERC 1.
Furthermore, the gene composition or the expression product thereof can be applied to the preparation of clinical drug gene detection preparations for postmenopausal osteoporosis.
Furthermore, the sample group suitable for the pills of six ingredients with rehmannia has high expression of TMED2 gene or TMED2 protein, or has high expression of ERC1 gene or ERC1 protein, or has low expression of CCDC72 gene or CCDC72 protein.
In order to achieve the purpose, candidate genes TMED2, CCDC72 and ERC1 are screened by a high-throughput sequencing combined bioinformatics method, the expression level of TMED2 and ERC1 genes is obviously reduced after patients with osteoporosis take Liuwei Dihuang pills, the expression level of CCDC72 genes is obviously increased, the genes are shown to be related to postmenopausal osteoporosis or function targets of Liuwei Dihuang pills, and the genes can be used for preparing postmenopausal osteoporosis diagnosis preparations and clinical drug gene detection targets and have important clinical application value.
Further, the test agent is used for testing the expression of TMED2, CCDC72 and/or ERC1 genes by one or more of the following methods: fluorescent quantitative PCR method, gene chip method, and high-throughput sequencing method.
Preferably, the gene chip comprises probes that hybridize to the nucleic acid sequences of the TMED2, CCDC72, and/or ERC1 genes.
Preferably, the fluorescent quantitative PCR method employs a specific primer pair selected from the group consisting of: a primer pair consisting of SEQ ID NO.1 and SEQ ID NO. 2; or a primer pair consisting of SEQ ID NO.3 and SEQ ID NO. 4; a primer pair consisting of SEQ ID NO.5 and SEQ ID NO. 6.
The invention aims to provide a postmenopausal osteoporosis detection kit, which is used for detecting the expression of TMED2, CCDC72 and/or ERC1 genes and proteins thereof. Furthermore, the kit also comprises other detection reagents.
The invention aims to provide a gene chip for detecting postmenopausal osteoporosis, which comprises a probe hybridized with a nucleic acid sequence of TMED2, CCDC72 and/or ERC1 genes.
The invention aims to provide a gene detection kit for clinical drugs for postmenopausal osteoporosis, which is used for detecting the expression of TMED2, CCDC72 and/or ERC1 genes and proteins thereof. The sample group with high expression of TMED2 and/or ERC1 gene and protein thereof recommends using Liuwei Dihuang pill, and the sample group with low expression of CCDC72 gene and/or CCDC72 protein recommends using Liuwei Dihuang pill.
The invention aims to provide a gene chip for detecting clinical medicine for postmenopausal osteoporosis, which comprises a probe hybridized with a nucleic acid sequence of TMED2, CCDC72 and/or ERC1 genes. The sample group with high expression of TMED2 and/or ERC1 gene and protein thereof recommends using Liuwei Dihuang pill, and the sample group with low expression of CCDC72 gene and/or CCDC72 protein recommends using Liuwei Dihuang pill.
The invention aims to provide application of a TMED2 inhibitor, an ERC1 inhibitor and/or a CCDC72 promoter in preparation of a medicament for treating postmenopausal osteoporosis.
Further, the medicament for treating postmenopausal osteoporosis inhibits the expression of TMED2 and/or ERC1 genes and/or promotes the expression of CCDC72 genes. It is well known to those skilled in the art that suppression of gene expression can generally be achieved by one or more of the following methods: the method comprises the steps of activating a suppressor gene of a TMED2 and/or ERC1 gene, activating a protein expressed by the suppressor gene of the TMED2 and/or ERC1 gene, inhibiting the expression of the TMED2 and/or ERC1 gene by adopting an RNA interference technology, activating microRNA for promoting the degradation of mRNA of the TMED2 and/or ERC1 gene, introducing a molecule for promoting the degradation of protein coded by the TMED2 and/or ERC1 gene, and inhibiting a factor for promoting the expression of the TMED2 and/or ERC1 gene and the expression of the protein. It is well known to those skilled in the art that promoting gene expression may generally be accomplished by one or more of the following methods: the CCDC72 gene is regulated by DNA level: including but not limited to increasing the copy number of the CCDC72 gene, transfecting an overexpression vector containing the CCDC72 gene; the CCDC72 gene is regulated by the transcriptional level: including but not limited to activating the expression of CCDC72 gene, activating the promoter for regulating the expression of CCDC72 gene, inhibiting the transcription factor for negatively regulating the expression of CCDC72 gene, and interfering the inhibitor for inhibiting the expression of CCDC72 gene by adopting RNA interference technology; the CCDC72 gene is regulated by post-transcriptional levels: including but not limited to, inhibiting the transcriptional expression of microRNA which promotes the mRNA degradation of the CCDC72 gene, and introducing microRNA which promotes the expression of the CCDC72 gene; the CCDC72 gene is regulated by post-translational levels: including but not limited to, the introduction of molecules promoting the protein coded by the CCDC72 gene, the inhibition of the protein negatively regulating the expression of the CCDC72 gene, the promotion of the expression of the CCDC72 gene and the expression of the protein.
RNA interference (RNAi) refers to the phenomenon that exogenous and endogenous double-stranded RNA induces mRNA specific degradation of homologous target genes in an organism to cause post-transcriptional gene silencing, and is a technology which uses small double-stranded RNA to efficiently and specifically block the expression of a certain specific gene in the organism, promote the mRNA degradation and enable cells to show a specific gene deletion phenotype. After the siRNA design is finished, a direct synthesis method or a constructed siRNA expression vector can be adopted, and the prepared siRNA can transfect cells by a calcium phosphate coprecipitation method, an electroporation method, a DEAE-dextran and polybrene method, a mechanical method such as microinjection or a gene gun, a cationic liposome reagent method and the like.
The invention aims to provide a medicine for treating postmenopausal osteoporosis, which inhibits the expression of TMED2 and/or ERC1 genes and proteins thereof and promotes the expression of CCDC72 genes and proteins thereof.
Furthermore, the medicine for treating postmenopausal osteoporosis also comprises a pharmaceutically acceptable carrier.
The pharmaceutically acceptable carrier included in the present invention is a carrier generally used in the preparation, and includes, but is not limited to, lactose (lactose), dextrose (dextrose), sucrose (sucrose), sorbitol (sorbitol), mannitol (mannitol), starch, gum arabic, calcium phosphate, alginate (alginate), gelatin (gelatin), calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone (polyvinylpyrrolidone), cellulose (cellulose), water, syrup, methyl cellulose (methyl cellulose), methyl hydroxybenzoate (methyl hydroxybenzoate), propyl hydroxybenzoate (propyl hydroxybenzoate), talc, magnesium stearate (magnesium stearate), mineral oil (mineral oil), and the like.
The composition of the present invention may contain, in addition to the above components, a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. The carrier and the preparation which can be accepted in pharmacy are described in the complete Remington pharmaceutical book in detail.
The composition of the present invention can be administered orally or parenterally, and when administered parenterally, it can be administered by intravenous injection, intranasal injection, local injection, intracerebroventricular injection, spinal cavity injection, subcutaneous injection, intraperitoneal injection, transdermal administration, etc.
The appropriate dose of the composition of the present invention can be prescribed in various ways depending on factors such as the method of preparation, the mode of administration, the age, body weight, sex, disease state, food, administration time, administration route, excretion rate and reaction sensitivity of the patient, and the effective dose for the desired treatment or prevention can be easily determined and prescribed by a skilled physician.
The invention aims to provide application of a reagent for detecting TMED2, CCDC72 and/or ERC1 genes and proteins thereof in preparation of a postmenopausal osteoporosis diagnosis preparation.
Further, the TMED2 and/or ERC1 genes and proteins thereof are highly expressed in postmenopausal osteoporosis samples, and the CCDC72 gene or proteins thereof are less expressed in postmenopausal osteoporosis samples.
Further, the postmenopausal osteoporosis diagnostic agent detects the expression of the TMED2, CCDC72 and/or ERC1 genes and proteins thereof in peripheral blood.
Further, the diagnostic agent for postmenopausal osteoporosis was tested for expression of the TMED2, CCDC72 and/or ERC1 genes using one or more of the following methods: fluorescent quantitative PCR method, gene chip method, and high-throughput sequencing method.
The fluorescence quantitative PCR method is characterized in that a PCR product is marked and tracked through a fluorescent dye or a fluorescence-marked specific probe, the reaction process is monitored on line in real time, the product can be analyzed by combining corresponding software, and the initial concentration of a sample template to be detected is calculated. The occurrence of fluorescence quantitative PCR greatly simplifies the quantitative detection process and truly realizes absolute quantification. The presence of multiple detection systems makes the assay more selective. The automatic operation improves the working efficiency, and the reaction is rapid, the repeatability is good, the sensitivity is high, the specificity is strong, and the result is clear.
Gene chips, also known as DNA microarrays, can be divided into three main types: 1) nucleic acid probes or cDNA fragments immobilized on the surface of a polymer substrate (nylon membrane, nitrocellulose membrane, etc.) are usually hybridized with an isotope-labeled target gene and detected by a radioimaging technique. 2) The detection is carried out by hybridization with a target gene labeled with fluorescence using a DNA probe array immobilized on a glass plate by spotting. 3) An oligonucleotide probe array synthesized directly on a hard surface such as glass is hybridized with a target gene labeled with fluorescence for detection. As an advanced, large-scale and high-throughput detection technology, the gene chip is applied to the diagnosis of diseases, and has the advantages of the following aspects: firstly, high sensitivity and accuracy; secondly, the method is quick, simple and convenient; thirdly, can detect a plurality of diseases simultaneously.
High-throughput sequencing (also called next generation sequencing) is a revolutionary change to the conventional sequencing, and sequences of hundreds of thousands to millions of DNA molecules are determined at one time, so that the sequencing efficiency is greatly improved. The large-scale sequencing technology greatly improves the reading speed of genetic information of a plurality of species, and provides guarantee for acquiring sequence information of all mRNA and decrypting mRNA maps. High throughput sequencing at the same time makes it possible to perform a detailed global analysis of the transcriptome and genome of a species and is therefore also referred to as deep sequencing. Representative of high throughput sequencing platforms are the 454 sequencer (Roch GSFLX sequencer) by Roche (Roche), the Solexa Genome Analyzer (Illumina Genome Analyzer) by Illumina, and the SOLiD sequencer (ABI SOLiD sequencer) by ABI.
The product for detecting TMED2, CCDC72 and/or ERC1 genes in postmenopausal osteoporosis by the fluorescent quantitative PCR method contains a primer pair for specifically amplifying TMED2, CCDC72 and/or ERC1 genes; the gene chip comprises a probe which is hybridized with the nucleic acid sequence of TMED2, CCDC72 and/or ERC1 genes.
Further, the diagnostic preparation for postmenopausal osteoporosis further comprises an immunological test for the expression of TMED2, CCDC72 and/or ERC1 proteins. Preferably, the immunological method detects expression of TMED2, CCDC72 and/or ERC1 proteins in postmenopausal osteoporosis as a western blot and/or ELISA and/or colloidal gold assay.
Enzyme-linked immunosorbent assay (ELISA) is a technique in which a known antigen or antibody is adsorbed on the surface of a solid phase carrier, and an enzyme-labeled antigen-antibody reaction is carried out on the surface of the solid phase. The technology can be used for detecting macromolecular antigens, specific antibodies and the like, and has the advantages of rapidness, sensitivity, simplicity, convenience, easy standardization of carriers and the like. The ELISA detection kit can be divided into indirect method, double antibody sandwich method, competition method, two-site one-step method, IgM antibody detection by capture method and ELISA using avidin and biotin according to the detection purpose and operation steps. The chromogenic substrate in the ELISA detection kit can be selected from horseradish peroxidase (HRP) or Alkaline Phosphatase (AP).
The commonly used detection technique of immune colloidal gold: (1) the immune colloidal gold optical lens staining method cell suspension smear or tissue section can stain with colloidal gold labeled antibody, or enhance labeling with silver developing solution based on the colloidal gold labeling, so that reduced silver atoms are deposited on the surface of the labeled gold particles, and the sensitivity of the colloidal gold labeling can be obviously enhanced. (2) The immune colloidal gold electron microscope staining method can combine the colloidal gold labeled antibody or anti-antibody with the negative staining virus sample or tissue ultrathin section, and then carry out negative staining. Can be used for observing virus morphology and detecting virus. (3) The dot immunogold filtration method uses microporous filter membrane as carrier, firstly, the antigen or antibody is spotted on the membrane, after closed, the sample to be detected is added, after washing, the corresponding antigen or antibody is detected by using colloidal gold-labeled antibody. (4) The colloidal gold immunochromatography fixes specific antigen or antibody on a membrane in a strip shape, a colloidal gold labeled reagent (antibody or monoclonal antibody) is adsorbed on a binding pad, when a sample to be detected is added on the sample pad at one end of a test strip, the sample moves forwards through capillary action, the colloidal gold labeled reagent on the binding pad is dissolved and then reacts with each other, when the sample moves to a region of the fixed antigen or antibody, a combination of the object to be detected and the gold labeled reagent is specifically combined with the sample to be detected and intercepted, and is gathered on a detection zone, and a color development result can be observed through naked eyes. The method is developed into a diagnostic test strip, and is very convenient to use.
Further, the ELISA method for detecting TMED2, CCDC72 and/or ERC1 protein is to use an ELISA detection kit. The antibodies in the kit can adopt commercially available TMED2, CCDC72 and ERC1 monoclonal antibodies. Further, the kit comprises: the kit comprises a solid phase carrier coated with TMED2, CCDC72 and/or ERC1 monoclonal antibodies, enzyme-labeled secondary antibodies, enzyme substrates, protein standard products, negative control products, diluent, washing liquid, enzyme reaction stopping liquid and the like.
Further, the colloidal gold method for detecting the TMED2, CCDC72 and ERC1 proteins uses a colloidal gold test strip, and the antibodies adopt commercially available TMED2, CCDC72 and ERC1 monoclonal antibodies. Further, the colloidal gold test strip adopts a colloidal gold immunochromatography technique or a colloidal gold percolation method. Furthermore, anti-TMED 2, CCDC72 and ERC1 monoclonal antibodies are sprayed on the detection area (T) on the nitrocellulose membrane of the colloidal gold test strip, and immunoglobulin IgG is sprayed on the quality control area (C).
The invention aims to provide a fluorescent quantitative PCR kit for detecting postmenopausal osteoporosis, which is characterized in that the kit detects genes TMED2, CCDC72 and/or ERC1 and adopts a specific primer pair.
Furthermore, the PCR kit is suitable for all types of fluorescent quantitative gene amplification instruments on the market at present, has high sensitivity, quick and accurate quantification and good stability, and has good application prospect.
Further, the fluorescent quantitative PCR kit comprises the following components: a specific primer pair, an internal reference primer and a fluorescent quantitative PCR reaction solution.
The kit also comprises an RNA extraction reagent. Preference is given to
Reagent performs sample RNA extraction.
The compositions of the present invention are formulated according to methods that can be readily practiced by those of ordinary skill in the art using pharmaceutically acceptable carriers and/or excipients, and can be prepared in unit dosage form or in multi-volume containers. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oily or aqueous medium, or may be in the form of a extract, powder, granule, tablet or capsule, and may further include a dispersant or stabilizer.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
Drawings
FIG. 1 is a graph showing relative expression amounts of TMED2, CCDC72 and ERC1 genes in peripheral blood before and after administration of LIUWEIDIHUANG pill to postmenopausal osteoporosis patients
FIG. 2 is a graph of the relative expression levels of the TMED2, CCDC72 and ERC1 genes in postmenopausal osteoporotic peripheral blood and healthy human peripheral blood
Detailed Description
The present invention is further illustrated below with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention. Those of ordinary skill in the art will understand that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents. The following examples are examples of experimental methods not indicating specific conditions, and the detection is usually carried out according to conventional conditions or according to the conditions recommended by the manufacturers.
Example 1 high throughput sequencing and analysis
Respectively collecting 3 cases of peripheral blood samples before and after taking Liuwei Dihuang pills for postmenopausal osteoporosis patients and 3 cases of healthy people contrast peripheral blood samples, carrying out RNA extraction, carrying out agarose gel electrophoresis after the RNA extraction, and preliminarily judging whether the quality of the extracted RNA sample is qualified or not from electrophoresis results, wherein the RNA sample can be used for further transcriptome analysis. And further detecting the extraction condition of the RNA sample by a NanoDrop1000 spectrophotometer, wherein the sample for RNA-seq sequencing requires: OD260/OD280 was 1.8-2.2.
The sequencing platform is a HiSeq 2500 high-throughput sequencing platform of Illumina company, high-throughput transcriptome deep sequencing is carried out, and after sequencing, the quality of sequencing data is integrally evaluated by using Fast-QC software, wherein the quality comprises the mass value distribution of bases, the position distribution of mass values, GC content, PCR amplification content, the frequency of kmer and the like. And during differential gene expression analysis, performing differential screening by adopting an internationally recognized algorithm EBSeq according to the obtained FPKM value. Wherein, during screening, LOG2FC is greater than 1 or < -1, and FDR is less than 0.05. To better understand the function of differentially expressed genes, we performed Gene ontology and signaling pathway analysis on the differentially expressed genes, and performed functional annotation and protein interaction network analysis on the differentially expressed genes, and in view of the results of the above data analysis, we screened the differentially expressed genes TMED2, CCDC72, and ERC1 in conjunction with the literature. TMED2 and ERC1 were highly expressed in the osteoporosis group and were low expressed after the administration of the six-ingredient rehmannia pill, and CCDC72 was low expressed in the osteoporosis group and were high expressed after the administration of the six-ingredient rehmannia pill.
Example 2 peripheral blood TMED2, CCDC72 and ERC1 Gene expression before and after taking Liuwei Dihuang pills for postmenopausal osteoporosis patients
Materials and methods
1. Material
22 postmenopausal osteoporosis patients were collected with consent, peripheral blood was collected at the initial stage of their examination, and half a year after taking liuwei di huang pills, and the peripheral blood was collected again, grouped and numbered.
2. Method of producing a composite material
2.1 extraction of Total RNA from postmenopausal osteoporotic peripheral blood and healthy human peripheral blood
By using
Reagent carries out sample RNA extraction, and the experimental operation is carried out according to the product instruction.
RNA quality determination criteria: the OD260/OD280 value of the RNA sample is between 1.8 and 2.2; the total RNA electrophoresis pattern has clear 28S and 18S bands; the electrophoresis pattern after the water bath heat preservation for 1 hour at 70 ℃ has no obvious difference with the pattern before the water bath heat preservation.
2.2 Synthesis of cDNA by reverse transcription
By using
III Reverse transcription of cDNA by Reverse transcription of Transcriptase (Invitrogen, cat # 18080-044), the experimental procedures were performed according to the product instructions, and the specific procedures were as follows:
using a reverse transcription kit, cDNA was synthesized by reverse transcription of l. mu.g of total RNA with reverse transcription buffer. A25-mu-l reaction system is adopted, 1 mu g of total RNA is taken from each sample as template RNA, and the following components are respectively added into a PCR tube:
5 Xreverse transcription buffer 5U l, 10mmol/l dNTP 1.25U l, 0.1mmol/l DTT 2.5U l, 30U mmol/l OligodT 2U l, 200U/l MMLV 1.25U l, template RNA 1U g, adding sterile water to the total system 25U l. Incubate at 42 ℃ for 1 hour, 72 ℃ for 10 minutes, and briefly centrifuge. The cDNA was stored in a freezer at-20 ℃ for future use.
2.3Real-Time PCR
2.3.1 Instrument and analytical method
ABI 7500 type fluorescent quantitative PCR instrument, 2- △△ CT method for relative quantitative analysis of data, 2.3.2 primer design
The template sequences were NM-001321445.1 (TMED2), NM-001301248.1 (ERC1) and NM-001329417.1 (CCDC72) using online primer design software, and the primers were synthesized by Invitrogen. The specific primer sequences are as follows:
TMED2 primer:
5’-CCATCAGTGTCAGCATTAG-3’(SEQ ID NO.1)
5’-CCATCATCAGAGAACAAGAG-3’(SEQ ID NO.2)
ERC1 primer:
5’-GGCTATCTTCACCTACTCTTCAG-3’(SEQ ID NO.3)
5’-AATCAGGAGACAGTTGGTTACAT-3’(SEQ ID NO.4)
CCDC72 primers:
5’-CTTACAGTGGCTCATCATC-3’(SEQ ID NO.5)
5’-AGACTCTGGCAAGATTCT-3’(SEQ ID NO.6)
the operation process is as follows:
reaction system: 2 × mix 10 μ l; 0.5. mu.l of each of the upstream primer (10uM) and the downstream primer (10 uM); 2 mul of template; sterile distilled water was added to make up to 25. mu.l.
By Power
Green PCR Master Mix (Invitrogen, cat # 4367659) was amplified and the experimental protocol was performed according to the product instructions.
The amplification procedure was: 95 ℃ 10min, (95 ℃ 15sec, 58 ℃ 60 sec). times.35 cycles.
Sample RealTimePCR assay: after 10-fold dilution of cDNA of each sample, 2 μ l of cDNA was used as a template, and the target gene primer and the reference gene primer were used for amplification. At the same time, the dissolution curve analysis is carried out at 60-95 ℃. Second, experimental results
The inflection point of the real-time quantitative PCR amplification curve is clear, the overall parallelism of the amplification curve is good, the amplification efficiency of each reaction tube is similar, the limit is flat without raising, the slope of the exponential phase of the curve is larger, and the amplification efficiency is higher; the dissolution curves of the sample amplification products are all unimodal, which indicates that only one amplification product is specifically amplified; according to a relative quantitative formula of qRT-PCR, TMED2, CCDC72 and ERC1 genes are compared with the TMED2, CCDC72 and ERC1 expression conditions in peripheral blood before and after taking Liuwei Dihuang pills for postmenopausal osteoporosis patients, and the TMED2, CCDC72 and ERC1 genes after taking Liuwei Dihuang pills have different expression amounts than before taking, namely 0.61, 1.31 and 0.58 (shown in figure 1) before taking respectively, and are consistent with the trend of high-throughput sequencing results.
Example 3 expression of TMED2, CCDC72 and ERC1 genes in peripheral blood of postmenopausal osteoporosis patients and healthy persons
1. Material
Peripheral blood was collected from 32 postmenopausal osteoporosis patients and 29 healthy people, and was grouped and numbered.
2. Method of producing a composite material
The specific procedure is as in example 3.
3. Results
The real-time quantitative PCR results show (see fig. 2 in particular): the qRT-PCR amplification result is stable, wherein the expression levels of TMED2, CCDC72 and ERC1 in postmenopausal osteoporosis peripheral blood are obviously different from the expression levels in healthy human peripheral blood, and the former of 3 genes is about 1.22 times, 0.83 times and 1.39 times of the latter, and the result verifies the result of high-throughput sequencing.
The invention screens postmenopausal osteoporosis pathogenic related genes TMED2, CCDC72 and ERC1 by adopting high-throughput sequencing, and further analysis shows that the genes are related to the administration effect of the Liuwei Dihuang pill and have important significance in the aspect of clinical guidance of medication, and the inventor combines molecular cell biology experiments to verify that the TMED2, the CCDC72 and the ERC1 have good correlation with postmenopausal osteoporosis. The invention provides a new diagnosis target point and a new medicine target point for clinical diagnosis and treatment of postmenopausal osteoporosis, and has good clinical application prospect.
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agactctggc aagattct 18
Claims (4)
1. The application of the gene composition or the expression product thereof in preparing the gene detection preparation for the postmenopausal osteoporosis pill of six ingredients with rehmannia is characterized in that the gene composition comprises one or more of the following genes: TMED2, CCDC72, and ERC 1.
2. The use of claim 1, wherein the TMED2 gene or TMED2 protein is highly expressed, or the ERC1 gene or ERC1 protein is highly expressed, or the CCDC72 gene or CCDC72 protein is lowly expressed in the sample group to which the liuwei dihuang pill is applied.
3. The use according to claim 1, wherein the test agent is tested for expression of the TMED2, CCDC72 and ERC1 genes by one or more of the following methods: fluorescent quantitative PCR method, gene chip method, and high-throughput sequencing method.
4. The use according to claim 1, wherein the gene chip comprises probes hybridizing to the nucleic acid sequences of the TMED2, CCDC72 and/or ERC1 genes; the fluorescent quantitative PCR method adopts a specific primer pair which is selected from the following group: a primer pair consisting of SEQ ID NO.1 and SEQ ID NO. 2; or a primer pair consisting of SEQ ID NO.3 and SEQ ID NO. 4; a primer pair consisting of SEQ ID NO.5 and SEQ ID NO. 6.
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| CN201711468676.3A CN108085383B (en) | 2017-12-29 | 2017-12-29 | Gene detection composition and application thereof |
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| CN201711468676.3A CN108085383B (en) | 2017-12-29 | 2017-12-29 | Gene detection composition and application thereof |
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| CN108085383B true CN108085383B (en) | 2020-04-10 |
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Non-Patent Citations (5)
| Title |
|---|
| Bioinformatics Analysis Reveals the Altered Gene Expression of Patients with Postmenopausal Osteoporosis Using Liuweidihuang Pills Treatment.;Rui Gong等;《Biomed Res Int.》;20190127;1-11 * |
| GenBank NM_001301248.1;Held RG等;《GenBank》;20171113;1-6 * |
| GenBank NM_001321445.1;Nagae M等;《GenBank》;20171003;1-3 * |
| GenBank NM_001329417.1;Song Z等;《GenBank》;20171004;1-2 * |
| 六味地黄丸对绝经后骨质疏松症肾阴虚证JAK/STAT信号通路基因的影响;谢丽华等;《中国骨质疏松杂志》;20140720;741-746 * |
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