CN111718992A - Application of RTN3 or mRNA or gene coding RTN3 protein as coronary heart disease inflammation detection marker - Google Patents

Abstract

The invention relates to application of RTN3 protein as a coronary heart disease inflammatory response detection marker, belonging to the field of biological medicine. The invention provides a new application of RTN3 protein or mRNA coding RTN3 protein or gene coding RTN3 protein in serving as a coronary heart disease inflammation detection marker. The amino acid sequence of the RTN3 protein is shown as SEQ ID No. 1. The sequence of the mRNA for coding the RTN3 protein is shown as SEQ ID No. 2. The CDS sequence of the gene coding the RTN3 protein is shown as SEQ ID No. 3. A large number of experiments prove that the possibility of generating the inflammatory response of the coronary heart disease of an individual is higher when the expression level of RTN3 is increased, and vice versa. Therefore, the content of the RTN3 protein can directly reflect the development process of the inflammatory reaction of the coronary heart disease to a certain extent.

Description

Application of RTN3 or mRNA or gene coding RTN3 protein as coronary heart disease inflammation detection marker

Technical Field

The invention relates to application of RTN3 as a coronary heart disease inflammation detection marker, belonging to the field of biological medicine.

Background

Coronary atherosclerotic heart disease (coronary heart disease) is a multifactorial induced disease that is the leading cause of morbidity and mortality among various diseases. The main cause of coronary heart disease is the formation of lipid plaques due to the accumulation of large amounts of apolipoprotein B-containing lipoproteins and other substances, and the accumulation of these lipid plaques in blood vessels leads to a complex series of cellular events in the coronary artery wall and the occurrence of chronic vascular inflammation, which in turn drives the development and enlargement of lipid plaques to form a vicious circle. In the later stage of coronary heart disease development, the plaque gradually increases and causes the coronary artery lumen to narrow, and because the existence of the plaque is unstable, once the plaque breaks, the plaque can cause thrombus and cause a plurality of serious diseases, so the development is extremely dangerous.

Vascular inflammation is an important pathological reaction in the process of coronary heart disease. Inflammation is part of the complex biological response of human tissue to harmful stimuli (e.g., pathogens, damaged cells, or irritants), and is a protective response involving immune cells, blood vessels, and molecular mediators. Inflammation functions to eliminate the original cause of cellular injury, to remove necrotic cells and tissue damaged during the original injury and inflammation, and to initiate tissue repair. Thus, in general, the inflammatory response is beneficial to the body. In some cases, however, the occurrence of an inflammatory response is detrimental. Some favorable factors in the inflammatory reaction may also be harmful to the body under certain pathological conditions. Inflammatory responses occur without involvement of cytokines. Inflammatory cytokine is a cytokine secreted from immune cells and some other cells promoting inflammation, including interleukins, tumor necrosis factor, interferons, granulocyte-macrophage colony stimulating factor, and the like. Inflammatory cytokines play an important role in the initiation of inflammatory responses and in the regulation of host defense against pathogens that mediate innate immune responses, [ Li Zhi, Wangwei, Zhang Guang, etc. ] the expression and significance of serum inflammatory cytokines in patients with coronary heart disease [ J ]. J.China J.Oakuchi J.2018, 38(7): 1559. 1560.] [ Youefi-Ahma dioxide A, Ebrahimi-Barou S, Niknia S, et al. therapeutic effects of combination of planar and subcutaneous tissue analysis in TNBS-induced collagen ratio [ J ]. Biomedicine & Pharmacotherapy,2020,125:109949.] [ Wumin, Wang. application of proinflammatory factors and anti-inflammatory factors in research of coronary heart disease [ J.J.Pharmacologne & Pharmacology, 87-90, 2010-IL 1.] and TNF. alpha.6. TNF [ II, 2010-6, etc. ], and anti-inflammatory cytokines such as IL-10 and TGF-beta, etc., play an important role in a variety of chronic inflammatory reactions including coronary heart disease inflammation, and the increase and decrease of their content can be used to reflect the degree of inflammation. The C-reactive protein is an acute protein which rises sharply in plasma when an organism is infected or tissues are damaged, the content of the C-reactive protein can be used as a non-specific inflammatory reaction marker, and the C-reactive protein is directly involved in cardiovascular diseases such as inflammation and coronary heart disease, so the C-reactive protein is a strongest powerful predictive factor and risk factor of the cardiovascular diseases.

The RTN3(Reticulon 3) protein, also known as endoplasmic reticulum-associated protein 3, belongs to a family of Reticulon (RTN) proteins that have a Reticulon Homology Domain (RHD) at their C-terminus and are relatively conserved among species. Among them, the RTN family is divided into four members, RTN1-4, in humans and mice. These family members are primarily localized to the ER and play an important role in various physiological processes of the cell. Although current research on RTN3 focuses primarily on the processes involved in functioning of the nervous system and playing a role in the development of various neurodegenerative diseases, the function of RTN3 is not limited to this.

Disclosure of Invention

The invention aims to provide a new application of RTN3 protein or mRNA coding RTN3 protein or gene coding RTN3 protein in serving as a coronary heart disease inflammation detection marker.

The coronary heart disease inflammation refers to an inflammatory reaction occurring in the occurrence and development process of coronary atherosclerotic heart disease.

The novel application provided by the invention is to predict the degree of occurrence and/or development of an inflammatory response of an individual by detecting the change of the expression level of RTN3 in a sample.

The amino acid sequence of the RTN3 gene coding protein is shown in SEQ ID No. 1.

The cDNA sequence of the protein coded by the RTN3 gene can be obtained by reverse transcription of the mRNA sequence coded by the RTN3 protein and can be directly translated into the protein amino acid sequence of the RTN3, so that the cDNA sequence of the protein coded by the RTN3 gene can also be used as a marker for detecting the coronary heart disease inflammation.

The sequence of the mRNA for coding the RTN3 protein is shown as SEQ ID No. 2.

The Ensembl number of the gene for coding the RTN3 protein is ENSG00000133318, and the CDS sequence of the gene is shown as SEQ ID No. 3.

Preferably, the detection marker is a serum diagnostic marker, a plasma diagnostic marker or a tissue cell diagnostic marker.

The invention also provides an application of the RTN3 or mRNA encoding RTN3 protein or gene encoding RTN3 protein in preparing an inflammation detection reagent, wherein the amino acid sequence of the RTN3 is shown as SEQ ID No. 1; the mRNA sequence of the coding RTN3 protein is shown as SEQ ID No. 2; the CDS sequence of the gene coding the RTN3 protein is shown as SEQ ID No. 3.

The invention also provides a kit for detecting coronary heart disease inflammation, which comprises: a reagent for specifically detecting the expression level of RTN3 protein, a reagent for specifically detecting the mRNA level of the coding RTN3 protein or a reagent for specifically detecting the gene level of the coding RTN3 protein.

Preferably, the detecting comprises: colloidal gold immunochromatography, immunoblotting, immunohistochemistry, immunofluorescence, flow cytometry, enzyme linked immunosorbent assay, nucleic acid probe assay or qPCR assay.

Preferably, the detector is selected from: specific antibody against RTN3 protein, specific primer for amplifying mRNA or gene encoding RTN3 protein, probe or chip for detecting mRNA or gene encoding RTN3 protein.

For example, a primer specific to the RTN3 gene was designed to detect the RTN3 gene using polymerase chain reaction, and a primer specific to the RTN3 gene was designed to detect the mRNA of RTN3 using reverse transcription polymerase chain reaction.

Preferably, the sequence of the specific primer for amplifying the mRNA or gene encoding the RTN3 protein is shown as SEQ ID No.4 and SEQ ID No. 5.

Preferably, the specific primers for amplifying the mRNA or gene encoding the RTN3 protein further comprise specific primers for fluorescent quantitative PCR amplification of an internal reference gene GAPDH, and the sequences are shown as SEQ ID No.6 and SEQ ID No. 7.

The detection method of the RTN3 gene or mRNA can be realized by a conventional gene chip or a nucleic acid probe or a technical method such as PCR amplification or gene sequencing. For example [ Huang M, Wang J, Torre E, et al. SAVER: genexpression recovery for single-cell RNA sequencing [ J ]. Nature methods,2018,15(7): 539-) ], [ Brich S, Bozzi F, Perron F, et al. Fluorescences In Simple Hybridization (FISH) properties of minute and intellectual BAP1, CDKN2A, and NF2 gene deletions in property synthesis [ J ]. model Pathology,2019:1-11 ], [ Chen C Y, an R W, Ma T, et al. effects of coding of probability of natural genes [ J ], [ 12W ], Ma T, et al. experiments of coding of sample of family of the same documents [ 113, 2016 ], [ 2016, et al, expression of family J ], (2016, expression of natural genes, expression of genes, and so on.

Preferably, the test sample is human serum sample, whole blood sample and tissue cell sample, and cell line of human other sources.

The invention also provides application of the kit in preparation of a coronary heart disease inflammation detection reagent.

The expression level of RTN3 according to the present invention includes the expression or content level of any one of the following (1), (2) and (3):

(1) RTN3 protein;

(2) mRNA encoding the RTN3 protein;

(3) a gene encoding the RTN3 protein.

The specific method comprises the following steps: collecting a sample of the subject and comparing the expression level of the RTN3 protein or the mRNA or the gene in a normal control or standard sample. According to the comparison result, whether the coronary heart disease inflammatory reaction exists in the sample to be detected or not is detected/predicted according to the following method: if the expression level of the RTN3 protein or the mRNA or the gene in the sample to be tested is higher than the normal expression level of the RTN3 protein or the mRNA or the gene in the normal control or the standard sample, the more likely the sample to be tested is to generate the inflammatory reaction of the coronary heart disease; on the contrary, when the expression level of the RTN3 protein or the mRNA or the gene in the sample to be tested is lower than or equal to the normal expression level of the RTN3 protein or the mRNA or the gene in the normal control or the standard sample, the sample to be tested does not have coronary heart disease inflammatory reaction or the degree of coronary heart disease inflammatory reaction is possibly low; the control is a normal cell line of healthy human or human origin; preferably, the sample to be tested is serum or normal cell lysate. Wherein, the sample of the testee comprises but is not limited to a serum sample, a whole blood sample, a tissue cell sample and a related cell lysate of the patient.

According to previous researches, RTN3 is found to be involved in a plurality of processes of coronary atherosclerotic heart disease, plays an important role in inflammatory reaction and macrophage autophagy, and can be used as an important marker for development of inflammatory reaction of coronary heart disease. By detecting the RTN3 level of the patient, the possibility and/or the degree of the coronary heart disease inflammation of the patient can be detected/predicted.

A large number of experiments prove that the higher the expression level of RTN3, the higher the possibility of generating the inflammatory reaction of the coronary heart disease of an individual is, and vice versa. Therefore, the content of the RTN3 protein can directly reflect the generation and development process of the inflammatory reaction of the coronary heart disease to a certain extent.

Drawings

FIG. 1 is a graph showing the statistics of RTN3 expression in peripheral blood mononuclear cells of patients with coronary heart disease and normal persons measured by flow cytometry; (. indicates p < 0.05; data are expressed as (Mean ± SEM).

FIG. 2 shows the detection of RTN3 expression in peripheral blood mononuclear cells of patients with coronary heart disease and normal persons by immunoblotting.

FIG. 3 is a statistical graph of the correlation of the log of serum C-reactive protein levels in clinically confirmed coronary heart disease patients with expression of RTN3 in peripheral blood mononuclear cells; the area within the dashed line represents the 95% confidence region of the regression curve.

FIG. 4 is a statistical chart of the results of partial inflammatory factor expression detection in HEK293 cells transfected with plasmids containing RTN3 coding fragments and negative control blank plasmid vectors by performing fluorescent quantitative PCR; (. x.) represents p <0.01, and (. x.) represents p < 0.001.

Detailed Description

The present invention will be described in detail with reference to examples. The experimental procedures used in the following examples are conventional unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The amino acid sequence of the RTN3 protein involved in the following examples is shown as SEQ ID No.1 in the sequence table, and the coding gene sequence thereof is shown as SEQ ID No.3 in the sequence table.

Example 1 results of detecting expression of RTN3 protein in peripheral blood mononuclear cells of patients with coronary heart disease and normal persons

The test person: clinically confirmed coronary heart disease patients and control group normal human peripheral blood mononuclear cells

Through flow cytometry and immunoblotting, the expression difference of RTN3 protein in peripheral blood mononuclear cells of clinically confirmed coronary heart disease patients and control group normal persons is respectively detected, and relevant clinical test information of the patients is collected. The specific implementation steps are as follows:

(1) procedure for the preparation of the

First, mononuclear cells in human peripheral blood were extracted:

20mL of blood from a patient is taken and added to a 50mL centrifuge tube. Adding 120 μ L heparin sodium, and mixing to prevent coagulation. Then adding PBS buffer solution into the blood for 1:1 dilution;

preheating the human lymphocyte separation liquid in a 37 ℃ constant-temperature water bath box;

thirdly, subpackaging the preheated lymphocyte separation fluid into 4 centrifuge tubes with the volume of 5mL each, wherein the volume of each centrifuge tube is 15 mL;

(iv) slowly and slantingly adding the diluted blood sample to each small centrifuge tube containing the separation solution (taking care not to break the layering);

fifthly, centrifuging 4 small centrifuge tubes with the density gradient for 25min at 2000rpm (slowly increasing to 2000 rpm);

sixthly, after the centrifugation is finished, the separated human lymphocytes are cloudy between the first layer and the second layer in the centrifugal tube. Taking a total of 4mL of the layer up and down by using a pipette, collecting the layers together, and then centrifuging the layers at 2500rpm for 10 min;

the sediment at the bottom of the tube which is finally centrifuged is the needed cells.

Subsequently, a portion of the monocytes was examined by flow cytometry:

resuspending the cells in FACS buffer (1 × PBS/FCS/10% sodium azide) to adjust the cell concentration to 1-3 × 106/mL;

diluting the RTN3 antibody with 1% BSA at a ratio of 1:200 to label the mononuclear cells, gently shaking and uniformly mixing, and incubating at room temperature for 1 h;

③ after washing twice with PBS, labeling mononuclear cells (1% BSA 1:100) with fluorescent secondary antibody, incubating for 30min at 4 ℃ in the dark;

and fourthly, after two times of PBS washing, resuspending and centrifuging for 5min at 1500rpm, twice, and detecting by an up-flow cytometer (Beckman-CoulterXL-MCL). The data obtained by measuring 10000 cells were expressed as the mean fluorescence intensity value of the sample. The excitation light source is a 2W argon ion laser (the excitation wavelength is 488nm), and the emission wavelength is 570 nm. The assay data was analyzed using EXP032ADC Analysis software available from Beckmann-Coulter. The expression level of the cell surface protein was expressed as the average fluorescence intensity.

Another portion of the monocytes was used for immunoblot detection:

preparation of related reagents:

formulation of SDS-PAGE-15% separation gel (lower gel):

formulation of SDS-PAGE-5% concentrated gel (top gel):

c.10 × gel electrophoresis buffer:

heating at 56 deg.C to dissolve, storing at room temperature, and diluting at 1X when using.

D. Electrophoresis transfer buffer:

E. blocking buffer:

preparing SDS-PAGE according to the formula;

thirdly, putting the sample and the protein Marker into a boiling water bath kettle to be heated for 2min, then taking out the sample, adding 20 mu L of sample into each hole, and performing electrophoresis for 2h at a constant voltage of 100V;

transferring the protein to a PVDF membrane by an electrotransfer method, and performing constant current electrophoresis for 1h at 200mA in a refrigerator at 4 ℃;

taking out the PVDF membrane, placing 50mL of blocking buffer solution in a refrigerator at 4 ℃ and sealing overnight by shaking;

sixthly, slightly washing the membrane by using a washing buffer TBST (10mM Tris-HCl pH8.0, 150mM NaCl, 0.05% Tween-20), sealing the membrane in a thin-film bag by using 2mL of RTN3 primary anti-dilution solution, and putting the thin-film bag on a shaking table to incubate in a refrigerator at 4 ℃ overnight;

seventhly, washing the TBST for 3 times for 10min each time;

sealing 2mL of anti-RTN 3 antibody secondary antibody diluent marked by horseradish peroxidase on the membrane in a film bag, and incubating for 2h on a shaking table;

ninthly, washing for 10min by TBST for 3 times;

the membrane was stained with ECL luminol and the bands were recorded using light sensitive X-ray film.

As a result, it was found that: as shown in figure 1-2, the expression level of RTN3 protein of peripheral blood mononuclear cells of patients with coronary heart disease is significantly increased compared with that of normal people in a control group through flow cytometry and immunoblotting detection, which indicates that the increase of RTN3 is closely related to the occurrence and development of coronary heart disease. Meanwhile, the data in fig. 3 show that, in patients with coronary heart disease, the logarithm of the content of the inflammatory marker C-reactive protein has a significant positive correlation with the expression of the RTN3 protein of peripheral blood mononuclear cells, i.e., the expression level of the RTN3 of the patients is increased, and the degree of the inflammatory reaction of the coronary heart disease in the patients is also enhanced. Therefore, the expression level of RTN3 is proved to be a detection marker of coronary heart disease inflammation, namely RTN3 protein or mRNA coding RTN3 protein or gene coding RTN3 protein can be used as a detection marker of coronary heart disease inflammation.

Example 2 detection of partial inflammatory indicators cytokines after overexpression of RTN3 in human embryonic Kidney cells 293(HEK293)

The test person: human HEK293 cell

The total RNA of the cells is extracted and the expression of the cell factor mRNA is detected by a cell transfection technology. The specific implementation steps are as follows:

(1) procedure for the preparation of the

The cultured cells were first transfected with a plasmid containing the coding fragment of RTN3 or a control plasmid blank vector, respectively, to allow overexpression of RTN3 in the experimental cells:

a transfection step may be performed for cells cultured in a 6-well cell culture plate when the density thereof grows to 70-80%;

changing the culture medium for each hole, and only reserving 2mL of new complete culture medium without the double antibody for each hole;

③ for each well of cells, prepare a sterilized 1.5mL centrifuge tube, add 150. mu.L of OPTI-MEM medium and 12. mu.L of Lipofectamine 2000 reagent into the tube, and blow the mixture gently;

fourthly, preparing a sterilized 1.5mL centrifuge tube, adding 150 mu L of OPTI-MEM culture medium and 14ug of plasmid DNA into the tube, and gently and evenly blowing;

fifthly, adding the diluted DNA-OPTI culture medium into the diluted Lipofectamine-OPTI culture medium, and gently and uniformly blowing;

standing and incubating for 5min at room temperature;

seventhly, the mixed DNA liposome is respectively added into each cell hole, is mixed evenly and is placed back into a constant temperature incubator at 37 ℃ to be cultured for 2 days continuously, and then the next experiment can be carried out.

Total RNA of the cells was then extracted:

adding a corresponding amount of beta mercaptoethanol (10 mu L of beta mercaptoethanol is added to every 1000 mu L of lysate and is added before each use) into a lysate (lysine Buffer), adding a corresponding amount of lysate into a cell hole, and scraping cells by using an enzyme-free cell scraper;

transferring the scraped cell lysate to an enzyme-free 1.5mL centrifuge tube by using an enzyme-free suction head, and putting the centrifuge tube into a 4 ℃ rotary instrument for rotary lysis for half an hour;

thirdly, adding 70 percent of absolute ethyl alcohol with the same volume into the centrifuge tube, shaking and mixing uniformly;

transferring the well mixed liquid into a centrifugal column, and centrifuging for 12,000 Xg for 15s at room temperature by using a centrifuge;

fifthly, discarding the waste liquid in the collecting pipe, adding 700 mu L of Wash Buffer I into a centrifugal column, and centrifuging for 12,000 Xg for 15s at room temperature by using a centrifuge;

sixthly, discarding the collecting pipe and the waste liquid therein, and replacing with a new collecting pipe; adding 500 μ L WashBuffer II (added with corresponding amount of absolute ethanol in advance) into the centrifugal column, and centrifuging at room temperature for 12,000 Xg for 15s by using a centrifuge;

seventhly, repeating the step one time;

eighthly, discarding the waste liquid in the collecting pipe, centrifuging the waste liquid at room temperature for 12,000 Xg by an empty adsorption column in a centrifuge, and drying an adsorption column membrane for 1 min;

ninthly, abandoning the collecting tube, sleeving the centrifugal column into a new 1.5mL RNA-free enzyme centrifugal tube, adding 50 mu L RNA-free enzyme water into the adsorption column, standing for 1min at room temperature, and centrifuging for 12,000 Xg for 2min at room temperature by using a centrifuge;

the liquid in the centrifugal tube is stored at the positive ion (R) and is the whole blood leucocyte RNA solution of the sample, and the sample is stored for a long time at minus 80 ℃.

Taking the RNA sample obtained above for reverse transcription to obtain a cDNA library sample:

establishing a reverse transcription synthesis reaction system (20 mu L system): the reaction system is as follows:

establishing a reverse transcription synthesis reaction program: the procedure was as follows:

and (3) taking out the cDNA library sample obtained above, and carrying out real-time fluorescent quantitative PCR amplification and detection:

at least 3 times of reaction systems (25 mu L systems) are needed for each sample, wherein the reaction system is as follows:

secondly, placing the reaction tube into a qPCR instrument, and setting corresponding groups and reaction procedures, wherein the procedures are as follows:

③ use 2^(-△△CT)The method is used for comparing the difference of the mRNA expression quantity of the related inflammatory cytokine genes in the test sample and the control group.

As a result, it was found that: as shown in FIG. 4, after the RTN3 is over-expressed, compared with cells transfected with blank plasmid vectors in a control group, the expression of proinflammatory cytokines such as IL-1 beta, IL-6 and TNF-alpha is remarkably increased, and the expression of inflammation-inhibiting cytokines such as TGF-beta and IL-10 is remarkably reduced, wherein the inflammatory factors are closely related to coronary heart disease inflammation. The data in fig. 4 suggest that coronary heart disease inflammatory response is significantly elevated after RTN3 is elevated.

The foregoing examples are set forth to illustrate the present invention more clearly and are not to be construed as limiting the scope of the invention, which is defined in the appended claims to which the invention pertains, as modified in all equivalent forms, by those skilled in the art after reading the present invention.

SEQUENCE LISTING

<110> university of south-middle school

<120> use of RTN3 or mRNA coding for RTN3 protein or gene coding for RTN3 protein as coronary heart disease inflammation detection marker

Application in recording

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ggtctgtgca cactgataaa gaatcatccg aagacatttc agagactaat gacaagcttt 1020

ttccactgag aaataaagag gcaggacgtt acccaatgtc tgcattgctc agtaggcagt 1080

tttcacacac aaatgcagca ctggaagagg tgtccagatg cgtgaatgat atgcataact 1140

ttactaacga aatactgact tgggatctgg ttccccaagt gaaacaacag accgataaat 1200

cttctgactg catcacaaaa actacaggac ttgacatgag tgaatataat tcagaaattc 1260

cagttgtaaa tcttaaaact agcactcatc agaaaactcc tgtatgttct attgatggga 1320

gcactcccat cactaaatca acaggtgatt gggcagaagc atctctccag caagaaaatg 1380

ctattactgg aaaacctgta cctgactctt tgaattccac aaaagaattc agtatcaaag 1440

gtgtgcaagg caatatgcag aaacaggatg acacacttgc agaattacct ggatctccac 1500

ctgagaaatg tgactctttg ggttctggag tggccacagt gaaagtggtt ttacctgatg 1560

accacctgaa agatgaaatg gactggcaga gctctgcatt gggagaaatc acagaagctg 1620

atagttctgg tgagtctgat gacacagtaa tagaggacat cacagcagat acatcatttg 1680

aaaataacaa aattcaggct gaaaaacctg tttccattcc aagtgctgtt gtaaaaacag 1740

gtgaaagaga aatcaaagag attcccagtt gtgagagaga agaaaaaaca tctaaaaact 1800

ttgaagaatt ggtcagtgac tctgagctgc atcaagatca gcctgatatt cttggaagga 1860

gtccagctag tgaggcagca tgttcaaaag tacccgatac gaatgtctcc ttagaagatg 1920

tgagtgaagt tgctcctgaa aagcctatta ctactgagaa ccccaaactt ccttcaacag 1980

tgtctccaaa tgtttttaat gagacagaat tctcattaaa tgtgacaaca tctgcctatt 2040

tggagtcatt acatgggaaa aatgttaaac atatagatga ttcctcccca gaggacctga 2100

tagcagcctt tacagaaacc agagataaag gaatagtaga tagtgaaaga aatgctttta 2160

aagcaatatc agagaagatg acagacttta aaacaactcc tcctgtagaa gtcttacatg 2220

aaaatgagtc cggtggttct gaaattaaag acattggaag caaatacagt gaacaaagca 2280

aagaaacaaa tggaagtgag cctctaggtg ttttccctac ccaaggtact ccagtagcat 2340

ctcttgactt agaacaagaa cagctcacaa ttaaggctct taaagaatta ggtgaaagac 2400

aggttgagaa gtcaacttct gcacagcgtg acgcagaatt gccttctgaa gaagtactga 2460

agcaaacttt cacatttgct ccagaatctt ggccacagag atcatatgac atcctagaac 2520

gtaatgtcaa gaatggatct gatcttggga tttcccagaa gcccatcact atcagagaaa 2580

ctactagggt agatgctgtt tccagcctta gcaagactga attggtaaaa aagcatgtcc 2640

tagcaagact tctgacagac ttctcagtgc acgatctgat tttctggaga gatgtgaaga 2700

agactgggtt tgtctttggc accacgctga tcatgctgct ttccctggca gctttcagtg 2760

tcatcagtgt ggtttcttac ctcatcctgg ctcttctctc tgtcaccatc agcttcagga 2820

tctacaagtc cgtcatccaa gctgtacaga agtcagaaga aggccatcca ttcaaagcct 2880

acctggacgt agacattact ctgtcctcag aagctttcca taattacatg aatgctgcca 2940

tggtgcacat caacagggcc ctgaaactca ttattcgtct ctttctggta gaagatctgg3000

ttgactcctt gaagctggct gtcttcatgt ggctgatgac ctatgttggt gctgttttta 3060

acggaatcac ccttctaatt cttgctgaac tgctcatttt cagtgtcccg attgtctatg 3120

agaagtacaa gacccagatt gatcactatg ttggcatcgc ccgagatcag accaagtcaa 3180

ttgttgaaaa gatccaagca aaactccctg gaatcgccaa aaaaaaggca gaataagtac 3240

atggaaacca gaaatgcaac agttactaaa acaccattta atagttataa cgtcgttact 3300

tgtactatga aggaaaatac tcagtgtcag cttgagcctg cattccaagc ttttttttta 3360

atttggtgtt ttctcccatc ctttcccttt aaccctcagt atcaagcaca aaaattgatg 3420

gactgataaa agaactatct tagaactcag aagaagaaag aatcaaattc ataggataag 3480

tcaatacctt aatggtggta gagcctttac ctgtagcttg aaaggggaaa gattggaggt 3540

aagagagaaa atgaaagaac acctctgggt ccttctgtcc agttttcagc actagtctta 3600

ctcagctatc cattatagtt ttgcccttaa gaagtcatga ttaacttatg aaaaaattat 3660

ttggggacag gagtgtgata ccttccttgg tttttttttg cagccctcaa atcctatctt 3720

cctgccccac aatgtgagca gctacccctg atactccttt tctttaatga tttaactatc 3780

aacttgataa ataacttata ggtgatagtg ataattcctg attccaagaa tgccatctga 3840

taaaaaagaa tagaaatgga aagtgggact gagagggagt cagcaggcat gctgcggtgg 3900

cggtcactcc ctctgccact atccccaggg aaggaaaggc tccgccattt gggaaagtgg 3960

tttctacgtc actggacacc ggttctgagc attagtttga gaactcgttc ccgaatgtgc 4020

tttcctccct ctcccctgcc cacctcaagt ttaataaata aggttgtact tttcttacta 4080

taaaataaat gtctgtaact gctgtgcact gctgtaaact tgttagagaa aaaaataacc 4140

tgcatgtggg ctcctcagtt attgagtttt tgtgatccta tctcagtctg ggggggaaca 4200

ttctcaagag gtgaaataca gaaagccttt ttttcttgat cttttcccga gattcaaatc 4260

tccgattccc atttgggggc aagttttttt cttcaccttc aatatgagaa ttcagcgaac 4320

ttgaaagaaa aatcatctgt gagttccttc aggttctcac tcatagtcat gatccttcag 4380

agggaatatg cactggcgag tttaaagtaa gggctatgat atttgatggt cccaaagtac 4440

ggcagctgca aaaagtagtg gaaggaaatt gtctacgtgt cttggaaaaa ttagttagga 4500

atttggatgg gtaaaaggta cccttgcctt actccatctt attttcttag ccccctttga 4560

gtgttttaac tggtttcatg tcctagtagg aagtgcattc tccatcctca tcctctgccc 4620

tcccaggaag tcagtgattg tctttttggg cttcccctcc aaaggacctt ctgcagtgga 4680

agtgccacat ccagttcttt tcttttgttg ctgctgtgtt tagataattg aagagatctt 4740

tgtgccacac aggatttttt ttttttttta agaaaaacct atagatgaaa aattactaat 4800

gaaactgtgt gtacgtgtct gtgcgtgcaa cataaaaata cagtagcacc taaggagctt 4860

gaatcttggt tcctgtaaaa tttcaaattg atgtggtatt aataaaaaaa aaaaaaa 4917

<210>3

<211>3099

<212>DNA

<213> Artificial Synthesis

<400>3

atggcggagc cgtcggcggc cactcagtcc cattccatct cctcgtcgtc cttcggagcc 60

gagccgtccg cgcccggcgg cggcgggagc ccaggagcct gccccgccct ggggacgaag 120

agctgcagct cctcctgtgc ggattccttt gtttcttcct cttcctctca gcctgtatct 180

ctattttcga cctcacaaga gggattgagc tctctttgct ctgatgagcc atcttcagaa 240

attatgactt cttcctttct ttcatcttct gaaatacata acactggcct tacaatacta 300

catggagaaa aaagccatgt gttagggagc cagcctattt tagccaaaga aggaaaagac 360

cacttggatc ttctagatat gaaaaagatg gaaaagcctc aggggaccag caacaacgta 420

tcagactctt cagtttctct tgcagcagga gttcattgtg accgtccttc tattccagcc 480

agtttcccag agcatcctgc ttttctctca aagaaaattg gtcaagtgga agagcaaata 540

gataaagaga ccaagaaccc aaatggggta tcaagtaggg aggctaaaac tgcattggat 600

gctgatgaca gattcacttt gctgacagcc cagaaaccac ctactgagta ctctaaggta 660

gaaggcattt atacatattc tttgtctcca tccaaagttt caggagatga tgttattgaa 720

aaggattccc ctgaatcacc atttgaagta attattgaca aagcagcatt tgacaaagaa 780

tttaaagact catataagga gagcacagat gattttggta gctggtctgt gcacactgat 840

aaagaatcat ccgaagacat ttcagagact aatgacaagc tttttccact gagaaataaa 900

gaggcaggac gttacccaat gtctgcattg ctcagtaggc agttttcaca cacaaatgca 960

gcactggaag aggtgtccag atgcgtgaat gatatgcata actttactaa cgaaatactg 1020

acttgggatc tggttcccca agtgaaacaa cagaccgata aatcttctga ctgcatcaca 1080

aaaactacag gacttgacat gagtgaatat aattcagaaa ttccagttgt aaatcttaaa 1140

actagcactc atcagaaaac tcctgtatgt tctattgatg ggagcactcc catcactaaa 1200

tcaacaggtg attgggcaga agcatctctc cagcaagaaa atgctattac tggaaaacct 1260

gtacctgact ctttgaattc cacaaaagaa ttcagtatca aaggtgtgca aggcaatatg 1320

cagaaacagg atgacacact tgcagaatta cctggatctc cacctgagaa atgtgactct 1380

ttgggttctg gagtggccac agtgaaagtg gttttacctg atgaccacct gaaagatgaa 1440

atggactggc agagctctgc attgggagaa atcacagaag ctgatagttc tggtgagtct 1500

gatgacacag taatagagga catcacagca gatacatcat ttgaaaataa caaaattcag 1560

gctgaaaaac ctgtttccat tccaagtgct gttgtaaaaa caggtgaaag agaaatcaaa 1620

gagattccca gttgtgagag agaagaaaaa acatctaaaa actttgaaga attggtcagt 1680

gactctgagc tgcatcaaga tcagcctgat attcttggaa ggagtccagc tagtgaggca 1740

gcatgttcaa aagtacccga tacgaatgtc tccttagaag atgtgagtga agttgctcct 1800

gaaaagccta ttactactga gaaccccaaa cttccttcaa cagtgtctcc aaatgttttt 1860

aatgagacag aattctcatt aaatgtgaca acatctgcct atttggagtc attacatggg 1920

aaaaatgtta aacatataga tgattcctcc ccagaggacc tgatagcagc ctttacagaa 1980

accagagata aaggaatagt agatagtgaa agaaatgctt ttaaagcaat atcagagaag 2040

atgacagact ttaaaacaac tcctcctgta gaagtcttac atgaaaatga gtccggtggt 2100

tctgaaatta aagacattgg aagcaaatac agtgaacaaa gcaaagaaac aaatggaagt 2160

gagcctctag gtgttttccc tacccaaggt actccagtag catctcttga cttagaacaa 2220

gaacagctca caattaaggc tcttaaagaa ttaggtgaaa gacaggttga gaagtcaact 2280

tctgcacagc gtgacgcaga attgccttct gaagaagtac tgaagcaaac tttcacattt 2340

gctccagaat cttggccaca gagatcatat gacatcctag aacgtaatgt caagaatgga 2400

tctgatcttg ggatttccca gaagcccatc actatcagag aaactactag ggtagatgct 2460

gtttccagcc ttagcaagac tgaattggta aaaaagcatg tcctagcaag acttctgaca 2520

gacttctcag tgcacgatct gattttctgg agagatgtga agaagactgg gtttgtcttt 2580

ggcaccacgc tgatcatgct gctttccctg gcagctttca gtgtcatcag tgtggtttct 2640

tacctcatcc tggctcttct ctctgtcacc atcagcttca ggatctacaa gtccgtcatc 2700

caagctgtac agaagtcaga agaaggccat ccattcaaag cctacctgga cgtagacatt 2760

actctgtcct cagaagcttt ccataattac atgaatgctg ccatggtgca catcaacagg 2820

gccctgaaac tcattattcg tctctttctg gtagaagatc tggttgactc cttgaagctg 2880

gctgtcttca tgtggctgat gacctatgtt ggtgctgttt ttaacggaat cacccttcta 2940

attcttgctg aactgctcat tttcagtgtc ccgattgtct atgagaagta caagacccag 3000

attgatcact atgttggcat cgcccgagat cagaccaagt caattgttga aaagatccaa 3060

gcaaaactcc ctggaatcgc caaaaaaaag gcagaataa 3099

<210>4

<211>20

<212>DNA

<213> Artificial Synthesis

<400>4

tggttgactc cttgaagctg 20

<210>5

<211>22

<212>DNA

<213> Artificial Synthesis

<400>5

gacaatcggg acactgaaaa tg 22

<210>6

<211>19

<212>DNA

<213> Artificial Synthesis

<400>6

acatcgctca gacaccatg 19

<210>7

<211>22

<212>DNA

<213> Artificial Synthesis

<400>7

tgtagttgag gtcaatgaag gg 22

Claims (10)

1. The application of RTN3 or mRNA or gene coding RTN3 protein in serving as a marker for detecting coronary heart disease inflammation is characterized in that the amino acid sequence of RTN3 is shown as SEQ ID No. 1; the mRNA sequence of the coding RTN3 protein is shown as SEQ ID No. 2; the CDS sequence of the gene coding the RTN3 protein is shown as SEQ ID No. 3.
2. 2. The use of claim 1, wherein the coronary heart disease inflammation is an inflammatory response that occurs during the development of coronary atherosclerotic heart disease.
3. 3. The use of claim 1, wherein said detectable marker is a serum diagnostic marker, a plasma diagnostic marker or a tissue cell diagnostic marker.
4. The application of RTN3 or mRNA or gene coding RTN3 protein in preparing a coronary heart disease inflammation detection reagent is characterized in that the amino acid sequence of the RTN3 is shown as SEQ ID No. 1; the mRNA sequence of the coding RTN3 protein is shown as SEQ ID No. 2; the CDS sequence of the gene coding the RTN3 protein is shown as SEQ ID No. 3.
5. 5. A kit for detecting coronary heart disease inflammation, the kit comprising: a reagent for specifically detecting the expression level of RTN3 protein, a reagent for specifically detecting the mRNA level of the coding RTN3 protein or a reagent for specifically detecting the gene level of the coding RTN3 protein.
6. 6. The kit of claim 5, wherein the detecting comprises: colloidal gold immunochromatography, immunoblotting, immunohistochemistry, immunofluorescence, flow cytometry, enzyme linked immunosorbent assay, nucleic acid probe assay or qPCR assay.
7. 7. The kit of claim 5, wherein the test agent is selected from the group consisting of: specific antibody against RTN3 protein, specific primer for amplifying mRNA or gene encoding RTN3 protein, probe or chip for detecting mRNA or gene encoding RTN3 protein.
8. 8. The kit according to claim 7, characterized in that the sequence of the specific primers for amplifying the mRNA or gene encoding the RTN3 protein is shown as SEQ ID No.4 and SEQ ID No. 5.
9. 9. The kit according to claim 7, wherein the specific primers for amplifying the mRNA or gene encoding the RTN3 protein further comprise specific primers for fluorescent quantitative PCR amplification of the reference gene GAPDH, and the sequences are shown as SEQ ID No.6 and SEQ ID No. 7.
10. 10. Use of the kit according to any one of claims 5 to 9 for the preparation of a reagent for the detection of inflammation in coronary heart disease.

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