CN108004313B - Early-onset coronary heart disease causative gene and its in vitro detection reagent, preparation or kit and application - Google Patents
Early-onset coronary heart disease causative gene and its in vitro detection reagent, preparation or kit and application Download PDFInfo
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Abstract
本发明涉及生物工程中早发冠心病相关基因相关技术领域，是一种早发冠心病致病基因及其体外检测的试剂、制剂或试剂盒和应用，该早发冠心病致病基因，即即c.987C/Del位点突变的Cyp17a基因，c.987C/Del位点突变的Cyp17a基因的核苷酸序列如序列表SEQ ID NO:1所示。本发明公开了早发冠心病相关基因以及其体外检测的试剂、制剂或试剂盒和应用，该体外检测早发冠心病相关基因的制剂或试剂盒除了可以用于体外检测冠心病相关基因的多态性外，还可以用于检测、预防、诊断或治疗早发冠心病，从而降低冠心病的发生率，对预防和治疗冠心病具有重大的意义。
The invention relates to the related technical field of early-onset coronary heart disease-related genes in bioengineering, and relates to a premature-onset coronary heart disease pathogenic gene and a reagent, preparation or kit and application for its in vitro detection. That is, the Cyp17a gene mutated at the c.987C/Del site, and the nucleotide sequence of the Cyp17a gene mutated at the c.987C/Del site is shown in SEQ ID NO: 1 in the sequence table. The invention discloses early-onset coronary heart disease-related genes and reagents, preparations or kits for in vitro detection thereof, and applications thereof. In addition to the state of coronary heart disease, it can also be used to detect, prevent, diagnose or treat premature coronary heart disease, thereby reducing the incidence of coronary heart disease, which is of great significance for the prevention and treatment of coronary heart disease.
Description
Technical Field
The invention relates to the technical field related to early-onset coronary heart disease related genes in bioengineering, in particular to an early-onset coronary heart disease pathogenic gene and an in-vitro detection reagent, a preparation or a kit thereof.
Background
Research currently shows that coronary atherosclerotic heart disease (coronary heart disease) is a polygenic disease whose genetic basis is not one pair of alleles, but multiple pairs of alleles. The interaction of these alleles contributes to individual differences in susceptibility to coronary heart disease. However, in the case of familial early-onset coronary heart disease (male <55 years old, female <65 years old), scholars consider it likely to be a monogenic genetic disorder. For example, Arya Mani1 et al found that LRP6 gene mutation is a pathogenic gene of early-onset coronary heart disease in a family of early-onset coronary heart disease, and shows autosomal dominant inheritance rule. The gene mutation was not detected in 400 other patients with coronary heart disease (the results of the studies were published in the journal of Science). In 2014, Keramati AR et al found that DYRK1B gene mutation is a pathogenic gene of familial metabolic syndrome and early-onset coronary heart disease, and missense mutation of the gene (R102C) causes abnormal gene expression and corresponding protein function of diseased individuals in families of the metabolic syndrome and the early-onset coronary heart disease, thereby causing the occurrence of the metabolic syndrome and increasing the susceptibility of the early-onset coronary heart disease, and presenting an autosomal dominant inheritance law (research results are published on New England Journal of Medicine). Our earlier studies also found that the gene RECQL5 is a pathogenesis-related gene of early coronary heart disease, and a new frame shift mutation of the gene can be detected in all affected individuals, but the mutation site is not found in unaffected individuals. Therefore, the linkage analysis based on the family is more helpful to discover new pathogenic genes of coronary heart disease.
The gene is located at 10q24.3, has 8 exons and 7 introns, and is mainly expressed in adrenal gland and gonad. The CYP17A gene encodes a P450c17 protein, which belongs to one of the cytochrome P450 superfamily of enzymes, catalyzes many reactions including drug metabolism and synthesis of cholesterol, steroids and other lipids, contains both 17 α -hydroxylase and 17, 20-carbon chain lyase enzyme activities, and is one of the key enzymes in the synthesis of adrenal and gonadal steroid hormones, including sex hormones.
Disclosure of Invention
The invention provides an early coronary heart disease pathogenic gene, a reagent, a preparation or a kit for in vitro detection of the early coronary heart disease pathogenic gene and application of the early coronary heart disease pathogenic gene.
One of the technical schemes of the invention is realized by the following measures: a coronary heart disease early-onset pathogenic gene, namely c.987C/Del site mutated Cyp17a gene, the nucleotide sequence of c.987C/Del site mutated Cyp17a gene is shown in sequence table SEQ ID NO: 1.
The second technical scheme of the invention is realized by the following measures: an in vitro detection technical scheme of the reagent for detecting the mutation of c.987C/Del locus of Cyp17a gene, which comprises the following primers:
an upstream primer: 5 'taggggacat ctttggggct g 3';
a downstream primer: 5 'tcactgatag ttggtgtgcg gc 3'.
The following is further optimization or/and improvement of the second technical scheme of the invention:
the reagent is used for combining polymerase chain reaction with restriction fragment length polymorphism analysis or combining polymerase chain reaction with direct sequencing.
The third technical scheme of the invention is realized by the following measures: the preparation or the kit containing the reagent for in vitro detection of the early coronary heart disease pathogenic gene is characterized by comprising PCR amplification enzyme and corresponding buffer solution.
The fourth technical scheme of the invention is realized by the following measures: an application of reagent for in vitro detecting pathogenic gene of early coronary heart disease in the preparation of preparation or reagent kit for in vitro detecting pathogenic gene of early coronary heart disease.
The following is further optimization or/and improvement of the fourth technical scheme of the invention:
the preparation or the kit comprises PCR amplification enzyme and corresponding buffer solution.
The invention discloses an early-onset coronary heart disease related gene, a reagent, a preparation or a kit for in vitro detection of the early-onset coronary heart disease related gene and application of the early-onset coronary heart disease related gene, wherein the preparation or the kit for in vitro detection of the early-onset coronary heart disease related gene can be used for detecting, preventing, diagnosing or treating the early-onset coronary heart disease besides polymorphism of the coronary heart disease related gene, thereby reducing incidence of the coronary heart disease and having great significance for preventing and treating the coronary heart disease.
Drawings
FIG. 1 is a sequencing map of the c.987C/Del site of the present invention, which shows that the genotype of the c.987C/Del site of the subject to be tested is GC/GC homozygote.
FIG. 2 is a sequencing map of the c.987C/Del site of the present invention showing that the c.987C/Del site of the subject is a GC/G-hybrid.
Detailed Description
In order that the invention may be more clearly understood, the invention will now be further described with reference to the following examples and the accompanying drawings, which are given by way of illustration only and are not to be construed as limiting in any way. Unless otherwise specified,% in the present invention are mass percentages; unless otherwise stated, the preparation process is carried out under normal temperature and normal pressure; reagents, methods and apparatus employed in the present invention are conventional in the art unless otherwise indicated; unless otherwise specified, the test conditions employed in the present invention are those conventional in the art; unless otherwise specified, all reagents used in the present invention are commercially available; unless otherwise specified, water in the present invention is deionized water; unless otherwise specified, the solutions in the present invention are aqueous solutions in which the solvent is water, for example, if not specified, the hydrochloric acid solution is an aqueous hydrochloric acid solution. The experimental procedures, for which specific conditions are not indicated in the examples, are generally carried out according to conventional conditions: molecular cloning as described in Sambrook et al: the conditions described in the handbook of experiments (New fork: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations.
Example 1, a nucleotide sequence of a c.987C/Del site mutated Cyp17a gene, a c.987C/Del site mutated Cyp17a gene, which is related to early onset coronary heart disease is shown in a sequence table SEQ ID NO: 1.
The test sample containing the gene for Cyp17a can be obtained from cells from an experimenter, such as cells from blood, urine, saliva, gastric juice, hair, biopsy, and autopsy material, preferably from blood.
Example 2, a reagent for in vitro detection of a pathogenic gene of early-onset coronary heart disease, which is used for detecting mutation at the c.987C/Del site of the Cyp17a gene, comprising the following primers:
an upstream primer (shown as SEQ ID No: 2 of a sequence table): 5 'taggggacat ctttggggct g 3';
the downstream primer (shown as SEQ ID No: 3 of the sequence table): 5 'tcactgatag ttggtgtgcg gc 3'.
As known to those of ordinary skill in the art, the mutation site described in the present invention is a Single Nucleotide Polymorphism (SNP) site, i.e., a single nucleotide change occurs in a genomic sequence; the difference of the nucleotide sequence can be expressed on the DNA level or the RNA level, so that the early-onset coronary heart disease related gene can be expressed on the DNA level, the RNA level, preferably on the DNA level, and more preferably on the genomic DNA.
Since early-onset coronary heart disease is a monogenic disease, a family linkage analysis method is often used. Therefore, the invention adopts family linkage analysis research. The family linkage analysis is to adopt the collected typical early coronary heart disease family to summarize and detect the mutation site of the gene.
After a large number of experiments in a family of early-onset coronary heart disease, the invention finally proves that the gene Cyp17a with the c.987C/Del site of the invention is the pathogenic gene of the early-onset coronary heart disease, and individuals carrying the mutant allele suffer from the coronary heart disease at an earlier age.
The reagent for in vitro detection of the early-onset coronary heart disease pathogenic gene described in the embodiment 2 can be used for in vitro detection of the Cyp17a gene with the c.987C/Del locus of the invention.
Example 3, as the above example 2 preferred, the reagent is used in polymerase chain reaction and direct sequencing method combined reagent.
Example 4, a formulation or kit containing a reagent for in vitro detection of early-onset coronary heart disease-associated genes, the formulation or kit comprising a PCR amplification enzyme and a buffer therefor.
The preparation or the kit for detecting the early-onset coronary heart disease related gene of the c.987C/Del locus can be used for detecting the polymorphism of the early-onset coronary heart disease related gene in vitro; c.987C/Del site mutated coronary heart disease related gene kit can be used for detecting, preventing, diagnosing or treating coronary heart disease; the method for detecting early-onset coronary heart disease related genes in vitro can be used for detecting, preventing, diagnosing or treating coronary heart disease.
Example 5, use of a reagent for in vitro detection of a causative gene of early coronary heart disease in the preparation of a formulation or kit for in vitro detection of a causative gene of early coronary heart disease.
The preparation or the kit for detecting the early-onset coronary heart disease related gene of the c.987C/Del site mutation can be used for detecting the mutation of the coronary heart disease related gene in vitro; c.987C/Del site mutated early-onset coronary heart disease related gene preparation or kit can be used for detecting, preventing, diagnosing or treating early-onset coronary heart disease; the method for detecting early-onset coronary heart disease related genes in vitro can be used for detecting, preventing, diagnosing or treating early-onset coronary heart disease.
Example 6, as the preference of the above example 5, the kit further comprises a PCR amplification enzyme and a buffer solution therefor.
Example 7 subjects selected three generation members of 1 family of early onset coronary heart disease, 11 of which all suffered from coronary heart disease before the age of 50. The control was an unaffected individual in the family, for a total of 24 cases.
The mutation of the c.987C/Del site of the early-onset coronary heart disease related gene is detected by a polymerase chain reaction-direct sequencing method.
The method comprises the following steps: PCR reaction (50. mu.l): 50ng of genome template DNA (from a blood sample, DNA in leucocyte is extracted by a phenol-atmosphere method or a salting-out method according to a conventional method), 25ul of 2 × powder Taq PCR master mix, 21ul of deionized water, 1ul of each of an upper primer and a lower primer, and PCR reaction is carried out on a 96-hole PCR automatic cycler, and PCR cycle parameters are as follows: pre-denaturation at 96 ℃ for 5 min; at 94 ℃ for 30 seconds, at 61.0 ℃ for 30 seconds and at 72 ℃ for 1 minute; extension at 72 ℃ for 10 min after 35 weeks of cycling. The primers are as follows:
an upstream primer: 5 'taggggacat ctttggggct g 3';
a downstream primer: 5 'tcactgatag ttggtgtgcg gc 3'.
The amplification product was 157 bases in length and the amplification was sequenced directly on a sequencer. The sequencing conditions were found in the manufacturer's operating manual.
And (3) sequencing results: as shown in figures 1 and 2, wherein the wild type individual shows a genotype at the c.987c/Del site as GC/GC homozygote; the mutant type shows that the c.987C/Del site of the tested individual is GC/G-heterozygote.
Example 8, a kit for in vitro detection of early coronary heart disease related gene, namely Cyp17a gene,
the kit comprises:
1) primers for amplification of c.987C/Del site:
an upstream primer: 5 'taggggacat ctttggggct g 3';
a downstream primer: 5 'tcactgatag ttggtgtgcg gc 3';
2) PCR amplification enzyme and corresponding buffer solution;
3) sequencing the corresponding map.
In conclusion, the invention discloses early-onset coronary heart disease related genes, and a reagent, a preparation or a kit for in vitro detection and application thereof, wherein the preparation or the kit for in vitro detection of the early-onset coronary heart disease related genes can be used for in vitro detection of polymorphism of the coronary heart disease related genes, and can also be used for detecting, preventing, diagnosing or treating the early-onset coronary heart disease, so that the incidence rate of the coronary heart disease is reduced, and the invention has great significance for preventing and treating the coronary heart disease.
The technical characteristics form an embodiment of the invention, which has strong adaptability and implementation effect, and unnecessary technical characteristics can be increased or decreased according to actual needs to meet the requirements of different situations.
Sequence listing
<110> first subsidiary Hospital of Xinjiang medical university
<120> early-onset coronary heart disease pathogenic gene, and reagent, preparation or kit for in vitro detection and application thereof
<130>
<160>3
<170>
<210> 1
<211> 1801
<212> mRNA
<213> human (Homo sapiens)
<400> 1
atgtgggagc tcgtggctct cttgctgctt accctagctt atttgttttg gcccaagaga 60
aggtgccctg gtgccaagta ccccaagagc ctcctgtccc tgcccctggt gggcagcctg 120
ccattcctcc ccagacacgg ccatatgcat aacaacttct tcaagctgca gaaaaaatat 180
ggccccatct attcggttcg tatgggcacc aagactacag tgattgtcgg ccaccaccag 240
ctggccaagg aggtgcttat taagaagggc aaggacttct ctgggcggcc tcaaatggca 300
actctagaca tcgcgtccaa caaccgtaag ggtatcgcct tcgctgactc tggcgcacac 360
tggcagctgc atcgaaggct ggcgatggcc acctttgccc tgttcaagga tggcgatcag 420
aagctggaga agatcatttg tcaggaaatc agtacattgt gtgatatgct ggccacccac 480
aacggacagt ccatagacat ctcctttcct gtcttcgtgg cggtaaccaa tgtcatctcc 540
ttgatctgct tcaatacctc ctacaagaat ggggaccctg agttgaatgt catacagaat 600
tacaatgaag gcatcataga caacctgagc aaagacagcc tggtggacct agtcccctgg 660
ttgaagattt tccccaacaa aaccctggaa aaattaaaga gccatgttaa aatacgaaat 720
gatctgctga ataaaatact tgaaaattac aaggagaaat tccggagtga ctctatcacc 780
aacatgctgg acacactgat gcaagccaag atgaactcag ataatggcaa tgctggccca 840
gatcaagact cagagctgct ttcagataac cacattctca ccaccatagg ggacatcttt 900
ggggctggcg tggagaccac cacctctgtg gttaaatgga ccctggcctt cctgctgcac 960
aatcctcagg tgaagaagaa gctctacgag gagattgacc agaatgtggg tttcagccgc 1020
acaccaacta tcagtgaccg taaccgtctc ctcctgctgg aggccaccat ccgagaggtg 1080
cttcgcctca ggcccgtggc ccctatgctc atcccccaca aggccaacgt tgactccagc 1140
atcggtgagt ttgctgtgga caagggcaca gaagttatca tcaatctgtg ggcgctgcat 1200
cacaatgaga aggagtggca ccagccggat cagttcatgc ctgagcgttt cttgaatcca 1260
gcggggaccc agctcatctc accgtcagta agctatttgc ccttcggagc aggacctcgc 1320
tcctgtatag gtgagatcct ggcccgccag gagctcttcc tcatcatggc ctggctgctg 1380
cagaggttcg acctggaggt gccagatgat gggcagctgc cctccctgga aggcatcccc 1440
aaggtggtct ttctgatcga ctctttcaaa gtgaagatca aggtgcgcca ggcctggagg 1500
gaagcccagg ctgagggtag cacctaaagg ctgtaactca cagcccctgt ccaccctatg 1560
tggccccaca acacagattt agagatacaa ccccccaccc ttctccgcca ttcttcccta 1620
ctcccaaccc actctgcctt ctttttcagc ttgtggcaat gccagtgatg tgcataaaca 1680
gttttttttt ttccaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1740
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1800
a 1801
<210> 2
<211>21
<212> mRNA
<213> Artificial sequence
<400> 1
taggggacat ctttggggct g 21
<210> 3
<211>22
<212> mRNA
<213> Artificial sequence
<400> 1
tcactgatag ttggtgtgcg gc 22
Claims (2)
1. An application of reagent for in vitro detecting early-onset coronary heart disease pathogenic gene in preparing a preparation or a kit for in vitro detecting early-onset coronary heart disease pathogenic gene is characterized in that the early-onset coronary heart disease related gene is Cyp17a gene with c.987C/Del site mutation, the nucleotide sequence of the Cyp17a gene with c.987C/Del site mutation is shown in a sequence table SEQ ID NO:1, the reagent is used for detecting the mutation of c.987C/Del site of the Cyp17a gene, and the reagent comprises the following primers:
an upstream primer: 5 'taggggacat ctttggggct g 3';
a downstream primer: 5 'tcactgatag ttggtgtgcg gc 3'.
2. The use of the reagent for in vitro assay of a pathogenic gene of early coronary heart disease according to claim 1 in the preparation of a formulation or kit for in vitro assay of a pathogenic gene of early coronary heart disease, wherein the formulation or kit comprises a PCR amplification enzyme and a buffer solution therefor.
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| CN112342293B (en) * | 2020-11-23 | 2024-02-06 | 新疆医科大学第一附属医院 | Family pathogenic coronary heart disease susceptibility gene, reagent for in vitro detection and application thereof |
| CN113136387B (en) * | 2021-04-01 | 2022-02-18 | 百世诺（北京）医疗科技有限公司 | Early-onset coronary heart disease related gene and detection reagent and application thereof |
| CN115851640A (en) * | 2022-12-30 | 2023-03-28 | 天津大学 | Module for avoiding path competition by mixed bacteria and application thereof |
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| GB9310842D0 (en) * | 1993-05-26 | 1993-07-14 | Imperial College | Gene identification |
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| CN103667301A (en) * | 2013-09-06 | 2014-03-26 | 新疆医科大学第一附属医院 | Gene related to coronary heart disease, and in-vitro detection reagent, preparation or kit and application thereof |
| CN104513828A (en) * | 2014-11-25 | 2015-04-15 | 新疆医科大学第一附属医院 | Coronary heart disease (CHD) related gene namely CYP17A1 gene, and in-vitro detection reagent, preparation or kit and application thereof |
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2017
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|---|---|---|---|---|
| GB9310842D0 (en) * | 1993-05-26 | 1993-07-14 | Imperial College | Gene identification |
| CN102732608A (en) * | 2011-03-31 | 2012-10-17 | 中国科学院上海生命科学研究院 | Marker for diagnosing liver cancer and application thereof |
| CN103667301A (en) * | 2013-09-06 | 2014-03-26 | 新疆医科大学第一附属医院 | Gene related to coronary heart disease, and in-vitro detection reagent, preparation or kit and application thereof |
| CN104513828A (en) * | 2014-11-25 | 2015-04-15 | 新疆医科大学第一附属医院 | Coronary heart disease (CHD) related gene namely CYP17A1 gene, and in-vitro detection reagent, preparation or kit and application thereof |
Non-Patent Citations (2)
| Title |
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| CYP17A1基因多态性与冠心病遗传易感性的研究;贾学文等;《中国循证心血管医学杂志》;20170228;第9卷(第2期);第187-198页 * |
| 罕见的CYP17A1基因外显子剪接突变导致17α-羟化酶缺陷的分子病因学研究;韩兵等;《中华内分泌代谢杂志》;20100306;第27卷(第11期);第911-915页 * |
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