CN1661049A - Relativity between gene of growth hormone 1 and essential hypertension - Google Patents
Relativity between gene of growth hormone 1 and essential hypertension Download PDFInfo
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Abstract
A process for testing the suscaptibility of primary hypertension includes detecting if there are variations in growth hormone 1 gene GH1, transcript and/or protein of an individual, and determining its high susceptability if there are. Its reagent kit is also disclosed.
Description
Technical field
The present invention relates to molecular biology and medical field.Relate more specifically to the growth hormone 1 gene (growthhormone 1, single nucleotide polymorphism GH1) (single nucleotide polymorphism, SNP) and with the dependency of essential hypertension.The invention still further relates to the method and the test kit that detect these SNP.
Background technology
Hypertension is meant one group of clinical symptom grouping that systolic pressure or diastolic pressure raise.The rising of blood pressure and coronary heart disease, renal tubal dysfunction, hypertensive heart disease and the concurrent cerebral apoplexy of hypertension have an apparent causal connection.The up-to-date Case definition of hypertension is systolic pressure 〉=19kpa (140mmHg) or diastolic pressure 〉=12kpa (90mmHg), and meeting wherein, a person can be diagnosed as hypertension.
Most hyperpietic only has slight subjective symptoms in early days in elevation of blood pressure, as headache, dizziness, insomnia, tinnitus, fidgety, the difficult concentrated fatigue etc. that also occurs easily of working and learning energy.Along with the development of the state of an illness, when particularly developing complications, symptom increases and obviously gradually, as numbness of the fingers and stiff, lower limb pain appears when walking more, or the nervous sense of nape portion sore muscle appears.Show when, shortness of breath nervous, uncomfortable in chest, precordialgia that heart gets involved, frequent micturition at night, diuresis occur, show when urine is light that kidney is got involved, the kidney arteriole hardens when occurring.If obnubilation appears suddenly in the hyperpietic, breathe promptings such as dull irregular, gatism hematencephalon may take place, if engender a side limb activity inconvenience, numbness even benumb, should suspect the formation whether cerebral thrombosis is arranged.
The early stage Non Apparent Abnormality sign of hypertension occurs.When appearring in vitals such as brain, the heart, kidney, the mild damage can have unusual sign to occur.The performance of common heart abnormality has that apex beat moves to left, pareordia is praised the sample pulsatory feeling, auscultation apical region of heart first heart sound strengthens, second sound of aortic area strengthens and systolic murmur and diastolic murmur are arranged, show arteriosclerosis and left ventricular hypertrophy take place, if listen in the apical region of heart and the galloping horse sample rhythm of the heart may show the appearance of central force depletion.Also common in addition ear-lobe folding line, capillary pulsation, radial artery occur and pulsus durus or acrotism and lower limb intermittent claudication etc. occur.
In addition, because the development of some risk factor or hypertension itself can cause the remarkable or hurried rising of some hyperpietic's blood pressures, simultaneously with vitals functional lesions such as brain, the heart, kidney, retinas, a series of clinical special signs appear in serious threat to life, are called hypertensive emergency.The sickness rate of hypertensive emergency accounts for 5% of Hypertensive Population, and common have hypertensive encephalopathy, hematencephalon, acute left heart failure, the acute withdrawal syndrome of clonidine, Acute Myocardial Infarction, a radical type malignant hypertension etc.
About 5% left and right sides no conscious sympton among the hyperpietic does not know when blood pressure raises, and does not more know to have produced when the complication of blood vessel and organ injury yet, some patient even just know after cardiovascular accident has taken place and oneself suffer from hypertension.So, find out the genetic cause of hypertension incidence, hypertensive morbidity can be effectively controlled in preventing and detecting early, and disease is reduced to minimum to the injury of human body.
(essential hypertension EH) also is essential hypertension to essential hypertension, is a kind of independently disease, and the cause of disease of oneself, rule and the clinical manifestation that the generation development lapses to are arranged.Mainly show as the rising of arteriotony clinically.Account for more than 90% of crowd hyperpietic, at present pathogeny is not clear fully as yet, mainly just can be diagnosed as essential hypertension (essential hypertension) after having got rid of the hypertension that other diseases causes.The rising of arteriotony mainly is because of due to periphery arteriole resistance increases, and in various degree Q volume of blood and kinemic increase are arranged simultaneously.Often cause late period internal organs such as the heart, brain, kidney to be got involved severe complications such as hypertensive heart disease, heart failure, renal tubal dysfunction, hematencephalon take place.The treatment of essential hypertension mainly is to bring high blood pressure down to prevent the generation of complication simultaneously.The primary hypertension patient cause of death is cerebrovascular accident, cardiovascular accident and renal insufficiency, and for seeing, in heart failure and uremia is taken second place more with cerebrovascular accident in China, and American-European countries sees with heart failure more, and cerebrovascular accident and uremia are taken second place.
Essential hypertension is as modal cardiovascular disorder, and its sickness rate rises year by year, and can cause the serious heart, brain, kidney complication, is the primary hazard factor of cerebral apoplexy and coronary heart disease.EH is a kind ofly interacted and the polygenic disease of morbidity by inherited genetic factors and environmental factors, and the genetic mechanism of seeking the EH genes involved and then illustrating hypertension incidence has become the focus of present research.
Though have many researchs about range gene polymorphism and essential hypertension, do not confirm the report of GH1 gene and essential hypertension dependency, more do not confirm the SNP of GH1 gene and the report of essential hypertension dependency.
In sum, for the final treatment hypertension that realizes, this area presses for seeks the essential hypertension tumor susceptibility gene, and develops method, test kit that detects essential hypertension and relevant medicine.
Summary of the invention
Purpose of the present invention just provides a kind of diagnosis (especially early diagnosis) hypertensive method and detection kit.
Another object of the present invention provides the hypertensive method of a kind of new treatment.
A further object of the present invention provides the hypertensive pharmaceutical composition of a kind of treatment.
In a first aspect of the present invention, a kind of method that the hypertension susceptibility of individuality is diagnosed is provided, it comprises step:
Detect this individual GH1 gene, transcript and/or albumen, and compare with normal GH1 gene, transcript and/or albumen,
There are differences and just show that this individuality suffers from hypertensive possibility and be higher than normal population.
In another preference, what detect in the described method is gene or the transcript of GH1, and with normal GH1 nucleotide sequence comparing difference.
In another preference, described difference is following single nucleotide polymorphism:
5158 G → A;
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 growth hormone 1 gene GH1, comprise step:
(a) with the GH1 gene of GH1 gene-specific primer amplification sample, obtain amplified production; With
(b) detect in the amplified production whether have following single nucleotide polymorphism;
5158 G → A;
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 100-3000bp, and contains among the SEQ IDNO:1 the 5158th.
In a third aspect of the present invention, provide a kind of detection hypertensive test kit, it comprises the primer of specific amplification GH1 gene or transcript, more preferably, it is 100-3000bp that described primer amplification goes out length, and contains among the SEQ ID NO:1 the 5158th 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 5158th sudden change bonded probe;
(b) the 5158th sudden change restriction enzyme among the identification SEQ ID NO:1.
In another preference, described sudden change is selected from following single nucleotide polymorphism:
5158 G → A;
Wherein, the nucleotide position numbering is based on SEQ ID NO:1.
In a fourth aspect of the present invention, a kind of pharmaceutical composition is provided, it contains the GH1 albumen and the pharmaceutically acceptable carrier of safe and effective amount.
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 part SNP and the hypertension of GH1 gene are closely related, and found its new function: the change of GH1 will cause hypertension, wherein the association study result shows, (5158 G → A) there is significant difference (P＜0.05) in the distribution in case and control group, therefore can be used as to detect hypertensive specificity SNP at the SNP of the 5158th of GH1.Finished the present invention on this basis.
The GH1 gene
(growth hormone 1, detailed sequence GH1) can be the nucleotide sequence (can referring to network address http://www.ncbi.nlm.nih.gov/) of J03071 referring to accession number to the growth hormone 1 gene, shown in SEQ ID NO:1.
The GH1 genes encoding produces growth hormone protein, and being that the prepituitary gland excretory is a kind of contains 191 amino acid whose polypeptide.The population difference, its structure and composition also are not quite similar.The molecular weight of human growth hormone is that (≈ 2.15 * 10 for 21 500D
4U), contain two disulfide linkage (Niall HD et al.Proc Natl Acad Sci USA.1971 Apr; 68 (4): 866-70.).Its most of effect is by IGF-1 (a kind of contain 70 amino acid whose basic polypeptides) mediation.It regulates the expression of rhIGF-1 (IGF1).
RhIGF-1 can promote the propagation of vascular smooth muscle cell.Be reported in the essential hypertension philtrum, the blood plasma level of IGF1 is just relevant with blood pressure.(Andromico G et al.J Hypertens1993 Oct; 11 (10): 1097-101) .IGF-1 is not the unique medium for GH, but such as GH also stimulating platelet source property somatomedin (PDGF) in expression in many tissues such as cardiac muscular tissue and proto-oncogene c-myc.
GH can cause the Q volume of blood expansion by stimulating kidney to absorb, reduce mechanism such as atrial natriuretic polypeptins secretion, activation RAA (RAA) system, the generation of increase red corpuscle again, and left chamber preload is increased.
Although GH can cause water-sodium retention and Q volume of blood expansion, it can not make arteriotony increase, and obviously descends at gigantosoma patient peripheral vascular resistance on the contrary, shows as a kind of hyperkinetic state.This of GH/IGF-1 kind of hemangiectasis effect is that endotheliocyte is dependent, may be relevant with the synthetic nitrogen protoxide (NO) of its promotion endotheliocyte.This indicates that with some researchs GH level lower result in the hypertension individuality is consistent (Ji Naijun etc., hypertension magazine 2000 Jun, 8 (2): 137-138).
The inventor checks order to the almost whole zone in the GH1 gene, has found many SNP, and wherein most of SNP and hypertension susceptibility are also uncorrelated, yet association study shows 5158 G → A but are and the very high SNP of hypertension susceptibility cognation.This SNP is positioned at the promoter region of GH1, points out influentially to the transcriptional level of GH1, and then causes influencing the interaction of itself and rhIGF-1 (IGF1), and the hypertension susceptibility that finally causes the carrier is apparently higher than normal population.
Based on new discovery of the present invention, GH1 albumen or polypeptide have many-sided new purposes.These purposes include, but is not limited to: the direct disease (as hypertension) due to the low or forfeiture and be used to screen the material that promotes the GH1 protein function as pharmacological agent GH1 protein function, and as antibody, polypeptide or other part.The peptide molecule that can stimulate people GH1 protein function that can be used for seeking therapeutic value with the recombinant human GH1 protein screening peptide library of expressing.
On the other hand, the present invention also comprises people GH1 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 GH1 gene product or fragment.Preferably, refer to that those can combine with people GH1 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 GH1, comprise that also those do not influence the antibody of people GH1 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 GH1 gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing human GH1 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 GH1 protein function and the antibody that does not influence people GH1 protein function.Each antibody-like of the present invention can utilize the fragment or the functional zone of people GH1 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 GH1 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 GH1 can be used in the immunohistochemistry technology, detect people GH1 in the biopsy specimen proteic what and/or whether suddenly change.A kind of preferred anti-GH1 antibody is the antibody of the normal GH1 of nonrecognition GH1 but identification suddenlys change, perhaps discerns normal GH1 but the antibody of nonrecognition sudden change GH1.Utilize these antibody, the hypertension susceptibility that can carry out protein level easily detects.
Utilize GH1 albumen of the present invention,, can filter out with GH1 albumen interactional material takes place, as inhibitor, agonist or antagonist etc. by various conventional screening methods.
GH1 albumen of the present invention and antibody thereof, inhibitor, agonist, antagonist etc. when using (administration) in treatment, can provide different effects.Usually, can these materials are formulated in nontoxic, inert and the pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5-8 usually, and preferably pH is about 6-8, although the pH value can be with being changed to some extent by preparation Substance Properties and illness to be treated.The pharmaceutical composition for preparing can carry out administration by conventional route, comprising (but being not limited to): intramuscular, intravenously or subcutaneous administration.
Normal GH1 polypeptide can be directly used in disease treatment, for example, is used for hypertension therapeutic.When using GH1 albumen of the present invention, also can use the hypertensive medicament of other treatment simultaneously.
The present invention also provides a kind of pharmaceutical composition, and it contains GH1 albumen of the present invention and the pharmaceutically acceptable carrier or the vehicle of safe and effective amount.This class carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Pharmaceutical preparation should be complementary with administering mode.Pharmaceutical composition of the present invention can be made into the injection form, for example is prepared by ordinary method with the physiological saline or the aqueous solution that contains glucose and other assistant agents.Pharmaceutical composition such as tablet and capsule can be prepared by ordinary method.Pharmaceutical composition such as injection, solution, tablet and capsule should be made under aseptic condition.The dosage of activeconstituents is the treatment significant quantity, for example every day about 0.1 microgram/kg body weight-Yue 10 mg/kg body weight.In addition, polypeptide of the present invention also can use with the other treatment agent.
When making pharmaceutical composition, be that the GH1 albumen of safe and effective amount or its antagonist, agonist are applied to Mammals, wherein this safe and effective amount is usually at least about 0.1 microgram/kg body weight, and in most of the cases be no more than about 10 mg/kg body weight, preferably this dosage is about 0.1 microgram/kg body weight-Yue 100 micrograms/kg body weight.Certainly, concrete dosage also should be considered factors such as route of administration, patient health situation, and these all are within the skilled practitioners skill.
The proteic polynucleotide of people GH1 also can be used for multiple therapeutic purpose.Gene therapy technology can be used for treating because cell proliferation, growth or the metabolic disturbance due to the proteic expression of GH1 of the proteic nothing expression of GH1 or unusual/non-activity.The method that structure carries the recombinant viral vector of GH1 gene is found in existing document (Sambrook, et al.).Recombinant human GH1 gene can be packaged in the liposome in addition, and then is transferred in the cell.
Polynucleotide imports tissue or intracellular method comprises: directly be injected into polynucleotide in the in-vivo tissue; Or external by carrier (as virus, phage or plasmid etc.) earlier with the polynucleotide transfered cell in, again cell is transplanted in the body etc.
The invention still further relates to the diagnostic testing process of quantitative and detection and localization people GH1 protein level.These tests are known in the art, and comprise ELISA etc.
Whether having the proteic method of GH1 in a kind of detection test sample is to utilize the proteic specific antibody of GH1 to detect, and it comprises: sample is contacted with the GH1 protein specific antibody; Observe whether form antibody complex, formed antibody complex and just represented to exist in the sample GH1 albumen.
The proteic polynucleotide of GH1 can be used for the diagnosis and the treatment of GH1 protein related diseases.Aspect diagnosis, the proteic polynucleotide of GH1 can be used for detecting the proteic expression of GH1 GH1 abnormal exprssion whether or under morbid state.Can be used for the hybridization of biopsy specimen to judge the proteic abnormal expression of GH1 as the GH1 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 GH1 albumen and also can detect the proteic transcription product of GH1.
The sudden change that detects the GH1 gene also can be used for diagnosing hypertension.Detection can be at cDNA, also can be at genomic dna.The form of GH1 protein mutation comprises that the point mutation compared with normal wild type GH1 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 GH1 gene with GH1 gene-specific primer amplification sample, obtains amplified production; Detect then and whether have following single nucleotide polymorphism in the amplified production: 5158 G → A, wherein, nucleotide position is numbered based on SEQ ID NO:1.
Should understand, after the present invention has disclosed the SNP and hypertensive dependency of GH1 gene 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 5158 G → A 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 100-3000bp usually, preferably is 150-2000bp, more preferably is 200-1000bp.These amplified productions all should contain among the SEQ ID NO:1 the 5158th.
Because SNP of the present invention and hypertension have very high cognation, therefore not only can be used in early days diagnosing primary hypertension more exactly, and can make the 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.
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
Because blood pressure is the quantitative character that is subjected to multiple such environmental effects that polygene participates in, the inevitable problem that when analyzing genes involved, will consider genetic heterogeneity.Select the relatively single isolated crowd of genetic background will help to reduce the influence of genetic heterogeneity, simultaneously because these colonies have comparison fixed living environment and more consistent living habit make Effect of Environmental reduce greatly as the material of research multigenic disease.Select such colony,, still therefrom choose family and carry out pedigree analysis, all help to improve the detection sensitivity in hypertension susceptible gene site no matter be to be used for the linkage disequilibrium analysis.Therefore selected in the present embodiment isolated population is as research object (isolated population has the relative uniformity of genetic background, the advantage that founder's number is fewer).
On the basis of informed consent random collecting 324 ages at the Han nationality's individuality more than 50 years old, all come from Dabie Mountain, Yuexi County, Anhui Province and ring the intestines town.80% individuality has only 6 surnames, and founder's number should be fewer, and other individuality of identity comes from same founder.The hypertension sample of selecting for use requires systolic pressure to be not less than 140mmHg, and diastolic pressure is not less than 90mmHg, measures blood pressure and averages for twice.
Select 11% being positioned at the top that blood pressure distributes (37 examples are individual wherein; SBP＞178mmHg) and 7% (22 individualities that are positioned at the lowermost end that blood pressure distributes are arranged; SBP＜104mmHg) is totally 59 individualities, carries out the detection of SNP by the method for direct order-checking.
1.2 experimental technique and result
1.2.1 DNA extraction
Extract DNA with conventional phenol chloroform method from people's blood, 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 GH1 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 | gggtggtatctctggctgac | ?????2 |
| Antisense primer | ccaaacctgagggttagtgc | ?????3 |
1.2.3 the pcr amplification of GH1 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 401bp.
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)) 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 (the http://droog.mbt.washington.edu/Polyphred.html of Washington, DC university) confirm.
As a result, find to exist following SNP:5158 position G → A.
1.2.5 SNP gene type and association analysis
Carry out the SNP gene type with direct unidirectional sequencing.Promptly in hypertensive patient and normal arterial pressure control group, carry out somatotype and association analysis.
Carry out chi square test after allelic frequency statistics is come out,, determine whether that tentatively allelic frequency and blood pressure level are chain by blood pressure the highest 11% and 7% minimum individual data items are compared.
The frequency of G type SNP in the hypertension individuality that found that among the SEQ ID NO:1 5158 is 13%, and the frequency in the ypotension crowd is 37.5%.Difference on the frequency between the two is 24.5%, has confirmed among the SEQ ID NO:1 5158 G/A type SNP and the hypertensive dependency that exists.
Embodiment 2
Essential hypertension susceptibility detection kit
As described in embodiment 1, sudden change and the high blood pressure disease of 5158 G → A 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 GH1 gene-specific primer with patient.
Prepare a test kit (100 person-times), it contains:
| Title | Sequence | Concentration |
| Adopted primer is arranged | SEQ?ID?NO：2 | 100pmol |
| Antisense primer | SEQ?ID?NO：3 | 100pmol |
| The PCR reaction solution | Contain Taq enzyme dNTP magnesium ion PCR reaction buffer | |
Extract male sex's patients'blood 3ml to be detected, use ordinary method (or using specific test kit) from blood, to extract DNA.PCR primer in the hypertension 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 5158 G → A.
Detected result, the hypertension susceptibility that contains the detected object of 5158 G → A is higher than normal population.
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〉Fudan University
Research Center of Shanghai Human Genome
＜120〉dependency of growth hormone 1 gene and essential hypertension
<130>040176
<160>3
<170>PatentIn?version?3.1
<210>1
<211>7140
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<400>1
gaattcctgg?gcctggggct?gtggcagctg?cctcgtccct?tcacctcctg?gcttattctc?????60
tccctccata?tcttagcaat?ttcttcatgg?gaatgtcccc?aattagaaat?ttctattata????120
ccattatatt?accaacatat?atatatatac?ctggccgggc?gcggtggctc?atgcctgtaa????180
tcccagcatt?ttggtaggcc?aaggcgggtc?ggatcacctg?aggtcaggag?ttcgagggcc????240
agcctgatga?ccatggtgaa?accccatctc?tactaaaaat?acaaaattaa?tcgggcatgg????300
tggcacatgc?ctgtaatccc?agctactcgg?gaggctgagg?caggagaatc?gcttaaaccc????360
aggaagtgga?ggtttcagtg?agctgagatt?gtgccattgc?actccagcct?gggtaacaag????420
agcaaaactc?catcaaaaaa?aataatatat?gtatatatat?attacaattt?tatatatata????480
tacacattat?gtaatttttt?ttaccatttt?atatatatac?attacgtaat?ggtaaatgtt????540
ttctctgccc?cccagtagat?tgttagctcc?agaagagaag?gatcatgtct?ttggtttatc????600
tagatatgtc?catcggcctg?gtacagtctc?tggcccatgt?tataggcaac?aactacttgt????660
agaatcggtg?aatgcatgaa?tagaagaatg?agtgaatgaa?tgaatagacg?aaaggcagaa????720
atccagcctc?aggccgggcg?tgggtggctc?acacctgtaa?tcccagcact?ttgggaggcc????780
gaggcgggtg?gatcacaaga?tcaagagatg?gagaccaacc?tggctaacac?agtgaaaccc????840
cgtctctact?aaaaatacaa?aacattagcc?gggcatggcg?gtaggctcct?gtagtccgag????900
ctactaggga?gcctgaggca?ggagaatggc?atgaacccag?gggggcggac?gttgcagtga????960
gccaagatcg?cgccactgca?ctccagcctg?ggcgacagag?caagactcgg?tctcaaaaaa???1020
aaaaaaaaaa?gaaatccagc?ctcaaagagc?ttacagtctg?gtaagaggaa?taaaatgtct???1080
gcaaatagcc?acaggacagg?tcaaaggaag?gagaggctat?ttccagctga?gggcacccca???1140
tcaggaaagc?accccagact?tcctacaatt?actagacaca?tctcgatgct?tttcacttct???1200
ctatcaatgg?atcgtctccc?tggagaataa?tccccaatgt?gaaattactt?agcacgtcaa???1260
gttaggtaga?tccttgtgta?cttcttggtt?gttcagagat?catcaccagt?gcaacatccc???1320
ccatcataca?cagcagtgtc?ttgcccctct?cctccccaag?ccttccgagg?cccttcctcc???1380
gtgcctgaac?cccctggaca?tatcatgtgg?caaactgaag?ggtccaacga?gatacaggaa???1440
gtgaaacacg?atgtacactg?aaacgtgcaa?tacaaatatg?cagcatgaag?tgcctcggtt???1500
cactaacccg?agctatgctg?ggtgcttctt?ttctaccact?ttccttaatg?cctatggaca???1560
cctcattctg?tggctgaagt?tccttgtgtt?caattccccc?catcttcatt?gaacatcctc???1620
tgtgcaggga?cttgacccct?gtcctgctag?ctttgcactg?aggcaagttt?tgtccatgcc???1680
tagtagtgcc?acatctttac?tagatgaggt?ttctaaagag?cttggcatgg?aaggaaagcc???1740
tggggggcct?tagaagccat?cacttaggaa?ctgggagagc?tccaggcaag?ccaccttatc???1800
tctttgggcc?tcagtatcct?cactcacatc?cccatggtca?gtagagtgcc?aagcacatgg???1860
tggacacgca?gaagtattga?tcatcttcct?cacctcccct?aggagagccc?ttggaatccc???1920
catataatat?tctctgaaga?cctcaatcca?atgagccgag?taattgactc?ggtagcaaac???1980
tcatggaaag?gacattagaa?gccaaaagga?ggtagagtat?gtcccagtac?aataaccagc???2040
ctctgatctc?aaggaagaaa?gaacagagct?gcaggtgaga?agtgtgcgcc?tcaaatcacc???2100
aaagtgagag?tggaaggagc?acccaccagt?cccttggagg?cgatccctaa?gaccggtgag???2160
aatggcttcg?aaaaatgtga?tcgtcctcaa?ccccacagtc?ttgaacctaa?caggagatct???2220
tgaagcctga?aagacaccac?tttaggatca?acagcagatc?tgtgactttc?cacagcaggc???2280
acacagaacc?ctagagttag?tcgaggttgg?acataggaag?ggcttccaaa?cacacagaga???2340
aattcatgga?tcctaaatta?taaagggagg?ttctttaaaa?gaaccaagat?gattctgaga???2400
tttatcctga?gctttttttt?tttttttttt?tttggatgca?gagtctcgct?ctgtcgccca???2460
ggctggagtg?cagtggcgtg?atctcggcta?actgcaacct?ccacctcgcg?ggttcaagca???2520
attccctgcc?tcggcctcct?gaatagctgg?gattacaggc?gcctgccacc?acacctggct???2580
aatttttgta?tttttagcag?agacgggatt?tcaccatctt?ggccaggctg?gtcttgaact???2640
cctgatctca?tgatccaccc?accttggcct?ccctaagtgg?tgggattaca?ggcgtgtgcc???2700
caacttttcc?tgagcctttt?gaggctgaca?ccagaggtag?aagcccagcc?tctccccact???2760
ggccatgtgg?ggagaggctc?cagcctgcag?caaccaggga?tctggcctca?agtgatgccc???2820
caacagtggg?cgacttccca?gtactgttag?gagaatccca?agtctaattc?aaagttgatt???2880
ttttactagc?aattaatgat?agacatggtc?tccattgctc?aaggctctgg?gaagatctag???2940
actagagaaa?acgatcaccg?acttctacca?cacctgtggg?cctcagttct?tccctctggt???3000
ccatggttac?cacagtaacc?ccttgtaaag?gtgtttcccc?aggggtccct?agagtcctct???3060
gagtcaccat?aattctgggt?caacagaaat?ggagaggtaa?gaggagacac?tcccctgccc???3120
tggctggtcc?cacgtgttca?tggcagtaac?ctgtctgggg?agcctcgcca?cccctgtgtc???3180
cacctgcaga?gttatgcagt?ggtccccaac?tagggcccct?gccaccctca?ttcctaaggg???3240
aggctggaga?cttcttccat?ggccgaaaat?ccacatctaa?gtccccggca?ctagcaaaaa???3300
ggccctgtca?tgggggctcc?ctgccccagt?tgagattgga?ggaacatgga?agaagctaaa???3360
ataactaaat?aactcagtag?catcacacta?cctgctctgg?agaaaaaaaa?atttaatatt???3420
attatttttt?gttttttgag?atggagttct?gctctgctgc?acaggctgga?gtgcagtggc???3480
gcaatcttgg?ctcacggcaa?cctctgcctc?ctgagggttc?aagtgattct?cccgcctcag???3540
cctcccgagt?agttgggatt?acagctcatg?ccaccacgcc?cagctaattt?ttgtactttt???3600
agtagagatg?gagttttgcc?atgttggcta?gtctggcctt?gaactcctga?cctcaagtga???3660
tccacccacc?tcaaagccac?ccaaagtttg?gggattacaa?gcgtgagcca?ctgtgtccgg???3720
cctggagaaa?ggactttaaa?tgacgcaatg?taggaagagc?aaggttgtgg?agatctgctg???3780
ccctggctga?ggtagctcat?gcaatcagtc?tctctgagcc?acagtctctt?gatctgtgaa???3840
atcggaagaa?aataatacct?ccttcacaag?acaagtggca?ggtcagatgt?gagaatgcac???3900
aggcaggccc?tcggcaactg?gaaaagctct?atacagatct?gaaaaggagg?aggagaaaaa???3960
agaggagggg?cttccatggc?tggacagggc?atctttcttt?ttctttttct?tttttttttt???4020
tttttttttt?ttgaggtgga?gtcttgctct?gttgccaagg?ttggagtgca?gcagcacgat???4080
ctccgctcac?tgcaagctct?gcctcccgga?ttcacgccat?tctcctgcct?cagcctcccg???4140
agtagctggg?aatacacggt?ccgccactac?gcccagctaa?cttttttgca?tttttagtac???4200
agagtggatt?tcacctggtt?agccaagatg?gtcttgatct?actgacctcg?tgatccgccc???4260
gcctcggcct?cccaaagtgc?tgggattaca?ggcatgagcc?accgcgccca?gcctgataga???4320
gcatctttcg?gcgtgatgtg?ttctgagttc?caaagctgag?gaagagactc?aaatcttcaa???4380
gagctcttct?aactttgaga?ttctctgatg?gtttcagggc?tatgggagga?agagcttgtg???4440
gtccgtgtct?gctcccggga?tttctgtttc?ttggtttgtg?tctctgctgc?aagtccaagg???4500
agctggggca?ataccttgag?tctgggttct?tcgtccccag?ggacctgggg?gagccccagc???4560
aatgctcagg?gaaaggggag?agcaaagtgt?ggggttggtt?ctctctagtg?gtcagtgttg???4620
gaactgcatc?cagctgactc?aggctgaccc?aggagtcctc?agcagaagtg?gaattcagga???4680
ctgaatcgtg?ctcacaaccc?ccacaatcta?ttggctgtgc?ttggcccctt?ttcccaacac???4740
acacattctg?tctggtgggt?ggaggttaaa?catgcgggga?ggaggaaagg?gataggatag???4800
agaatgggat?gtggtcggta?gggggtctca?aggactggct?atcctgacat?ccttctccgc???4860
gttcaggttg?gccaccatgg?cctgcggcca?gagggcaccc?acgtgaccct?taaagagagg???4920
acaagttggg?tggtatctct?ggctgacact?ctgtgcacaa?ccctcacaac?actggtgacg???4980
gtgggaaggg?aaagatgaca?agccaggggg?catgatccca?gcatgtgtgg?gaggagcttc???5040
taaattatcc?attagcacaa?gcccgtcagt?ggccccatgc?ataaatgtac?acagaaacag???5100
gtgggggcaa?cagtgggaga?gaaggggcca?ggtataaaaa?gggcccacaa?gagaccagct???5160
caaggatccc?aaggcccaac?tccccgaacc?actcagggtc?ctgtggacag?ctcacctagc???5220
ggcaatggct?acaggtaagc?gcccctaaaa?tccctttggg?cacaatgtgt?cctgagggga???5280
gaggcagcga?cctgtagatg?ggacgggggc?actaaccctc?aggtttgggg?cttctgaatg???5340
tgagtatcgc?catgtaagcc?cagtatttgg?ccaatctcag?aaagctcctg?gtccctggag???5400
ggatggagag?agaaaaacaa?acagctcctg?gagcagggag?agtgctggcc?tcttgctctc???5460
cggctccctc?tgttgccctc?tggtttctcc?ccaggctccc?ggacgtccct?gctcctggct???5520
tttggcctgc?tctgcctgcc?ctggcttcaa?gagggcagtg?ccttcccaac?cattccctta???5580
tccaggcttt?ttgacaacgc?tatgctccgc?gcccatcgtc?tgcaccagct?ggcctttgac???5640
acctaccagg?agtttgtaag?ctcttgggga?atgggtgcgc?atcaggggtg?gcaggaaggg???5700
gtgactttcc?cccgctggga?aataagagga?ggagactaag?gagctcaggg?tttttcccga???5760
agcgaaaatg?caggcagatg?agcacacgct?gagtgaggtt?cccagaaaag?taacaatggg???5820
agctggtctc?cagcgtagac?cttggtgggc?ggtccttctc?ctaggaagaa?gcctatatcc???5880
caaaggaaca?gaagtattca?ttcctgcaga?acccccagac?ctccctctgt?ttctcagagt???5940
ctattccgac?accctccaac?agggaggaaa?cacaacagaa?atccgtgagt?ggatgccttc???6000
tccccaggcg?gggatggggg?agacctgtag?tcagagcccc?cgggcagcac?agccaatgcc???6060
cgtccttccc?ctgcagaacc?tagagctgct?ccgcatctcc?ctgctgctca?tccagtcgtg???6120
gctggagccc?gtgcagttcc?tcaggagtgt?cttcgccaac?agcctggtgt?acggcgcctc???6180
tgacagcaac?gtctatgacc?tcctaaagga?cctagaggaa?ggcatccaaa?cgctgatggg???6240
ggtgagggtg?gcgccagggg?tccccaatcc?tggagcccca?ctgactttga?gagctgtgtt???6300
agagaaacac?tgctgccctc?tttttagcag?tcaggccctg?acccaagaga?actcacctta???6360
ttcttcattt?cccctcgtga?atcctccagg?cctttctcta?caccctgaag?gggagggagg???6420
aaaatgaatg?aatgagaaag?ggagggaaca?gtacccaagc?gcttggcctc?tccttctctt???6480
ccttcacttt?gcagaggctg?gaagatggca?gcccccggac?tgggcagatc?ttcaagcaga???6540
cctacagcaa?gttcgacaca?aactcacaca?acgatgacgc?actactcaag?aactacgggc???6600
tgctctactg?cttcaggaag?gacatggaca?aggtcgagac?attcctgcgc?atcgtgcagt???6660
gccgctctgt?ggagggcagc?tgtggcttct?agctgcccgg?gtggcatccc?tgtgacccct???6720
ccccagtgcc?tctcctggcc?ctggaagttg?ccactccagt?gcccaccagc?cttgtcctaa???6780
taaaattaag?ttgcatcatt?ttgtctgact?aggtgtcctt?ctataatatt?atggggtgga???6840
ggggggtggt?atggagcaag?gggcaagttg?ggaagacaac?ctgtagggcc?tgcggggtct???6900
attcgggaac?caagctggag?tgcagtggca?caatcttggc?tcactgcaat?ctccgcctcc???6960
tgggttcaag?cgattctcct?gcctcagcct?cccgagttgt?tgggattcca?ggcatgcatg???7020
accaggctca?gctaattttt?gtttttttgg?tagagacggg?gtttcaccat?attggccagg???7080
ctggtctcca?actcctaatc?tcaggtgatc?tacccacctt?ggcctcccaa?attgctggga???7140
<210>2
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>2
gggtggtatc?tctggctgac????????????????????????????????????????????????20
<210>3
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>3
ccaaacctga?gggttagtgc????????????????????????????????????????????????20
Claims (10)
1. whether a vitro detection sample exists the method for the single nucleotide polymorphism of growth hormone 1 gene GH1, it is characterized in that, comprises step:
(a) with the GH1 gene of GH1 gene-specific primer amplification sample, obtain amplified production; With
(b) detect whether there is following single nucleotide polymorphism in the amplified production:
5158 G → A;
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 100-3000bp, and contains among the SEQ ID NO:1 the 5158th.
4. one kind is detected hypertensive test kit, it is characterized in that it comprises the primer of specific amplification GH1 gene or transcript, and it is 100-3000bp that described primer amplification goes out length, and contains among the SEQ ID NO:1 the 5158th 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 5158th sudden change bonded probe;
(b) the 5158th 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:
5158 G → A;
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 hypertension susceptibility of individuality is diagnosed is characterized in that it comprises step:
Detect this individual GH1 gene, transcript and/or albumen, and compare with normal GH1 gene, transcript and/or albumen,
There are differences and just show that this individuality suffers from hypertensive possibility and be higher than normal population.
9. method as claimed in claim 8 is characterized in that, detection be gene or the transcript of GH1, and with normal GH1 nucleotide sequence comparing difference.
10. method as claimed in claim 9 is characterized in that, described difference is following single nucleotide polymorphism:
5158 G → A;
Wherein, the nucleotide position numbering is based on SEQ ID NO:1.
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|---|---|---|---|
| CN 200410016520 CN1661049A (en) | 2004-02-25 | 2004-02-25 | Relativity between gene of growth hormone 1 and essential hypertension |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410016520 CN1661049A (en) | 2004-02-25 | 2004-02-25 | Relativity between gene of growth hormone 1 and essential hypertension |
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| Publication Number | Publication Date |
|---|---|
| CN1661049A true CN1661049A (en) | 2005-08-31 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200410016520 Pending CN1661049A (en) | 2004-02-25 | 2004-02-25 | Relativity between gene of growth hormone 1 and essential hypertension |
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| Country | Link |
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| CN (1) | CN1661049A (en) |
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