CN100338228C - Method and reagent for predicting diabetes II susceptibility - Google Patents

Abstract

The present invention discloses a method for predicting the susceptibility of diabetes II. The susceptibility of detected people on the diabetes II is predicted through extracting host cell genome DNA and detecting the genotype of the 91st bit basic group polymorphism site of a TNFRSF9 gene intron non-coding region of the detected people; when the TNFRSF9 genotype is T/T homozygote, the susceptibility of the detected people is minimum; when the TNFRSF9 genotype is C/C homozygote, the susceptibility of the detected people is high; when the TNFRSF9 genotype is C/T heterozygote, the susceptibility of the detected people is maximum. The TNFRSF9 gene single nucleotide polymorphism (SNP) in the present invention has the C base substitution of a nucleotide sequence shown by SEQ ID NO. 1 positioned on the 91st bit (rs161810) site positioned in the intron 1 non-coding region arranged in a 1p36 region, and the C base substitution is one site with high risk associated with the pathogenesis of the diabetes II. The present invention has the advantages that correlation between the TNFRSF9 gene polymorphism site and T2DM is enucleated in the first time, the method for predicting the T2DM susceptibility is provided, and the method can be used for the prevention, the auxiliary diagnosis and the treatment for T2DM and can also be used for novel medicine development.

Description

A kind of reagent of predicting the diabetes B susceptibility

Technical field

The present invention relates to a kind of method and reagent of predicting the diabetes B susceptibility, more specifically say so by measuring the susceptibility of people TNFRSF9 gene pleiomorphism prediction experimenter for diabetes B, this method can be used for auxiliary diagnosis, treatment and the new drug development of disease, belongs to biological technical field.

Background technology

Diabetes B (T2DM) is a kind of chronic metabolic disease of common pilosity, is subjected to the double influence of h and E factor, has complicated pathogenesis, and the morbidity in the crowd is about 5%～15%.Because its a high proportion of great vessels complication, directly cause the heart, cerebrovascular disease to cause death, and the deformity that causes of microvascular complication, one of former diseases of the whole world serious danger side of body life and health have been become, consumed every year for this reason a large amount of human and material resources and financial resources (money is honourably obtained, Yang Ze, the diabetes control in Tong Zhifu chief editor .21 century. press of Henan Medical Univ., Zhengzhou, 2000.).Up to now, the cause of disease about T2DM is still unclear, but promptly toward studies show that in a large number, T2DM all presents height genetic identity (Zimmet P at pedigree analysis, twin study, Alberti KG, Shaw J.Globaland societal implications of the diabetes epidemic.Nature, 2001; 414:782-787.).The importance of inherited genetic factors in pathogenesis has obtained the proof of a lot of crowd's evidences, as karyomit(e) 2q (NIDDM ₁) site is the American related locus of Mexico, karyomit(e) 12q (NIDDM ₂) in the Finland crowd, find, in Pima American Indian autosomal gene group scanning discovery several LOD mark high dyeing body region (3q24,9q21 and 22q12) (Walker M.Gene detection in type 2 diabetes.International DiabetesMonitor, 1998; 10:14-15).

Carry out the research of T2DM inherited pathogenic factor at present, the association analysis methods that adopt SNPs as genomic marker more, be effective, obtained proof (Horikawa Y, Oda N, Cox NJ, et al.Genetic variation in the geneencoding calpain-10 is associated with type 2 diabetes mellitus.Nat Genet, 2000; 26:163-175.).SNP is meant the dna sequence polymorphism that single nucleotide diversity causes on the chromogene group level, frequency in the crowd needs＞1%, SNPs is a biallelic marker, have 70.1% to be the conversion between the homotype base during this single base changes: as G/A or T/C, 29.1% for occurring in the transversion between purine and the pyrimidine.C (cytosine(Cyt)) is the most labile site in the human genome, because great majority are the cytosine(Cyt)s that methylate, can be converted to T (thymus pyrimidine) by spontaneous deaminizating, and SNPs has comprised 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 (Brookes AJ.Theessence of SNPs.Gene, 1999; 234:177-186.).SNPs is with its density height, and average every 1kb just has 1; Representative strong, the SNPs that is positioned at gene inside may directly influence protein structure or expression level; Genetic stability is good, with microsatellite polymorphism comparatively speaking; Be easy to automated analysis, because of SNPs is biallelic marker in the crowd, can be simply with "+/-or 1/0 " direct somatotype, become good genetic marker.(Collins FS，Brooks LD，Chakravarti A.A DNA polymorphism discovery resource for research on human geneticvariation.Genome Res，1998；8：1229-1231)。

Be positioned at the molecule protein of the TNFRSF9 genes encoding tumor necrosis factor receptor super family component 9 of karyomit(e) 1p36, have important function, comprise the promotion immune response, apoptosis and the vital role in autoimmune response.TNFRSF9 is also referred to as CD137 or ILA, and 255 the amino acid whose transmembrane proteins of encoding are divided into the rich halfcystine sequence of extracellular region; Stride film district and short nitrogen-end born of the same parents oar district and contain phosphorylation signal conduction site.Limited research is pointed out, when lymphocyte and monocyte activation, stimulates and has started the TNFRSF9 expression, and then TNFRSF9 presents the propagation that suppresses activated T lymphocytes and lures idioblast the effect of programmed cell death to occur.(Schwarz H，BlancoFJ，VonKempis J，et al.ILA，a member of the tumor necrosis factor family，is located onchromosome 1p36，in a cluster of related genes，and colocotizes with several malignancies，Biochem Biophys，Res Commun 1997；235：699-703)。Owing in the T2DM pathogenesis, exist low-level immune response, comprise cytokines such as IL-6, TNF α, C-reactive protein and resistin in circulation and the phenotype position that takes place of focus all can detect.If focus occurs in liver, muscle and fatty tissue, can produce insulin resistant, if occurring in pancreas, focus can cause that amount of insulin secretion reduces (Ferandez-Real JM, RicartW, Insulin resistance and Chronic cardiovascular in flammatory syndrome, Endocr Rev2003; 34:278-301).The mechanism of TNF α has obtained proof in T2DM, and therefore, TNFRSF9 necessarily plays a significant role therein as acceptor.

Through existing domestic and foreign literature retrieval, do not see the research report that has TNFRSF9 to be associated with T2DM, do not see the report that the TNFRSF9 gene polymorphism sites is associated with T2DM yet.

Summary of the invention

Main purpose of the present invention provides a kind of method of predicting the diabetes B susceptibility.

Second purpose of the present invention provides a kind of reagent of predicting diabetes B, comprises PCR primer and the test kit that contains this primer.

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

A kind of method of predicting the diabetes B susceptibility, by extracting the genomic dna of host cell, measure the genotype of experimenter's TNFRSF9 gene intron non-coding region the 91st bit base pleomorphism site, predict the susceptibility of experimenter to diabetes B: when the TNFRSF9 genotype was the T/T homozygote, experimenter's susceptibility was minimum; When the TNFRSF9 genotype was the C/C homozygote, experimenter's susceptibility was higher; When the TNFRSF9 genotype was the C/T heterozygote, experimenter's susceptibility was the highest.

The invention provides a kind of isolating nucleic acid, have the base sequence shown in the SEQ ID NO 1, is C at the 91st.This nucleotide sequence is TNFRSF9.Fig. 1 is positioned at the TNFRSF9 gene structure of 1p36 and the synoptic diagram in polymorphic variation site thereof, includes 8 exons in the accompanying drawing, and the rs161810 site is marked on the corresponding position of introne 1 in the TNFRSF9 gene map.

The invention provides one group of allele specific primer, has the base sequence shown in SEQ ID NO 2 and the SEQ ID NO 3, length is 19-22bp, and can amplify shown in the SEQ ID NO:1 amplified production of the 91st SNP in the sequence specifically.

The invention provides a kind of diagnostic kit of the T2DM of detection susceptibility, the primer that wherein contains specific amplification TNFRSF9 gene of the present invention to the conventional assembly that is used for the test kit that pcr amplification detects, reagent, damping fluid etc., those skilled in the art know these conventional assembly and detection methods.Detection amplified production and normal the 91st SNP of TNFRSF9 gene contrast when whether having variation mutually, and required chemical reagent also comprises specificity restriction endonuclease etc.Whole components, content, source and using method in the test kit of the present invention are as follows:

Test kit of the present invention detects for 10 person-portions and uses,

Its component, content and source comprise:

60ul 10X PCR damping fluid (Pharmacia),

50ul 10mM dNTP mixed solution (Pharmacia),

10ul (2unit/ul) Taq archaeal dna polymerase (Takara),

Each 12ul (50pmol/ul) F1 (SEQ ID NO.2) and R1 (SEQ ID NO.3) primer (self-control),

1.5ml pure water (self-control),

20ul 10X restriction enzyme reaction buffer (Biolab),

10ul (2unit/ul) restriction enzyme Mn1I (Biolab).

Using method:

1) exons 1 and the introne 1 part fragment by pcr amplification TNFRSF9 gene, in brief, the preparation mixed solution, add genomic dna solution 100ng, 5ul 10X PCR damping fluid, 4ul 10mM dNTP, 1.25 Taq of unit archaeal dna polymerases and 50pmolF1 and R1 and be respectively adopted primer and antisense primer, every kind of primer of embodiment 2 narrations.Then, add pure water, making cumulative volume is 50ul.Be reflected at 95 ℃ 0.5 minute, 58 ℃ 0.5 minute, 72 ℃ 0.5 minute, carry out 30 circulations.After reaction is finished, with every kind of PCR reaction solution of 10ul electrophoresis detection on 7.5% polyacrylamide gel, as the fragment of the 253bp that increased, obtained single band, be and increase successfully.

2) by measure the polymorphism of TNFRSF9 gene 5 '-non-coding region with restriction enzyme treatment PCR reaction product.Add 1ul restriction enzyme reaction buffer, 1ul restriction enzyme Mn1I in the above-mentioned amplification PCR reaction product of 10ul, and in 37 ℃ of water-baths, digest and spend the night.Afterwards, electrophoresis on 7.5% polyacrylamide gel.

3) polymorphism typing: handle fragment with restriction enzyme Mn1I, when the 91st base is T allelotrope, the band of 151bp and 102bp occurs.As when being C allelotrope, band of 253bp only appears.

Measuring method of the present invention has been measured the genomic dna that derives from the people, sample without limits as, body fluid (as blood, ascites and urine), histocyte (as hepatic tissue) etc.Can prepare genomic dna by extraction and these samples of purifying.

From genomic dna, can increase contains the dna fragmentation of TNFRSF9 transgenation point, with the great amount of samples that obtains to be used to measure.Thisly contain the sample that the dna fragmentation of TNFRSF9 transgenation point obtains, be particularly suitable for as measuring material by amplification.For example, can be by PCR method, use primer to increase, this primer through design rationally so that only amplification contains the part of the non-coding region of TNFRSF9 gene pleiomorphism.This primer can be through conventional method preparation.The base length in district to be amplified is unrestricted.In the ordinary course of things, the about 100bp to 500bp of base length.When by preparation primer of the present invention, the working sample that can obtain to suit, it is as the dna fragmentation of amplification, and has specificity length.

When carrying out the PCR-RFLP method, the DNA of desire amplification is designed to contain and can be applicable to the specificity restriction enzyme site of RFLP method after this.

Unrestricted to this design, as long as different in two kinds of situations of mutator gene and wild type gene with the fragment length of enzyme cutting DNA district acquisition.Can use by the sudden change generation of TNFRSF9 gene or the restriction enzyme sites of losing.

Therefore, the required dna fragmentation according to the PCR method amplification is designed by the restriction enzyme of above-mentioned choose reasonable.The fragment that enzyme is cut generation confirms that by electrophoresis they show specific band.According to the banding pattern that in aforesaid method, obtains, can measure the TNFRSF9 gene pleiomorphism.

When carrying out gene diagnosis of the present invention, preferably be used to measure diagnostic reagent according to the mutation type existence of TNFRSF9 gene, diagnostic reagent comprises the particular agent as neccessary composition, it is corresponding to the method that is used to measure the TNFRSF9 gene mutation type.Specific reagent is suitably selected by the measuring method that adopts.The feature of reagent is, composition measuring is necessary by the means of the mutation type of TNFRSF9 gene definition, as, dna fragmentation/and/or as the special restriction enzyme that is used to measure.Reagent, the primer that is used for the pcr amplification step of specific preparation for example, this step is used to comprise the specific amplified fragments of the catastrophe point of TNFRSF9 gene, is not considered to the neccessary composition of diagnostic reagent of the present invention, and they also are contained among the diagnostic reagent of the present invention.

Advantage of the present invention is: the present invention has illustrated the dependency of TNFRSF9 gene polymorphism sites and T2DM first, and a kind of method of the T2DM of prediction susceptibility is provided, and this method can be used for prevention, auxiliary diagnosis and the treatment of T2DM, can also be used for new drug development.

Below in conjunction with the drawings and specific embodiments the present invention being done further narration, so that the public has more deep understanding to summary of the invention, is not limitation of the present invention.

Description of drawings

Fig. 1 is positioned at the TNFRSF9 gene structure of 1p36 and the synoptic diagram in polymorphic variation site thereof.

Embodiment

The english abbreviation that is used for the following example expression reagent is as follows.

When needing, with pressure kettle (120 ℃, 20 minutes) sterilization

EDTA: disodium ethylene diamine tetraacetate

SDS: sodium lauryl sulphate

TE：10mM Tris-HCl(pH7.5)，1mM EDTA(pH8.0)

10 * PCR damping fluid: 100mM Tris-HCl (pH8.3), 500mM KCl, 15mM magnesium chloride (MgCl2) 0.01% (W/V) gelatinum

DNTP: deoxynucleoside triphosphate

Methane amide pigment: 95% deionization first phthalein amine, 0.05% blue dextran

APS: ammonium persulphate → 10 * TBE:Tris (108g), boric acid (55g), EDTA2Na (9.3g) is dissolved in the pure water, and cumulative volume is 1 liter.

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

By WHO (1999) the standard MethodsThe cases enrolled of clarifying a diagnosis, must be through empty stomach 8-12 hour the oral 75g dextrose anhydrous that dissolves in the 300ml warm water, the diabetic subject who detects its plasma glucose value 〉=11.1mmol/L or clarified a diagnosis via the above rank in counties and cities hospital in the past after 2 hours, collect from Heilungkiang altogether, affinity-less relation T2DM patient 690 examples in Beijing, Dalian and Yinchuan, 49 years old ± 21.2 years old mean age, the male sex's 300 examples wherein, with geographic normal healthy controls volunteer's 710 examples, 47 years old ± 19.6 years old mean age, the wherein male sex's 325 examples.All persons under inspection are Han nationality, and the signature Informed Consent Form, and this research has also obtained the approval of Ethics Committee of our unit.

According to following method, prepare genomic dna with human peripheral.In the presence of antithrombotics EDTA, at 2500rpm, centrifugation removed serum deprivation in 30 minutes with the 10ml human peripheral collected.Then add 0.2%NaCl solution, making cumulative volume is 50ml.Vibrate gently solution 5-6 time, and it was positioned over 15 minutes on ice.After this, at 2500rpm centrifugation 30 minutes, collecting precipitation thing whereby.NaCl solution with 0.2% washs in the mode that is similar to the front again.In the throw out that so obtains, add 10mMTris-HCl (pH8.0) and 10mM EDTA (4ml), with this throw out that suspends.With 10%SDS, the Proteinase K of 25mg/ml and the RNaseA of 10mg/ml add in the suspension, and its add-on is respectively 4ml, 16ul and 20ul, and the suspension that then turns upside down mixes gently.Then, at 37 ℃ of incubation suspensions that spend the night.After spending the night, add 4ml phenol/Tris solution, the mixture of the mixture mixing gained that turns upside down.Carry out centrifugation with 3000rpm and removed water layer in 10 minutes.Water layer and 4ml phenol/chloroformic solution are mixed, then against mixing and with 3000rpm centrifugation 10 minutes.Remove water layer.At last, with chloroform extraction twice, obtaining water, toward wherein adding 1/10,3M NaAC (pH5.2), the cold dehydrated alcohol of doubling dose makes the DNA precipitation.Washing with alcohol with 70% is to obtain genomic dna.Make the DNA of such acquisition, genomic dna is dissolved among the TE, and the quantitative assay mixture is in the specific absorption of 260nm then.DNA working fluid concentration correction is put-20 ℃ of refrigerators and is preserved to 30ng/ul.

The identification of embodiment 2SNP is determined

The present invention adopts PCR-RFLP and PCR sequencing technologies simultaneously the 91st the SNP site (its loci is to being T/C) of the introne 1 of TNFRSF9 gene to be detected.

1) primer determine be positioned at TNFRSF9 genetic transcription starting point upstream promoter district-11bp to introne 1 non-coding region+242bp, total length 253bp, determined positive-sense strand (11--+8bp) and antisense strand (222--242bp) Auele Specific Primer as follows:

F1：5′-CAG AAG CCT GAA GAC CAAG～3′(SEQ ID NO：2)

R1：5′-TTG AGA TGT AAG TTT GTA TGG～3′(SEQ ID NO：3)

2) exons 1 and the introne 1 part fragment by pcr amplification TNFRSF9 gene, in brief, the preparation mixed solution adds genomic dna solution 100ng, 5ul 10X PCR damping fluid, 4ul 10mMdNTP, 1.25 Taq of unit archaeal dna polymerases and the 50pmol above-mentioned steps 1 of the foregoing description 1 preparation) every kind of primer of narration.Then, add pure water, making cumulative volume is 50ul.Be reflected at 95 ℃ 0.5 minute, 58 ℃ 0.5 minute, 72 ℃ 0.5 minute, carry out 30 circulations.After reaction is finished, every kind of PCR reaction solution of 10ul electrophoresis on 7.5% polyacrylamide gel has been obtained single band, observed, used Gel Doc 2000 programs at the BioRad imager.

3) use F1 and R1 to be respectively adopted primer in brief by the polymorphism of measuring TNFRSF9 gene 5 '-non-coding region with restriction enzyme treatment PCR reaction product and antisense primer carries out PCR.As a result, the increased fragment of 253bp.In the above-mentioned amplification PCR reaction product of 10ul, add 1ul restriction enzyme reaction buffer, 1ul restriction enzyme Mn1I, and spend the night 37 ℃ of digestion.After this, electrophoresis on 7.5% polyacrylamide gel.Being determined as of polymorphism: handle fragment with restriction enzyme Mn1I, when the 91st base is T allelotrope, the band of 151bp and 102bp occurs.As when being C allelotrope, band of 253bp only appears.

Embodiment 3TNFRSF9 gene SNP is relevant with T2DM's

Statistical method: the loci that utilizes Pearson chi square test calculating TNFRSF9 gene pleiomorphism in STATA8.0 and the SPSS11.0 software, genotype frequency, the Hardy-Weinberg balance detection, carry out continuous correction and one-sided asymptotic probability analysis, statistical significance level is set at P＜0.05.Adopt single factor Logistic regression analysis to calculate ill risk OR value and 95% credibility interval (CI) thereof of T2DM.

The result

1) healthy people TNFRSF9 gene pleiomorphism distributes

Measured 710 healthy people's gene pleiomorphism by the method for embodiment 1 and 2.584 people have polymorphism at the 91st bit base, find that 231 people have the homozygote (32.65%) of T, and 353 people have the heterozygote (49.66%) of T and C, and 126 people have the homozygote (17.69%) of C.

2) patient T2DM TNFRSF9 gene pleiomorphism distributes

The method of pressing embodiment 1 and 2 is measured 690 healthy people's gene pleiomorphism.481 people have polymorphism at the 91st bit base, find that 136 people have the homozygote (19.70%) of T, and 345 people have the heterozygote (50.00%) of T and C, and 209 people have the homozygote (30.30%) of C.

3) T2DM case and normal healthy controls group are relatively

T2DM case and normal healthy controls group compare the rs161810 on its TNFRSF9 gene, and its loci sees table 1 for details to being the frequency distribution of T/C polymorphism.

The genotype and the comparison of gene frequency risk in T2DM and healthy normal population in table 1TNFRSF9 gene rs161810 site

	Sample number	Genotype (%)			Allelotrope (%)
							C/C	C/T	T/T	C	T
		2 type DM people normal peoples	690 710	209 30.30 126 17.69	345 50.00 353 49.66	136 19.70 231 32.65							763 55.30 604 42.53	617 44.70 816 7.48
P OR(95％CI)							(df＝2) 0.0001			0.0026(df＝1) 2.458(1.345-4.492)

Table 1 as seen, the A loci in the common SNP site (rs161810) that the TNFRSF9 gene is the 91st, on its DNA complementary strand, be the T loci promptly, when sporting C, when being G on the complementary strand, distribution frequency in patient colony is much higher than the frequency in healthy normal population, differs about 12%, and significance difference (P=0.0026) is arranged.And the frequency of OR value reflection mutational site C exceeds 2.46 times of normal peoples in T2DM, and 95%CI lower limit＞1 shows that all this is the allelotrope of an ill high risk of T2DM.With SNP (rs161810) site of T2DM significant correlation, be positioned at the shearing point side of introne 1 near exons 1, may pass through to influence the space structure of DNA, thereby this mutational site influences transcribing and shearing of DNA.

Embodiment 4 detection kit

Preparation detects the test kit of T2DM relevant risk, and it is right to include the primer that can amplify TNFRSF9 gene SNP (rs161810) site, and the PCR-RFLP corresponding reagent, and composition and content are as follows, in-20 ℃ of preservations:

60ul 10X PCR damping fluid (Pharmacia),

50ul 10mM dNTP mixed solution (Pharmacia),

10ul (2unit/ul) Taq archaeal dna polymerase (Takara),

Each 12ul (50pmol/ul) F1 (SEQ ID NO.2) and R1 (SEQ ID NO.3) primer (self-control),

1.5ml pure water (self-control),

20ul 10X restriction enzyme reaction buffer (Biolab),

10ul (2unit/ul) restriction enzyme Mn1I (Biolab).

The present invention has the illustration of practicality:

1) detection method of TNFRSF9 gene pleiomorphism of the present invention can be used for the T loci of the common SNP (rs161810) on the TNFRSF9 gene in analyst's euchromosome 1p36 district, whether is the A loci on its DNA complementary strand promptly, being applied in to the complementary diagnosis of T2DM with to individuality has the ill risk of great T2DM to assess.Be beneficial to carry out early intervention and the treatment of T2DM.

2) utilize the present invention to set forth the variation of TNFRSF9 gene the 91st bit base, as one of biomarker, the screening of the molecular target of useful as drug design is regulated the bioactive molecule that TNFRSF9 expresses to help to seek to have, and promotes the T2DM new drug development.

3) nucleotide sequence and the T2DM related locus of the detection TNFRSF9 gene pleiomorphism of the present invention's foundation, but highly sensitive is applied to the test kit that the T2DM gene diagnosis is used specifically.

As mentioned above, reach a conclusion, the TNFRSF9 gene is at the polymorphism and the T2DM tool significant correlation of the 91st bit base.Therefore, according to the present invention, measure this polymorphism and can be used for carrying out gene diagnosis.

The present invention has narrated the relevant new mutant point of TNFRSF9 gene T2DM, and a kind of method of the TNFRSF9 of mensuration gene pleiomorphism is provided.And, according to the present invention, only need a small amount of DNA sample just to be enough to measure the polymorphism of TNFRSF9 gene.As a result, the invention provides a kind of gene diagnosis method of the T2DM of mensuration related gene polymorphism.

Sequence table

＜110〉Beijing Hospital

＜120〉a kind of method and reagent of predicting the diabetes B susceptibility

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agaccaagga gtggaaagtt ctccggcagc cctgagatct caaggtctgt ccatctgggg 60

gagtgggtgg gggcactgag aaggggtgag cttggaactt ttgctccctt tgcccatttc 120

tagacttttt ctcctaatat gtaataactt ctcttcaata gccctttaaa taaacatcaa 180

taacttagcc tgaagagttt ttcactcctt agttttgtta ccatacaaac ttcacatctc 240

aaaacatgtc tctgtttaag aaaatgtgtt agtgagttga acagggaggt ctttccacat 300

gttagtagtt aggtgttcag ctaaaggggg aagagtgatt atgtgatagc ttcttcttga 360

actgaattgt ctgatgcccc tgacagattc tctttgtaag gagtttattt caggggcaat 420

aagtaattgg cattattgct ggttggtact gcaaagtacc tatgaaagtc cccaaaagtt 480

cttgctattg ttatttctgc attttggcag aacatgatgg aaaatgcacc ctcaaacttt 540

ggcaaaccgg cacaaagctg tgtgtttaat cacgcctgcc ttgtcctagt ggtttctatg 600

aatctgctac ttttccgtaa tattgcatca ttaattgttc ctgaaaaacc ctgagttatc 660

ctcttataga attgtataag taatgattgc aatatagata attttgaaag gagaaaccac 720

ctttccttgg aaatgtttat cttttgcaga gtgacatttg tgagaccagc taatttgatt 780

aaaattctct tggaatcagc tttgctagta tcatacctgt gccagatttc atcatgggaa 840

acagctgtta caacatagta gccactctgt tgctggtcct caactttgag aggacaagat 900

cattgcagga tccttgtagt aactgcccag ctggtgagta cccagttatc atgtgcattt 960

gatctgctct gttggaagta tggttcagtt agtctagtag tcagggctaa cgagctccct 1020

tttaaggaaa ggaaaatgaa aattcattca tttacaaatg tttattggat gctacaacct 1080

agctgtgtga acacagcaaa gtcattcaac ctcttgtgcc ttgactttct catctgggga 1140

taataagaga acctgtttta taggatggct gggaggatca aatgaagggc ttagaacagt 1200

gcatggcaca aggcaagact tcaataaatg ttagttttgt gtgtagggct ttgtgctccg 1260

actgggggca tagcagcgag taagcgcgta gtaaagggct taacagagtg gggacggtca 1320

gtcgcattta aattttagtg taggacattg atgtcctcct ggatccagtc atattcatct 1380

cctacatcaa tcaagataat cattttgttt tattcaatag ataaagtatt ttctttctgc 1440

tgatttattt gttacaatat ctggtttttt tgtttttttt gtgtgtgtat gttttttttt 1500

ttttttttga gacacagtct cactctgtcg cccaggctgg agtgcagtgg cacgatctca 1560

gctcactgca accttggcct cccaggttca agcaattctc ctgcctcagc ctcccgagta 1620

gctgggatta caggtgtgca ccactgcgcc tggctaattt ttgtattttt agtagagaca 1680

ggatttcacc atgttggcca agctggtctt gaactgctga cctcaggtga tctgcccgct 1740

tcggcctccc aaagtgctgt gattacaggc gtgagccact gcacctggcc tgtattttgt 1800

tatttctaat tctgtgatta cacaaagaat aatcttgcaa atgtattgtt ttaggaagtc 1860

aatcaaacta atgtccacca ctgtggcatc tagaaaactc atcattcttt tataatctta 1920

ttttcttgta aacttcggaa tgccttgggt gacgattgcc cattgtcaaa cgtgtaccac 1980

tattgtctgc ctttcttagg tacattctgt gataataaca ggaatcagat ttgcagtccc 2040

tgtcctccaa atagtttctc cagcgcaggt ggacaaagga cctgtgacat atgcaggcag 2100

tgtaaaggta tgagtt 2116

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ttgagatgta agtttgtatg g 21

Claims (3)

1. an isolating nucleic acid has the base sequence shown in the sequence table SEQ ID NO.1, is C at the 91st.

2. the primer of one group of prediction diabetes B susceptibility, its length is 19～22bp, can amplify the amplified production of the 91st SNP in the sequence shown in the SEQ ID NO.1 that contains tumor necrosis factor receptor super family component 9 genes specifically, have the base sequence shown in sequence table SEQ ID NO.2 and the SEQ ID NO.3.

3. a test kit of predicting the diabetes B susceptibility is characterized in that, contains each 12ul of the described primer sequence of claim 2 and the following reagent of 50pmol/ul, comprising: 60ul 10X PCR damping fluid; 50ul 10mMdNTP mixed solution; 10ul Taq archaeal dna polymerase, 2unit/ul; 1.5ml pure water; 20ul 10X restriction enzyme reaction buffer; 10ul restriction enzyme MnlI, 2unit/ul; This test kit detects for 10 person-portions and uses, and the storage temperature of test kit is-20 ℃.

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