CN1661063A - Relationship between haptoglobin gene and essential hypertension - Google Patents

Abstract

A process for testing the susceptibility of primary hypertension includes detecting if their are variations in haptoglobin gene HP, transcript and/or protein of an individual, and determining its high susceptability of there are. Its reagent kit is also disclosed.

Description

Relationship between haptoglobin gene and essential hypertension

technical field

The present invention relates to the fields of molecular biology and medicine. More specifically, it involves single nucleotide polymorphism (single nucleotide polymorphism, SNP) of haptoglobin gene (haptoglobin, HP) and its correlation with essential hypertension. The present invention also relates to methods and kits for detecting these SNPs.

Background technique

Hypertension refers to a group of clinical syndromes with elevated systolic or diastolic blood pressure. There is an obvious causal relationship between the increase of blood pressure and the occurrence of coronary heart disease, renal dysfunction, hypertensive heart disease and hypertension complicated with stroke. The latest diagnostic criteria for hypertension are systolic blood pressure ≥ 19kpa (140mmHg) or diastolic blood pressure ≥ 12kpa (90mmHg), and those who meet one of them can be diagnosed as hypertension.

Most hypertensive patients only have mild subjective symptoms in the early stage of high blood pressure, such as headache, dizziness, insomnia, tinnitus, irritability, difficulty in concentrating work and study, and fatigue. With the development of the disease, especially when complications occur, the symptoms gradually increase and become more obvious, such as numbness and stiffness of fingers, pain in the lower limbs when walking more, or muscle pain and tension in the neck and back. When there is palpitation, shortness of breath, chest tightness, and precordial pain, it indicates that the heart has been involved. When there is nocturnal frequent urination, polyuria, and light urine, it indicates that the kidneys are involved and the renal arterioles are hardened. If a hypertensive patient suddenly develops confusion, deep and irregular breathing, and incontinence, etc., it may indicate cerebral hemorrhage. If one side of the body gradually becomes inconvenient, numb or even paralyzed, it should be suspected whether there is a cerebral thrombosis.

There were no obvious abnormal signs in the early stage of hypertension. Abnormal signs may appear when the brain, heart, kidney and other vital organs are slightly damaged. Common heart abnormalities include leftward shift of the apical beat, lifting-like pulsation sensation in the precordial area, enhanced first heart sound in the apical area, enhanced second heart sound in the aortic valve area, and systolic murmurs and diastolic murmurs. Arteriosclerosis and left ventricular hypertrophy, and a galloping rhythm in the apex may indicate heart failure. In addition, earlobe creases, capillary pulsation, hard or pulseless radial artery, and intermittent claudication of the lower extremities are also common.

In addition, due to certain predisposing factors or the development of hypertension itself, some hypertensive patients may have a significant or sudden increase in blood pressure, accompanied by damage to the brain, heart, kidney, retina and other important organs, which is seriously life-threatening, and a series of Special clinical signs are called hypertensive emergencies. The incidence of hypertensive emergencies accounts for 5% of the hypertensive population, common hypertensive encephalopathy, cerebral hemorrhage, acute left heart failure, clonidine acute withdrawal syndrome, acute myocardial infarction, rapidly progressive malignant hypertension, etc.

About 5% of hypertensive patients have no symptoms, do not know when blood pressure rises, and do not know when complications of blood vessel and organ damage have occurred. have high blood pressure. Therefore, finding out the genetic cause of hypertension, early prevention and detection can effectively control the incidence of hypertension and minimize the damage of the disease to the human body.

Essential hypertension (essential hypertension, EH), also called hypertension, is an independent disease with its own etiology, occurrence, development, outcome, and clinical manifestations. Clinically, it is mainly manifested as an increase in arterial blood pressure. Accounting for more than 90% of hypertensive patients in the population, the pathogenesis is not fully understood at present, and it is mainly diagnosed as essential hypertension (hypertension) only after the hypertension caused by other diseases has been ruled out. The increase in arterial blood pressure is mainly due to the increase in the resistance of peripheral small arteries, and at the same time there are varying degrees of increase in blood volume and cardiac output. In the late stage, the heart, brain, kidney and other organs are often involved, and serious complications such as hypertension, heart disease, heart failure, renal dysfunction, and cerebral hemorrhage occur. The treatment of essential hypertension is mainly to lower blood pressure while preventing the occurrence of complications. The causes of death in patients with essential hypertension are cerebrovascular accident, cardiovascular accident and renal insufficiency. In my country, cerebrovascular accident is more common, followed by heart failure and uremia. In European and American countries, heart failure is more common, and cerebrovascular accident is more common. and uremia followed.

As the most common cardiovascular disease, the incidence of essential hypertension is increasing year by year, and it can cause serious heart, brain, and kidney complications. It is a major risk factor for stroke and coronary heart disease. EH is a polygenic disease caused by the interaction of genetic factors and environmental factors. Finding EH-related genes and clarifying the genetic mechanism of hypertension has become a research hotspot.

Although there have been many studies on various gene polymorphisms and essential hypertension, there is no report confirming the correlation between HP gene and essential hypertension, and there is no confirmation that the SNP of HP gene is associated with essential hypertension sex reports.

To sum up, in order to finally realize the treatment of hypertension, there is an urgent need in this field to find essential hypertension susceptibility genes, and to develop methods, kits, and related therapeutic drugs for detecting essential hypertension.

Contents of the invention

The object of the present invention is to provide a method and detection kit for diagnosing (especially early diagnosis) hypertension.

Another object of the present invention is to provide a new method for treating hypertension.

Another object of the present invention is to provide a pharmaceutical composition for treating hypertension.

In a first aspect of the present invention, a method of diagnosing susceptibility to hypertension in an individual is provided, comprising the steps of:

detecting the individual's HP gene, transcript and/or protein, and comparing it with normal HP gene, transcript and/or protein,

A difference indicates that the individual is more likely to have high blood pressure than the normal population.

In another preferred example, the HP gene or transcript is detected in the method, and the difference is compared with the normal HP nucleotide sequence.

In another preferred example, the difference is the following single nucleotide polymorphism:

4884 bits G → C;

Wherein, the nucleotide position numbers are based on SEQ ID NO:1.

In a second aspect of the present invention, there is provided a method for detecting whether a single nucleotide polymorphism of the haptoglobin gene HP exists in a sample, comprising the steps of:

(a) amplifying the HP gene of the sample with HP gene-specific primers to obtain an amplified product; and

(b) Detect whether the following single nucleotide polymorphisms exist in the amplification product:

4884 bits G → C;

Wherein, the nucleotide position numbers are based on SEQ ID NO:1.

In another preferred example, the gene-specific primers have the sequences of SEQ ID NO: 2 and 3.

In another preferred example, the length of the amplified product is 100-3000bp, and contains the 4884th position in SEQ ID NO:1.

In the third aspect of the present invention, a kit for detecting hypertension is provided, which includes primers for specifically amplifying HP genes or transcripts, more preferably, the amplified length of the primers is 100-3000bp, And contain the amplified product at the 4884th position in SEQ ID NO:1.

In another preferred embodiment, the kit also contains reagents selected from the following group:

(a) a probe that binds to the mutation at position 4884 in SEQ ID NO: 1;

(b) Recognition of the mutant restriction enzyme at position 4884 in SEQ ID NO:1.

In another preferred example, the mutation is selected from the following single nucleotide polymorphisms:

4884 bits G → C;

Wherein, the nucleotide position numbers are based on SEQ ID NO:1.

In the fourth aspect of the present invention, a pharmaceutical composition is provided, which contains a safe and effective amount of HP protein and a pharmaceutically acceptable carrier.

Detailed ways

After intensive and extensive research, the inventors have determined and analyzed the SNPs of a large number of candidate genes. For the first time, it was discovered and proved that part of the SNP of the HP gene is closely related to hypertension, and its new function was discovered: the change of HP will lead to hypertension. C) There is a significant difference in the distribution between the case and the control group (P<0.05), so it can be used as a specific SNP for detecting hypertension. The present invention has been accomplished on this basis.

HP gene

Haptoglobin is also called haptoglobin, and the detailed sequence of its gene (haptoglobin, HP) can refer to the nucleotide sequence whose accession number is M69197 (see the website http://www.ncbi.nlm.nih.gov/) , as shown in SEQ ID NO:1.

The protein encoded by the HP gene is an α2-glycoprotein with the ability to bind Hb protein. In 1982, Chapelle et al. found that the HP gene was strongly correlated with the occurrence of myocardial infarction (Chapelle et al. New Eng. J. Med. 307: 457-463, 1982). By analyzing the HP genotypes of 496 myocardial infarction patients, they found that the number of patients with HP2-2 protein was far more than that of patients with HP1-1 and HP2-1 (p<0.05). In the control comparison of serum enzyme activities, it was confirmed that the activities of total creatine kinase, creatine kinase isoenzyme MB fragment, aspartate aminotransferase and lactate dehydrogenase were significantly higher in patients with HP2-2 phenotype . These patients also had a significantly increased risk of left ventricular complications. This result shows that the mutation of the gene HP is an important reason for aggravating myocardial infarction, and also shows that the HP gene has a very important influence and effect on the cardiovascular system.

In 2002, Koda et al. found that HP gene polymorphisms were associated with diabetic microangiopathy in the Japanese population (Koda et al.Diabetologia.45(7):1039-40, 2002).

The inventors sequenced almost the entire region of the HP gene and found many SNPs, most of which are not associated with susceptibility to hypertension, but association studies have shown that 4884 G→C are associated with susceptibility to hypertension Very high SNP. The SNP is located in intron No. 5 of HP, suggesting that it affects the post-transcriptional splicing process of HP, which in turn affects the expression of haptoglobin, and ultimately leads to a significantly higher susceptibility to hypertension in carriers than in normal populations.

Based on the new discovery of the present invention, the HP protein or polypeptide has many new uses. These uses include (but are not limited to): direct use as a drug to treat diseases caused by hypofunction or loss of HP protein (such as hypertension), and screening for substances that promote the function of HP protein, such as antibodies, polypeptides or other ligands. Screening the polypeptide library with the expressed recombinant human HP protein can be used to find therapeutically valuable polypeptide molecules that can stimulate the function of the human HP protein.

On the other hand, the present invention also includes polyclonal antibodies and monoclonal antibodies, especially monoclonal antibodies, specific to human HP DNA or polypeptides encoded by fragments thereof. Here, "specificity" means that the antibody can bind to human HP gene products or fragments. Preferably, it refers to those antibodies that can bind to human HP gene products or fragments but do not recognize and bind to other irrelevant antigenic molecules. Antibodies in the present invention include those molecules capable of binding and inhibiting human HP protein, as well as those antibodies that do not affect the function of human HP protein.

The present invention includes not only complete monoclonal or polyclonal antibodies, but also immunologically active antibody fragments, such as Fab' or (Fab) ₂ fragments; antibody heavy chains; antibody light chains; genetically engineered single-chain Fv molecules; or chimeric antibodies.

Antibodies of the present invention can be prepared by various techniques known to those skilled in the art. For example, purified human HP gene products, or antigenic fragments thereof, can be administered to animals to induce polyclonal antibody production. Similarly, cells expressing human HP protein or antigenic fragments thereof can be used to immunize animals to produce antibodies. Various adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant and the like.

Antibodies of the invention may also be monoclonal antibodies. Such monoclonal antibodies can be prepared using hybridoma technology. The antibodies of the present invention include antibodies capable of blocking the function of human HP protein and antibodies that do not affect the function of human HP protein. Various antibodies of the present invention can be obtained by conventional immunization techniques using fragments or functional regions of human HP gene products. These fragments or functional regions can be prepared using recombinant methods or synthesized using a polypeptide synthesizer. Antibodies that bind to unmodified forms of human HP gene products can be produced by immunizing animals with gene products produced in prokaryotic cells (e.g., E. coli); antibodies that bind to post-translationally modified forms (such as glycosylated or phosphorylated Proteins or polypeptides), which can be obtained by immunizing animals with gene products produced in eukaryotic cells (such as yeast or insect cells).

The antibody against human HP protein can be used in immunohistochemical technique to detect the amount and/or mutation of human HP protein in the biopsy specimen. A preferred anti-HP antibody is an antibody that does not recognize normal HP but recognizes mutant HP, or an antibody that recognizes normal HP but not mutant HP. Using these antibodies, the detection of hypertension susceptibility at the protein level can be conveniently performed.

Using the HP protein of the present invention, substances that interact with the HP protein, such as inhibitors, agonists or antagonists, can be screened out through various conventional screening methods.

When the HP protein of the present invention and its antibody, inhibitor, agonist, antagonist, etc. are administered (administered) therapeutically, various effects can be provided. Generally, these materials can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is usually about 5-8, preferably about 6-8, although the pH value can be changed according to the Depending on the nature of the substance formulated and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intramuscular, intravenous, or subcutaneous administration.

Normal HP polypeptides can be directly used in the treatment of diseases, for example, in the treatment of hypertension. When using the HP protein of the present invention, other agents for treating hypertension can also be used simultaneously.

The present invention also provides a pharmaceutical composition, which contains a safe and effective amount of the HP protein of the present invention and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to: saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of injection, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods. Pharmaceutical compositions such as injections, solutions, tablets and capsules are preferably manufactured under sterile conditions. The active ingredient is administered in a therapeutically effective amount, for example about 0.1 microgram/kg body weight to about 10 mg/kg body weight per day. In addition, the polypeptides of the invention can also be used with other therapeutic agents.

When using the pharmaceutical composition, the HP protein of safe and effective amount or its antagonist, agonist is administered to mammal, wherein the safe and effective amount is usually at least about 0.1 microgram/kg body weight, and in most cases no more than about 10 mg/kg body weight, preferably the dosage is about 0.1 μg/kg body weight to about 100 μg/kg body weight. Of course, factors such as the route of administration and the health status of the patient should also be considered for the specific dosage, which are within the skill of skilled physicians.

Polynucleotides of human HP protein can also be used for various therapeutic purposes. Gene therapy technology can be used to treat cell proliferation, development or metabolic abnormalities caused by the non-expression of HP protein or the expression of abnormal/inactive HP protein. The method for constructing the recombinant viral vector carrying the HP gene can be found in the existing literature (Sambrook, et al.). In addition, the recombinant human HP gene can be packaged into liposomes and then transferred into cells.

The methods for introducing polynucleotides into tissues or cells include: directly injecting polynucleotides into tissues in the body; or first introducing polynucleotides into cells in vitro through vectors (such as viruses, phages, or plasmids, etc.) , and then transplant the cells into the body, etc.

The invention also relates to a diagnostic test method for quantitatively and locally detecting human HP protein level. These assays are well known in the art and include ELISA and the like.

A method for detecting whether there is HP protein in a sample is to use an HP protein-specific antibody for detection, which includes: contacting the sample with an HP protein-specific antibody; observing whether an antibody complex is formed, and the formation of an antibody complex means HP protein is present in the sample.

The polynucleotide of HP protein can be used for the diagnosis and treatment of HP protein-related diseases. In terms of diagnosis, the polynucleotide of HP protein can be used to detect the expression of HP protein or the abnormal expression of HP protein in disease state. For example, HP DNA sequence can be used for hybridization of biopsy specimens to determine the abnormal expression of HP protein. Hybridization techniques include Southern blotting, Northern blotting, in situ hybridization, and the like. These technical methods are all open and mature technologies, and relevant kits are available from commercial sources. Part or all of the polynucleotides of the present invention can be immobilized as probes on microarrays or DNA chips (also known as "gene chips") for analysis of differential expression of genes in tissues and gene diagnosis. RNA-polymerase chain reaction (RT-PCR) in vitro amplification with HP protein-specific primers can also detect HP protein transcripts.

Detection of mutations in the HP gene can also be used to diagnose hypertension. Detection can be against cDNA or genomic DNA. The forms of HP protein mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared with normal wild-type HP DNA sequences. Mutations can be detected using established techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression, so Northern blotting and Western blotting can be used to indirectly determine whether a gene has a mutation.

The most convenient method for detecting the SNP of the present invention is to amplify the HP gene of the sample with HP gene-specific primers to obtain the amplified product; then detect whether there is the following single nucleotide polymorphism in the amplified product: 4884 position G → C, wherein the nucleotide position numbering is based on SEQ ID NO:1.

It should be understood that after the present invention first revealed the correlation between the SNP of the HP gene and hypertension, those skilled in the art can easily design an amplification product that can specifically amplify the position of the SNP, and then through methods such as sequencing Determine if there are 4884 bits G→C. Usually, the length of the primer is 15-50bp, preferably 20-30bp. Although it is preferred that the primer is completely complementary to the template sequence, those skilled in the art know that it can also be specifically amplified (that is, only amplify the desired fragment). Kits containing these primers and methods using these primers are within the scope of the present invention, as long as the amplified product amplified by the primers contains the corresponding position of the SNP of the present invention. A preferred primer pair has the sequences of SEQ ID NO: 2 and 3.

Although the length of the amplified product is not particularly limited, generally the length of the amplified product is 100-3000 bp, preferably 150-2000 bp, more preferably 200-1000 bp. These amplified products should all contain the 4884th position in SEQ ID NO:1.

Because the SNP of the present invention has a very high correlation with hypertension, it can not only be used for early and more accurate diagnosis of essential hypertension, but also can take precautions to make the carrier take reasonable preventive measures before the onset of the disease (such as in the Therefore, it has extremely important application value and social benefits.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate specific conditions in the following examples, usually according to conventional conditions such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's instructions suggested conditions.

Example 1

1.1 Research object

Since blood pressure is a quantitative trait that is affected by multiple genes and is affected by various environmental factors, it is necessary to consider the issue of genetic heterogeneity when analyzing related genes. Selecting isolated populations with a relatively single genetic background as materials for the study of polygenic diseases will help reduce the impact of genetic heterogeneity. reduce. Selecting such a group, whether it is used for linkage disequilibrium analysis or selecting families for family analysis, will help to improve the detection sensitivity of hypertension susceptibility loci. Therefore, in this embodiment, the isolated population is selected as the research object (the isolated population has the advantages of relative consistency of genetic background and relatively small number of founders).

On the basis of informed consent, 324 individuals of Han nationality over the age of 50 were randomly collected from Xiangchang Town, Dabie Mountain, Yuexi County, Anhui Province. 80% of individuals have only 6 surnames, the number of founders should be relatively small, and individuals of the same gender come from the same founder. The selected hypertensive samples require that the systolic blood pressure is not lower than 140mmHg, and the diastolic blood pressure is not lower than 90mmHg, and the blood pressure is measured twice to take the average value.

11% of them are located at the top of the blood pressure distribution (37 individuals; SBP>178mmHg) and 7% are located at the bottom of the blood pressure distribution (22 individuals; SBP<104mmHg), a total of 59 individuals were selected by direct sequencing method Perform SNP detection.

1.2 Experimental methods and results

1.2.1 DNA extraction

DNA was extracted from human blood by the conventional phenol-chloroform method, and the concentration was corrected to 20ng/ul for conventional PCR amplification.

1.2.2 Design of PCR and sequencing primers

According to the genome sequence of HP in GenBank, the following primers were designed and synthesized. The specific primers are shown in Table 1 below.

Table 1 Primer sequence list Primer name Sequence (5'-3') SEQ ID NO: sense primer tcggttcgctaccagtgtaa 2 antisense primer ccttagagcagtgccaggac 3

1.2.3 PCR amplification of HP gene

Using the extracted DNA as a template, PCR amplification was performed on a GeneAmp 9700 PCR instrument with the Touchdown program using Taq enzyme. The reaction conditions are: pre-denaturation at 94°C for 2 minutes, denaturation at 94°C for 30 seconds, annealing at 63°C for 40 seconds, extension at 72°C for 40 seconds, a total of 10 cycles, and the annealing temperature decreases by 0.5°C for each cycle; after denaturation at 94°C for 30 seconds, Anneal at 58°C for 40 seconds, extend at 72°C for 40 seconds, a total of 30 cycles; finally extend at 72°C for 7 minutes. PCR amplification products were verified by agarose gel electrophoresis.

As a result, an amplification product of 401 bp was obtained.

1.2.4 SNP discovery and detection

After the PCR products were purified by Resin resin, the ABI-PRISM ^TM 377 DNA sequencer (appliedbiosystems (ABI) of the United States) was used for bidirectional sequencing of the fluorescence-labeled terminal termination method, and the Polyphred software (University of Washington, USA http://droog. mbt.washington.edu/Polyphred.html) for sequence interpretation and SNP confirmation.

As a result, the following SNP was found to exist: G→C at position 4884.

1.2.5 SNP genotyping and association analysis

SNP genotyping by direct one-way sequencing. That is, typing and correlation analysis were carried out in hypertensive patients and normotensive control groups.

After the allele frequencies are counted, chi-square test is carried out. By comparing the individual data of the highest 11% with the lowest blood pressure of 7%, it is preliminarily determined whether the allele frequencies are linked with the blood pressure level.

It was found that the 4884 G-type SNP in SEQ ID NO: 1 had a frequency of 4% in hypertensive individuals and 17% in hypotensive individuals. The frequency difference between the two is 13%, which confirms that there is a correlation between the 4884th G/C type SNP in SEQ ID NO: 1 and the occurrence of hypertension.

Example 2

Essential Hypertension Susceptibility Detection Kit

As described in Example 1, the mutation of G→C at position 4884 in SEQ ID NO: 1 is closely related to hypertension. Therefore, based on this mutation, HP gene-specific primers can be designed and amplified using the patient's DNA as a template for detection.

Prepare a test kit (100 person-times), which contains: name sequence concentration sense primer SEQ ID NO: 2 100pmol antisense primer SEQ ID NO: 3 100pmol PCR reaction solution Containing Taq Enzyme dNTP Magnesium Ion PCR Reaction Buffer

Take 3ml of blood from the male patient to be tested, and use a conventional method (or use a specific kit) to extract DNA from the blood. Dilute the PCR primers in the high blood pressure detection kit to 1μmol/μl, and use the extracted DNA as a template to perform a PCR reaction with the provided primers. After the PCR products were purified, ABI-PRISM ^TM 377 DNA sequencer was used for bidirectional sequencing by fluorescence-labeled end termination method, and Polyphred software was used for sequence interpretation and SNP confirmation.

Alternatively, the chromatographic analysis of the amplified product and the normal control is performed with a denaturing high-performance liquid chromatograph (DHPLC), and the 4884-position G→C can also be detected.

As a result of the test, the susceptibility to hypertension of the test subjects containing 4884 G→C was higher than that of the normal population.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Sequence Listing

<110> Fudan University

  Shanghai Human Genome Research Center

<120> Correlation between haptoglobin gene and essential hypertension

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tgcccaaatt caggcagcct gtaagaggca gagtcaggat ttgaaccctg agccctccct 3540

gtactgcttg gctgtgaccg ccatgaccac agtgtgttct gctgggctta actggtgtcc 3600

aggcacttgg cttccagcac agcactcttt cccttcctcc ttctcatatt ctctctcctt 3660

tctcccttcc tgtctgcctc ctttcttctt cttcttttta attcttctcc ttaaatgcct 3720

tctcactctg ctctgggtgc agacttgact tttcctttgg ctcatttctt gccttttgtt 3780

tcaggagtat acaccttaaa tgataagaag cagtggataa ataaggctgt tggagataaa 3840

cttcctgaat gtgaagcagg tgggtgctga gcactgagca cttaagagag caggcaggcg 3900

tccagcgggg aacgtcctag aggcacagcc ttccagtgcg gcttcctctg agcacacaag 3960

agccaggagg agggatgtgg gagaaccgca gctggccagg gagagactta agcagttagg 4020

tgatgactcc ctaagggtca ccaagggtct tgttcattgg ggcctgaagg gcactggctg 4080

aatccactgt cggcactgcc cacagatcag gagagcctgt gcatacagag agcctgctag 4140

aaagccctgg gtctaaggag aagcaagctc cagggagaac aagtcaagga atgacataaa 4200

atcttaatcc atggaagcct agcaggaggc tggacatggg ctggaactcc tgcttctcgt 4260

tattaggagg agctgttgct ctctcctttc attctcagaa ccagaggcaa agacccagcc 4320

tcttctgctc ttactggtgt ggaaatgcca acctgcctcg tattaactgc accatctaca 4380

aaatctgagc tccagccagt gctgctctag attcatcttt ctttagagag aatgaattat 4440

tgtagcccct agccctttca atgaatttca gggaattgtg gaaattcctt tattgggata 4500

attgtttaaa tataatacag ttcgcgagct tctattcggg gtggaaggag attgatgtgc 4560

agagcagctc ccgctcatct gacttttcac ggttcactgg gaacaatttc caaatagcaa 4620

actctctggc ttctctctct ttgcagatga cggctgcccg aagccccccg agattgcaca 4680

tggctatgtg gagcactcgg ttcgctacca gtgtaagaac tactacaaac tgcgcacaga 4740

aggagatggt aagatgtgga caactgtctc catgccctac atacaaccccc cttctctgac 4800

attccatga tgggtggtgc tgaggtgatt cgccagaaag ttcgttgctc tccttggagc 4860

caggagattt agattctaat aagggttttg tcgccagtag ccatggccct ttgggcagac 4920

taacttttgt cagcctcaag ttttctgttt tgttaagggg aggcgatgcc atgcagccta 4980

cctcatgtaa atctcagagt cagattaca tctccagcag atgtgggaaa agaaggaatg 5040

ctgatgatga tgtcaccctc acctagtgag tcttgctgtc ctggcactgc tctaagggct 5100

ttatacttat ttgctcactt agtcctcaca gtatccctct gaacagagtt tattgttttc 5160

actttgctga taaggaaact gaggcacaga caggttgagt atcttgccca aattcaggca 5220

gcctgtaaga ggcagagtca ggatttgaac cctgagccct ccctgtactg cttggctgtg 5280

accgccatga ccacagtgtg ttctgctggg ctaactggc atccaggcac ttggcttcca 5340

gcacagcact ctttcccttc ctccttctca tatactctct ccttttcccc ttcctttttg 5400

tccccttttc ctcttccttt tagttcttct ctttaaatgc cttctcactc tgcacggggt 5460

ctagacttga cttctccttt ggctcacttc ttgccttttg tttcaggagt gtacaccctta 5520

aacaatgaga agcagtggat aaataaggct gttggagata aacttcctga atgtgaagca 5580

ggtgggtgct gagcacttaa gagagcaggc aggcgtccag cggggaacgt cctagaggca 5640

cagccttcca gtgcggcttc ctctgagcac acaagagcca ggaggaggga tgtgggagaa 5700

ccgcagctgg ccagggag acttaagcag ttaggtgatg actccctaag ggtcaccaag 5760

ggtcttgttc attagggcct gaagggcact ggctgaatcc attgtctaca tcgcccacag 5820

attaggagag cctgtgcata cagagagcct gctagagagc cctgggtcta aggagaagca 5880

agctccaggg agaacaagtc aaggaatgac ataaaatctt aatccatgga agcctagcag 5940

gaggctggac atgggctgga actcctgctt ctcgtttatta gggagagctg ttgctctctc 6000

ctttcattct cagaacaaga ggcaaaggcc cagcctcttc tgctcttact ggtgtggaaa 6060

tgccaacctg cctcgtatta actgcaccat ctacaaaatc tgagctccag ccagtgctgc 6120

tctagattca tctttcttta gagagaatga attattgtag cccctagccc tttcaatgaa 6180

tttcagggaa ttgtggaaat tcctttatg ggataattgt ttaaatataa tacagttcac 6240

cagccagggc tcaaaaatct cagtatttcc cacttccttt gttagaaaag tgggaaatag 6300

agctttttgt aatgtaaaca atttaaaaaa cagaattatt ttaaaactgc aactattgga 6360

aatgagatca gcaggtggta agggcaaagc atttaaatct ttctacttta cgcagcagtg 6420

acagccgccc atgctttcac ccctttctca gatggaaagg ctcttgcaca tttccactca 6480

cgagtgtctt gctctccttg acagtatgtg ggaagcccaa gaatccggca aacccagtgc 6540

agcggatcct gggtggacac ctggatgcca aaggcagctt tccctggcag gctaagatgg 6600

tttcccacca taatctcacc acaggtgcca cgctgatcaa tgaacaatgg ctgctgacca 6660

cggctaaaaa tctcttcctg aaccattcag aaaatgcaac agcgaaagac attgccccta 6720

ctttaacact ctatgtgggg aaaaagcagc ttgtagagat tgagaaggtt gttctacacc 6780

ctaactactc ccaggtagat attgggctca tcaaactcaa acagaaggtg tctgttaatg 6840

agagagtgat gcccatctgc ctaccttcaa aggattatgc agaagtaggg cgtgtgggtt 6900

atgtttctgg ctgggggcga aatgccaatt ttaaatttac tgaccatctg aagtatgtca 6960

tgctgcctgt ggctgaccaa gaccaatgca taaggcatta tgaaggcagc acagtccccg 7020

aaaagaagac accgaagagc cctgtagggg tgcagcccat actgaatgaa cacaccttct 7080

gtgctggcat gtctaagtac caagaagaca cctgctatgg cgatgcgggc agtgcctttg 7140

ccgttcacga cctggaggag gacacctggt atgcgactgg gatcttaagc tttgataaga 7200

gctgtgctgt ggctgagtat ggtgtgtatg tgaaggtgac ttccatccag gactgggttc 7260

agaagaccat agctgagaac taatgcaagg ctggccggaa gcccttgcct gaaagcaaga 7320

tttcagcctg gaagagggca aagtggacgg gagtggacag gagtggatgc gataagatgt 7380

ggtttgaagc tgatgggtgc cagccctgca ttgctgagtc aatcaataaa gagctttctt 7440

ttgacccatt tctgtgttgt gttcagtctt gagtcttttt tatttgctcc tttatggtcc 7500

agggtagtca gaaggtatag agtctactgg gagtatggca gaaaacaccc taaacccact 7560

ggaaatcccg aaggtgatac aaactcttcc accttaggga atcatgctca ctgattgagt 7620

gcctattgaa tgctaggtcc cagaaagtta actgttgtcc ttgttttaca gacaaggaaa 7680

cagagactca gagatggtaa gtgagttgct taaggttaca tagctatgaa acagggaagc 7740

agaactttga acccaggtct gtttgataca aactcagagg tcctttcact gcatgctgtt 7800

gcctcctcaa agtgaattag gagaaaaggc atgggcctgg ttgaggaaga ggctagctca 7860

aaatgggatg gggaaaaagt gttttaacac agacagtaat tcagagtttg ggatctaacc 7920

taccagcact ccagaaaaca aaagacaatg atgtaaaggc aggatctctg gacttactga 7980

gtccaaatca cagaatggcc cataaatttg ctaggtaatt taggt 8025

<210>2

<211>20

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> Primer

<400>2

tcggttcgct accagtgtaa 20

<210>3

<211>20

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> Primer

<400>3

ccttagagca gtgccaggac 20

Claims (10)

1. a method for in vitro detection of whether there is a single nucleotide polymorphism of haptoglobin gene HP in a sample, characterized in that, comprising steps: (a) amplifying the HP gene of the sample with HP gene-specific primers to obtain an amplified product; and (b) Detect whether the following single nucleotide polymorphisms exist in the amplification product: 4884 bits G → C; Wherein, the nucleotide position numbers are based on SEQ ID NO:1. 2. The method of claim 1, wherein the gene-specific primer has the sequences of SEQ ID NO: 2 and 3. 3. The method according to claim 1, wherein the length of the amplified product is 100-3000bp, and contains the 4884th position in SEQ ID NO:1. 4. A test kit for detecting hypertension, characterized in that it includes primers for specific amplification of HP genes or transcripts, and the amplified length of the primers is 100-3000bp, and contains SEQ ID NO: 1 Amplification product at position 4884. 5. The kit of claim 4, further comprising reagents selected from the group consisting of: (a) a probe that binds to the mutation at position 4884 in SEQ ID NO: 1; (b) Recognition of the mutant restriction enzyme at position 4884 in SEQ ID NO:1. 6. test kit as claimed in claim 5, is characterized in that, described mutation is following single nucleotide polymorphism: 4884 bits G → C; Wherein, the nucleotide position numbers are 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. A method for diagnosing an individual's susceptibility to hypertension, characterized in that it comprises the steps of: detecting the individual's HP gene, transcript and/or protein, and comparing it with normal HP gene, transcript and/or protein, A difference indicates that the individual is more likely to have high blood pressure than the normal population. 9. The method according to claim 8, characterized in that the gene or transcript of HP is detected, and the difference is compared with the normal HP nucleotide sequence. 10. The method of claim 9, wherein said difference is the following single nucleotide polymorphism: 4884 bits G → C; Wherein, the nucleotide position numbers are based on SEQ ID NO:1.