CN103882110B - A kind of reagent detecting susceptibility of ankylosing spondylitis - Google Patents

Abstract

The invention discloses a kind of nucleotide sequence detecting susceptibility of ankylosing spondylitis, belong to biological technical field.Detecting a nucleotide sequence for susceptibility of ankylosing spondylitis, is nucleotide sequence shown in sequence table SEQ ID No.1.Advantage of the present invention is: the dependency illustrating AGER gene polymorphism sites and mandatory spondylitis first, provide a kind of method and the detection reagent of predicting mandatory spondylitis susceptibility, the method can be used for auxiliary diagnosis and the new drug development of mandatory spondylitis.

Description

A kind of reagent detecting susceptibility of ankylosing spondylitis

Technical field

The present invention relates to a kind of reagent detecting susceptibility of ankylosing spondylitis, predict the susceptibility of experimenter for ankylosing spondylitis by measuring with the polymorphism of AS genes involved AGER in particular, the method can be used for the auxiliary diagnosis of disease, treatment and new drug development, belongs to biological technical field.

Background technology

Ankylosing spondylitis (Ankylosing spondylitis, AS) is a kind of autoimmune disorder, and be mainly in the person between twenty and fifty in 16-40 year, the ill ratio of men and women is about 4 ~ 10:1.Pathology Chang Shouxian betides articulatio sacroiliaca, and minority patient with severe symptoms shows as whole rigid spine.In addition, some patients is with the outer pathology of the backbones such as hip joint in various degree, eye, lung, cardiovascular, kidney.The sickness rate of AS in white race crowd is about 1% ~ 3%, China's AS morbidity is about 0.2-0.6%, wherein the patient of more than 60% gets involved with hip joint, cause more than 20% AS patient disabilities, inflammation mainly involves the bone attachment point of joint capsule, tendon and ligament, cause local joint accretion tetanic, limitation of activity.So far the medicine can obviously alleviating and control disease progression is still lacked clinically.AS belongs to multigenic disease, has obvious genetic predisposition, although it has been generally acknowledged that heredity and immune factor play a leading role in AS falls ill, definite cause and onset of disease mechanism is still unclear.

Carry out the research of AS inherited pathogenic factor at present, the SNP that adopts, as the association analysis method of genomic marker, is effective, is proven more.SNP refers to the DNA sequence polymorphism that in chromogene group level, single nucleotide diversity causes, frequency in crowd needs >1%, SNPs is biallelic marker, 70.1% is had for the conversion between homotype base: as G/A or T/C, 29.1% for occurring in the transversion between purine and pyrimidine in this single base change.C(cytosine(Cyt)) be the most labile site in human genome because great majority are methylated cytosines, can spontaneous deaminizating be converted to T(thymus pyrimidine), SNP contains the 80-90% of known polymorphism, is modal heritable variation.

Because the selective pressure of existence causes the distribution of SNP in term single gene and whole genome to be ununiformity.SNPs is 4 times of coding region in the quantity of gene non-coding region, and sum can reach 300 ten thousand.SNP with its density high (average every 1kb just have 1), representative strong (SNP being arranged in gene internal may directly affect protein structure or expression level), genetic stability good (with microsatellite polymorphism comparatively speaking), be easy to the features such as automated analysis (because SNP mostly is biallelic marker crowd, can simply with " +/-or 1/0 " direct somatotype) and become good genetic marker.

1973, first Brewerton etc. found and the Human leukocyte antigen-B27 of AS strong correlation (HLA-B27).Along with progress of research, other tumor susceptibility genes relevant to AS such as tumor necrosis factor-alpha (TNF-α), il-1 (IL-1) are identified successively.

Advanced Glycation Endproducts (advanced glycosylation end product-specific receptor, AGER) gene is positioned at 6q21.3, total length 28933870bp, containing 11 exons, its coded product is a kind of cell membrane surface receptors, this assignment of genes gene mapping, in human leucocyte antigen III region, occurs to develop etc. closely related with immune response and self property Immunological diseases.

There is no any result of study be associated with AS about AGER gene at present.

Summary of the invention

Main purpose of the present invention is to provide a kind of method of susceptibility of ankylosing spondylitis gene.

Second object of the present invention is to provide a kind of reagent detecting susceptibility of ankylosing spondylitis gene, comprises PCR primer and the test kit containing this primer.

For achieving the above object, the present invention is by the following technical solutions:

Detecting a nucleotide sequence for susceptibility of ankylosing spondylitis, is nucleotide sequence shown in sequence table SEQ ID No.1.This nucleotide sequence is that the 8th of AGER gene includes subarea Partial Fragment, and namely its+206 be variant sites, and indicate with letter " R ", this site is positioned at AGER gene+879, i.e. Chr6:g.32149745G>A.Fig. 1 is the schematic diagram in AGER gene structure and polymorphic variation site thereof, includes 11 exons, and Chr6:g.32149745G>A site to be marked in AGER gene map the corresponding position that the 8th includes subarea.

When the genotype of described mononucleotide polymorphism site Chr6:g.32149745G>A is GG, the susceptibility of experimenter is minimum; Carry A allelotrope, the susceptibility of experimenter raises.

The primer of one group of detection susceptibility of ankylosing spondylitis, can increase and obtain the nucleotide sequence of described detection susceptibility of ankylosing spondylitis.

The nucleotide sequence of described primer is respectively shown in sequence table SEQ ID No.2 and sequence table SEQ ID No.3.

A detection method for susceptibility of ankylosing spondylitis gene, comprises the steps:

(1) genomic dna of extracting sample, the 8th of amplification AGER gene includes the Partial Fragment that subarea comprises mononucleotide polymorphism site Chr6:g.32149745G>A;

(2) genotype of mononucleotide polymorphism site Chr6:g.32149745G>A in detecting step (1) product, when genotype is GG, the susceptibility of experimenter is minimum; Carry A allelotrope, the susceptibility of experimenter raises.

Nucleotide fragments in described step (1) is the nucleotide sequence shown in sequence table SEQ ID No.1, and mononucleotide polymorphism site Chr6:g.32149745G>A is positioned at+206 of this nucleotide sequence.

The 8th of described amplification AGER gene includes the Partial Fragment that subarea comprises mononucleotide polymorphism site Chr6:g.32149745G>A, and the nucleotide sequence of one group of primer of use is respectively shown in sequence table SEQ ID No.2 and SEQ ID No.3.

The invention provides a kind of test kit detecting ankylosing spondylitis tumor susceptibility gene, wherein include the primer pair that subarea comprises mononucleotide polymorphism site Chr6:g.32149745G>A and the general components, reagent, damping fluid etc. of test kit detected for pcr amplification containing the 8th of specific amplification AGER gene of the present invention, those skilled in the art know these general components and detection method.Whole components in test kit of the present invention, content, source and using method are as follows:

Predict a test kit for ankylosing spondylitis, detect application for 10 person-portions, be made up of following reagent:

30 μ L10 × PCR damping fluids (purchased from Pharmacia);

5 μ L10mM dNTP mixed solution (purchased from Pharmacia);

5 μ L Taq archaeal dna polymerases (2unit/ μ L) (purchased from Takara);

2.5 μ L F1 primers, be the nucleotide sequence shown in SEQ ID NO.2, concentration is 10pmol/ μ L;

2.5 μ L R1 primers, be the nucleotide sequence shown in SEQ ID NO.3, concentration is 10pmol/ μ L;

235 μ L pure water.

Using method:

(1) pcr amplification: include subarea Partial Fragment by the 8th of pcr amplification AGER gene, prepare mixed solution: 10 × PCR reaction buffer 3 μ L, 10mM/L dNTP0.5 μ L, Taq DNA polymerase 0.5 μ L, 10pM/L F1 primer 0.5 μ L, 10pM/L R1 primer 0.5 μ L, genomic dna 2 μ L, adds pure water to 30 μ L.PCR reaction conditions is 95 DEG C of denaturation 5min, 95 DEG C of sex change 30s, and 65 DEG C of annealing 30s, 72 DEG C extend 25s, altogether 35 circulations, 72 DEG C of total elongation 2min.In each system, the paraffin oil of 20 μ L is added, to prevent from evaporating before PCR.

(2) genotype judges: by PCR primer direct Sequencing, and the difference according to fluorescent signal judges genotype.

AGER gene the 8th includes the purposes of subarea mononucleotide polymorphism site Chr6:g.32149745G>A in the reagent preparing diagnosis or treatment ankylosing spondylitis or medicine.

Measuring method of the present invention determines the genomic dna deriving from people, and sample source is unrestricted, as: body fluid (blood, ascites and urine etc.), histocyte (as hepatic tissue) etc.Genomic dna can be prepared by these samples of Isolation and purification.The concentration of adjustment genomic dna, makes it consistent as much as possible.Take genomic dna as template, the nucleic acid fragment containing AGER gene mutation site can be amplified, to obtain the great amount of samples of mensuration.This sample obtained containing the DNA fragmentation of AGER genovariation point by amplification, is particularly suitable for as mensuration material.

When carrying out gene auxiliary diagnosis, the present invention is preferably applied in the auxiliary diagnostic measuring and exist according to AGER gene mutation type (the such as the 8th includes subarea mononucleotide polymorphism site Chr6:g.32149745G>A suddenlys change), auxiliary diagnostic comprises the particular agent as neccessary composition, and it corresponds to the method for measuring rs4714476 gene mutation type.Suitable particular agent is selected, as DNA fragmentation and/or the primer for pcr amplification step by the measuring method adopted.

Advantage of the present invention is: the present invention illustrates the dependency of AGER gene polymorphism sites and mandatory spondylitis first, provide a kind of method and the detection reagent of predicting mandatory spondylitis susceptibility, the method can be used for auxiliary diagnosis and the new drug development of mandatory spondylitis.

Below in conjunction with the drawings and specific embodiments, the present invention is further described; so that the public has a better understanding summary of the invention; not limitation of the present invention, the equivalent replacement of all any this areas of doing according to the disclosure of invention, all belongs to protection scope of the present invention.

Accompanying drawing explanation

Fig. 1 is the schematic diagram in AGER gene structure and polymorphic variation site thereof

Fig. 2 is the sequencer map in AGER genovariation site

Embodiment

As follows for representing the english abbreviation of reagent in the following example:

10 × PCR damping fluid: 10mM Tris-HCI (pH=8.3), 500mM Repone K (KCl), 10mM magnesium chloride (MgCl ₂), 0.01% (W/V) gelatinum

DNTP: deoxynucleoside triphosphate

EDTA: disodium ethylene diamine tetraacetate

TE：10mM Tris-HCl(pH=7.5)，1mM EDTA(pH=8.0)

Embodiment 1: the extraction of blood sample collection and genomic dna:

One. case is selected in:

By the New York Revised diagnostic criteria MethodsThe cases enrolled of 1984, choose altogether from Jilin Area consanguinity-less relation AS patient 72 example (age: 14-44 year, average 24 years old), with area normal healthy controls volunteer 111 example (age: 39-72 year, average 46 years old).All persons under inspection are Han nationality and signature Written informed consent, this research obtains Beijing Hospital, the accreditation of ethics audit committee of Gerontological Research Center institute of the Ministry of Health, meets " World Medical Association's Declaration of Helsinki ": the ethic principle of human medical research.

Two. according to following method, preparation human gene group DNA.

1. in the 1.5mLEP pipe of label, add 1000 μ L erythrocyte cracked liquids, after add 400 μ LEDTA anticoagulations (anticoagulation puts upside down mixing 3-5 time before adding), put upside down mixing, standing 10 minutes of room temperature;

2.13000rpm, after centrifugal 30 seconds, removes supernatant liquor;

3. in gained precipitation, add 480 μ L nucleic acid cleavage liquid, attack tube wall, fully adding 20 μ L Proteinase Ks (diluting 20 times of diluted protein enzyme K with splitting karyolymph) after mixing, putting upside down mixing, 65 DEG C of incubators 10 minutes, (period mixes frequently up and down, guarantees without grumeleuse);

4. be down to room temperature after taking out, add 300 μ L albumen precipitation liquid, fully put upside down mixing, leave standstill 10 minutes, centrifugal 2 minutes of 13000rpm;

5. moved to by supernatant liquor in new EP pipe, add the Virahol of 670 μ L precoolings, fully put upside down mixing (more than 10 times), visible linear DNA forms little agglomerate gradually, centrifugal 2 minutes of 13000rpm;

6. abandon supernatant liquor and guarantee that precipitation is stayed in EP pipe, adding 670 μ L70% ethanol, mixing of turning upside down, centrifugal 2 minutes of 13000rpm;

7. abandon supernatant, make ethanol volatilization in pipe clean;

8. add TE solution (400 μ L), fully dissolve, the genomic dna extracted is carried out to the analysis of concentration and purity, draw part DNA solution as working fluid, concentration correction to 20ng/ μ L, be placed in 4 DEG C for subsequent use, residue genomic dna put-20 DEG C of Refrigerator stores.

The identification qualification of embodiment 2SNP

The present invention adopts PCR-sequencing analysis method to include+879 sites in subarea to the 8th of AGER gene the, and namely its allelotrope of Chr6:g.32149745G>A(is G/A) genotype detect.

The determination of one .PCR-HRM primer

From Genebank, look into the DNA base sequence (SEQ ID No.1) got near Chr6:g.32149745G>A, design of primers completes under Oligo7.0 software.Object fragment is positioned at AGER gene the 8th and includes subarea, total length 342bp, determines positive-sense strand F1(+34bp-+295bp) and antisense strand R1(+15bp-+309bp), specific primer sequence is as follows:

F1：5’-CAATCTATGCCTCCTGGGTTCAAG-3’（SEQ ID NO.2）

R1：5’-GCCAAGAGAGCAGCCAAGCCT-3’（SEQ ID NO.3）

Two .PCR reaction system and reaction conditionss

Subarea Partial Fragment is included by pcr amplification AGER gene the 8th, PCR reaction system is: 10 × PCR reaction buffer 3 μ L, 10mM/LdNTP0.5 μ L, Taq DNA polymerase 0.5 μ L, 10pM/L upstream primer 0.5 μ L, 10pM/L downstream primer 0.5 μ L, genomic dna 1 μ L, adds deionized water to 30 μ L.In each system, add 20 μ L paraffin oils during PCR, prevent from evaporating.PCR reaction conditions is 95 DEG C of denaturation 5min, 95 DEG C of sex change 30s, and 65 DEG C of annealing 30s, 72 DEG C extend 25s, altogether 35 circulations, 72 DEG C of total elongation 2min.

Three. order-checking judges genotype

PCR primer detects through 8% polyacrylamide gel electrophoresis, send Hua Da gene sequencing portion to carry out sequence verification after gel imaging system observation is qualified.Fig. 2 is the sequencer map in AGER genovariation site.

The dependency of embodiment 3 gene SNP and mandatory spondylitis (AS)

One. statistical method:

Use the populational representation of Hardy-Weinberg balance check research sample.Pearson chi square test in SPSS11.0 software is utilized to calculate allelotrope, the distribution frequency of genotype between AS case group and Normal group in AGER gene C hr6:g.32149745G>A site, the risk OR value of AS and 95%CI credibility interval thereof take P<0.05 as significance of difference standard.

Two. result:

On the AGER gene being positioned at karyomit(e) 6p21.3 region, the genotype in Chr6:g.32149745G>A site and the distribution of gene frequency between case and control group refer to table 1.

The genotype in table 1:AGER (Chr6:g.32149745G>A) site and the distribution of gene frequency between case-control group

Note: OR: odds ratio; CI: credibility interval.* A allelotrope is the risk allelotrope of easily suffering from AS.Experimenter is divided into risk allelotrope (GA) carrier of AS, and non-risk allelotrope (GG) carrier.

From table 1, AGER(Chr6:g.32149745G>A site) A allelotrope, namely be T allelotrope on its DNA complementary strand, distribution frequency in PATIENT POPULATION is significantly higher than its genotypes distribution and allele frequencies (0.08vs.0.009) in healthy normal population, there is significant difference (P=0.000), and the OR value in A site is 3.752,95%CI; In risk allelotrope (GA) carrier and non-risk allelotrope (GG) carrier of AS, the distribution frequency of risk genotype in case group is significantly higher than (P < 0.05) in control group, all show AGER gene C hr6:g.32149745G>A site and AS is ill is proportionate, the risk of AS morbidity may be increased.Therefore, when the genotype of described mononucleotide polymorphism site Chr6:g.32149745G>A is GG, the susceptibility of experimenter is minimum; Carry A allelotrope, when namely genotype is GA, the susceptibility of experimenter raises.

Implement 4 detection kit

Preparation detects the test kit of ankylosing spondylitis (AS) relevant risk, includes the primer pair that can amplify AGER gene SNP+879 site, and other PCR-HRM corresponding reagent.Test kit of the present invention detects application for 10 person-portions, and keep in Dark Place in-20 DEG C, its component, content and source comprise:

30 μ L10 × PCR damping fluids (Pharmacia)

5 μ L10mMdNTP mixed solutions (Pharmacia)

5 μ L Taq DNA polymerase (2U/ μ L) (Takara)

μ L F1 primer 2.5 (SEQ ID No.2) (10pM/ μ L)

μ L R1 primer 2.5 (SEQ ID No.3) (10pM/ μ L)

235 μ L pure water.

After PCR order-checking detects, can detect that AGER gene the 8th includes subarea Chr6:g.32149745G>A polymorphism easily.

Proof test: adopt this test kit, random selecting AS clinical samples 10 example, control group sample 10 example, detects AGER gene the 8th through PCR order-checking and includes subarea Chr6:g.32149745G>A loci polymorphism.

One. method:

1.PCR amplification: include subarea Partial Fragment by pcr amplification AGER gene the 8th, PCR reaction system is: 10 × PCR reaction buffer 3 μ L, 10mM/LdNTP0.5 μ L, Taq DNA polymerase 0.5 μ L, 10pM/L F1 primer 0.5 μ L, 10pM/L R1 primer 0.5 μ L, genomic dna 1 μ L, adds deionized water to 30 μ L.In each system, add 20 μ L paraffin oils during PCR, prevent from evaporating.PCR reaction conditions is 95 DEG C of denaturation 5min, 95 DEG C of sex change 30s, and 65 DEG C of annealing 30s, 72 DEG C extend 25s, altogether 35 circulations, 72 DEG C of total elongation 2min.

2. order-checking judges genotype

PCR primer detects through 8% polyacrylamide gel electrophoresis, send Hua Da gene sequencing portion to carry out sequence verification after gel imaging system observation is qualified.Fig. 2 is the sequencer map in AGER genovariation site.

Two. result:

Result shows, and AS clinical samples AGER gene the 8th includes 3 examples that subarea Chr6:g.32149745G>A loci polymorphism genotype is GA, GG7 example; Control group genotype is all GG.Present method can effectively detect Chr6:g.32149745G>A loci polymorphism: during GG, and the susceptibility of experimenter is minimum; Carry A allelotrope, the susceptibility of experimenter raises.

The present invention has the illustration of practicality:

The detection method of AGER gene pleiomorphism of the present invention can be used for the T allelotrope of the rare variant sites on the AGER gene in analyst's euchromosome 6p21.3 district, namely be A allelotrope on its DNA complementary strand, be applied to the complementary diagnosis of AS and the individual AS risk of assessment how, be beneficial to early intervention and the treatment of carrying out AS.

The present invention is utilized to set forth the nucleotide variation in AGER gene C hr6:g.32149745G>A site, as one of biomarker, can be used as the screening of the molecular target of medicinal design, to help to find the bioactive molecule having and regulate AGER to express, promote AS new drug development.

The nucleotide sequence of detection AGER gene pleiomorphism that the present invention sets up and AS related locus, can highly sensitive, the specific test kit being applied to AS gene auxiliary diagnosis.

As above told, reached a conclusion, the polymorphism in AGER gene C hr6:g.32149745G>A site and AS tool significant correlation.Therefore, measure this gene pleiomorphism according to the present invention, can be used for the gene auxiliary diagnosis of AS.

Invention describes the new mutant site that AGER Gene A S-phase is closed, and provide a kind of method measuring AGER gene pleiomorphism, and, according to the present invention, only need a small amount of DNA sample to be just enough to measure gene polynorphisms.

As a result, the invention provides a kind of method measuring AS related gene polymorphism.

Claims (2)

1. one group is detected the primer of susceptibility of ankylosing spondylitis, it is characterized in that: the nucleotide sequence of described primer is respectively shown in sequence table SEQ ID No.2 and sequence table SEQ ID No.3.

2. detect a test kit for ankylosing spondylitis tumor susceptibility gene, it is characterized in that being made up of following reagent:

30 μ L 10 × PCR damping fluids;

5 μ L 10mM dNTP mixed solutions;

5 μ L Taq archaeal dna polymerases, 2unit/ μ L;

2.5 μ L F1 primer, be the nucleotide sequence shown in SEQ ID NO.2, concentration is 10pmol/ μ L;

2.5 μ L R1 primer, be the nucleotide sequence shown in SEQ ID NO.3, concentration is 10pmol/ μ L;

235 μ L pure water.