CN1661070A - Relativityl between converzyme gene of angiotensin I and essential hypertension - Google Patents

Abstract

A process for testing the susceptibility of primary hypertension includes detecting if there are variations in angiotonin I converzyme gene ACE, transceipt and/or protein of an individual, and determining its high susceptibility if there are. Its reagent kit is also disclosed.

Description

The dependency of angiotensin i-converting enzyme gene and essential hypertension

Technical field

The present invention relates to molecular biology and medical field.Relate more specifically to the angiotensin i-converting enzyme gene (angiotensin I converting enzyme, single nucleotide polymorphism ACE) (singlenucleotide 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 ACE gene and essential hypertension dependency, more do not confirm the SNP of ACE 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 ACE gene, transcript and/or albumen, and compare with normal ACE 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 ACE, and with normal ACE nucleotide sequence comparing difference.

In another preference, described difference is following single nucleotide polymorphism:

5525 C → G;

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 angiotensin i-converting enzyme gene A CE, comprise step:

(a) with the ACE gene of ACE gene-specific primer amplification sample, obtain amplified production; With

(b) detect whether there is following single nucleotide polymorphism in the amplified production:

5525 C → G;

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 5525th.

In a third aspect of the present invention, provide a kind of detection hypertensive test kit, it comprises the primer of specific amplification ACE 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 5525th 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 5525th sudden change bonded probe;

(b) the 5525th sudden change restriction enzyme among the identification SEQ ID NO:1.

In another preference, described sudden change is selected from following single nucleotide polymorphism:

5525 C → G;

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 ACE protein inhibitor 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 ACE gene are closely related, and found its new function: the change of ACE will cause hypertension, wherein the association study result shows, (5525 C → G) 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 5525th of ACE.Finished the present invention on this basis.

The ACE gene

Angiotensin i-converting enzyme gene (angiotensin I converting enzyme, ACE) detailed sequence can be the nucleotide sequence (can referring to network address http://www.ncbi.nlm.nih.gov/) of AF118569 referring to accession number, shown in SEQ ID NO:1.

The angiotensin i-converting enzyme of angiotensin i-converting enzyme coded by said gene (being also referred to as " Zinc metallopeptidase Zace1 ") has and promotes angiotensin I to be converted into to have active phosphorylation polypeptide angiotensin II.Angiotensin II is that a kind of effective blood vessel boosts and the polypeptide of aldosterone Class Activation, and it can controlling blood pressure and mobile electrobalance.This gene plays a part crucial in the system of feritin-Angiotensin (RAS).

RAS is the english abbreviation of Renin-Angiotensin System (Renin-Angiotensin-System), and its major function is the stability of regulating human blood-pressure, moisture, ionogen and maintenance human internal environment.RAS both had been present in the recycle system, also was present in the tissues such as vessel wall, heart, maincenter, kidney and suprarenal gland, and fellowship is to the adjusting of target organ.Feritin is mainly from kidney in the circulation of blood, it can be converted into angiotensin I to the proangiotensin that is mainly derived from liver, under the effect of angiotensin-converting enzyme, be converted into Angiotensin II (AngII) again, then by organizing the nervous plain II acceptor of medium vessels to play a role.There is local RAS in many tissues, in the function of corresponding organ, tissue, cell is regulated, plays an important role.If the generation of nervous plain II is too much, in circulation, just show as hypertension.Cause then that in tissue structure reinvents, reinvent when the weave construction of certain organ and to develop into to a certain degree, the physiological function that this organ just can't be brought into normal play, in the clinical myocardial infarction that will show corresponding symptom such as heart, the uremia of kidney, the palsy of brain, eyes blind or the like.Therefore, Angiotensin II is not only and is caused hypertensive reason, also is the immediate cause that causes organ injuries such as the heart, brain, kidney.

The existence that the inner 287bp Alu repeated sequence of this gene is paid close attention in many tests of having carried out whether, on the dependency of cardiovascular physiopathology.2002, people such as Procopciuc found a SNP on No. 2 exons of ACE gene, M235T (Procopciuc et al.J Cell Mol Med.2002Apr-Jun; 6 (2): 245-50).This SNP has caused a transformation from the methionine(Met) to the Threonine.In 38 essential hypertension people and 21 normotensive people's contrast, they find that M235T is 81.57%, and is 66.66% in normal control in patient crowd.Because there is frequency difference in M235T in patient crowd and normal population are compared, illustrated that the ACE gene is hypertensive candidate gene.

Animal experiment shows that the mouse of ACE genetically deficient has ypotension, kidney disorder, female sterile isophenous (Ramaraj et al.J.Clin.Invest.102:371-378,1998).

The inventor checks order to the almost whole zone in the ACE gene, has found many SNP, and wherein most of SNP and hypertension susceptibility are also uncorrelated, yet association study shows 5525 C → G but are and the very high SNP of hypertension susceptibility cognation.This SNP is arranged in No. 4 intron of ACE, and prompting is influential to the montage process after the transcribing of ACE, and then causes influencing the activity of ACE, and the hypertension susceptibility that finally causes the carrier is apparently higher than normal population.

Based on new discovery of the present invention, ACE albumen or polypeptide have many-sided new purposes.These purposes include, but is not limited to: be used to screen the material that promotes the ACE protein function, as antibody, polypeptide or other part.The peptide molecule that can stimulate people ACE protein function that can be used for seeking therapeutic value with the recombinant human ACE protein screening peptide library of expressing.

On the other hand, the present invention also comprises people ACE 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 ACE gene product or fragment.Preferably, refer to that those can combine with people ACE 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 ACE, comprise that also those do not influence the antibody of people ACE 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 ACE gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing human ACE 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 ACE protein function and the antibody that does not influence people ACE protein function.Each antibody-like of the present invention can utilize the fragment or the functional zone of people ACE 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 ACE 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 ACE can be used in the immunohistochemistry technology, detect people ACE in the biopsy specimen proteic what and/or whether suddenly change.A kind of preferred anti-ACE antibody is the antibody of the normal ACE of nonrecognition ACE but identification suddenlys change, perhaps discerns normal ACE but the antibody of nonrecognition sudden change ACE.Utilize these antibody, the hypertension susceptibility that can carry out protein level easily detects.

Utilize ACE albumen of the present invention,, can filter out with ACE albumen interactional material takes place, as inhibitor, agonist or antagonist etc. by various conventional screening methods.

ACE 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 ACE inhibitor can be directly used in disease treatment, for example, is used for hypertension therapeutic.When using ACE protein inhibitor 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 ACE protein inhibitor 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 ACE 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 ACE 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 ACE of the proteic nothing expression of ACE or unusual/non-activity.The method that structure carries the recombinant viral vector of ACE gene is found in existing document (Sambrook, et al.).Recombinant human ACE 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 ACE protein level.These tests are known in the art, and comprise ELISA etc.

Whether having the proteic method of ACE in a kind of detection test sample is to utilize the proteic specific antibody of ACE to detect, and it comprises: sample is contacted with the ACE protein specific antibody; Observe whether form antibody complex, formed antibody complex and just represented to exist in the sample ACE albumen.

The proteic polynucleotide of ACE can be used for the diagnosis and the treatment of ACE protein related diseases.Aspect diagnosis, the proteic polynucleotide of ACE can be used for detecting the proteic expression of ACE ACE abnormal exprssion whether or under morbid state.Can be used for the hybridization of biopsy specimen to judge the proteic abnormal expression of ACE as the ACE 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 ACE albumen and also can detect the proteic transcription product of ACE.

The sudden change that detects the ACE gene also can be used for diagnosing hypertension.Detection can be at cDNA, also can be at genomic dna.The form of ACE protein mutation comprises that the point mutation compared with normal wild type ACE 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 ACE gene with ACE gene-specific primer amplification sample, obtains amplified production; Detect then and whether have following single nucleotide polymorphism in the amplified production: 5525 C → G, 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 ACE 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 5525 C → G 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 5525th.

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 ACE 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	gcctgtgtctcagatctcacc	????2
Antisense primer	cagaggcaaagaggagcatc	????3

1.2.3 the pcr amplification of ACE 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 395bp.

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:5525 position C → G.

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 5525 is 22%, and the frequency in the ypotension crowd is 11%.Difference on the frequency between the two is 11%, has confirmed among the SEQ ID NO:1 5525 G/C 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 5525 C → G 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 ACE 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 5525 C → G.

Detected result, the hypertension susceptibility that contains the detected object of 5525 C → G 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 angiotensin i-converting enzyme gene and essential hypertension

<130>040158

<160>3

<170>PatentIn?version?3.1

<210>1

<211>24070

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>1

tgcccagaca?gccttatctc?tttcctacct?ttcaaggtta?ttgtaaatac?caaattagat??????60

tgtttataca?caagaattta?gcactaaagc?actatacaaa?tgtaagctat?ttatttctat?????120

ttatccttct?ccttcatgaa?taagacccta?aaaatagaag?atatttttaa?tttttactca?????180

ctgggctcaa?ggttgcagtg?tctgtattat?tgcaaattcc?aaattaatga?agtctggctc?????240

ttcttatatt?attcctgcaa?aaggctgtgt?gctcaccccc?cggagtgtga?atacgagtgt?????300

gggtcatttc?ctctttcctc?tgcacccttc?cttcgatgag?gttttgccct?gtgctaggca?????360

ccatgctaaa?ctctgagaaa?accacagaga?acaaggcaga?cgccatccct?gccctccagt?????420

aacatacaat?ttagtgatga?agctggggga?tttaacaagc?cattctaata?aagcgttaca?????480

gtgagggagg?tgcagggtga?tgcctccagc?agccctggtg?tcagctgctc?caggtccagg?????540

ggaagacttc?cattatcttc?caaccctgtc?ggaagtggag?gcggaggctg?tttattgctg?????600

acacagtccc?taacccaggg?tctatagaca?ttgtggaaat?gccttggagt?cagacgggag?????660

aatgaaccag?cagaagcaat?gcccgccctc?caccctcctg?aagagggttc?tcaggaactc?????720

tttggaggcg?aggccagcct?ctggctgagg?gcctctggat?acaggttagg?cctcaggctc?????780

ttctcctctc?tactcatctc?tcctcccttg?gcccctcctt?cagaggctga?cagagcccca?????840

ctctcatctc?ttccccaccc?aagcctcttt?ccacagaaag?actgcttcct?cccaggagac?????900

agcagctcat?ttgcacacag?acacccacag?ccctcaaagc?ctggaaggcc?aagctgttag?????960

gacccctgag?agcagggtgg?ctcctgggag?gagagcccag?gccaccacct?tgccctccct????1020

ggcccctggc?cttcgatggg?gctgctctga?tcacaaacgt?ccaccaacgt?agccggccca????1080

gaagtgcacc?catgtcctct?ggtatccact?ggctctccaa?gccaaactgg?gcagggagga????1140

gttgtgaggg?aaaactgcag?gtcaggaggg?aggctggcaa?agcgggccag?ggccaggcct????1200

gaccccagct?ctcctctccc?ggccccactg?ccggccagtg?tttaacaagg?ccctgccttc????1260

tccctctagt?gctagggaca?gccaccttct?tcctctcccc?accgccccct?ctcccctgca????1320

acacgtcatc?tgacaagtca?gtgcgatctc?actggaggtg?catctcacag?gaacgcgggg????1380

tcacagcctc?ctgcacacac?tccatgctgc?acagcaaggt?gcacgtgtcc?ctcagagccc????1440

cagacaccat?cccccactca?cccagaagcc?caagtgattc?ccaacagccc?ccagcagcct????1500

aatgggttgg?ggtcttggga?gcagctgtcc?ctggctcctt?ccctgatccc?accgcccagc????1560

ctcaccccac?ggttcctcca?ttgccccacc?tcccactgcg?ccgccgggcc?tctgccaggg????1620

tcaaggggct?tcccccctct?ggcagcagac?gccatggtgc?cgaggtggcc?tccacaaccg????1680

ccctgtgcgc?caataggaca?agactgtcct?ccctccccca?cacttgtcac?tttgagggac????1740

acgtggatga?gacaggaaaa?cacaggggag?tgtggagacc?tgaggtgact?tggagcaagc????1800

ctctcaacct?gagcggcaat?ttcttcatct?gtaaaatgag?ggggttgttc?tcatctctga????1860

ggctttgtgt?cgctctcaaa?gcctgctagc?ctcgggttct?aggactctgt?tgggatcgtg????1920

tgtgatgttt?tctgctgagc?gactggcagc?ctgtgtcctc?ggggggaaag?agggcaggcg????1980

ctccaaagct?cctgcgctct?gtggctcccc?ctccctcgca?gccccaagcc?ccaggtgtgc????2040

cggccgccct?gagcccctcc?agcacctccc?ggaggcgcct?gcaagacacc?taaggtcccc????2100

gcctccctcc?tctccccccc?gccacacccc?tacccccggc?aggcgacgtc?cccgcccctc????2160

gaccatggcc?tggtgaagaa?gccggccagg?cccgatcagc?cccatccccg?ccgcacgagc????2220

ggcgcctgcg?gacagctcct?ggggccccgg?ccttgtcact?ccggaggcgg?gaggctccgg????2280

ggggtcgggc?tgggaagatc?gagccggagg?ccgctaggct?cccaggcccc?ggccgaggct????2340

gcgcggccgc?acggtgggca?ggctcgggtg?ttccggcaaa?ctgccgggtc?cccatcttca????2400

aaagagagga?ggccctttct?ccagcttcct?ctgcgggagc?ccgacccagc?cccatcccgc????2460

cacccccggg?ctgcacctcg?gcccctcccc?ggcccgcgcc?cctgcccggg?gcgggccagg????2520

aacctcggcc?cgcgccgctg?gggactttgg?agcggaggag?gaagcgcggc?ggggcggggg????2580

cgggggtgtg?tcgggtttta?taacccgcag?ggcggccgcg?gcgcaggaga?aggggcagag????2640

ccgagcaccg?cgcaccgggt?catgggggcc?gcctcgggcc?gccgggggcc?ggggctgctg????2700

ctgcccctgc?cgctgctgtt?gctgctgccg?ccgcagcccg?ccctggcgtt?ggaccccggg????2760

ctgcagcccg?gcaacttttc?tgctgacgag?gccggggcgc?agctcttcgc?gcagagctac????2820

aactccagcg?ccgaacaggt?gctgttccag?agcgtggccg?ccagctgggc?gcacgacacc????2880

aacatcaccg?cggagaatgc?aaggcgccag?gtgggcgccc?gggcccgggc?gggggcgggg????2940

cggggccgcg?gcggccaatc?acagcacgcg?gccggcttgt?ggggcgggca?ggctggcgcc????3000

cccgacccga?accccacccc?gaccccggac?cctcgccccg?acagtcagcc?gcggggcccg????3060

agcgccgggc?tgcgcgcacg?gcctacgctc?ccagcatgca?cgagttggat?ggatgaaggt????3120

ggctgctccc?aggccgcgcc?cgccttcgcc?gaaggtgctg?ggcttggctc?tggggccccc????3180

gcgctctcgg?gcagctgcct?tctcacctcc?ggacgctgtc?gctgtcaccg?tcaccgcact????3240

gcactgtcca?tccaccctcc?actcgcccgg?cctcttttct?ggtcccaatt?tctgctccac????3300

cattcccatg?aggcagattc?cctccagaag?gaggaagcgc?ggcttccgca?aactaaggtc????3360

tcccgcaggg?atctccccag?ggcccgggct?ctggaagccc?ttggccttcc?tcccctcccc????3420

cagcaccgtg?gcttctcctt?tatggcctgc?atctaagcag?ggtcctacac?cctccctgcc????3480

ctcctggtgc?ccaataggag?gaagcagccc?tgctcagcca?ggagtttgcg?gaggcctggg????3540

gccagaaggc?caaggagctg?tatgaaccga?tctggcagaa?cttcacggac?ccgcagctgc????3600

gcaggatcat?cggagctgtg?cgcaccctgg?gctctgccaa?cctgcccctg?gctaagcggc????3660

agcaggtggg?ctgagggctg?aggcagagct?cgggggcggc?ctcctagtgc?cccatcgtgg????3720

gggtcggggg?agagcagccc?atcagggagg?gaggaaccct?gggatccaca?tgggccctga????3780

cagaagggaa?agcccaggta?agcacagaat?ggctttctga?gcattgattt?ttcttggaga????3840

tggggtgggg?agttactttc?tgttaaagga?agcattctgg?agtaggaagc?caaattcaaa????3900

tacacttctc?cctaggctgg?tttatgagct?tctttggaag?agttgagaag?ggctgggcgt????3960

ggtggctcaa?gcctgtaatc?ccagcacttt?gggaggctga?ggtgggcgca?tcgcttgagc????4020

ccaggagttc?aagaccagcc?tggccaacat?ggcaaaacct?cgtctctaca?aaaaaaaaat????4080

agctgggctt?ggtggtgcgt?gcacctacag?tcccagctac?tcttgaaact?gagggggaag????4140

gatcacctga?gcccaggagg?tcaaggctac?agtgagctgt?gattgcacta?ctgcacccca????4200

gcctgcgtga?cagagtgaga?cctcccccca?aaaaaaagag?agagagaaaa?ggttgagaaa????4260

gactgggaag?tcaccaaagc?cagagaatgg?gagggatctg?ccctcactgc?agggtggtgc????4320

caagctggga?cttgacccag?accctgactt?tcaggactcc?tgtcccccac?tccacaggct????4380

gcctccactg?gcaggggact?cagaagtgat?ccggtcacac?taagtgacac?ttagtgatca????4440

gaagtgcccc?ggtgccactg?agtggccttg?tccaagctac?atccactctg?tgggctcctc????4500

cttgtagcag?cgaggggagg?gcagatgtcc?caggggctgg?tcactggagc?attcctcccc????4560

tctgactccc?cagtacaacg?ccctgctaag?caacatgagc?aggatctact?ccaccgccaa????4620

ggtctgcctc?cccaacaaga?ctgccacctg?ctggtccctg?gacccaggta?cggcccttgc????4680

agctcccctc?tcggcggtgc?cctagtgttc?ccacattgcc?ctgctgcact?ccggaccatg????4740

cagttgtgta?gggtctgtgg?agacagcagg?taaacccaaa?ggtgttgccc?tccaactggg????4800

gctggacggt?gcagataccc?ccacgccctg?cttctcttgg?caagtggact?tccggaatct????4860

ccagctgcag?cccccacttc?tgtgtgtacc?tcggcctctc?ccatcacccc?taggccttcc????4920

tcctggctgc?ctggtttccc?ctttcgtggg?tcctctcatg?ttccccaaga?gccctcaggc????4980

cagggacccc?tcgtggcttc?cccttaaacc?ccgctccagc?cccctttatg?agcagcttcg????5040

aggaaggcac?tccatccaat?aggccgctaa?gtgtctgtct?gggttttggc?ctttgggtgt????5100

ccccttggtg?tcagccacct?taggtggtca?tgtctctggg?gcaggggccc?tgcctgggtg????5160

tttctgtagc?tcccagcccc?ctcccaccag?gcctgtaggt?ggcccctgtc?tctgggggca????5220

ccgtgatgtt?caggaagctg?gtgggagcag?taaggactgg?tgcaggctct?ggtgaaggcc????5280

gttgaagact?tcaacgtgga?ggcctcctca?ccgaccctgc?ctgcctgtgt?ctcagatctc????5340

accaacatcc?tggcttcctc?gcgaagctac?gccatgctcc?tgtttgcctg?ggagggctgg????5400

cacaacgctg?cgggcatccc?gctgaaaccg?ctgtacgagg?atttcactgc?cctcagcaat????5460

gaagcctaca?agcaggacgg?tgagcaggcc?tctccctgtc?caggaaccac?gccaggtgtc????5520

ctctctcagc?tgtctcccca?gagtcccagc?ccagagtcag?gcagagcagc?tggtatgaca????5580

attccagcag?gccctgagtt?tcccagaaag?tggaggtggg?accggcctgc?acccagtgtg????5640

cctggacttt?gctgctggcc?tgccccacgt?ggccatcctg?ctgtcactcc?tggccctgat????5700

gctcctcttt?gcctctggga?acctccagga?tctgtttagc?tggctgtagc?taattagaaa????5760

ttgtagagtg?gcaaccccca?agccaatttt?ccagctagct?gcagatccac?gggcctcgag????5820

ccagtggaag?agccgactta?cagctgagag?gctgaggtcc?gagcctttgg?cctgagctac????5880

atacctcacc?cccacgcccc?caggcttcac?agacacgggg?gcctactggc?gctcctggta????5940

caactccccc?accttcgagg?acgatctgga?acacctctac?caacagctag?agcccctcta????6000

cctgaacctc?catgccttcg?tccgccgcgc?actgcatcgc?cgatacggag?acagatacat????6060

caacctcagg?ggacccatcc?ctgctcatct?gctgggtaag?gacctggcct?cgcctccaca????6120

tgagtcccac?ggaagtgtgg?gtcccgaggt?aggggtgggg?gatgtccagg?gtaagggaag????6180

gtgggttgtg?accctcacat?ctcacatgtg?tggggcatca?tactgtttgc?ttcacatgca????6240

ggagaccatt?cgtgttccca?ctttacaggt?ggggaccctg?aggcttaggg?tcgtgaggga????6300

cttagtggtc?agagagctag?gggccaaacc?aaaggctctg?gccctgggtc?cagtggggga????6360

gccatcagcc?tagctcatgc?ccaaggaaac?aagcactgtg?gccctgcctc?aggattgagt????6420

ggctggggcc?tggcacagcc?agaaatgaca?gtggcagcat?cttgcagccc?caggacatgt????6480

ggccctcgga?ggagtgtggg?tgggactgat?gtgtgagatt?tctggcccta?agccaggcct????6540

gcagcccttg?agggccccag?ggtacaggtg?ccggccccag?ggtgccactc?agcgatgcat????6600

gaagaagcag?gcacagccag?gcagggagcc?aagctgtccc?cttccttcct?tatctaggag????6660

acatgtgggc?ccagagctgg?gaaaacatct?acgacatggt?ggtgcctttc?ccagacaagc????6720

ccaacctcga?tgtcaccagt?actatgctgc?agcaggtaag?ctctgggctc?aagcctgggg????6780

tggtgggggt?cgggggtggg?gcgcaaaaaa?agggagtcac?agatgggcac?aggggcggga????6840

aggtttcggg?tactgagcag?cagcctggtg?tgtctgtagg?agcagtgagc?tggggtcggc????6900

cccctcagtg?aggtgccagc?tcctccctcc?aggctccaca?gtggcaggat?gagagcaaca????6960

acgcactttc?actcatctgc?tgtgggagtg?agggccctgc?ctctgggaat?ggtggccaca????7020

gagcagagaa?gctttcatgc?acagggagtt?gacccgagat?ggggacccca?gccctgtccc????7080

caggccagcc?agagtgggct?ccccctgacc?tggctccaca?cccctcctcc?agggctggaa????7140

cgccacgcac?atgttccggg?tggcagagga?gttcttcacc?tccctggagc?tctcccccat????7200

gcctcccgag?ttctgggaag?ggtcgatgct?ggagaagccg?gccgacgggc?gggaagtggt????7260

gtgccacgcc?tcggcttggg?acttctacaa?caggaaagac?ttcaggttca?gacatgggaa????7320

gagcacgttc?tggggttccc?cggttctggg?gcccggggaa?aggcaggcag?cccaggcgca????7380

gggaagctgg?ttcccaggcc?tgcctctacc?ctaccccagc?actggttgga?ggctgggtct????7440

gttccagggc?tagggggtat?aggaggccta?ttagtccacc?ttctctggca?gctttgacaa????7500

atagtcactt?ctataccttg?gaatggagga?agaaggccca?agtggtggtg?agccagggca????7560

gggtaaagaa?tttgcttgtt?tctgccaggc?acggtggtca?cacctgtaat?cccagcactt????7620

tgggaggtca?aggcgggtgg?atcacctgag?gccaggagtt?cgagaccagc?ctggccaaca????7680

tggcgaaacc?ccgtctctac?taaaaataca?aaaataaatt?agccaggggt?gatggcgggc????7740

gcctgtaatc?ccagctactc?aggaggctga?ggcaggagaa?tctcttgaac?ccgggaggcg????7800

gaggttgcag?tgagctgaga?ttgtgccact?gcaggccagc?ctgtgcaaaa?gagtgagacg????7860

ctgtctcaaa?aaaaaaaaga?aaaaaagaag?ttacttgttt?ctactgcggc?ttcatgcccc????7920

agggcagctc?cctcctcatt?cctgtctttc?aggtgccaat?ctgccctgtg?ccctggccct????7980

gccctgttct?gtccatccgt?cactctcacc?ctcgccctct?ctacgcccca?ggatcaagca????8040

gtgcacacgg?gtcacgatgg?accagctctc?cacagtgcac?catgagatgg?gccatataca????8100

gtactacctg?cagtacaagg?atctgcccgt?ctccctgcgt?cggggggcca?accccggctt????8160

ccatgaggcc?attggggacg?tgctggcgct?ctcggtctcc?actcctgaac?atctgcacaa????8220

aatcggcctg?ctggaccgtg?tcaccaatga?cacgggtatg?ggagggctga?gaggccccca????8280

cccagcctca?cctaaacccc?gctccacccc?acagcaggac?ctcacttgcc?ccactcagct????8340

ctgcccttct?ttctgcctcc?cggccccagg?tcaggcaggg?ttcgggatcc?tcctagagcc????8400

tcacggtgca?cactgcgccc?agctcagcac?acctgggggt?cctcttccaa?gcagggccca????8460

gggtctcgag?ggccagccat?accttctctg?catctccctg?gcctcacttt?ctgctgcccc????8520

gccagcccac?actcttaggg?gaccctcttc?tccctctgac?ctcttccctc?tcctttcatc????8580

tcatctccca?acagaaagtg?acatcaatta?cttgctaaaa?atggcactgg?aaaaaattgc????8640

cttcctgccc?tttggctact?tggtggacca?gtggcgctgg?ggggtcttta?gtgggcgtac????8700

ccccccttcc?cgctacaact?tcgactggtg?gtatcttcgg?tgagaggagg?gatagaaaag????8760

ccttcgcccc?agctagccct?ccccagcctc?ctggacagcc?aggcgcctcc?tgccccagcc????8820

agttctagcc?tctcctctct?aatgatgtcc?cccgctgtga?cccaccgcct?tctcctttcc????8880

tgcctgaaac?tccctcttcc?aggaagtctt?ccccagttcc?tcaggatggg?gaagggttgc????8940

cgggtggaaa?tgccttttct?acaaaagcta?aatccatctg?tttgcaacct?ctaggcccta????9000

agacaattta?accatccttt?tccagaacca?agtatcaggg?gatctgtcct?cctgttaccc????9060

gaaacgaaac?ccactttgat?gctggagcta?agtttcatgt?tccaaatgtg?acaccataca????9120

tcaggtatta?gcgcccccac?cccacccacc?cccagtactg?tcacaccctc?aatccacttc????9180

tcctcctgtg?atcctagctg?cctcatcccc?agggcttgtc?cccatgctcc?tccagacctc????9240

aaaggcctgg?agttagagtg?gcccactctc?ctgagcctgt?cttgggtctc?ccttctcccc????9300

caagatagct?tctggtccag?cctctgccct?gcaggaagct?ggatggtgcc?tgggtaagga????9360

acccctgttc?ctggcccccc?atgatcttcc?ctgactccca?ccctgtgcct?gcaggtactt????9420

tgtgagtttt?gtcctgcagt?tccagttcca?tgaagccctg?tgcaaggagg?caggctatga????9480

gggcccactg?caccagtgtg?acatctaccg?gtccaccaag?gcaggggcca?agctccggtg????9540

tgtggtggga?agccggggga?agtgggaggc?agagaggagc?ggctggcaaa?gggtgtggca????9600

ggaggtgtct?ggctgctctg?atggggtggg?gggcaccaac?cacagagctg?gactgatgtg????9660

gatgcctgtc?tcctcgctat?gtcatcaaat?atttattgag?tgggccttct?ggctggcatg????9720

gggcgacaca?aatgccccct?gccaccatca?gagagatccc?aggccccagg?gtcttattgc????9780

cacagtttct?gcagtccatt?ggggggcgga?agtggccagg?ggcatgtggg?ccggggtcca????9840

ggagcagact?ccagcctgag?tcccctgtgc?ccatggtacc?cactctgccc?accaggaagg????9900

tgctgcaggc?tggctcctcc?aggccctggc?aggaggtgct?gaaggacatg?gtcggcttag????9960

atgccctgga?tgcccagccg?ctgctcaagt?acttccagcc?agtcacccag?tggctgcagg???10020

agcagaacca?gcagaacggc?gaggtcctgg?gctggcccga?gtaccagtgg?cacccgccgt???10080

tgcctgacaa?ctacccggag?ggcataggta?aagccctgag?tgaggatggt?gtggggctaa???10140

ggtgggtcct?caactctggg?cttggcccag?gccccaggtt?cctggtcagc?tcctaccagc???10200

tgagccctgg?taccctgtcc?tggagggcca?ggcagccccc?caagctcatc?agcagggcct???10260

gcgagtgggg?acaggcatgt?ctttccccca?gcatcctaga?gagggtgtgc?tcagacctga???10320

gggcccctcc?ccttccagag?gaagccagac?acaaggctct?gtgaggtcac?actgcgggct???10380

ccgctcttat?tggccagggg?acggtagctg?caggactctg?ctctcctgcg?gccatgggcc???10440

agggttgggc?tactgcagga?cttcccagcc?tcctcttcct?gctgctctgc?tacgggcacc???10500

ctctgctggt?ccccagccag?gaggcatccc?aacaggtgac?agtcacccat?gggacaagca???10560

gccaggcaac?aaccagcagc?cagacaacca?cccaccaggc?gacggcccac?cagacatcag???10620

cccagagccc?aagtgggacc?atgcagggga?ggggcagggt?gccaggggtg?ggagaggcgg??10680

ggccgggtag?ggacagggca?gggtacaagg?gagtgcgaga?gggataatgg?cttctggtga??10740

gaccacaaac?ctggagaggg?gaggcagagg?tttgtctgtt?tccctgcact?ctgtcccaca??10800

gacctggtga?ctgatgaggc?tgaggccagc?aagtttgtgg?aggaatatga?ccggacatcc??10860

caggtggtgt?ggaacgagta?tgccgaggcc?aactggaact?acaacaccaa?catcaccaca??10920

gagaccagca?agattctggt?gggagccacc?tccccacccc?caaacctgag?catgtgcata??10980

cacacagaga?tgctgtcccg?ctcaccacac?agtggggctg?ccaccacatt?ttaaattgaa??11040

tatttaaaac?aatactcaat?ttcgggccgg?gcgcggtggc?tcacgcctgt?aatcccagca??11100

ctttgggagg?gggaggcggg?cggatcacga?ggtcagatca?agaccatcct?ggctaacacg??11160

gtgaaacccc?atctctagta?aaaatacaaa?aattagccgg?gtgtggtggc?gagcacctgt??11220

agtcccagta?ctcaggaggc?tgaggcagga?gaatggcatg?aacccgggag?gcagagcttg??11280

cagtgagccg?agatggcacc?actgcactcc?agcctgggcg?acagagcgag?actccatcaa??11340

aaaaaaaaaa?aaaaaaaact?caatttcaga?ttttgatgaa?catttactca?atgcctgagc??11400

aattcttctt?tccttaaaaa?tcagtctctg?ggaggcctag?gtgggaggat?cacttgaagc??11460

caggagttgg?agactagcct?gggcaacata?gcaagatccc?atctctattc?aaacaaacaa??11520

ataaacaaaa?atcaatctct?agtaacagaa?taatttgtac?ataaataagt?ggtgctcaag??11580

tcgtttttta?aaagattgaa?agcctctgtt?tgtctcctct?acaaaagggg?ctacacttcc??11640

tctttaccct?cattccctgc?ctatttggct?gagcacaaat?tatgccactg?agccacacac??11700

tgttactgtt?ccttggcact?ttgatctgtt?gcctcatctt?tttctcaaca?gccttgcaaa??11760

attggtgagc?ttattcccat?tttacagatg?ggatttgata?ttaactctga?ggttcagaaa??11820

ggccacagag?ctaataccaa?gctggctcct?tcctaagggc?ctttaagaca?cttgggggtc??11880

ttctcttctc?tgcccctgcc?tggatatgtg?ttgcttgacc?gcaggcatcc?agggagggtg??11940

agtactgcat?ccaggacgtt?atcagcgtcc?agcttgcaga?gagtcttata?ggcaaaggtt??12000

gcaacttaat?tccactgccc?cctcaccacc?acctccagcc?ctcagctccc?acttggggcc??12060

tcccgctcag?aggctgctct?ggagctcctg?ggccctgtga?caccatcccc?ctgtgccctc??12120

agctgcagaa?gaacatgcaa?atagccaacc?acaccctgaa?gtacggcacc?caggccagga??12180

agtttgatgt?gaaccagttg?cagaacacca?ctatcaagcg?gatcataaag?aaggttcagg??12240

acctagaacg?ggcagcactg?cctgcccagg?agctggagga?ggtgtgtggc?tcgcaaggta??12300

cagggagagg?ggaatcctgg?ggcagtgagc?ccaacacagg?gtctggcctg?gccttcacgc??12360

tgcttcctct?tcctcgttgt?atcaagtcat?ggcatctgcc?atgcgatgtg?cacctcagaa??12420

ctgctgagag?ggcagcgctc?cccagctccc?tggctcccca?cctgccagcc?catggggcct??12480

gggggtagtg?caggccccag?agagaccaag?tgcaaaggag?tacagctcat?tgcctctcct??12540

tcctcctgca?gtacaacaag?atcctgttgg?atatggaaac?cacctacagc?gtggccactg??12600

tgtgccaccc?gaatggcagc?tgcctgcagc?tcgagccagg?tgagagctca?tgtgcaggct??12660

gagtgagagg?cgagggctgg?gactggcatg?gggcccgggg?gtgctgggtg?agagcacaga??12720

gttgggttcc?cctcgctctt?ggggtcagcg?tgcccaggaa?atgccctttc?ttgttttcca??12780

cgaggggggc?ttctctgccc?cactgagagc?cggcacctac?ttcataccat?gccccgatca??12840

gctgcccctc?cctcagaacc?gccctctgct?taagggtgtc?cactctctcc?tgtcctctct??12900

gcatgccgcc?cctcagagca?gcgggatctc?aaagttatat?ttcatgggct?tggactccaa??12960

atggggggaa?ctcggggaca?ctagctcccc?ccggcctcct?ttcgtgaccc?tgcccttgac??13020

ttcctcacct?tctctgtctt?tcctgagccc?ctctcccagc?atgtgactga?taaggaaatt??13080

gagtcacaca?gcccctgaaa?gcgccagact?agaacctgag?cctctgattc?ctctcacttc??13140

cctcacctac?cctgccactt?cctactggat?agaagtagac?agctcttgac?tgtcctcttt??13200

tctccccact?ggctggtcct?tcttacccca?gcccgtttga?aagagctcac?ccccgacaca??13260

aggacccgca?cacagatacc?tcccagctcc?ctctcaaccc?accctttcca?gggttggaga??13320

acttgaggca?taaacttgct?tccatgagga?atctccaccc?agaaatgggt?ctttctggcc??13380

cccagcccag?ctcccacatt?agaacaatga?caaatagaag?gggaaatgga?aaataaacag??13440

gagaaacggt?tttcccagga?cagggtttgg?cctacaagtt?gtggatgtgg?gtacccatgc??13500

caagtgtgag?gggaggctgg?ccgggtgtgg?tggctcatgc?ctctaatccc?agcactttgg??13560

gaggccaagg?tgagtagatc?acttgaggcc?gggagtttga?gaccagcctg?gccaacatgg??13620

tgaaacccca?tctgtactaa?aaatacaaaa?gttagctggg?cgtggtggta?gatgcctgta??13680

gtcccagcta?cttgggaggc?tgaggcatga?gaatcgcttg?agcccagcca?gggcaataca??13740

gcaagacccc?gtctctacaa?ataaaataca?aaaaattagt?tggatgtggt?ggtgcatgcc??13800

tgtagtccta?gctgctaggg?aggctgagat?ggaaggattg?cttgagcctg?ggaggtcaag??13860

gctgcagtga?gccgagatgg?cgccactgca?ctccagcctg?ggcaacagag?tgagaccctg??13920

tctcagaaag?aaaaaaaaaa?aaaaaggaga?ggagagagac?tcaagcacgc?ccctcacagg??13980

actgctgagg?ccctgcaggt?gtctgcagca?tgtgccccag?gccggggact?ctgtaagcca??14040

ctgctggaga?ccactcccat?cctttctccc?atttctctag?acctgctgcc?tatacagtca??14100

cttttttttt?ttttttgaga?cggagtctcg?ctctgtcgcc?caggctggag?tgcagtggcg??14160

ggatctcggc?tcactgcaag?ctccgcctcc?cgggttcacg?ccattctcct?gcctcagcct??14220

cccaagtagc?tgggaccaca?ggcgcccgcc?actacgcccg?gctaattttt?tgtattttta??14280

gtagagacgg?ggtttcaccg?ttttagccgg?gatggtctcg?atctcctgac?ctcgtgatcc??14340

gcccgcctcg?gcctcccaaa?gtgctgggat?tacaggcgtg?atacagtcac?ttttatgtgg??14400

tttcgccaat?tttattccag?ctctgaaatt?ctctgagctc?cccttacaag?cagaggtgag??14460

ctaagggctg?gagctcaagg?cattcaaacc?cctaccagat?ctgacgaatg?tgatggccac??14520

atcccggaaa?tatgaagacc?tgttatgggc?atgggagggc?tggcgagaca?aggcggggag??14580

agccatcctc?cagttttacc?cgaaatacgt?ggaactcatc?aaccaggctg?cccggctcaa??14640

tggtgagtcc?ctgctgccaa?catcactggc?acttgggtcc?cttcattttc?ctcaaagagg??14700

tgctgtgaaa?ccccaagcct?aggaaaaggt?agatccctgg?aggaggcagg?taatgtggtg??14760

ttgggagagc?ctggctgtgt?cccctctgta?ggctatgtag?atgcagggga?ctcgtggagg??14820

tctatgtacg?agacaccatc?cctggagcaa?gacctggagc?ggctcttcca?ggagctgcag??14880

ccactctacc?tcaacctgca?tgcctacgtg?cgccgggccc?tgcaccgtca?ctacggggcc??14940

cagcacatca?acctggaggg?gcccattcct?gctcacctgc?tgggtaaggg?cacatgtcgg??15000

gccttgagga?gggtaaagac?ggaccacagt?gtgagtgagg?gttgggacag?ggctgactag??15060

agggtaggga?gcaggctggg?gactgagaga?ctccagccct?gtgggggatg?gttgcccagg??15120

ctggaggggg?gtgggcgctg?ggagtgggga?gccccccact?tgcatctggt?gccacattca??15180

ctgcagatct?atgtcgggca?agtcaccatg?gatgggggaa?gaagttaata?atcttgtcca??15240

ggagaccacg?gcacccatca?caacattgtg?tgatcttaga?gggcgaggaa?gaggctgtga??15300

gtgggagctg?gggaggcttt?gccaagaggt?ggcctgtgag?cagggcctcg?gaagatgaca??15360

gggtttgaca?gatgggaagt?gggggatgag?aggacagacg?cagtgttcag?gccaagggaa??15420

ctggaacaaa?gaagaacctg?agaatgtaaa?tctacttcaa?ccctggaccc?tcctttgcca??15480

agggctgcaa?tctcagatgc?cctgaatgtg?tgaagtaggc?ggtgaggaca?gtaagggatg??15540

gtagggagta?aggcaaagca?gaggctactg?gttctctgtc?cctgatgggc?tgttaggaac??15600

actttcctgg?agcagagaga?ccagacaggc?cctcagacca?tttagaaact?ataagggagg??15660

ccccagagga?cggcctggct?gtgggtctag?ctcccacaca?ggctgggagt?ccagccctct??15720

tcagcccctc?tctggtgaga?ccaaagaaca?tctggtgatg?tcacagtgga?cgtcagttca??15780

ccaactggga?gacacaggcc?ccgggaagaa?aagcaacatg?cccagcgtgg?cctgggagct??15840

ggggcagagc?tggccttaga?actcagcccc?tgacaattgg?taaaagggga?aaggggagca??15900

acctaacact?gatgcgctct?ctgtctctct?ctctggctct?ctccctggct?ctctccctct??15960

tctctctcat?gttctctcca?tcactcatcg?ctctaaccct?ctctcactgg?tttgcacttt??16020

agacttcatc?tgatgtcagc?cgaagcttca?cctcacttgg?ctaggaaaga?gccttgagtc??16080

caaatctgtt?tctgagcctt?ccattcatcc?tgagtttctt?ccttttcctc?tgtcgtggag??16140

aactaggctc?ttttcttaca?ctaaactcag?aggcatcagc?ctctcctgaa?ggagacggct??16200

ggttcctgtc?agagttgctg?agctgcagac?accgacctca?ggtggtgcgg?aggggacatg??16260

gcagagtggc?tggtgaagag?agcagcctgc?cagcctttca?atcccagccc?tgccacttag??16320

gagccgtggg?cccccggcga?ggggggcggt?cacttaactc?tccagcctgt?ttcctttact??16380

agccaatggg?aatcgtgaca?gtacctgggt?gcagacagga?ttgaaagtga?attcacacaa??16440

tgttcttggt?gcagagccaa?taagaggtgg?ccaccggggt?gtaggtgttc?tggggacctg??16500

taatgtcctc?acatgtcagc?agttgctagt?cacattggtc?tccactgctc?acggacagtg??16560

aagaccacct?ggattccctg?ataaccagca?aggcccccac?ctagagccag?gcagtaatga??16620

cctactgggc?tggatatgca?caccaaagat?gatgtgtgcc?tcaaagcttg?caaacagtag??16680

gtgctcaaga?aatgccacta?tgattagcag?gactgggatc?tggagcgctc?ttcctgcagg??16740

agggcattga?gcctaagtaa?catttgtctt?tcctctctct?gccgtccccc?acactcgcct??16800

ccagggaaca?tgtgggcgca?gacctggtcc?aacatctatg?acttggtggt?gcccttccct??16860

tcagccccct?cgatggacac?cacagaggct?atgctaaagc?aggtccgcac?cagcccaggg??16920

gcagggaggc?cccgccggga?tgggagggac?cctctgattc?aggagttccc?tccagtttag??16980

ccctcccccg?ggatccccac?ggcagcacgc?agtctgtccc?cggaaccccc?agtttgggca??17040

gaactccctc?tgcttgcagg?gctggacgcc?caggaggatg?tttaaggagg?ctgatgattt??17100

cttcacctcc?ctggggctgc?tgcccgtgcc?tcctgagttc?tggaacaagt?cgatgctgga??17160

gaagccaacc?gacgggcggg?aggtggtctg?ccacgcctcg?gcctgggact?tctacaacgg??17220

caaggacttc?cggtacatcc?agctagggct?caggtctcgt?tcctgagccc?cacgggcaag??17280

ggaaatgaac?caagcaaagg?gtccactact?gtcccccagc?tggagccagc?agggcaggat??17340

ggggacaggg?ccagagtttg?ggactgagtg?tctagagagg?tgttggcttc?tggcaggaaa??17400

accccatccg?cctgatgggg?acttctgaag?cacgcaacag?ctctgtcagc?ctggccgctg??17460

ggaagtgctc?aaggtcccag?tcctgggttt?gagcatggta?ggctgccccg?cgtccctcct??17520

tgggagcagc?ccctgcatgg?agctggcctc?tccctggggg?cacatgctgt?gacacaggga??17580

ggcacacgag?gatgttgggt?gctctgtaca?gatccactct?cacccctgac?aggctcagaa??17640

gctgccttcc?ttggaggatg?gcgttttagt?tacctattgc?tgtgcaacca?agcaccccag??17700

agcttagcct?tacgaaacaa?ccagttgatt?ttgcttatga?ttttatgtgc?caggaattca??17760

ggcagtacac?agtggaaatg?gcctttctct?acagggcccc?ctctgttggg?ggcagctctc??17820

acagccggga?tggctcaatg?ggggccatac?gtccagagcc?ccagttctgg?ctgtcggttg??17880

aggtcctcgg?tttttcccat?gtggcagatg?ctggggcaca?tgttcccagt?ggcctcttgg??17940

ctcacatgtt?gggtgcttgg?ggtgagatgg?ctggaacggc?tggaggtggg?tcaggcatct??18000

ctccaggcta?gctcgggcgc?cctcccagcg?tggaatctga?ggtgggcaga?tttacctggc??18060

agccagcatc?ccccacagca?actactgacg?cagccagttc?tcaaggctag?gtccaaaact??18120

ggcccagagt?cacttctgcc?atgttttatt?ggctagaaca?agtcacaagt?tcacccagat??18180

tcaagagaag?agaaaaaagt?ccctcccact?tgggagaagt?ggcaaagacc?atctgtcaca??18240

gctgaagaag?tgtctcttac?aaggagaaca?gacacgggga?gcctgaaaca?aaacccgatg??18300

ggattccctg?ggctgtgcag?gcccttccag?gcatgaggac?tcagccacag?ggctgagagg??18360

agacaggatc?tgggggatga?gagcccttgt?ggggtcttcc?cttttatggg?gagtcagagg??18420

agaagctgga?tagatcccca?gccttgtggc?caggatgctg?ggcagctctt?ccttccccct??18480

ccccgatgag?aatgacagaa?aaacaggatt?cacctgagcc?aaaaggcttc?cagttagatc??18540

caagagagaa?tttcccgcag?tttgaattgg?tttgctaaac?aacaaggaag?ggctgggtgc??18600

ggtggctcac?acctgtaatc?tcagcacttt?gggaagccga?ggcaggaggt?ctgcttgagc??18660

tcaggggttc?gagaccatcc?tgggcaacat?agcgagaccc?catttcataa?aaaataaata??18720

agtaaatgag?aacaaggaag?gactgacgag?agacggtaga?accttctggt?ttgggcagct??18780

ctgcagctgc?cattcatcct?ggccataaga?attcttgggg?tgaataagtt?tgtcgctgtt??18840

gggccgcatg?agatgcagaa?tcgcccactc?tcacccctga?cagaaacagt?tgtttccttc??18900

agggagcctc?catcttggga?gataaagcat?gtgtacatgg?gaacccactg?gccacacatt??18960

ctctagaaag?tacacaatgt?cccagtgcct?ctagagcaag?cactttgtac?agtcagaaag??19020

caacaggtgg?tgggggctgg?agtcattcag?gaaaatggga?ggcagaggaa?tggcctgaac??19080

ggcccgatgc?taggggcttc?tgcccccaga?ttccctctta?cgcacactca?gtggttgccc??19140

ttcccctccc?tccccacagt?gctgtgtccc?ctgcatgctg?cagtgctggg?gtctgccctg??19200

ggtatagcaa?ggcccactgt?tcccttatgc?ccagggcttc?tcactgtcct?ctcccaacac??19260

cctctccccc?actccactat?tcctaggatc?aagcagtgca?ccaccgtgaa?cttggaggac??19320

ctggtggtgg?cccaccacga?aatgggccac?atccagtatt?tcatgcagta?caaagactta??19380

cctgtggcct?tgagggaggg?tgccaacccc?ggcttccatg?aggccattgg?ggacgtgcta??19440

gccctctcag?tgtctacgcc?caagcacctg?cacagtctca?acctgctgag?cagtgagggt??19500

ggcagcgacg?gtgagagaga?agcgggaggc?cctggtgggc?tgaggaccaa?gaaagggtgg??19560

tgagcttggg?aggtgggaaa?ggggcactta?gtggcccatg?ggcagaggtg?tggggcagag??19620

caatcggaag?gaagggagcc?acccagacca?tcccaggagg?caggtcacag?ggcccaaaag??19680

gtacagcacc?cccacccctc?caccatcaca?ggcacaccag?ggccaagccg?ctaggaccct??19740

gggtctgaca?gctgggctcc?cttcccttgc?agagcatgac?atcaactttc?tgatgaagat??19800

ggcccttgac?aagatcgcct?ttatcccctt?cagctacctc?gtcgatcagt?ggcgctggag??19860

ggtatttgat?ggaagcatca?ccaaggagaa?ctataaccag?gagtggtgga?gcctcaggtt??19920

ctggaacact?cccacgggat?gcgggctggg?ggatctctgc?gagtgtctgc?atgtgcctgg??19980

gtgtctggat?gggccagggt?aggggagtgt?gtgtgtgtgt?gtacactatt?gtgtctgtgc??20040

atatgatgtg?tgtggtgtaa?gtgatgggga?aaacggggtg?attgtgcaca?gaggcccagc??20100

acgcaggaga?atggggtgcc?cagtatagcc?ccaagtgcag?ggaccctccc?tcaagtcaaa??20160

aatgccaccc?ccagcctggt?tctccccaaa?ctcatcttcc?aacatatatt?cccactcgac??20220

aggctgaagt?accagggcct?ctgcccccca?gtgcccagga?ctcaaggtga?ctttgaccca??20280

ggggccaagt?tccacattcc?ttctagcgtg?ccttacatca?ggtaacggga?aaggcaggag??20340

ggcacattgt?gaggggcagt?acccacagct?ttgtgtttca?actgcggcca?ctgcccggtc??20400

cacaagctct?gtcagtcagg?gcagacccgg?gggagccggc?cgcacggtgc?aggtgcctgg??20460

gcccactcac?actgccaagg?ctgatgggtt?ttttcttgaa?cattcttttg?atgagagtct??20520

gtaccatcca?aacagttgaa?cacagaaact?caacctaata?attggctaat?ggttaccaga??20580

ccttggttaa?gtagttaaca?ttaaccacga?ctcatggctg?gatcatgagc?tctgcactgt??20640

tttgttttgc?ttttaaaaca?agactgtgat?tcttttacta?ttattgaaca?ttgtctgcga??20700

tacaatttga?attgtacctg?gaagcccttc?tagacactaa?aatgtaggat?tggagatcgg??20760

ttaaggtggg?aggcagggtt?gctggggcaa?gttacagtca?caggctgggg?tcagacagaa??20820

ctgggttcaa?accccgtctc?cattactttg?tttccttgag?aaaattcctc?aatttctgtg??20880

agcttccatt?tcctgacctg?tgaaccccat?ttcacaggat?gcacgatggc?taacttctta??20940

gcattctgtc?tcatacacag?cctcctcagg?gaggggtggc?caggacccca?ctattcatca??21000

ctctctagtg?gaatgtagct?gcacactagg?tctgcaggtc?acatggccac?agatgagtgt??21060

gcccaatgca?gcccctctcc?ttctgtgtgc?cccgggagag?cacttgctga?gggctagcaa??21120

ggctgtttgt?gatccgggag?gctccctggg?aggctggggg?ctagagagac?ctcaggctgg??21180

agttccaggt?gcccccgggc?tacaggtagc?ccaggccacc?cccagagggc?tgtggctgcc??21240

tctggccctg?gcctcccgtg?gttcctggaa?gcccagcaag?ggcagggccc?atgcccacct??21300

tgcctcctgg?cacctgggat?gatgccagca?catcatcaag?tgctaataac?tgattgtggg??21360

atggatgaag?tctgtcccca?gagtccagga?agagggcatc?cctggagcac?ctcagatagg??21420

cctgacctag?acggtgctcc?agagatgaca?cttaggacag?ggctcccgcc?tgcctgctgg??21480

agtggtccct?ggggttccca?gccggcgctg?gctctcaccc?gggagccagc?tggtgtgatg??21540

gctagcttcc?cagcttaatg?cagacaattc?tccaaacagg?ggtgggcaaa?ggagacttgg??21600

ctgctctaga?aaaacattcc?ggattctggc?cagcagcctt?cacaaagcac?ttttaggaaa??21660

gaccagggaa?ctaggtggta?catgcttgca?cccagcattc?aaagtgagag?gcctgtgcca??21720

ctggctcagg?acatttaaaa?cctcttcaga?ctttaagctg?gggagaatcc?tccagccttg??21780

actggcagat?ttctaccagg?gaattcgtga?tgctttggat?aagatcatgt?aggactggct??21840

tccctgccca?gaccacccta?gtagatccac?acggcccatt?tggccacacc?ttgcctctac??21900

ttgcatatac?cccagggatg?gagacctcac?tgcctccaaa?gccacctgcc?agatctctga??21960

aggctctgcc?ctgaccccat?ggagcaggcc?ctcctgagtt?gtggaggcag?ctctgtgggt??22020

gggaggcatc?tacacaggca?cggctaggaa?gaggctcaga?caatgctaag?agctggggtg??22080

ggggagctca?ccctgatagc?tgtgggcaga?gttggggggc?cttggctctg?ctgtgcgcat??22140

gtgacttagc?acacatcaca?tgtgatgtgc?agaagggcct?ggggcccagt?ggcacaaggc??22200

cctcaaccaa?ctccgccccg?ggccacggcc?tcgctctgct?ccaggtactt?cgtcagcttc??22260

atcatccagt?tccagttcca?cgaggcactg?tgccaggcag?ctggccacac?gggccccctg??22320

cacaagtgtg?acatctacca?gtccaaggag?gccgggcagc?gcctggcgtg?agtgtcctcc??22380

agccctcctt?tgtttccatg?ctctggcctg?cgcccctggg?ccttgagggg?tctgtccact??22440

ggagcttttg?tgggaacact?tgccattttg?agccgggaac?tcccacctgc?agcgtgggcc??22500

aggcctgatt?gccatctcct?taggcacctg?gagccctggg?gccctgggac?aagtttcagc??22560

tgggagtggg?tatggagagt?ggatgtcagg?tgggggcaag?aggggccatg?tccttctgac??22620

tctgcctccc?tgtctcatgc?ctccccagga?ccgccatgaa?gctgggcttc?agtaggccgt??22680

ggccggaagc?catgcagctg?atcacgggcc?agcccaacat?gagcgcctcg?gccatgttga??22740

gctacttcaa?gccgctgctg?gactggctcc?gcacggagaa?cgagctgcat?ggggagaagc??22800

tgggctggcc?gcagtacaac?tggacgccga?actccggtac?cgccacccac?cccacctcca??22860

gccttgggtc?ttaaccccct?ccccaggctg?ggcagccatg?cggctgacct?cggagcctgg??22920

ccctgccccg?cacccttgcc?ctgccctgcc?ctgccctgcc?catgctgtct?ccttgcttcc??22980

cactcagctc?gctcagaagg?gcccctccca?gacagcggcc?gcgtcagctt?cctgggcctg??23040

gacctggatg?cgcagcaggc?ccgcgtgggc?cagtggctgc?tgctcttcct?gggcatcgcc??23100

ctgctggtag?ccaccctggg?cctcagccag?cggctcttca?gcatccgcca?ccgcagcctc??23160

caccggcact?cccacgggcc?ccagttcggc?tccgaggtgg?agctgagaca?ctcctgaggt??23220

gacccggctg?ggtcggccct?gcccaagggc?ctcccaccag?agactgggat?gggaacactg??23280

gtgggcagct?gaggacacac?cccacacccc?agcccaccct?gctcctcctg?ccctgtccct??23340

gtccccctcc?cctcccagtc?ctccagacca?ccagccgccc?cagccccttc?tcccagcaca??23400

cggctgcctg?acactgagcc?ccacctctcc?aagtctctct?gtgaatacaa?ttaaaggtcc??23460

tgccctcccc?atctgagtct?gtgtccctca?cagggaagcc?agggacaggg?acaggctgct??23520

ttcctgcctc?ctggcagtca?agtgggtccc?gttactaggt?ttgttcctcc?atcctccttc??23580

aggagccggg?gaggatcccc?agagctctgc?cccagcacct?cctggcgctg?gcgcctgtct??23640

tccctccagc?ccaggcagcc?cgccactgtc?ctgccaccgc?aggcagcccc?tgtctggccc??23700

aagcactgac?ccacgcggac?tctgggaagc?agacatcctg?ggctgctggc?ctcacatttc??23760

cactggcagt?ggagcctttc?cctgctccac?aaatggccag?gtccccccag?gggaaggctt??23820

ccggctgtta?tcggctgcct?cagggggcga?gtaccttgga?gggcctgctt?caaggagggt??23880

gccccctgga?gggcacacac?cagcctagtg?cttaccttgg?ctcctgcctg?taccagctcc??23940

atgactctct?gctcgggtga?acagccttgg?ctctcagaca?gccattctaa?cactgccagt??24000

gcagaggggc?ctcagacgct?ggagtgtagc?agtggctgca?cctgcacagg?gattagctgc??24060

cagcagccac?????????????????????????????????????????????????????????24070

<210>2

<211>21

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>2

gcctgtgtct?cagatctcac?c????????????????????????????????????????????21

<210>3

<211>20

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>3

cagaggcaaa?gaggagcatc????????????????????????????????????????????????20

Claims (10)

1. whether a vitro detection sample exists the method for the single nucleotide polymorphism of angiotensin i-converting enzyme gene A CE, it is characterized in that, comprises step:

(a) with the ACE gene of ACE gene-specific primer amplification sample, obtain amplified production; With

(b) detect whether there is following single nucleotide polymorphism in the amplified production:

5525 C → G;

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 5525th.

4. one kind is detected hypertensive test kit, it is characterized in that it comprises the primer of specific amplification ACE gene or transcript, and it is 100-3000bp that described primer amplification goes out length, and contains among the SEQ ID NO:1 the 5525th 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 5525th sudden change bonded probe;

(b) the 5525th 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:

5525 C → G;

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 ACE gene, transcript and/or albumen, and compare with normal ACE 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 ACE, and with normal ACE nucleotide sequence comparing difference.

10. method as claimed in claim 9 is characterized in that, described difference is following single nucleotide polymorphism:

5525 C → G;

Wherein, the nucleotide position numbering is based on SEQ ID NO:1.