阅读说明：本技术 检测galnt4基因的产品在制备用于冠心病的诊断和/或预后的工具中的应用 (Application of product for detecting GALNT4 gene in preparation of tool for diagnosis and/or prognosis of coronary heart disease ) 是由 黄榕翀 叶智帅 徐鸣悦 李泽亚 于 2021-08-11 设计创作，主要内容包括：本发明公开了检测GALNT4基因的产品在制备用于冠心病的诊断和/或预后的工具中的应用。根据该应用,通过检测受试者中GALNT4的表达,可以判断受试者是否患有冠心病、或者判断受试者是否存在患有冠心病的风险、或判断受试者的预后是否良好,从而指导临床医师为受试者提供有效的预防手段或者治疗方案,有利于提供受试者生存率。(The invention discloses an application of a product for detecting GALNT4 gene in preparing a tool for diagnosing and/or prognosing coronary heart disease. According to the application, by detecting the expression of GALNT4 in the subject, whether the subject has coronary heart disease or not can be judged, whether the subject has the risk of having coronary heart disease or not can be judged, or whether the prognosis of the subject is good or not can be judged, so that a clinician is guided to provide an effective preventive means or treatment scheme for the subject, and the survival rate of the subject is favorably provided.)

1. Application of a product for detecting GALNT4 gene in preparing a tool for diagnosis and/or prognosis of coronary heart disease.

2. The use of claim 1, wherein the product for detecting GALNT4 gene comprises a reagent for detecting the expression level of GALNT4 gene;

the reagent includes a reagent capable of quantifying GALNT4 gene mRNA; and/or, the agent comprises an antibody or fragment thereof capable of quantifying GALNT4 protein.

3. The use of claim 2, wherein the reagents comprise primers that specifically amplify GALNT4 gene and/or probes that specifically recognize GALNT4 gene;

the primer for specifically amplifying the GALNT4 gene comprises the nucleotide sequence shown as SEQ ID NO: 1 and the nucleotide sequence of the forward primer shown as SEQ ID NO: 2, or a reverse primer nucleotide sequence shown in figure 2.

4. The use of claim 1, wherein the product for detecting GALNT4 gene is a kit, chip, strip, or high throughput sequencing platform.

5. The use of claim 1, wherein the sample for the detection of the GALNT4 gene is blood of a subject.

6. A tool for the diagnosis and/or prognosis of coronary heart disease, comprising a product which detects GALNT4 gene.

7. The kit of claim 6, wherein the product for detecting the GALNT4 gene comprises an agent for detecting the expression level of GALNT4 gene;

the reagent includes a reagent capable of quantifying GALNT4 gene mRNA; and/or, the agent comprises an antibody or fragment thereof capable of quantifying GALNT4 protein.

8. The tool of claim 7, wherein the reagents comprise primers that specifically amplify the GALNT4 gene and/or probes that specifically recognize the GALNT4 gene;

the primer for specifically amplifying the GALNT4 gene comprises the nucleotide sequence shown as SEQ ID NO: 1.

9. The tool of claim 7 or 8, wherein the tool is a kit, a chip, a strip, or a high throughput sequencing platform.

10. The kit of claim 6, wherein the sample for detection of the GALNT4 gene is blood of a subject.

Technical Field

The invention relates to the technical field of gene diagnosis and biological pharmacy, in particular to application of a polypeptide acetylgalactosamine aminotransferase 4(pp-GalNAc-T4, GALNT4) gene in a tool for diagnosing and prognosing coronary heart disease.

Background

Coronary atherosclerotic heart disease (coronary heart disease) is one of the main diseases threatening human health, and has the characteristics of high morbidity and high mortality. According to statistics, the number of cases of interventional therapy of the coronary heart disease in China in 2019 is estimated to exceed 110 thousands, and although a series of new technologies represented by percutaneous coronary artery interventional therapy technology are developed and popularized, the prevalence rate and the fatality rate of the coronary heart disease population in China are not reduced in the last years, so that the deep understanding of the etiology mechanism of the coronary heart disease has important significance for preventing and treating the coronary heart disease. Atherosclerosis is an important pathological change of coronary heart disease, and is basically represented by: lipid deposition of the intima of arteries, intimal fibrosis, formation of atheromatous plaque, hardening of the vessel wall, narrowing of the vessel lumen, and simultaneously, secondary intra-plaque hemorrhage, thrombosis and rupture are combined to cause ischemic change of the corresponding organs.

A large number of epidemiological studies prove that the coronary heart disease is a complex multi-factor combined-action disease, and besides the traditional risk factors of smoking, diabetes, hyperlipidemia, hypertension, obesity and age as the coronary heart disease, other non-traditional risk factors, genetic factors and environmental factors play important roles in the coronary heart disease. Coronary heart disease and other chronic diseases are complex polygenic diseases with polygenic combined action, and because the causes of coronary heart disease are various and the pathogenesis is not completely clarified, the occurrence of myocardial infarction cannot be accurately predicted and prevented until now, and a more effective detection means is needed.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides the application of a product for detecting GALNT4 gene in the preparation of a tool for the diagnosis and/or prognosis of coronary heart disease.

Specifically, the product for detecting the GALNT4 gene comprises a reagent for detecting the expression level of the GALNT4 gene.

The above-mentioned reagents include a reagent capable of quantifying mRNA of GALNT4 gene.

Such reagents include reagents capable of quantifying antibodies or fragments thereof to GALNT4 protein.

Specifically, the above-mentioned reagent capable of quantifying GALNT4 gene mRNA may comprise a primer specifically amplifying GALNT4 gene and/or a probe specifically recognizing GALNT4 gene.

In one embodiment of the present invention, the primer for specifically amplifying GALNT4 gene comprises the nucleotide sequence shown in SEQ ID NO: 1.

Specifically, the above-mentioned reagent capable of quantifying GALNT4 protein may comprise a substance (e.g., an antibody or a fragment thereof) capable of specifically binding to GALNT4 protein.

The above-mentioned product for detecting GALNT4 gene may be a kit, a chip, a strip or the like, which contains a reagent capable of quantifying GALNT4 gene mRNA (e.g., a primer specifically amplifying GALNT4 gene and/or a probe specifically recognizing GALNT4 gene) and/or a reagent capable of quantifying GALNT4 protein (e.g., a substance (e.g., an antibody) capable of specifically binding GALNT4 protein).

The above-described GALNT4 gene detection product may be a high-throughput sequencing platform that detects GALNT4 gene using a reagent capable of quantifying GALNT4 gene mRNA (e.g., a primer specifically amplifying GALNT4 gene and/or a probe specifically recognizing GALNT4 gene) and/or a reagent capable of quantifying GALNT4 protein (e.g., a substance (e.g., an antibody) capable of specifically binding GALNT4 protein).

The product for detecting GALNT4 gene of the present invention can exert its function based on a known method using a nucleic acid molecule: such as Polymerase Chain Reaction (PCR), Southern blot hybridization, Northern blot hybridization, dot hybridization, Fluorescence In Situ Hybridization (FISH), DNA microarray, high throughput sequencing platform, etc., in particular PCR methods, such as real-time fluorescence quantitative PCR methods.

The product for detecting the GALNT4 gene is a kit, which comprises a real-time fluorescent quantitative PCR reaction system, and specifically comprises: primers for specifically amplifying GALNT4 gene, SYBR Primix Ex Taq, nuclease-free double distilled water, ROX Reference Dye II and cDNA template; wherein, the primer comprises the nucleotide sequence shown as SEQ ID NO: 1 and the forward primer shown as SEQ ID NO: 2, or a reverse primer as shown in figure 2.

The real-time fluorescent quantitative PCR reaction conditions comprise: pre-denaturation at 94 ℃ for 30 seconds; 40 cycles: denaturation at 94 ℃ for 5 seconds, annealing at 60 ℃ for 30 seconds, and extension at 72 ℃ for 30 seconds; the dissolution curve and the amplification curve were 95 ℃ for 15 seconds, 60 ℃ for 1 minute, 95 ℃ for 15 seconds; GAPDH gene is used as an internal reference gene.

The product for detecting GALNT4 protein of the present invention can exert its function based on a known method using an antibody: for example, ELISA, radioimmunoassay, immunohistochemistry, Western blotting, etc. may be used.

Specifically, the coronary heart disease may be chronic Coronary Artery Disease (CAD) (including stable angina, ischemic cardiomyopathy, latent coronary heart disease, etc.), Acute Coronary Syndrome (ACS) (including unstable angina, non-ST elevation myocardial infarction, ST elevation myocardial infarction), and especially chronic coronary artery disease.

Specifically, the sample used for the detection of the above-mentioned GALNT4 gene may be, for example, a tissue sample or fluid obtained from a biopsy subject, for example, tissue, blood, plasma, serum, lymph, urine, serosal cavity fluid, spinal fluid, synovial fluid, aqueous humor, tears, saliva or the like or a fraction thereof or a processed material.

The sample is blood of a subject, particularly peripheral blood.

The invention also provides a tool for the diagnosis and/or prognosis of coronary heart disease, comprising a product for detecting the GALNT4 gene.

Specifically, the product for detecting the GALNT4 gene comprises a reagent for detecting the expression level of the GALNT4 gene.

The above-mentioned reagents include a reagent capable of quantifying mRNA of GALNT4 gene.

Such reagents include those capable of quantifying GALNT4 protein.

Specifically, the above-mentioned reagent capable of quantifying GALNT4 gene mRNA may comprise a primer specifically amplifying GALNT4 gene and/or a probe specifically recognizing GALNT4 gene.

In one embodiment of the present invention, the primer for specifically amplifying GALNT4 gene comprises the nucleotide sequence shown in SEQ ID NO: 1 and the nucleotide sequence shown as SEQ ID NO: 2.

Specifically, the above-mentioned reagent capable of quantifying GALNT4 protein may comprise a substance (e.g., an antibody or a fragment thereof) capable of specifically binding to GALNT4 protein.

The above-mentioned product for detecting GALNT4 gene may be a kit, a chip, a strip or the like, which contains a reagent capable of quantifying GALNT4 gene mRNA (e.g., a primer specifically amplifying GALNT4 gene and/or a probe specifically recognizing GALNT4 gene) and/or a reagent capable of quantifying GALNT4 protein (e.g., a substance (e.g., an antibody) capable of specifically binding GALNT4 protein).

The above-described GALNT4 gene detection product may be a high-throughput sequencing platform that detects GALNT4 gene using a reagent capable of quantifying GALNT4 gene mRNA (e.g., a primer specifically amplifying GALNT4 gene and/or a probe specifically recognizing GALNT4 gene) and/or a reagent capable of quantifying GALNT4 protein (e.g., a substance (e.g., an antibody) capable of specifically binding GALNT4 protein).

The product for detecting GALNT4 gene of the present invention can exert its function based on a known method using a nucleic acid molecule: such as Polymerase Chain Reaction (PCR), Southern blot hybridization, Northern blot hybridization, dot hybridization, Fluorescence In Situ Hybridization (FISH), DNA microarray, high throughput sequencing platform, etc., in particular PCR methods, such as real-time fluorescence quantitative PCR methods.

The product for detecting the GALNT4 gene is a kit, which comprises a real-time fluorescent quantitative PCR reaction system, and specifically comprises: primers for specifically amplifying GALNT4 gene, SYBR Primix Ex Taq, nuclease-free double distilled water, ROX Reference Dye II and cDNA template; wherein, the primer comprises the nucleotide sequence shown as SEQ ID NO: 1 and the forward primer shown as SEQ ID NO: 2, or a reverse primer as shown in figure 2.

In an embodiment of the present invention, the real-time fluorescent quantitative PCR reaction conditions include: pre-denaturation at 94 ℃ for 30 seconds; 40 cycles: denaturation at 94 ℃ for 5 seconds, annealing at 60 ℃ for 30 seconds, and extension at 72 ℃ for 30 seconds; the dissolution curve and the amplification curve were 95 ℃ for 15 seconds, 60 ℃ for 1 minute, 95 ℃ for 15 seconds; GAPDH gene is used as an internal reference gene.

The product for detecting GALNT4 protein of the present invention can exert its function based on a known method using an antibody: for example, ELISA, radioimmunoassay, immunohistochemistry, Western blotting, etc. may be used.

Specifically, the coronary heart disease may be chronic Coronary Artery Disease (CAD) (including stable angina, ischemic cardiomyopathy, latent coronary heart disease, etc.), Acute Coronary Syndrome (ACS) (including unstable angina, non-ST elevation myocardial infarction, ST elevation myocardial infarction), and especially chronic coronary artery disease.

Specifically, the sample used for the detection of the above-mentioned GALNT4 gene may be, for example, a tissue sample or fluid obtained from a biopsy subject, for example, tissue, blood, plasma, serum, lymph, urine, serosal cavity fluid, spinal fluid, synovial fluid, aqueous humor, tears, saliva or the like or a fraction thereof or a processed material.

In one embodiment of the present invention, the sample is blood of a subject, in particular peripheral blood.

Specifically, the tool may be a kit, a chip, or a strip, which includes the product for detecting GALNT4 gene, or a high throughput sequencing platform, which uses the product for detecting GALNT4 gene to detect GALNT4 gene.

The invention also provides a method for diagnosing coronary heart disease or prognosis of coronary heart disease, which comprises the step of detecting the expression level of GALNT4 gene in a subject.

In one embodiment of the present invention, the step of detecting the expression level of GALNT4 gene in the subject comprises a real-time fluorescent quantitative PCR reaction step.

In an embodiment of the present invention, the real-time fluorescent quantitative PCR reaction conditions include: pre-denaturation at 94 ℃ for 30 seconds; 40 cycles: denaturation at 94 ℃ for 5 seconds, annealing at 60 ℃ for 30 seconds, and extension at 72 ℃ for 30 seconds; the dissolution curve and the amplification curve were 95 ℃ for 15 seconds, 60 ℃ for 1 minute, 95 ℃ for 15 seconds; GAPDH gene is used as an internal reference gene.

Specifically, the method may include the steps of:

(1) obtaining a sample of a subject;

(2) detecting the level of GALNT4 gene expression in a sample from the subject;

(3) correlating the measured expression level of GALNT4 gene with the presence or absence of disease in the subject.

In particular, an elevated level of GALNT4 gene expression compared to a normal control, the subject may be diagnosed with or at high risk of developing coronary heart disease, or the subject may be determined to have a poor prognosis. The clinical needs to be comprehensively evaluated by the clinician in combination with other detection indexes of the subject, such as the risk, the severity and the prognosis of a specific disease.

Specifically, the coronary heart disease may be chronic Coronary Artery Disease (CAD) (including stable angina, ischemic cardiomyopathy, latent coronary heart disease, etc.), Acute Coronary Syndrome (ACS) (including unstable angina, non-ST elevation myocardial infarction, ST elevation myocardial infarction), and especially chronic coronary artery disease.

Specifically, the sample used for the detection of the above-mentioned GALNT4 gene may be, for example, a tissue sample or fluid obtained from a biopsy subject, for example, tissue, blood, plasma, serum, lymph, urine, serosal cavity fluid, spinal fluid, synovial fluid, aqueous humor, tears, saliva or the like or a fraction thereof or a processed material.

The sample is blood of a subject, particularly peripheral blood.

According to the application of the invention, whether the subject has coronary heart disease or not can be judged, whether the subject has risk of having coronary heart disease or not can be judged, or whether the prognosis of the subject is good or not can be judged by detecting the expression of GALNT4 in the subject, so that a clinician is guided to provide an effective prevention means or treatment scheme for the subject, and the survival rate of the subject is favorably provided.

Drawings

FIG. 1 shows the amplification curve of fluorescent quantitative PCR of GALNT4 gene.

FIG. 2 shows the melting curve of the PCR-specific primers for fluorescent quantitation of the GALNT4 gene.

FIG. 3 is a graph showing the comparison of the expression levels of GALNT4 gene mRNA in peripheral blood of coronary heart disease and control population.

Detailed Description

Unless defined otherwise, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

In the present invention, "expression level" refers to a measurable amount of the GALNT4 gene product in a sample, wherein the gene product may be a transcription product or a translation product. Thus, the expression level is related to a nucleic acid gene product (e.g., mRNA or cDNA) or a polypeptide gene product.

In the present invention, "primer" generally refers to a linear oligonucleotide that is complementary to and anneals to a target sequence. The lower limit on primer length is determined by the ability to hybridize, since very short primers (e.g., less than 5 nucleotides) do not form thermodynamically stable duplexes under most hybridization conditions. The primer length typically varies from 8 to 50 nucleotides. In certain embodiments, the primer is between about 15-25 nucleotides. In the present invention, the "forward primer" refers to an oligonucleotide that anneals to one specific strand of a target DNA, and the "reverse primer" refers to an oligonucleotide that anneals to the opposite strand of the target DNA. In summary, the forward and reverse primers are typically oriented on the target DNA sequence in a manner similar to the PCR primers such that their 3 'ends are closer to the target sequence than their 5' ends. Naturally occurring nucleotides (particularly guanine, adenine, cytosine, and thymine, hereinafter referred to as "G", "A", "C", and "T"), as well as nucleotide analogs, are useful as primers in the present invention.

In the present invention, a primer can be appropriately designed by referring to known information using a method known to those skilled in the art and prepared by chemical synthesis; the probe may be appropriately designed by referring to known information using a method known to those skilled in the art and prepared by chemical synthesis, or may be prepared by preparing a gene containing a desired nucleic acid sequence from a biological material and amplifying it using a primer designed to amplify the desired nucleic acid sequence.

In the present invention, the antibody or fragment thereof may be monoclonal or polyclonal. An antibody fragment refers to a portion of an antibody (partial fragment) or a peptide comprising a portion of an antibody that retains the binding activity of the antibody to an antigen, and may include F (ab')₂Fab', Fab, single chain fv (scfv), disulfide-bonded fv (dsfv) or polymers thereof, dimerized V regions (diabodies), or CDR-containing peptides. The product for detecting GALNT4 protein of the present invention may include an isolated nucleic acid encoding the amino acid sequence of an antibody or encoding a fragment of an antibody, a vector comprising the nucleic acid, and a cell carrying the vector. Antibodies can be prepared by methods well known to those skilled in the artObtained by the method. For example, mammalian cell expression vectors that retain all or part of the target protein or incorporate polynucleotides encoding them are prepared as antigens. After immunizing an animal with an antigen, immune cells are obtained from the immunized animal and myeloma cells are fused to obtain hybridomas. The antibody is then collected from the hybridoma culture. Finally, a monoclonal antibody against GALNT4 protein can be obtained by subjecting the obtained antibody to antigen-specific purification using GALNT4 protein or a part thereof used as an antigen. Polyclonal antibodies can be prepared as follows: an animal is immunized with the same antigen as above, a blood sample is collected from the immunized animal, serum is separated from the blood, and then antigen-specific purification is performed on the serum using the above antigen. The antibody fragment can be obtained by treating the obtained antibody with an enzyme or by using sequence information of the obtained antibody.

In the present invention, the "GALNT 4 gene" includes a polynucleotide of GALNT4 gene itself and any functional equivalent of GALNT4 gene, such as a DNA sequence having 70% or more (e.g., 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more) homology with the DNA sequence of GALNT4 gene in the current GeneBank of the international public nucleic acid sequence database, and encoding the same functional protein.

In the present invention, "diagnosing coronary heart disease" includes both determining whether a subject has suffered from coronary heart disease and determining whether a subject is at risk of suffering from coronary heart disease.

In the present invention, "prognosis" refers to a process or a result of a coronary heart disease patient after suppressing or alleviating the coronary heart disease by a surgical treatment or the like. In the present description, prognosis may be the inhibition or alleviation of the vital state at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 years or more after coronary heart disease by surgical treatment. Prognosis is predicted by examining biomarkers, i.e., GALNT4 gene or GALNT4 protein. The prognosis prediction can be performed by: determining whether the prognosis of the patient is good or poor, or determining the probability of a good or poor prognosis, based on the presence or absence, or increase or decrease, of the biomarker.

In the present invention, "good prognosis" means that the patient has no critical condition for a long period of time (e.g., 3, 5, 6, 7, 8, 9, 10, 15, 20 years or more) after the patient suppresses or alleviates the coronary heart disease by surgical treatment or the like. The most preferred state for good prognosis is long-term disease-free survival.

In the present invention, "poor prognosis" means that a patient develops a fatal condition within a short period of time (e.g., 1, 2, 3, 4, 5 years or less) after the suppression or alleviation of coronary heart disease by surgical treatment or the like.

Predicting prognosis refers to predicting the course or outcome of a patient's condition and does not mean that the course or outcome of the patient's condition can be predicted with 100% accuracy. Predictive prognosis refers to determining whether a certain process or result is more likely than not, and does not mean determining the likelihood of the certain process or result occurring by comparison with a case where the certain process or result does not occur. As used herein, a particular process or outcome is more likely to be observed in a patient having an elevated or reduced level of GALNT4 gene or GALNT4 protein of the invention than in a patient not displaying that characteristic.

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1: case collection

1. Selected group of people

Patients who were hospitalized and received coronary angiography in the Beijing friendship hospital affiliated with the university of capital medical science from 8 months to 2017 and 11 months in 2019 are admitted to 58 patients with coronary heart disease and 55 control groups who excluded the diagnosis of coronary heart disease in the same period.

2. Detection standard

The standard for diagnosing coronary heart disease is that the diameter of any section of the left main artery and 3 epicardial inferior coronary arteries (anterior descending branch, left circumflex branch and right coronary artery) and major branches thereof is narrowed to be more than or equal to 50%.

3. Inclusion criteria

(1) The age is 18-80 years (including 18 and 80 years);

(2) the LVEF is more than or equal to 45 percent proved by the ultrasonic of the chest and the heart;

(3) voluntarily receive all examinations required by the study protocol;

(4) subjects (or legal guardians) understand the study requirements and treatment procedures and sign written informed consent before performing the prescribed examinations or procedures of the protocol.

4. Exclusion criteria

(1) Patients with rheumatic heart disease, coronary arteritis, hypertrophic cardiomyopathy, and dilated cardiomyopathy;

(2) combined with malignant tumor or accompanied with cancer, immune system diseases, blood system diseases, people taking glucocorticoid medicine recently (within 2 weeks), people taking immunosuppressant, and cerebral infarction;

(3) acute and chronic infection, operation and trauma exist in the last 1 month;

(4) secondary hypertension, severe hepatic insufficiency (transaminase elevation more than 3 times the upper limit of normal), and severe renal function (eGFR)<30ml/(min·1.73m²))；

(5) Patients with thyroid dysfunction and allergy to iodine agents;

(6) refusing the blood-taking person;

(7) sign an informed consent or withdrawal from the study was denied.

Example 2: detection of GALNT4 gene expression content in peripheral blood of coronary heart disease and control group population

1. Collecting peripheral blood, extracting mRNA

(1) Reserving 5ml of fasting peripheral venous blood of each study object in the morning, and centrifugally collecting blood cells;

(2) adding Trizol reagent to grind the cell or tissue fully until the cell or tissue is pasty;

(3) adding 1/5 volumes of chloroform, shaking up and down violently and mixing uniformly for 15s, standing for 15min at room temperature, 12000g, centrifuging for 15min at 4 ℃;

(4) carefully taking out the EP tube from the centrifuge, and dividing the homogenate into 3 layers at the moment to obtain colorless supernatant which is RNA, a middle white egg layer and a lower organic phase with color; carefully suck the upper aqueous phase and transfer to another new EP tube, do not suck the white middle layer;

(5) adding isopropanol with the same volume into the supernatant, turning upside down, mixing, and standing on ice box for 10 min;

(6)12000g, centrifuging for 10min at 4 ℃, generating precipitate at the bottom of the test tube after centrifugation, discarding supernatant, and taking white precipitate;

(7) carefully discarding the supernatant, slowly adding 500 μ L of 75% ethanol prepared from RNA-Free water along the wall of the EP tube, and washing the wall of the EP tube by slightly inverting the solution;

(8)12000g, centrifuging for 5min at 4 ℃, carefully removing ethanol, and taking the precipitate;

(9) drying at room temperature for 5-10min, adding about 30 μ L RNA-Free water to dissolve to constant volume;

(10) the RNA concentration was measured using a nucleic acid instrument.

2. Reverse transcription PCR to obtain cDNA

(1) Reactions for removing genomic DNA

The reaction system and reaction conditions are shown in tables 1 and 2, respectively.

TABLE 1 reaction System

TABLE 2 reaction conditions

(2) Reverse transcription reaction to obtain cDNA

The reverse transcription reaction system and reaction conditions are shown in tables 3 and 4, respectively.

TABLE 3 reverse transcription System (20. mu.L)

TABLE 4 reverse transcription reaction conditions

The cDNA solubility was determined.

3. Real-time fluorescent quantitative PCR

The primer design, reaction system and RT-PCR reaction conditions involved in this part of the experiment are shown in tables 5-7, respectively.

TABLE 5 primer design

TABLE 6 reaction system (20. mu.L)

TABLE 7RT-PCR reaction conditions

Recording the Ct value of the sample, and using the obtained data as 2^-ΔΔCtThe method is used for analyzing and calculating, GAPDH is used as an internal reference, and the relative expression quantity of the target gene is presented in a fold form. The formula is multiple 2^-ΔΔCtWherein Δ Δ Ct ═ is (Ct value of gene of order experimental group-Ct value of reference gene in experimental group) - (Ct value of gene of order control group-Ct value of reference gene in control group).

4. Statistical analysis

Data analysis was performed using SPSS 21.0 statistical analysis software, the classification data are expressed in numbers and percentages, and the chi-square test was used for comparison between groups. The continuous data were expressed as mean ± Standard Error (SEM) following a normal distribution, and the means between the two groups were compared using the t-test. Continuous data, not normally distributed, are described as median (lower quartile-upper quartile), group comparisons are using the Kruskai-Wallis rank sum test, and bilateral P <0.05 is considered statistically different.

5. Comparison of clinical baseline data

Results of clinical baseline data for the enrolled patients are shown in table 8, which indicate: the level of low density lipoprotein (LDL-C) and hypersensitive C-reactive protein (hs-CRP) is statistically different between patients with Coronary Heart Disease (CHD) and control group (p is less than 0.05); the age and sex, and the risk factors of coronary heart disease such as hypertension, diabetes, blood fat, smoking, blood pressure and the like have no statistical difference.

Table 8 results of clinical baseline data for selected patients

6. GALNT4 gene real-time fluorescent quantitative PCR amplification product identification

The real-time fluorescence quantitative PCR detection result of peripheral blood RNA is shown in figure 1, wherein: the GALNT4 gene amplification curve is a significantly smooth "sigmoid". The dissolution curve is shown in FIG. 2, which shows a single dissolution peak, and the amplified product has high specificity.

7. Increase of peripheral blood GALNT4 gene expression content of coronary heart disease population

The delta Ct values of GALNT4 gene mRNA level relative expression were averaged over 3 replicates for a single sample, as compared between coronary heart disease and control groups. As shown in FIG. 3, the relative expression level of GALNT4 gene mRNA in peripheral blood of patients with coronary heart disease was significantly higher than that of the control group, and was 1.34 times higher than that of the control group, and the p-value was 0.0021, which was statistically different.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and the like that are within the spirit and principle of the present invention are included in the present invention.

The listing herein of method steps in a certain order does not constitute any limitation on the order of the method steps.