CN101294198A - coronary disease testing method and reagent kit - Google Patents
coronary disease testing method and reagent kit Download PDFInfo
- Publication number
- CN101294198A CN101294198A CNA2007100401007A CN200710040100A CN101294198A CN 101294198 A CN101294198 A CN 101294198A CN A2007100401007 A CNA2007100401007 A CN A2007100401007A CN 200710040100 A CN200710040100 A CN 200710040100A CN 101294198 A CN101294198 A CN 101294198A
- Authority
- CN
- China
- Prior art keywords
- itga2
- gene
- seq
- atherosclerosis
- primer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for detecting atherosclerosis and/or coronary heart disease susceptibility, which includes detecting whether variation exists in individual integrin Alpha 2 (ITGA2), transcript and/or albumen in comparison with normal. The existence of variation shows that the possibility that an individual contracts atherosclerosis and/or coronary heart disease is higher than that of a normal person. The invention also discloses a corresponding detection kit.
Description
Technical field
The present invention relates to molecular biology and medical field.Relate more specifically to beta 2 integrin alpha 2 (Integrinalpha 2, single nucleotide polymorphism ITGA2) (single nucleotide polymorphism, SNP) and with the dependency of atherosclerosis and/or coronary heart disease.The invention still further relates to the method and the test kit that detect these SNP.
Background technology
Atherosclerosis is a kind of common carrying out property artery disease; pathology is mainly involved medium sized flesh layer artery; the endarterium lipidosis; smooth muscle cell proliferation; form the limitation patch; can make the arterial wall hardening; plaque rupture causes thrombus, embolism, hemorrhage when serious; the tube chamber of getting involved is partially or completely inaccessible; clinical manifestation is the generation of atherosclerotic blood vessel complication, among the gerontal patient the most common and the most serious vascular complication be atherosclerosis and/or coronary heart disease (unstable angina pectoris and myocardial infarction) and cerebral apoplexy.
Four incremental pathologic processes have mainly been experienced in the generation of atherosclerotic lesion and development:
1. lipid soaks into membrane change in early stage: endothelial injury
2. lipid striped: foam cell, T lymphocyte are assembled smooth muscle cell proliferation.
3. fibrous plaque: smooth muscle fibersization forms fibrous cap.
4. compound pathology: calcify plaque, break, ulcer, middle film pathology.
The atherosclerotic cause of disease and pathogenesis are very complicated, be the result of a series of complexing actions between cell, extracellular matrix, blood ingredient (particularly monocyte, thrombocyte and LDL), regional flow's kinetics, environment and the inherited genetic factors of arterial wall, thereby the cause of disease is not to be single factors.
Atherosclerosis is as multigenic disease, and pathogenic factor not only relates to the different gene of a plurality of effects, but also relates to the interaction of gene-gene, gene-environment.Theory about incidence of atherosclerosis mechanism has much now, as infiltrating lipid theory, tunica intima damage theory, inflammation reaction theory, oxidation antioxidation theory and thrombosis theory.All these theories all relate to different biological pathways, but now also do not have a kind of theory can obtain complete explanation on genetics and molecular biology.
For many years, based on to the studying for a long period of time of atherosclerosis pathogenic risk factor, scientists has been carried out a large amount of work at these Hazard Factor.So far, some insertion, disappearance and the sudden change that has had been found that tonin (ACE) and MTHFR may make atherosclerosis and/or the morbific latent dangerous factor of coronary heart disease.The former makes one of enzyme of performance critical function in the blood pressure regulation, and the latter is a kind of enzyme relevant with the halfcystine metabolism.
In addition, Germany scientist confirms that recently AT1 receptor expression level and vascular injury process are closely connected: ACE inhibitor can reduce the activation of AT1 acceptor, thus improve vascular function bad, effectively suppress atherosclerotic generation and development.
But for a long time, study the sudden change and the expression that concentrate on genes involved in the lipid metabolism path both at home and abroad morely, as: secondary oxygenase, lipoprotein lipase, hepatic lipase etc.In the research to endogenous nitric oxide synthetic enzyme (eNOS), more existing now research work show eNOS and atherosclerotic tight association.Other has U.S. scientist to propose the effect of inflammation mechanism in atherosclerotic formation, and has obtained certain confirmation in mouse test.
In recent years, the extensive utilization in multigenic disease research of single nucleotide polymorphism (SNP) and haplotype (haplotype) notion is for the generation of studying atherosclerotic on molecular level and the mechanism of development have been opened up brand-new thinking.Simultaneously, high-throughput, the continuous appearance of SNP detection means cheaply also provide possibility for the extensive fast typing of SNP.
The Japan scientist utilizes large-scale case-control study that more than 90,000 genes involved SNPs have been carried out association analysis, the haplotype zone of having found a 50KB is closely related with myocardial infarction, comprising LTA (lymphotoxin-α), a plurality of SNP site of NFKBIL1 and BAT1 etc.
In another research, though middle S290N of palatelet-selectin (P-selectin) and N562D do not have independent dependency with myocardial infarction, haplotype and myocardial infarction significant correlation that their constitute separately.
In addition, the existence of SNP whether and gene frequency have the difference of ethnic group and region, this just points out the existence of multigenic disease genetic heterogeneity, promptly same disease or proterties may be that different inherited genetic factorss causes in different crowds.In research to the factor V gene, fail to find the external VLeiden sudden change that has dependency with the morbidity of white man women's myocardial infarction that report in the Chinese population, found that but a new SNP-G/A1628 (Arg485Lys) sudden change and APCR and atherosclerosis and/or coronary heart disease are relevant, the factor V gene may be the atherosclerotic tumor susceptibility gene of Chinese population.
In addition, this species diversity that the type of SNP and haplotype thereof and frequency exist in the different crowd may with different crowd reactive different closely related to medicine and environmental factors.
In the past over more than 50 year, large-scale epidemiology survey and genetic research have been done a series of research to this complex disease of coronary atherosclerosis, found that some tumor susceptibility genes are relevant with atherosclerosis, for example ABCA1, CYBA, ApoE, LDLR etc.More relevant continuation with atherosclerosis is in finding and verifying.
Atherosclerosis and/or coronary heart disease are interacted and the polygenic disease of morbidity by inherited genetic factors and environmental factors as a kind of, seek its genes involved and then illustrate atherosclerosis and/or the genetic mechanism of incidence of coronary heart disease has become the focus of present research.
Though it is existing many about range gene polymorphism and atherosclerosis and/or Study on Coronary Heart Disease, but do not confirm the report of ITGA2 gene and atherosclerosis and/or coronary heart disease dependency, more do not confirm the SNP of ITGA2 gene and the report of atherosclerosis and/or coronary heart disease dependency.
In sum, for diagnosing atherosclerotic and/or coronary heart disease as early as possible, this area presses for seeks atherosclerosis and/or coronary heart disease tumor susceptibility gene, and exploitation detects the method and the test kit of atherosclerosis and/or coronary heart disease.
Summary of the invention
Purpose of the present invention just provides the method and the detection kit of a kind of auxiliary diagnosis (especially early diagnosis) atherosclerosis and/or coronary heart disease.
Another object of the present invention provides a kind of new treatment atherosclerosis and/or the method for coronary heart disease.
In a first aspect of the present invention, a kind of method that the atherosclerosis and/or the coronary disease susceptibility of individuality are diagnosed is provided, it comprises step:
Detect this individual ITGA2 gene, transcript and/or albumen, and compare with normal ITGA2 gene, transcript and/or albumen,
The possibility that there are differences with regard to showing this individuality trouble atherosclerosis and/or coronary heart disease is higher than normal population.
In another preference, what detect in the described method is gene or the transcript of ITGA2, and with normal ITGA2 nucleotide sequence comparing difference.
In another preference, described difference is following single nucleotide polymorphism:
301 G → C;
Wherein, the nucleotide position numbering is based on SEQ ID NO:1.
In a second aspect of the present invention, provide a kind of test sample whether to have the method for the single nucleotide polymorphism of beta 2 integrin alpha 2 (ITGA2), comprise step:
(a) with the ITGA2 gene of ITGA2 gene-specific primer amplification sample, obtain amplified production; With
(b) detect whether there is following single nucleotide polymorphism in the amplified production:
301 G → C;
Wherein, the nucleotide position numbering is based on SEQ ID NO:1.
In another preference, described gene-specific primer has the sequence of SEQ ID NO:2 and 3.
In another preference, the length of described amplified production is 100-2000bp, and contains among the SEQ IDNO:1 the 301st.
In a third aspect of the present invention, a kind of test kit that detects atherosclerosis and/or coronary heart disease is provided, it comprises the primer of specific amplification ITGA2 gene or transcript, more preferably, it is 100-2000bp that described primer amplification goes out length, and contains among the SEQ ID NO:1 the 301st amplified production.
In another preference, described test kit also contains the reagent that is selected from down group:
(a) with SEQ ID NO:1 in the 301st sudden change bonded probe;
(b) the 301st sudden change restriction enzyme among the identification SEQ ID NO:1.
In another preference, described sudden change is selected from following single nucleotide polymorphism:
301 G → C;
Wherein, the nucleotide position numbering is based on SEQ ID NO:1.
Embodiment
The inventor is through deeply and extensive studies, and the SNP of a large amount of candidate genes is measured and analyzes.Find first and proved that the genome sequence of ITGA2 and atherosclerosis and/or coronary heart disease are closely related, wherein the association study result shows, (there is significant difference (P＜0.05) in the distribution of 301 G → C) (be designated as RS3212476) in case and control group to the 301st SNP in the SEQ of ITGA2 ID NO:1, therefore can be used as the specificity SNP of complementary detection atherosclerosis and/or coronary heart disease (or its susceptibility).Finished the present invention on this basis.
The ITGA2 gene
Beta 2 integrin alpha 2 (Integrin alpha 2, sequence ITGA2) is known, its detailed sequence and some relevant informations can be referring to network address http://www.ncbi.nlm.nih.gov/Genebank/; Http:// www.ncbi.nlm.nih.gov/SNP.For convenience's sake, provide nucleotide sequence relevant among the ITGA2 at SEQ ID NO:1 with SNP of the present invention.
Beta 2 integrin alpha 2 is also referred to as CD49B or VLA-2 acceptor α 2 subunits (CD49B, alpha 2subunit of VLA-2 receptor).The ITGA2 gene belongs to the Integrins gene family, and the gene of this family is some surface receptor proteins relevant with cell adhesion, and has been considered to participate in the signal conduction of cell surface.The acting in conjunction body (alpha-2/beta-1) of the ITGA2 of platelet surface and ITGB1 has been regulated the initial deposition on collagen of thrombocyte, and has influence on formation (the Siljander PR et al.Blood.2004 of next step thrombus; 103 (4): 1333-41).Some polymorphisms of ITGA2 gene can have influence on density (the Kritzik M et al.Blood.1998 of the alpha-2/beta-1 of platelet surface; 92 (7): 2382-8).And then this influence can be related to generation (the Santoso S et al.Blood.1999 of the myocardial infarction of young individuality; 93 (8): 2449-53; Carlsson LE et al.Blood.1999; 93 (11): 3583-6).And the ITGA2 gene also is considered to have effect in the cardiovascular thrombosis, and (Charakida M etal.Ital Heart J.2003; 4 (1): 17-22).
The inventor checks order to the almost whole zone in the ITGA2 gene, many SNP have been found, wherein most of SNP and atherosclerosis and/or coronary disease susceptibility are also uncorrelated, yet association study shows that 301 G → C but are and atherosclerosis and/or the very high SNP of coronary disease susceptibility cognation among the SEQID NO:1.
Particularly, the present invention has disclosed the single nucleotide polymorphism (SNP) of No. 4 intron of ITGA2 gene and the dependency of this polymorphism and coronary atherosclerosis.SNP of the present invention is that the 301st G/C of sequence shown in the SEQ ID NO:1 is polymorphic, and it is positioned at No. 4 intron of people ITGA2 gene, and the frequency of heterozygote GC in coronary atherosclerosis patient group will be higher than the frequency in the normal control crowd.
Based on new discovery of the present invention, ITGA2 albumen or polypeptide have many-sided new purposes.These purposes include, but is not limited to: be used for complementary diagnosing atherosclerotic and/or coronary heart disease, or be used to screen the material that promotes the ITGA2 protein function, as antibody, polypeptide or other part.
On the other hand, the present invention also comprises people ITGA2 DNA or the polypeptide of its fragment coding has specific polyclonal antibody and monoclonal antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into people ITGA2 gene product or fragment.Preferably, refer to that those can combine with people ITGA2 gene product or fragment but nonrecognition and be incorporated into the antibody of other irrelevant antigen molecule.Among the present invention antibody comprise those can in conjunction with and suppress the proteic molecule of people ITGA2, comprise that also those do not influence the antibody of people ITGA2 protein function.
The present invention not only comprises complete mono-clonal or polyclonal antibody, but also comprises having immunocompetent antibody fragment, as Fab ' or (Fab)
2Fragment; Heavy chain of antibody; Light chain of antibody; Genetically engineered strand Fv molecule; Or chimeric antibody.
Antibody of the present invention can be prepared by the known various technology of those skilled in that art.For example, the people ITGA2 gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing human ITGA2 albumen or its have antigenic segmental cell and can be used to immune animal and produce antibody.Multiple adjuvant can be used for the enhancing immunity reaction, includes but not limited to freund's adjuvant etc.
Antibody of the present invention also can be monoclonal antibody.This type of monoclonal antibody can utilize hybridoma technology to prepare.Antibody of the present invention comprises the antibody that can block people ITGA2 protein function and the antibody that does not influence people ITGA2 protein function.Each antibody-like of the present invention can utilize the fragment or the functional zone of people ITGA2 gene product, obtains by the routine immunization technology.These fragments or functional zone can utilize recombinant methods or utilize Peptide synthesizer synthetic.Can come immune animal and produce with the gene product of producing in the prokaryotic cell prokaryocyte (for example E.Coli) with the unmodified form bonded antibody of people ITGA2 gene product; With posttranslational modification form bonded antibody (as the albumen or the polypeptide of glycosylation or phosphorylation), can come immune animal and obtain with the gene product that produces in the eukaryotic cell (for example yeast or insect cell).
The proteic antibody of anti-people ITGA2 can be used in the immunohistochemistry technology, detect people ITGA2 in the biopsy specimen proteic what and/or whether suddenly change.A kind of preferred anti-ITGA2 antibody is the antibody of the normal ITGA2 of nonrecognition ITGA2 but identification suddenlys change, perhaps discerns normal ITGA2 but the antibody of nonrecognition sudden change ITGA2.Utilize these antibody, the atherosclerosis and/or the coronary disease susceptibility that can carry out protein level easily detect.
Utilize ITGA2 albumen of the present invention,, can filter out with ITGA2 albumen interactional material takes place, as inhibitor, agonist or antagonist etc. by various conventional screening methods.
The invention still further relates to the diagnostic testing process of quantitative and detection and localization people ITGA2 protein level.These tests are known in the art, and comprise ELISA etc.
Whether having the proteic method of ITGA2 in a kind of detection test sample is to utilize the proteic specific antibody of ITGA2 to detect, and it comprises: sample is contacted with the ITGA2 protein specific antibody; Observe whether form antibody complex, formed antibody complex and just represented to exist in the sample ITGA2 albumen.
The proteic polynucleotide of ITGA2 can be used for the auxiliary diagnosis of ITGA2 protein related diseases.Aspect diagnosis, the proteic polynucleotide of ITGA2 can be used for detecting the proteic expression of ITGA2 ITGA2 abnormal exprssion whether or under morbid state.Can be used for the hybridization of biopsy specimen to judge the proteic abnormal expression of ITGA2 as the ITGA2 dna sequence dna.Hybridization technique comprises the Southern blotting, Northern blotting, in situ hybridization etc.These technological methods all are disclosed mature technologies, and relevant test kit all can obtain from commercial channels.Part or all of polynucleotide of the present invention can be used as probe stationary on microarray (microarray) or DNA chip (being called " gene chip " again), is used for analyzing the differential expression analysis and the gene diagnosis of tissue gene.Carry out RNA-polymerase chain reaction (RT-PCR) amplification in vitro with the special primer of ITGA2 albumen and also can detect the proteic transcription product of ITGA2.
Detection can be at cDNA, also can be at genomic dna.The form of ITGA2 protein mutation comprises that the point mutation compared with normal wild type ITGA2 dna sequence dna, transposition, disappearance, reorganization and other are any unusual etc.Available existing technology such as Southern blotting, dna sequence analysis, PCR and in situ hybridization detect sudden change.In addition, sudden change might influence proteic expression, therefore can judge indirectly that with Northern blotting, Western blotting gene has or not sudden change.
The method of the detection SNP of the present invention of most convenient is by the ITGA2 gene with ITGA2 gene-specific primer amplification sample, obtains amplified production; Detect then and whether have following single nucleotide polymorphism in the amplified production: 301 G → C, wherein, nucleotide position is numbered based on SEQ ID NO:1.
Should understand, after the present invention has disclosed the dependency of the SNP of ITGA2 gene and atherosclerosis and/or coronary heart disease first, but those skilled in the art can design the amplified production that specific amplification goes out to contain this SNP position easily, determine whether to exist 301 G → C by methods such as order-checkings then.Usually, the length of primer is 15-50bp, preferably is 20-30bp.Though the complete complementation of primer and template sequence is preferred, one skilled in the art will appreciate that at primer and template to have under the situation of certain not complementary (especially 5 of primer ' end) also can increase specifically (promptly only amplifying required fragment).The method that contains the test kit of these primers and use these primers is all within the scope of the invention, as long as the amplified production that this primer amplification goes out contains the correspondence position of SNP of the present invention.A kind of preferred primer is to having the sequence of SEQ ID NO:2 and 3.
Though the length of amplified production is not particularly limited, the length of amplified production is 100-2000bp usually, preferably is 150-1500bp, more preferably is 200-1000bp.These amplified productions all should contain among the SEQ ID NO:1 the 301st.
Because SNP of the present invention and atherosclerosis and/or coronary heart disease have very high cognation, therefore not only can be used for early stage complementary diagnosing atherosclerotic and/or coronary heart disease, and can make some carrier just take reasonable precautions (as on diet, taking corresponding control) against a rainy day at premorbid not, thereby improve carrier's lifetime and life quality, therefore have earth shaking using value and social benefit.Certainly, finally should make a definite diagnosis with the conventional sense method.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:ColdSpring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1
1.1 research object
Be diagnosed as among the atherosclerotic patient at the underwent coronary radiography, select number of stenosed coronary vessel more than 2 or 2, and the pathology stenosis the patient more than 50% as case.Other is diagnosed as and does not have atherosclerotic normal people in contrast at the coronarography of learning from else's experience, and chooses age, sex and native place and the case group is complementary as far as possible.
On the basis of informed consent random collecting the peripheral blood sample of 181 patients and 183 normal control individualities.
1.2 experimental technique and result
1.2.1 DNA extraction
Extract DNA with conventional phenol chloroform method from people's peripheral blood sample, concentration correction is used for conventional pcr amplification to 20ng/ul.
1.2.2 the design of PCR and sequencing primer
According to the genome sequence of ITGA2 among the GenBank, design and synthetic following primer.Concrete primer is as shown in table 1 below.
Table 1 primer sequence table
| The primer title | Sequence (5 '-3 ') | SEQ ID NO： |
| Adopted primer is arranged | tttataactg attcacaact ttaca | 2 |
| Antisense primer | agttttctgc agttccttcc attca | 3 |
1.2.3 the pcr amplification of ITGA2 gene
DNA with extraction is a template, uses the Taq enzyme, carries out pcr amplification with the Touchdown program on GeneAmp 9700 PCR instrument.Reaction conditions is: 94 ℃ of pre-sex change 2 minutes, and 94 ℃ of sex change 30 seconds, 63 ℃ of annealing 40 seconds, 72 ℃ were extended totally 10 circulations, 0.5 ℃ of each cycle annealing lapse of temperature 40 seconds; Later 94 ℃ of sex change 30 seconds were annealed 40 seconds for 58 ℃, and 72 ℃ were extended totally 30 circulations 40 seconds; Last 72 ℃ were extended 7 minutes.Pcr amplification product is verified through agarose gel electrophoresis.As a result, obtain the amplified production of ITGA2.
1.2.4 the discovery of SNP and detection
The PCR product is used ABI-PRISM behind the Resin resin purification
TM377, dna sequencing instrument (appliedbiosystems of u.s.a. applied biosystem company (ABI)) carries out the two-way order-checking of the terminal cessation method of fluorescent mark, and the interpretation and the SNP that carry out sequence with Polyphred software (the http://droog.mbt.washington.edu/Polyphred.html of Washington, DC university) confirm.
As a result, find to have several SNP, comprising 301 G → C among the following SNP:SEQ ID NO:1.
1.2.5 SNP gene type and association analysis
Carry out the SNP gene type with direct unidirectional sequencing.Promptly in atherosclerosis and/or coronary heart disease patient and normal control group, carry out somatotype and association analysis.
To the statistical study of being described property of gene type result, contingency table carries out chi square test.Observe genotype and between patient and contrast, whether have difference.
Analytical results shows, in 181 patients altogether and 183 normal controls, there is significant difference in 301 the SNP (being designated as RS3212476) of ITGA2 between patient group and control group: for the single nucleotide polymorphism of No. 4 intron of ITGA2 gene, the frequency of its heterozygote GC is 51.4% in patient group, be 41.0% in contrast, both have significant difference (p=0.10) in 90% fiducial interval.
The above results has confirmed among the SEQ ID NO:1 dependency that exists of 301 SNP and atherosclerosis and/or coronary heart disease.
Embodiment 2
Atherosclerosis and/or reagent kit for detecting coronary heart disease susceptibility
As described in embodiment 1, the sudden change of 301 G → C and atherosclerosis and/or coronary heart disease disease are closely related among the SEQ ID NO:1.Therefore, can be that template increases and detects at DNA based on this sudden change design ITGA2 gene-specific primer with patient.
Prepare a test kit (100 person-times), it contains:
Whether the test group that 100 people of random choose constitute is suffered from atherosclerotic object, known trouble atherosclerosis and/or coronary heart disease patient and is not had atherosclerotic normal people after testing comprising the unknown.
Extract the peripheral blood 3ml of object to be detected in the test group, use ordinary method (or using specific test kit) from blood, to extract DNA.PCR primer in atherosclerosis and/or the coronary heart disease detection kit is diluted to 1 ì mol/ ì l, is that template is carried out the PCR reaction with the primer that is provided with the DNA that is extracted.Behind the PCR product purification, use ABI-PRISM
TM377 dna sequencing instrument carry out the two-way order-checking of the terminal cessation method of fluorescent mark, and the interpretation and the SNP that carry out sequence with Polyphred software confirm.
Perhaps, amplified production and normal control are carried out stratographic analysis with sex change high performance liquid chromatograph (DHPLC), also can detect 301 G → C.
Detected result:
For the object of 301 G → C of ITGA2 existence, further detect to confirm whether to suffer from the atherosclerosis situation by ordinary method.Detected result shows that the atherosclerosis and/or the coronary disease susceptibility ratio that contain the detected object (especially GC heterozygote) of 301 G → C are the normal population (exceeding at least about 40%) of GG apparently higher than genotype.
Therefore, this shows by detecting 301 G → C of ITGA2, can carry out atherosclerotic complementary detection and/or early diagnosis.
Embodiment 3
The complementary detection of atherosclerosis and/or coronary disease susceptibility
Repeat the detection of embodiment 2, difference be picked at random 80 people (not knowing before the detection whether the atherosclerosis shape is arranged) detect.
Prepare a test kit (100 person-times), it contains:
Extract the peripheral blood 3ml of object to be detected, use ordinary method (or using specific test kit) from blood, to extract DNA.PCR primer in atherosclerosis and/or the coronary heart disease detection kit is diluted to 1 ì mol/ ì l, is that template is carried out the PCR reaction with the primer that is provided with the DNA that is extracted.Behind the PCR product purification, use ABI-PRISM
TM377 dna sequencing instrument carry out the two-way order-checking of the terminal cessation method of fluorescent mark, and the interpretation and the SNP that carry out sequence with Polyphred software confirm.
The result has confirmed that equally the atherosclerosis ratio that contains the detected object (especially GC heterozygote) of 301 G → C is the detected object of GG apparently higher than this site.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
＜110〉Research Center of Shanghai Human Genome
＜120〉coronary disease testing method and test kit
<130>072062
<160>3
<170>PatentIn version 3.1
<210>1
<211>601
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<400>1
tttataactg attcacaact ttacataacg ttaaattctg taggcatttt ttaggatctt 60
aattagattt ctcttttgca cttgtctcct ttttcatgta tcaattttaa agttatttaa 120
tctaatgttt cttgtgtcat ttaatcatga tttaatcagg agtttttggc attcaaggac 180
tcctgaaaag gagagaattg ggattgcatt aatggagata ttaaggattg gagcaggtat 240
aatgtgagca agaagacaag tgccattcag cgttagtgac tgcagttaat tgggaagtgg 300
gcaaagttaa aaactggtag tcagtgaacc tcatttgtgt ttgcttagca tatggagtaa 360
ttttgaactg aattttcaca caacatttgc cctctcgact ctcttttaca aattagggaa 420
tctggcaaca caacctattt catacccgtg ggcaggcaga aaacatagtg gtctgtacat 480
aggcatgggt tcttcattca ccatgatgtc tatacctgac cccaagccct cttcaagcgt 540
tactgccatt aggtttatgg tttctgctga gcagtatgaa tggaaggaac tgcagaaaac 600
t 601
<210>2
<211>25
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>2
tttataactg attcacaact ttaca 25
<210>3
<211>25
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>3
agttttctgc agttccttcc attca 25
Claims (10)
1. whether a vitro detection sample exists the method for the single nucleotide polymorphism of beta 2 integrin alpha 2 (ITGA2), it is characterized in that, comprises step:
(a) with the ITGA2 gene of ITGA2 gene-specific primer amplification sample, obtain amplified production; With
(b) detect whether there is following single nucleotide polymorphism in the amplified production:
301 G → C;
Wherein, the nucleotide position numbering is based on SEQ ID NO:1.
2. the method for claim 1 is characterized in that, described gene-specific primer has the sequence of SEQID NO:2 and 3.
3. the method for claim 1 is characterized in that, the length of described amplified production is 100-2000bp and contains among the SEQ ID NO:1 the 301st.
4. test kit that detects atherosclerosis and/or coronary heart disease, it is characterized in that, it comprises the primer of specific amplification ITGA2 gene or transcript, and described primer amplification goes out length to be 100-2000bp and to contain among the SEQ ID NO:1 the 301st amplified production.
5. test kit as claimed in claim 4 is characterized in that, it also contains the reagent that is selected from down group:
(a) with SEQ ID NO:1 in the 301st sudden change bonded probe;
(b) the 301st sudden change restriction enzyme among the identification SEQ ID NO:1.
6. test kit as claimed in claim 5 is characterized in that, described sudden change is following single nucleotide polymorphism:
301 G → C;
Wherein, the nucleotide position numbering is based on SEQ ID NO:1.
7. test kit as claimed in claim 4 is characterized in that described primer has the sequence of SEQ ID NO:2 and 3.
8. method that the atherosclerosis and/or the coronary disease susceptibility of individuality are diagnosed is characterized in that it comprises step:
Detect this individual ITGA2 gene, transcript and/or albumen, and compare with normal ITGA2 gene, transcript and/or albumen,
Wherein, the possibility that there are differences with regard to showing this individuality trouble atherosclerosis and/or coronary heart disease is higher than normal population.
9. method as claimed in claim 8 is characterized in that, detection be gene or the transcript of ITGA2, and with normal ITGA2 nucleotide sequence comparing difference.
10. method as claimed in claim 9 is characterized in that, described difference is following single nucleotide polymorphism:
301 G → C;
Wherein, the nucleotide position numbering is based on SEQ ID NO:1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100401007A CN101294198A (en) | 2007-04-27 | 2007-04-27 | coronary disease testing method and reagent kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100401007A CN101294198A (en) | 2007-04-27 | 2007-04-27 | coronary disease testing method and reagent kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101294198A true CN101294198A (en) | 2008-10-29 |
Family
ID=40064732
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2007100401007A Pending CN101294198A (en) | 2007-04-27 | 2007-04-27 | coronary disease testing method and reagent kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101294198A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102239595A (en) * | 2008-12-08 | 2011-11-09 | 株式会社日立制作所 | Process for producing lithium secondary battery |
| WO2024001669A1 (en) * | 2022-06-27 | 2024-01-04 | 昱言科技(北京)有限公司 | Antibodies targeting itga2 and antibody-drug conjugates comprising same |
-
2007
- 2007-04-27 CN CNA2007100401007A patent/CN101294198A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102239595A (en) * | 2008-12-08 | 2011-11-09 | 株式会社日立制作所 | Process for producing lithium secondary battery |
| CN102239595B (en) * | 2008-12-08 | 2014-11-05 | 株式会社日立制作所 | Process for producing lithium secondary battery |
| WO2024001669A1 (en) * | 2022-06-27 | 2024-01-04 | 昱言科技(北京)有限公司 | Antibodies targeting itga2 and antibody-drug conjugates comprising same |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Eeles et al. | The genetic epidemiology of prostate cancer and its clinical implications | |
| Tejedor et al. | Reliable low-density DNA array based on allele-specific probes for detection of 118 mutations causing familial hypercholesterolemia | |
| Svobodova et al. | Apolipoprotein E gene polymorphism in the Mongolian population | |
| Castro et al. | Screening of the endothelin1 gene (EDN1) in a cohort of patients with essential left ventricular hypertrophy. | |
| JP2002527079A (en) | Genes for assessing cardiovascular status and compositions for their use | |
| CN101294201A (en) | coronary disease testing method and reagent kit | |
| CN101294195A (en) | Coronary disease testing method and reagent kit | |
| CN101525656B (en) | Method for testing susceptibility of ankylosing spondylitis and kit | |
| CN101294198A (en) | coronary disease testing method and reagent kit | |
| WO2008118969A2 (en) | Methods and agents for evaluating inflammatory bowel disease, and targets for treatment | |
| CN101294197A (en) | coronary disease testing method and reagent kit | |
| CN104419748A (en) | Method and kit for detecting susceptibility of ankylosing spondylitis by using genotype | |
| EP2459740A2 (en) | Processes and methods for diagnosis of alzheimer's disease | |
| CN101525658B (en) | Method and kit for detecting susceptibility of ankylosing spondylitis | |
| JP4688492B2 (en) | Determination method of inflammatory diseases | |
| CN104232650A (en) | New pathogenic gene of idiopathic basal ganglia calcification, and coding protein and application thereof | |
| CN101294210A (en) | coronary disease testing method and reagent kit | |
| CN101294200A (en) | coronary disease testing method and reagent kit | |
| CN101294199A (en) | coronary disease testing method and reagent kit | |
| CN101294214A (en) | coronary disease testing method and reagent kit | |
| CN101294209A (en) | coronary disease testing method and reagent kit | |
| CN101294206A (en) | coronary disease testing method and reagent kit | |
| CN101294204A (en) | coronary disease testing method and reagent kit | |
| CN101294205A (en) | coronary disease testing method and reagent kit | |
| CN101294203A (en) | coronary disease testing method and reagent kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Open date: 20081029 |