CN101173278A - Ankylosing spondylitis pathopoiesia correlation gene IRS-1 mutant gene, detecting method and reagent kit thereof - Google Patents

Abstract

The invention discloses an IRS-1 mutant to relative gene of ankylosing spondylitis pathogenesis, which comprises IRS-1 gene series and 2759C to G heterozygous mutation. The invention also discloses a method of detecting the IRS-1 mutant, which comprises the comparison between the nucleotide sequence from samples under testing and the sequence of IRS-1 normal gene and checking that mutating is arranged at 2750 situs of the IRS-1 gene and mutated into G from basic group C; the invention also discloses a kit detecting the IRS-1 mutant, which comprises a PCR primer designed at neighboring coding area aiming at IRS-1 gene or No. 2759 basic group of the IRS-1 gene and other components. The invention has the advantages of benefitting for further studying pathogenesis theory of ankylosing spondylitis, developing work of IRS-1 mutation screening for ankylosing spondylitis patients, providing relative information for reference for prenatal diagnosis of patients with family history, predicting, screening and assuring disease of family patients and sporadic patients and providing service for diagnosis and therapy for ankylosing spondylitis.

Description

Ankylosing spondylitis pathogenic related gene IRS-1 mutator gene and detection method and test kit

Technical field

The invention belongs to biomedicine field, relate to the haplotype zone at Disease-causing gene place of a kind of detection ankylosing spondylitis (AS) and method, test kit and the IRS-1 mutator gene of the relevant IRS-1 mutator gene that causes a disease in the district specifically.

Background technology

Ankylosing spondylitis belongs to the rheumatosis category, is a kind of in the seronegative spondyloanthropathy.At present, ankylosing spondylitis is that a kind of reason is still not really clear and definite, the same disease or HLAB 27 positives are arranged in the family history, with the backbone is the chronic disease of major lesions, and pathology is mainly involved articulatio sacroiliaca, causes rigid spine and fibrosis, cause bend over, Ambulatory Activity is limited, and the pathology of in various degree eye, lung, muscle, bone can be arranged, the disorder of autoimmune function is also arranged, so belong to autoimmune disorder again.

People such as Sun have separated one section cDNA in 1991, the Tyrosine O-phosphate albumen of coding 160-185kD, and this albumen is considered to participate in the insulin signaling conduction as a substrate of insulin receptor tyrosine kinase.This albumen, called after IRS-1 (IRS-1) is thought at present at present, and IRS-1 is the important molecule of conduction in the insulin signaling born of the same parents, be the intermediate of the multiple biological regulating effect of Regular Insulin, in to many cells of insulin response and tissue, all can find.It does not show the activity of inherent enzyme, but believe it is as a docking protein, combination and activate other signal transduction molecule after by its tyrosine of insulin receptor kinase phosphorylation.

People such as Stoffel have cloned the IRS-1 gene in the gene pool of human male's placenta.Application is PCR from the DNA of people mouse somatocyte mosaic hybrid, and they locate this gene in No. 2 karyomit(e)s, utilize the in situ hybridization fluorescence technique, and they fix on 2q35-36.1 with the zone.Utilize gene clone and in situ hybridization fluorescence technique, people such as Nishivama 1994 the IRS-1 assignment of genes gene mapping at 2q36.People such as Araki in 1993 equally this assignment of genes gene mapping in this zone, the homologous gene of locating mouse simultaneously is positioned at karyomit(e) No. 1.People such as Stoffel have also identified a dna polymorphism that helps the simple polyphone tumor-necrosis factor glycoproteins (STR) of gene studies in 1993.Because the central dogma of signal transduction path, IRS-1 can be used as a candidate point of insulin function defect sites in non-insulin-dependent diabetes mellitus (NIDDM) (NIDDM).

Cause the phosphorylation of subunit IRS-1 and IRS2 in the tenuigenin after Regular Insulin and its receptors bind, this process is with several to comprise the regional albumen of Src homologous region 2 (SH2) relevant.In order to differentiate unique conjugated protein of IRS-1, people such as Ogihara have been scanned human heart cDNA library in 1997 with reorganization IRS-1, and they have obtained the hypotype of 14-3-3 (YWHA) protein family, respectively called after 14-3-3-epsilon and 14-3-3-zeta.14-3-3 albumen is verified relevant with IRS-1 in the cerebral tissue of L6 myotube cell, HepG2 hepatoma cells, Chinese hamster ovary cell and ox.IRS-2 is the albumen with the IRS-1 structural similitude, also shows to form mixture with 14-3-3 albumen in the expression system of baculovirus.The quantity of the 14-3-3 relevant with IRS-1 is not influenced by the Regular Insulin activated, but at okadaic acid, a kind of serine/threonine phospholipase inhibitor significantly increases after the processing.The polymorphic inhibition experiment of carrying out with the polypeptide that contains phosphoserine of IRS-1 shows, IRS-1 comprised 3 14-3-3 protein binding sites (Ser-270, Ser-374, Ser-641).In these three sites, the sequence that centers on 270 Serines is positioned at Tyrosine O-phosphate combination (PTB) zone of IRS-1, and this zone is relevant with the interaction of insulin receptor (INSR).But experiment shows the deletion mutantion of IRS-1 still has the ability in conjunction with 14-3-3 of still keeping in PTB zone in the actual upper body.Effect after people such as Ogihara have also studied 14-3-3 albumen and the IRS-1 of insulin-mediated phosphorylation combines.Phosphorylated amino acid studies show that, on tyrosine and serine residue, the IRS-1 of contrast and anti-IRS-1 antibody mediated immunity post precipitation is difficult to by the Regular Insulin activating phosphataseization with IRS-1 behind the anti-14-3-3 antibody coimmunoprecipitation.Author prompting thus, thus relatedly may in process, play the part of a part of role with 14-3-3 is proteic by blocking-up insulin receptor and IRS-1 relation adjusting insulin sensitivity.

So far, except judging ankylosing spondylitis, also there is not laboratory diagnosis reagent to be used for diagnosis that ankylosing spondylitis is determined from clinical symptom and imaging data.

Summary of the invention

The technical problem that the present invention need solve provides the haplotype zone at ankylosing spondylitis Disease-causing gene place and the relevant IRS-1 mutator gene that causes a disease in the district.

The technical scheme that solves the problems of the technologies described above is as follows:

A kind of ankylosing spondylitis pathogenic related gene IRS-1 mutator gene, this IRS-1 mutator gene contains the IRS-1 gene order, and contains 2759C → G heterozygous mutant.

Further, this IRS-1 mutator gene also contains 2727C → T, 3419T → A, rs6725556T → C ,-3637A → T ,-3295G → T, rs6436635G → A, rs3731597G → A, 3 ' UTR 707G → T, one or more in-2990C → T or rs1801278A → G heterozygosis or the homozygous mutation.

The encoded protein matter of above-mentioned ankylosing spondylitis pathogenic related gene IRS-1 mutator gene has 580R → G (mutational site of Arg-＞Gly).

Another technical problem that the present invention need solve provides a kind of method that detects above-mentioned ankylosing spondylitis pathogenic related gene IRS-1 mutator gene.

A kind of method that detects ankylosing spondylitis pathogenic related gene IRS-1 mutator gene; the sequence of nucleotide acid sequence that obtains from testing sample and IRS-1 normal gene compares; checking suddenlys change is positioned at 2759 sites of IRS-1 gene, sports bases G by C.

Described detection method mainly may further comprise the steps,

1) DNA in the extraction sample to be detected;

2) be template with this DNA, carry out the PCR reaction, carry out the PCR reaction, obtain the PCR reaction product with PCR primer near the design of the coding region IRS-1 gene or the 2759th base of IRS-1 gene;

3) base sequence of measuring the PCR reaction product is formed;

4) sequence with resulting sequence and IRS-1 normal gene compares, and determines that 2759 sport bases G.

Preferably, the based composition of the primer in the PCR reaction is:

IRS-1-F：CGGATGAGTATGGCTCCAGT

IRS-1-R：ACCTCCAATGTCAGGAGAGC。

The base sequence of the described PCR of measuring reaction product form can be with the PCR reaction product the sequenator order-checking or with the specific probe sequencing by hybridization.

Further, by the BLAST comparison, translate to determine whether to exist R580G amino acid mutation site by normal single open reading frame.

Sample to be detected can be blood, body fluid or tissue etc.In addition, also available additive method is realized detecting of mutational site, comprise: quantitative fluorescent PCR, sex change high performance liquid chromatography (dHPLC) technology, restriction fragment length polymorphism (RestrictionFragment Length Polymorphism, RFLP), and the ligase enzyme detection reaction (ligase detection reaction, LDR) etc.

Another object of the present invention provides a kind of test kit that is used for diagnosing ankylosing spondylitis.

A kind of test kit that is used for diagnosing ankylosing spondylitis pathogenic related gene IRS-1 mutator gene mainly includes:

With PCR primer, TaqDNA polysaccharase and corresponding damping fluid near the design of the coding region IRS-1 gene or the 2759th base of IRS-1 gene.

Another object of the present invention provides the application in the medicine of ankylosing spondylitis pathogenic related gene IRS-1 mutator gene in preparation treatment disease.

The application of described ankylosing spondylitis pathogenic related gene IRS-1 mutator gene in the medicine of preparation treatment ankylosing spondylitis

Contain the virus vector of wild-type IRS1 gene by structure, be prepared into medicine, it is imported in patients with ankylosing spondylitis body, the immune inflammation reaction that improves AS patient's abnormal bone metabolism and bring out thus, thus reach the purpose of treatment.

The present invention chooses the IRS-1 gene as research object, by (8 AS patients of ankylosing spondylitis family at three generations's pedigree, 18 non-patients), 97 are distributed among AS patient and 122 normal healthy controls persons and carry out examination, discovery in the family patient, all patients all have the IRS-1 gene new mutational site (2759C → G, R580G), this mutational site in other non-patient of family all less than finding, illustrate this mutational site and ankylosing spondylitis be divided into from.And other 3 of this site is distributed among the AS patient and is verified, and other 122 normal healthy controls persons do not find this mutational site, have further confirmed the dependency of this mutational site and AS.

The present invention shows that by full genome scanning and linkage analysis this zone is ankylosing spondylitis susceptible zone, and the gene IRS-1 in this zone is the disease related gene of ankylosing spondylitis.IRS-1 mutator gene of the present invention is relevant with ankylosing spondylitis, finds also in distributing the patient that in addition a plurality of mutational sites are relevant with AS.This mutator gene with and related detecting method and test kit will help carrying out clinically the IRS-1 Mutation Screening work of patients with ankylosing spondylitis, can be in AS family clinically and distribute patient's primary dcreening operation or assist diagnosis to provide relevant information for your guidance, and the while also provides necessary means for the exploitation of related drugs.IRS-1 also is expected the target spot as pharmacological agent, for example gene therapy.In addition, therapeutic strategy also comprises: the synthetic mutagenic compound, and the induced mutation reparation, making the mutant allele reparation is wild-type, is the gene therapy of target with the catastrophe point; Synthetic IRS1 polypeptide, compensatory treatment etc.

In general, IRS-1 mutator gene of the present invention and detection method will help further studying the pathogenesis of ankylosing spondylitis, carry out the IRS-1 Mutation Screening work of patients with ankylosing spondylitis clinically, for patients with ankylosing spondylitis diagnosis and treatment provide relevant information that can reference.

Figure of description

Fig. 1 is the haplotype collection of illustrative plates of the AS family of Cyrillic software building;

Fig. 2 makes up family collection of illustrative plates experiment flow synoptic diagram;

Fig. 3 is the PCR reaction conditions synoptic diagram of embodiment 2;

Fig. 4 is the PCR product sequencing reaction condition synoptic diagram of embodiment 2.

Embodiment

Embodiment 1

Collect the AS family in China, (8 AS patients, 18 non-patients), 97 are distributed among AS patient and 122 normal healthy controls persons and carry out examination.All patients meet the New York standard of revision in 19994 all through the making a definite diagnosis of Rheumatism Dept. expert.Utilize Cyrillic 2.0 software building family collection of illustrative plates.By to behind the family pedigree analysis, can find following characteristics 1) continuously the three generations patient is all arranged, transmit continuously.2) the existing women of patient has the male sex again, and the men and women is ill has equal opportunities.So find to meet the autosomal dominant inheritance pattern two familys.

Experiment flow is referring to Fig. 2.

The selection of microsatellite locus and design of primers

1. the selection and the design of primers of full genome scanning microsatellite locus:

Select the microsatellite marker primer in the PRISMRLinkageMapping Sets-MD10 Version of ABI company 2.0 test kits for use, have 400 microsatellite marker primers, cover 22 euchromosomes (382) box X chromosomes (18).400 marks all come from 5264 microsatellite markers [4] (Dib et al, 1996) that French Genethon human inheritance center of diversity (CEPH) is delivered.This cover mark is two nucleic acids and repeats.Average heterozygosity in CEPH colony is more than 70%, and has all obtained the genetic stability checking in the CEPH family.

400 genetic markers are that ABI company selects from above-mentioned 5264 marks delivering specially, all carry out stdn by PCR and detect, and the primer that part is used for PCR has passed through adjustment and redesign.The annealing temperature of PCR is between 55-63 ℃.Owing to two nucleotide sequences when amplification easily because the low repairing activity of Taq archaeal dna polymerase produces the situation that the product end adds A, the said firm also specialized designs special modification that this effect is reduced to is minimum.

382 genetic markers all adopt pcr amplification to detect, and the fragment length of the PCR product of detection is generally in the 90-400bp scope.One 5 ' end of a pair of primer that is used for increasing has passed through fluorescent mark.Because ABI377 type dna sequencing instrument can detect 4 kinds of fluorescence (ROX or TAMARA, FAM, HEX, NED) simultaneously, therefore every primer that is labeled has all passed through FAM (blueness), HEX (green) or NED (yellow) mark (red ROX or TAMARA are used for molecular weight marker in the swimming lane).

Multiplex PCR amplification microsatellite marker

Divided into groups according to product range size, fluorescent mark color in the site of desire amplification, every group of 10-16 site, majority is 12-14, bad for expanding effect in multiple system, can organize separately and be new group or single amplification, if unit point amplification does not still have PCR product or product peak type bad, then abandon further test.

PCR product capillary electrophoresis, data gathering are used ABI PRISMR 3700 Data Collection Software v2.0.

Linkage analysis

1. the used software of linkage analysis

(1) adopt the LINKAGE5.1 software package that family is carried out linkage analysis.

The core of LINKAGE software package is the maximum or the likelihood ratio of a series of estimation recombination fractions, calculates the LOD value, and the program of analyzing the genetic risk rate.The whole software bag is divided into two groups.One group is used for common family and common pathogenic sites.Another group is used for three generations's family and codominant inheritance site, is mainly used in the genetic map that makes up nuclear family.The first parts that adopt in the practical application more.

2.. when carrying out Non-Parametric Linkage Analysis Methods, do not need to set hereditary pattern and genetic parameter, search in the main analysis family patient's blood relationship consistence (IBD) is arranged, calculate chain score value (NPL) of distribution free and corresponding p value, o'clock there is statistical significance p value＜0.05, and p＜0.01 o'clock just can confirm that site and this disease reported are chain.And on karyomit(e), determine a new pathogenic related gene site, and p＜0.0002 could illustrate it is meaningful chain, p＜0.0074 explanation is that support is chain.(Lander E, Kruglyak L.Genetic dissection of complex traits:guidelines for interpreting and reporting linkageresults.Nat Genet 1995,11:241-247.) need two files in the calculating process of GENEHUNTER, family file (Linkped.pre) and Parameter File (Linkloci.dat), these two files are realized by Checkped software.The selection of Fine Mapping microsatellite locus and design of primers:

Full genome scanning result calculates the LOD value of each microsatellite marker with the LINKAGE software package, obtains the Primary Location result of Disease-causing gene desmic region, then by database from the more microsatellite markers of desmic region selection.For the required microsatellite marker of Fine Mapping, the information of inquiry microsatellite marker in http://research.marshfieldclinic.org/ website, comprise its genetic distance, segment size and heterozygosity, be chosen in according to these information that genetic distance is about 1cM in the locating area, heterozygosity is at the microsatellite marker more than 7.5, on http://www.ncbi.nlm.nih.bov/ website, find the primer sequence of these microsatellite markers then, and further find out the physical distance of these microsatellite markers, when primer is synthetic according to a segment mark FAM or the HEX fluorescent mark of segment size at primer.

Haplotype makes up

Utilize Cyrillic software,, produce a family collection of illustrative plates, can show the somatotype information of a plurality of genetic markers of each member in each generation in the family in the collection of illustrative plates analyzing genotype result's input of gained.Carrying out manual regulation for genetic affinity can arrange out each individual haplotype up and down to these information bases.According to the situation that haplotype is shared, can find the generation that exchanges and then the zone of one section minimum that can obtain finally sharing by all patients in individual patients.

The result

1. full genome scanning result

The data file that comprises each Marker information computing on LINKAGE5.2 software of All Ranges is calculated the LOD SCORE (recombination fraction is respectively θ=0.0, θ=0.1, θ=0.2, θ=0.3, θ=0.4, θ=0.5) of each Marker.

2. Fine Mapping result

Selecting polymorphic micro-satellite Marker to carry out genescan and gene type at the D2S1371-D2S377 that is selected and two heredity zones of D2S1349-D2S2158 according to http://research.marshfieldclinic.org/ genetic map information.

3. haplotyping result

Used the Cyrillic software building in proper order the haplotype of this family (Fig. 1) according to the genetic marker on the Genome Database collection of illustrative plates.Get involved whether the exchange recombination event is arranged in the individuality by analysis, the Disease-causing gene of this disease is positioned between the D2S377-D2S1349.

In this AS family, all AS patients all find the change point of C/C-＞C/G, cause the 580th amino acid to become glycine (2759 SNP, C/C-＞C/G from arginine; 580R → G (the BLOSUM80 value of Arg-＞Gly) :-4, the 74-100PAM value :-1.0, residue replace ability matrix:-0.11, hydrophobicity: R 0.60, G 0.07.This newfound change point distributes at other 3 and obtains among the AS patient repeating confirming.

In addition, have 4 patients to find 2 change points in this gene in addition among AS patient and other familys AS patient distributing of expansion, they are respectively: 1) 2727 unknown SNP, C/C-＞C/T, cause amino acid to change: proline(Pro)-＞leucine, PAM100:-2.2, BLOSUM80:-5.2) rs1801278, G/G＞G/A, glycine＞arginine.

Embodiment 2

The detection of ankylosing spondylitis pathogenic related gene IRS-1 mutator gene:

Be used for the test kit of diagnosing ankylosing spondylitis pathogenic related gene IRS-1 mutator gene, mainly include:

Amplification contains 2759 locus genes segmental PCR primer, Taq archaeal dna polymerase and the corresponding damping fluid of IRS-1.

The PCR primer is as follows:

Upstream primer: IRS-1-F:CGGATGAGTATGGCTCCAGT

Downstream primer: IRS-1-R:ACCTCCAATGTCAGGAGAGC.

One, the preparation of object blood sample DNA to be measured

1. research object: all have the rheumatisant of inflammation low back pain clinically, perhaps the patient of doubtful ankylosing spondylitis.

2. extracting genome DNA: using modified Miller method

A. get Trisodium Citrate or EDTANa2 anticoagulated whole blood 3-5ml (as far as possible without anticoagulant heparin), place 50.0ml to cover centrifuge tube;

B. add 5-8 times of volume (about 40ml, fill it up with get final product centrifuge tube) cold distilled water, put upside down mixing repeatedly, 3600rpm at low temperatures, centrifugal 20 minutes;

C. slowly topple over supernatant, in precipitation, add 0 of precooling.1%Triton-X 100 (volume is the same), soft mixing precipitation, as above centrifugal, abandon supernatant;

D. add the 2.0ml lysate in the precipitation, thoroughly smash precipitation, add 10%SDS100.0ul, mixing then;

E. add 10mg/ml Proteinase K 40.0ul, mixing, 53-44 ℃, 3 hours, or 37-38 ℃, digested overnight (to spend the night) for good;

F. add 6.0MNaCL 0.7 left and right sides ml, thermal agitation;

G.3550-3650rpm centrifugal 40 minutes, supernatant is moved to another cover in the centrifuge tube;

H. the dehydrated alcohol or 95% ethanol that add about 2 times of volumes are put upside down mixing repeatedly to cotton-shaped DNA precipitation occurring, choose in DNA to the Eppendorf pipe;

I.80% washing with alcohol DNA twice, adds TE dissolving (200ul TE/5ml whole blood, 400ul TE/10ml whole blood) after the DNA extraction, and 4 ℃ leave standstill 7 days with abundant dissolving.

J. use 1% agarose electrophoresis and spectrophotometer and detect quantitative DNA, packing is preserved.

Two, zone, the mutational site of IRS-1 gene pcr amplification

1. primer sequence

Upstream primer: IRS-1-F:CGGATGAGTATGGCTCCAGT

Downstream primer: IRS-1-R:ACCTCCAATGTCAGGAGAGC

2.PCR reaction system

Table: the PCR reaction system of IRS-1 gene

Reaction conditions (see figure 3): 94 ℃ of pre-sex change of 5min; 94 ℃ of 30s sex change, 60 ℃ of 30s annealing, 72 ℃ of 1min extend 30 circulations; 72 ℃ of 7min fully extend; 4 ℃ of preservations.

Three, the detection of PCR product

Sepharose with 1% carries out electrophoresis to pcr amplification product, so that whether be purpose segment and estimate its concentration if detecting it.

The PCR product does not have assorted band through the single band of electrophoresis showed, then points out the PCR product component single, inclusion-free.If the band position is positioned at the position of the suitable size shown in the marker, then is the target segment.Purpose fragment PCR products according to above-mentioned primer amplification is 749bp, and promptly electrophoretic band is positioned at the position that is equivalent to the 750bp shown in the Marker approximately.

The purifying of PCR product and order-checking

Utilize ABI3730 sequenator (u.s.a. applied biosystem company), pcr amplification product is directly checked order.This sequencing reaction the primer the primer when carrying out pcr amplification is identical, for guaranteeing that longer sequence is connected smoothly, adopts positive and negative two-way order-checking.

Sample is prepared: DNA (2ul)+sample-loading buffer (6ul) is 8ul * 96 altogether

Get one 96 hole point templates, every hole adds sample damping fluid 6ul earlier, and from the chamber plate that the PCR product is housed, PCR product (2ul) is shifted out with the volley of rifle fire in every hole, transfer on the point template, mixing, with whizzer centrifugal (chamber plate hole number corresponding one by one) with 96 hole point templates.

1.PCR product purification

(1). in 96 orifice plates that the PCR product is housed, add 50 microlitre sterilized waters, mixing.

(2). it is transferred in the Millipore purifying plate, be put on the vacuum pump suction filtration about 3 minutes, see in the purifying plate not having water to get final product.

(3). in the purifying plate, add the deionized water of 50 microlitres once more, continue suction filtration, in the purifying plate, do not have water till.

(4). the purifying plate is taken off from vacuum pump, in plate, add the deionized water of 20 microlitres, left standstill 15 minutes, shook again 15 minutes, be drawn onto then in new 96 orifice plates.

(5). it is quantitative that the purifying after product is got 2 microlitre electrophoresis, is kept in-20 degree refrigerators.

Getting above purified product 2 μ l is template, uses ABI PRIMERTM, Big-Dye end mark test kit, and reacting a side primer (dilution is 2 μ M) with PCR is that sequencing primer carries out sequencing reaction.Reaction system 10 μ l, each component following (table 2-7):

Table 10 μ l sequencing reaction system

The sequencing reaction condition is seen Fig. 4

2. the purifying of sequencing reaction product

(1). every hole adds 60 μ l75% ethanol, mixing 30 seconds;

(2) .4000RPM is centrifugal 30 minutes, gets rid of supernatant;

(3). every hole adds 100 μ l75% ethanol, and centrifugal 10 minutes of 4000RPM gets rid of supernatant;

(4). repeat above-mentioned steps;

(5). be inverted on the thieving paper, centrifugally stop to 300RPM;

(6). placed the vacuum drier cryopump dry 4 minutes;

(7). every hole adds 20 μ l ddH2O (3700 sequenator) before going up sample;

3. starting sequenator checks order;

Seek morbific base mutation:

The standard sequence (with reference to Genebank NM_005544.) of the sequence that obtains and IRS-1 normal gene compared determine IRS-1 mutational site (2759C → G).

Further, the sequence that obtains is compared by BLAST, translated by normal single open reading frame, the amino acid after translating by normal single open reading frame with the IRS-1 normal gene is formed contrast, to determine whether to exist R580G amino acid mutation site.

Gene therapy is meant that using DNA to shift treats even prevent human illness.Exactly, it refers to the transfer of the relevant specific DNA sequences of genetic information, is highly integrated, a comprehensive highly difficult biotechnology, now has been widely used in treatment of diseases researchs such as tumour.The efficient gene treatment depends on exogenous gene high-efficient and stable expression.Utilize the virus vector mediated gene to shift, with its high transfection efficiency and good target and become most widely used method in the gene therapy, comprising retrovirus, adenovirus (AV), adeno-associated virus (AAV), hsv (HSV), vaccinia virus carriers such as (VV).

Separate and clone the IRS-1 gene of wild-type, choosing slow virus is carrier, with wild-type IRS-1 gene by on gene recombination technology assembling and the virus, remove to infect the somatocyte of patients with ankylosing spondylitis then with the virus that contains the IRS-1 gene of this reorganization, make in patient's body the albumen with normal function of IRS-1 transcription and translation that can the continuous expression wild-type, thereby reach the purpose of treatment.

Claims (11)

1. an ankylosing spondylitis pathogenic related gene IRS-1 mutator gene is characterized in that it contains the IRS-1 gene order, and contains 2759C → G heterozygous mutant.

2. IRS-1 mutator gene according to claim 1, it is characterized in that, this IRS-1 mutator gene also contains 2727C → T, 3419T → A, rs6725556T → C,-3637A → T,-3295G → T, rs6436635G → A, rs3731597G → A, 3 ' UTR 707G → T, one or more in-2990C → T or rs1801278A → G heterozygosis or the homozygous mutation.

3. IRS-1 mutator gene according to claim 1 is characterized in that, the encoded protein matter of described IRS-1 mutator gene has 580R → G mutational site.

4. method that detects IRS-1 mutator gene as claimed in claim 1; it is characterized in that; the sequence of nucleotide sequence that obtains from testing sample and IRS-1 normal gene compares, and checks 2759 sites that sudden change is positioned at the IRS-1 gene, and C sports G by base.

5. according to the method for the described detection of claim 4 IRS-1 mutator gene, it is characterized in that described detection method mainly may further comprise the steps:

1) DNA in the extraction sample to be detected;

2) be template with this DNA, carry out the PCR reaction, carry out the PCR reaction, obtain the PCR reaction product with PCR primer near the design of the coding region IRS-1 gene or the 2759th base of IRS-1 gene;

3) nucleotide sequence of measuring the PCR reaction product is formed;

4) sequence with nucleotide sequence and IRS-1 normal gene compares, and determines whether 2759 sport bases G.

6. according to the method for the described detection of claim 4 IRS-1 mutator gene, it is characterized in that, also comprise step:, translate to determine whether to exist R580G amino acid mutation site by normal single open reading frame by database BLAST comparison function.

7. according to the method for the described detection of claim 4 IRS-1 mutator gene, it is characterized in that the based composition of the primer in the PCR reaction is:

IRS-1-F：CGGATGAGTATGGCTCCAGT

IRS-1-R：ACCTCCAATGTCAGGAGAGC。

8. according to the method for the described detection of claim 4 IRS-1 mutator gene, it is characterized in that, the nucleotide sequence of the described PCR of measuring reaction product form can be with the PCR reaction product the sequenator order-checking or with the specific probe sequencing by hybridization.

9. test kit that is used to detect ankylosing spondylitis pathogenic related gene IRS-1 mutator gene, mainly include: with PCR primer, Taq archaeal dna polymerase and corresponding damping fluid near the design of the coding region IRS-1 gene or the 2759th base of IRS-1 gene.

10. method that detects haplotype in the D2S377-D2S1349 zone, it is characterized in that: the method for little satellite of determining according to genetic distance in the zone with capillary electrophoresis detected the family patient or distribute patient's sample, determine whether the peak type of sizes related, thereby determine whether to have and the consistent haplotype in haplotype zone that has detected.

11. the application of ankylosing spondylitis pathogenic related gene IRS-1 mutator gene as claimed in claim 1 in preparation treatment ankylosing spondylitis medicine.

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