CN1948504A - Adipocyte differentiation related protein gene and relativity with diabetes type2 - Google Patents

Abstract

The invention discloses a method to test susceptibility of 2-type diabetes. Compared with normal, variability of protein gene ADRP, transcript and/or protein about adipose cells differentiation of individuals are tested. It is indicated that the individual has more probability to suffer 2-type diabetes than others if there is variability. The invention also discloses the reagent boxes.

Description

The dependency of white gene of lipocyte differentiation associated protein and diabetes B

Technical field

The present invention relates to biology field.Relate more specifically to the white gene of people's lipocyte differentiation associated protein (Adipose Differentiation-Related Protein, ADRP) single nucleotide polymorphism (singlenucleotide polymorphism, SNP) and with the dependency of diabetes B.The invention still further relates to the method and the test kit that detect these SNP.

Background technology

It is the syndrome of being made up of the different pathological physiological change of feature with the abnormal carbohydrate metabolism that diabetes are one group, have bigger heterogeneity between the different patients, and its two kinds of main pathophysiological changes is hypoinsulinism and insulin resistant.

Diabetes B be a kind of complicated disease of multifactorial inheritance (O ' Rahilly S, Wainscoat JS, Turner RC.Type 2 (non-insulin-dependent) diabetes mellitus Newgenetics for old nightmares.Diabetologia, 1988,31:407-14; Yukio Horikawa, Naohisa Oda, Nancy J, et al.Genetic variation in gene encoding calpain-10is associated with type 2diabetesmellitus, Nature genetics, 2000,26:163-175), genetic background has a major gene to add a plurality of minor genes or is concured by some minor genes, and environmental factors has a significant effect to phenotype.Finishing of human genome examining order sketch is for the method research diabetes B Disease-causing gene that adopts positional cloning has been created good condition.(Single Nucleotide Polymorphism SNP) is a kind of genetic marker on the karyomit(e) to single nucleotide polymorphism, refers in genome substituting, insert or disappearance and the molecule that forms is polymorphic of single or multiple Nucleotide on certain nucleotide position.SNP quantity is many, density is big, topped whole genome, and average 500 base pairs just have a SNP, and are more stable than microsatellite genetic marker, are easy to automated operation, is the highly effective genetics instrument of the complicated multigenic disease of research.The SNP of quite a few can change gene expression dose or function, itself may be exactly pathogenic sites.

Though carried out the research of some diabetes genes involveds and polymorphism, before the present invention, do not confirm the report of ADRP gene pleiomorphism and diabetes dependency, more there is not report with the diabetes B dependency.

In view of diabetes have a strong impact on the healthy disease of people, in order to diagnose and treat diabetes B as early as possible, this area presses for seeks the diabetes B tumor susceptibility gene, and method, the test kit of exploitation detection diabetes B, or relevant medicine.

Summary of the invention

Purpose of the present invention just provides the method and the detection kit of a kind of diagnosis (especially early diagnosis or auxiliary diagnosis) diabetes B.

In a first aspect of the present invention, a kind of method that the diabetes B susceptibility of individuality is diagnosed is provided, it comprises step:

Detect this individual ADRP gene, transcript and/or albumen, and compare with normal ADRP gene, transcript and/or albumen,

The possibility that there are differences with regard to showing this individuality trouble diabetes B is higher than normal population.

In another preference, detection be gene or the transcript of ADRP, and with normal ADRP nucleotide sequence comparing difference.

In another preference, described difference is the single nucleotide polymorphism that is selected from down group:

697 A → G;

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

In a second aspect of the present invention, provide a kind of test sample whether to have the method for the single nucleotide polymorphism of ADRP gene, comprise step:

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

(b) detect whether there is the single nucleotide polymorphism that is selected from down group in the amplified production:

697 A → G;

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

In another preference, described single nucleotide polymorphism is selected from down group: 697 A → G.

In a third aspect of the present invention, a kind of test kit of complementary detection diabetes B is provided, it comprises the primer and the specification sheets of specific amplification ADRP gene or transcript.

In another preference, described test kit also contains the reagent that is selected from down group:

(a) with mutable site bonded probe;

(b) restriction enzyme in identification mutational site.

In another preference, described sudden change is the single nucleotide polymorphism that is selected from down group:

697 A → G;

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

In another preference, the primer in the described test kit is the primer of sequence shown in SEQ ID NO:2 and 3.

In a fourth aspect of the present invention, a kind of ADRP gene and proteic purposes are provided, it is used to prepare the reagent (as Auele Specific Primer) of complementary detection diabetes B.

Description of drawings

Fig. 1 has shown PCR product electrophoretogram.

Fig. 2 has shown the image of diabetes B family (No. 118) ADRP gene the 8th exon sequencing result after the identification of CHROMAS software.

Fig. 3 has shown the SNP somatotype of diabetes B family (No. 118) ADRP gene the 8th exon.

Fig. 4 has shown the image of diabetes B family (No. 25) sequencing result after the identification of CHROMAS software

Fig. 5 has shown diabetes B family (No. 25) ADRP gene SNP somatotype.

Embodiment

The inventor is through research for many years, find first and proved that ADRP gene and diabetes B are closely related, and found its new function: the change of ADRP will cause diabetes B, wherein the association study result shows, there is significant difference (P＜0.05) in the 697th the distribution of SNP (A/G) in case and control group in sequence shown in the SEQ of the ADRP ID NO:1, and this SNP is polymorphic to cause missense mutation.Finished the present invention on this basis.

Particularly, the inventor uses the PCR-sequence measurement, promotor, the exon of ADRP gene and the intron that closes on are checked order normal population, diabetes B crowd respectively, determine whether this gene is relevant with the Chinese East China diabetes B people of Han nationality.The result shows, 5003 bases of ADRP gene fragment total length of surveying have 23 SNP, and its medium-high frequency, low frequency are respectively 5,18.There were significant differences in the genotype of diabetes B crowd, normal population for a SNP (P/-407) of promotor (X2=6.65, P＜0.05); Two missense mutation Ser251Pro, Thr396Ala transmit altogether with two diabetes B familys respectively.These results suggest, ADRP is relevant with East China the Hans' diabetes B.

As used herein, term " ADRP " and " lipocyte differentiation associated protein is white " are used interchangeably, and all refer to protein molecular or nucleic acid molecule that lipocyte differentiation associated protein is white.

The detailed sequence of ADRP gene can be the nucleotide sequence (can referring to network address http://www.ncbi.nlm.nih.gov/) of Hs.3416 referring to accession number, and full-length gene is 11814bp.The sequence that has comprised upstream 5 '-untranslated zone is shown in SEQ ID NO:1, altogether 12184bp.

Based on new discovery of the present invention, ADRP albumen or polypeptide have many-sided new purposes.These purposes include, but is not limited to: the disease that complementary detection is relevant with ADRP (as diabetes B), or the material of screening promotion ADRP protein function, and as antibody, polypeptide or other part.The peptide molecule that can stimulate people ADRP protein function that can be used for seeking therapeutic value with the recombinant human ADRP protein screening peptide library of expressing.

On the other hand, the present invention also comprises people ADRP DNA or the polypeptide of its fragment coding has specific polyclonal antibody and monoclonal antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into people ADRP gene product or fragment.Preferably, refer to that those can combine with people ADRP gene product or fragment but nonrecognition and be incorporated into the antibody of other irrelevant antigen molecule.Among the present invention antibody comprise those can in conjunction with and suppress the proteic molecule of people ADRP, comprise that also those do not influence the antibody of people ADRP protein function.

The present invention not only comprises complete mono-clonal or polyclonal antibody, but also comprises having immunocompetent antibody fragment, as Fab ' or (Fab) ₂Fragment; Heavy chain of antibody; Light chain of antibody; Genetically engineered strand Fv molecule; Or chimeric antibody.

Antibody of the present invention can be prepared by the known various technology of those skilled in that art.For example, the people ADRP gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing human ADRP albumen or its have antigenic segmental cell and can be used to immune animal and produce antibody.Multiple adjuvant can be used for the enhancing immunity reaction, includes but not limited to freund's adjuvant etc.

Antibody of the present invention also can be monoclonal antibody.This type of monoclonal antibody can utilize hybridoma technology to prepare.Antibody of the present invention comprises the antibody that can block people ADRP protein function and the antibody that does not influence people ADRP protein function.Each antibody-like of the present invention can utilize the fragment or the functional zone of people ADRP gene product, obtains by the routine immunization technology.These fragments or functional zone can utilize recombinant methods or utilize Peptide synthesizer synthetic.Can come immune animal and produce with the gene product of producing in the prokaryotic cell prokaryocyte (for example E.Coli) with the unmodified form bonded antibody of people ADRP gene product; With posttranslational modification form bonded antibody (as the albumen or the polypeptide of glycosylation or phosphorylation), can come immune animal and obtain with the gene product that produces in the eukaryotic cell (for example yeast or insect cell).

The proteic antibody of anti-people ADRP can be used in the immunohistochemistry technology, detects the people ADRP albumen in the biopsy specimen.A kind of preferred anti-ADRP antibody is the antibody of the normal ADRP of nonrecognition ADRP but identification suddenlys change, perhaps discerns normal ADRP but the antibody of nonrecognition sudden change ADRP.Utilize this antibody to the proteic specificity difference of normal and unusual ADRP, the diabetes B susceptibility that can carry out protein level easily detects.

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

ADRP albumen of the present invention and antibody thereof, inhibitor, agonist, antagonist etc. when using (administration) in treatment, can provide different effects.Usually, can these materials are formulated in nontoxic, inert and the pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5-8 usually, and preferably pH is about 6-8, although the pH value can be with being changed to some extent by preparation Substance Properties and illness to be treated.The pharmaceutical composition for preparing can carry out administration by conventional route, comprising (but being not limited to): intramuscular, intravenously or subcutaneous administration.

The present invention also provides a kind of composition (as pharmaceutical composition), and it contains ADRP albumen of the present invention (or its antibody, inhibitor, agonist, antagonist) and the pharmaceutically acceptable carrier or the vehicle of safe and effective amount (as 0.001-99.99wt%).This class carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Pharmaceutical preparation should be complementary with administering mode.Pharmaceutical composition of the present invention can be made into the injection form, for example is prepared by ordinary method with the physiological saline or the aqueous solution that contains glucose and other assistant agents.Pharmaceutical composition such as tablet and capsule can be prepared by ordinary method.Pharmaceutical composition such as injection, solution, tablet and capsule should be made under aseptic condition.The dosage of activeconstituents is the treatment significant quantity, for example every day about 0.1 microgram/kg body weight-Yue 10 mg/kg body weight.In addition, polypeptide of the present invention also can use with the other treatment agent.

When making pharmaceutical composition, be that the ADRP albumen of safe and effective amount or its antagonist, agonist are applied to Mammals, wherein this safe and effective amount is usually at least about 0.1 microgram/kg body weight, and in most of the cases be no more than about 10 mg/kg body weight, preferably this dosage is about 0.1 microgram/kg body weight-Yue 100 micrograms/kg body weight.Certainly, concrete dosage also should be considered factors such as route of administration, patient health situation, and these all are within the skilled practitioners skill.

The invention still further relates to the diagnostic testing process of quantitative and detection and localization people ADRP protein level.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.

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

The proteic polynucleotide of ADRP can be used for the diagnosis and the treatment of ADRP protein related diseases.Aspect diagnosis, the proteic polynucleotide of ADRP can be used for detecting the proteic expression of ADRP ADRP abnormal exprssion whether or under morbid state.Can be used for the hybridization of biopsy specimen to judge the proteic abnormal expression of ADRP as the ADRP dna sequence dna.Hybridization technique comprises the Southern blotting, Northern blotting, in situ hybridization etc.These technological methods all are disclosed mature technologies, and relevant test kit all can obtain from commercial channels.Part or all of polynucleotide of the present invention can be used as probe stationary on microarray (microarray) or DNA chip (being called " gene chip " again), is used for analyzing the differential expression analysis and the gene diagnosis of tissue gene.Carry out RNA-polymerase chain reaction (RT-PCR) amplification in vitro with the special primer of ADRP albumen and also can detect the proteic transcription product of ADRP.

The sudden change that detects the ADRP gene also can be used for diagnosing diabetes B.Detection can be at cDNA, also can be at genomic dna.The form of ADRP protein mutation comprises that the point mutation compared with normal wild type ADRP dna sequence dna, transposition, disappearance, reorganization and other are any unusual etc.It is SNP that one class is preferably suddenlyd change.Available existing technology such as Southern blotting, dna sequence analysis, PCR and in situ hybridization detect sudden change.In addition, sudden change might influence proteic expression, therefore can judge indirectly that with Northern blotting, Western blotting gene has or not sudden change.

Major advantage of the present invention is:

The used experiment confirm first change of ADRP and the closely related property of diabetes B, and then the method and the test kit of complementary diagnosis diabetes B (or susceptibility) are provided.The present invention can provide very valuable complementary reference index for clinical diagnosis (especially early diagnosis) diabetes B, thereby benefits diagnosis morning, prevention early, the morning that help realizing diabetes B.

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:ColdSpring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment 1

One, method

1. the extracting of peripheral blood DNA:

Remove behind the blood plasma deshydremia 500ul and add behind the 500ulPBS piping and druming mixing 10, centrifugal 3 minutes of centrifugal 1 minute of 000rpm or 8000rpm remove broken red corpuscle; Add 800ul 0.025M KCl hypotonic medium, thorough mixing, 37 ℃ of temperature were bathed 20 minutes, 10,000 centrifugal 1 minute, abandon supernatant, repeat 2～3 times, remove red corpuscle to precipitation and turn white, add 500ul TE, 4ul 10mg/ml Proteinase K, 25ul 20%SDS uses forced oscillation, 65 ℃ of temperature were bathed 30 minutes, add the saturated phenol of 500ul, 37 ℃ vibrated 12 10 minutes, centrifugal 10 minutes of 000rpm, get supernatant, add the 1ml chloroform, 12, centrifugal 10 minutes of 000rpm, get supernatant, add the 3M sodium-acetate of 1/10 volume and the dehydrated alcohol of 2 volumes, mix the back and spend the night in-20 ℃ of precipitations; 12, centrifugal 10 minutes of 000rpm removes supernatant, control is done, and adds 500ul 75% washing with alcohol, 12, centrifugal 5 minutes of 000rpm, remove supernatant, drying at room temperature adds the 50ulTE dissolving, behind agarose gel electrophoresis, read the dna content of each sample on the gel imaging instrument ,-20 ℃ of preservations are got a part of DNA and markization final concentration to 1.5ng/ul.

2. design of primers:

That the software that design of primers adopts adopts is the Primer3 (http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi) of web interface.Design of primers mainly is that promoter region (Exon1 upstream 1.5-2kb scope) and the exon at gene carries out, the primer parameter setting is: fragment length is 300-500bp, the primer optimum length is 21bp, and best denaturation temperature is 62 ℃, and primer concentration is 0.3 μ M.The primer of design is also as sequencing primer, so the sequence that the upstream and downstream primer comprises is avoided polymer (repeat number N should be less than or equal to 8) as much as possible; Primer is initial or clearing end 50bp at least from exon.ADRP gene PCR primer designed in the present embodiment sees Table 1.

Table 1 primer sequence table

The zone	Forward primer (5 '～3 ')	SEQ ID NO：	Reverse primer (5 '～3 ')	SEQ ID NO：
					Promotor	AGTTCTATTCTGAGGACTAACGCCG		2	AAAGGCGAAGAGCAGGCG	3
Promotor	TGTTGGCCAGGCTGGTGAT	4	TGTGGTCGGGGTCCTGAGA	5
					Promotor	CTGGCGCAACTTGTCTGCTC	6	AAGTTTTGTGGCGGCTGGG	7
Exons 1	CGAGGCGGGGTTTATAGCC	8	ATGCGGTTTTCTAACGCGTTT	9
					Exon 2	ATGAATTTGGGAGAGTCTTATCCCG	10	GCTCGCCACTGACCAGTCC	11
Exon 3	CCCAGCCCATTATTGCCATT	12	CAGGAAGTAAGGAAGTGGCCATT	13
					Exon 4	GCTGTGCTGCCTTAGTTAAGACCTTA	14	CTGGATTGCAGTGATGCTTGC	15
Exon 5	GACTGCATGATTGCTAAGTGATTGG	16	AGTATTTGACCCCTCAGAGCGAG	17
					Exon 6	CATCTGCAAACAGGCTAACGTG	18	TGTACCCATTATGAGGGCAAGG	19
Exon 7	GTGGTGGACACATAACCAGCC	20	CCCAATTTAGGGTTGCCTAGCA	21
					Exon 8	CCTCAACTGGCTGGTAGGTCC	22	TCACACCAGAGCAGACACCAGT	23

3.DNA amplification in vitro:

For the trouble of saving sample DNA and reducing the PCR condition optimizing, present embodiment has adopted the method for multiple heminested PCR.

PCR (Long-PCR) increases in advance to dna profiling for the first time: 1) consult the primer data, the length of understanding Long fragment (being generally 1-1.5kb) with and the Tm value of upstream and downstream primer, find comparatively close but nonoverlapping 5-6 Long fragment to form an individual system.2) form dimer for fear of between the primer, adopt the warm start Taq enzyme of QiaGen company, (10 μ l) is as follows for reaction system: 10 * PCR damping fluid, 1 μ l, primer (20 μ M) 0.1 μ l * 2 * 5, dNTP (10mM) 0.3 μ l, MgCl ₂5mM) 0.6 μ l, Hotstar ^Taq 0.06 μ l, ddH ₂O 6.04 μ l, dna profiling (1.5-3ng/ μ l) 1 μ l. adopt the PCR response procedures of Touch-down: 95 ℃ 15 minutes, 94 ℃ 40 seconds, 63 ℃ of-0.5 ℃/circulations in 1 minute, 72 ℃ 1 minute 20 seconds, 10 circulations; 94 ℃ 40 seconds, 58 ℃ 40 minutes, 72 ℃ 1 minute 20 seconds, 30 circulations; 72 ℃ 8 minutes; 4 ℃ of termination reactions.

For the second time PCR (Nest--PCR) with the first time PCR product be amplification template, only use a pair of primer: 1) adding 30 μ lddH in first time PCR product ₂O, vibration evenly.2) with common Taq enzyme, reaction system (25 μ l) is: 10 * damping fluid, 2.5 μ l, primer (20 μ M) 0.375 μ l * 2, dNTP mixture (10mM) 0.5 μ l, MgCl ₂(25mM) 3 μ l, Taq 0.2 μ l, ddH ₂O 17.05 μ l, dna profiling (for the first time PCR product) 1 μ l. adopts the PCR response procedures of Touch-down: 95 ℃ 2 minutes; 94 ℃ 30 seconds, 63 ℃ 1 minute-0.5 ℃/circulation, 72 ℃ 40 seconds, 10 circulations; 94 ℃ 30 seconds, 58 ℃ 40 minutes, 72 ℃ 40 seconds, 30 circulations; 72 ℃ 3 minutes; 4 ℃ of termination reactions.

4.PCR product electrophoresis:

After PCR finished, every row extracted 3 samples, draws 3 μ l and adds sample on 2 μ l, 2 * sample-loading buffer volley of rifle fire, 120V voltage 20min in 1.5% agarose gel.The gel video camera is taken, and prints (the results are shown in Figure 1).

5.PCR product purification

The purification reaction step

1) the screen plate right side of face of using at purifying sticks adhesive tape, writes the sample name then in the above

2) add 150 μ l Resin resins (available from peace Pharmacia Corp), and PCR product (25-50 μ l all can), build sticking paper, after the concussion, left standstill 10 minutes.

3) remove sticking paper, 2200 left the heart 2 minutes, removed waste liquid.

4) add 150 μ l, 80% Virahol.

5) 2200 leave the heart 2 minutes, remove waste liquid.

6) add 150 μ l, 80% Virahol.

7) 2200 leave the heart 2 minutes, remove waste liquid.

8) do not add Virahol, 2200 changeed 3 minutes so that thoroughly remove clean residual Virahol.

9) vacuum drain 8 minutes (from the pressure pointer indicate began in nearly 0 o'clock the meter)

10) add deionized water 20 μ l, leave standstill dissolving 10 minutes after the concussion.

11) change 96 orifice plates, the good sample title of mark, mark position is corresponding with mark on the screen plate, and 2200 left the heart 5 minutes.

12) get 3 μ l purified products and 3 μ l sample-loading buffers and mix 100 volts of 1.5% agarose electrophoresis voltages.

6. the PCR fragment is checked order

The sequencing reaction operation steps:

1)-20 takes out PCR purified product, MIX, 0.8uM primer (not having then now to join) in ℃ refrigerator with the mother liquor of 10uM or 50uM

2) get 96 hole Sptting plates, put on template name (being PCR purified product name), date, add primer 2 ul one by one, add template 2ul with the 12 hole volley of rifle fires then with continuous sample injector, add mix 2ul one by one with continuous sample injector at last and on whizzer, get rid of, reach about 1300 rev/mins and promptly stop.

3) pcr amplification: (9700 types reaction instrument is available from PE biosystem company).Reaction conditions is as follows: 96 ℃ 10 seconds; 96 ℃ 10 seconds, 50 ℃ 5 seconds, 60 ℃ 4 minutes, totally 35 circulations; 60 ℃ 4 minutes.

4) after amplification finished, adding 70% ethanol (add-on is 9: 1 with the ratio of reaction system) with continuous sample injector was to precipitate in room temperature about 50ul, and the time must not be above 24 hours more than 15 minutes.

5) 4000 rev/mins, 4 ℃ centrifugal 30 minutes.

6) remove supernatant liquor gently, the inversion of 96 orifice plates is centrifugal, reach 800 rev/mins and promptly stop.

7) every hole adds the sample-loading buffer of 2-3ul, 2000 rev/mins of 1min.

8) 90 ℃ of sex change are 2 minutes, put immediately on ice, treat sample.

7.SNP the search in site:

The graphic file that Sequence Analysis3.3 software (ABI company) analyze is produced reaches and has Phredphrap, on the unix host of Polyphred and Consed software (University of Washington).Create four sub-directory chromat_dir earlier, edit_dir, poly_dir and phd_dir, the sequencer map shape file with each sample moves under the chromat_dir catalogue then, successively moves phredphrap and polyphred under edit_dir.Polyphred can put on cue such as shades of colour with the SNP site that software is suspected, the credibility that the different colours representative is different, and final affirmation need be opened figure and with the naked eye be evaluated.

Judge by following standard whether a site is the SNP site: the both sides sequence that 1) shows the base of heterozygosis is recognizable unimodal, and background is lower; 2) heterozygous individual and homozygous individual sequencer map are compared, in the figure of heterozygous individual, the base peak trend that significantly decreases.For the segment of inserting or lacking is arranged, the very sequencer map of feature is arranged---in heterozygous individual, the order-checking peak shape can begin to become in a mess from inserting or lack the position.By what contrast that two kinds of homozygous sequences of difference can determine to insert or lack is any base.

Two, result

5003 bases of ADFP gene fragment length overall of surveying are found 23 SNP altogether, and the result of SNP somatotype shows that high and low frequency SNP is respectively 5 and 18.The result who high frequency SNP site is carried out patient, normal controls research shows, high frequency SNP all is distributed in promoter region, their gene type sees Table (2), and wherein this SNP site of P/-407 is at the genotype of diabetes B crowd and normal population there were significant differences (X ²=6.65, P＜0.05).

Table 2.ADRP gene SNP somatotype result

In addition, find that also two missense mutation transmit altogether with two diabetes B familys respectively.No. 8 exon of ADFP gene has a missense mutation (Thr397Ala), and in 8 people of No. 118 familys, 4 diabeticss are A/G all; The people that other has 1 age is 45 years old impaired glucose tolerance is A/G, and age is that 29 years old normal people also is A/G.Two normal peoples that this family is surveyed are A/A (Fig. 2,3).

A missense mutation (Ser252Pro) of No. 6 exon of ADFP gene, three patients all are C/T in 4 people of No. 25 diabetes B familys that the present invention studies, a normal people is T/T (Fig. 4,5).It should be noted that these two polymorphisms only find in these two familys, distribute among diabetic individual and 200 normal peoples 200 of institute's examination and all not find this two polymorphisms, show that these two polymorphisms are these two peculiar sudden changes of family, but not SNP.

ADFP albumen finds to have very big similarity between different plant species after the BLAST software processes.Though at sudden change Ser252Pro place, not too cautious between the different plant species, the Thr397Ala regional area that the present invention discovers is suitable guarding during evolution, at species explicitly known, that can find at NCBI, genetic code in this site The Threonine of all encoding.This shows that the structural domain that amino acid constituted of this regional alkali yl coding that suddenlys change plays very large effect to the space structure of lipotropins (adipophilin), the performance of physiological function.

It is 82% identical that whole ADFP albumen has between ox and people, below protein sequence be these two the coded amino acid whose near zones of sudden change:

It is 82% identical that whole ADFP albumen has between Sus scrofa and people, and following protein sequence is these two the coded amino acid whose near zones of sudden change:

It is 76% identical that whole ADFP albumen has between mouse and people, and following protein sequence is these two the coded amino acid whose near zones of sudden change:

Though it is 55% identical that whole ADFP albumen has only between Xenopus laevis and people, these two the coded amino acid whose near zone protein sequences of sudden change are still quite conservative:

Three. discuss

In recent years, fat, the effect of fat toxicity in the diabetes B morbidity paid much attention to.(Adipose Differentiation-Related Protein, ADFP) the gene wide expression is in tissues such as liver, pancreas islet, skeletal muscle, kidney, lung, small intestines for fat differentiation associated protein.The protein of ADFP genetic expression---lipotropins (adipophilin) is the main component that forms lipid within endothelial cells droplet vesica, also be scavenger cell, the sedimentary sign of adipocyte inner lipid (Heid HW, Schnolzer M, Keenan TW.Adipocytedifferentiation-related protein is secreted into milk as a constituent of milk lipidglobule membrane, Biochem J, 1996,320 (Pt3): 1025-30; Buechler C, Ritter M, Duong CO et al.Adipophilin is a sensitive marker for lipid loading in human bloodmonocytes., Biochim Biophys Acta, 2001,1532 (1-2): 97-104.).

Previous research is positioned at No. 9 chromosomal and D9s171 to East China the Hans' diabetes B genes involved by full genome scanning and connects trivial zone.It is candidate gene that the present invention studies the ADRP gene of selecting to be positioned at the D9s171 near zone, detects and gene type by SNP, analyzes the dependency of ADRP and diabetes B.

The ADFP wide expression is in many tissues: liver, pancreas islet, skeletal muscle, kidney, lung, intestines, lymph etc.ADFP is an adipocyte differentiation phase sign (early than fatty acid binding protein, lipoprotein lipase) the earliest, in a day of preceding adipocyte differentiation, and its mRNA about 100 times (Biochem J 1996Dec 15 that can raise; 320 (Pt3): 1025-30Adipocyte differentiation-related protein is secreted intomilk as a constituent of milk lipid globule membrane.Heid HW, Schnolzer M, Keenan TW).The protein of ADFP genes encoding---adipophilin is the main component that forms the lipid within endothelial cells vesica, also is scavenger cell, the sedimentary sign of adipocyte inner lipid.The adipophilin of normal physiological expression amount and free fatty acid, cholesterol, cholesteryl ester level have nothing to do.LDL induced monocyte 2 hours, can cause that ADFPmRNA obviously raises, and adipophilin can promote the absorption of lipid acid, finally caused the interior free fatty acid of cytoplasm to increase.Some lipid acid is part (the 7.Buechler C of PPAR γ in the body, Ritter M, Duong CO et al.Adipophihn is a sensitive marker for lipid loading inhuman blood monocytes.Biochim Biophys Acta.2001 May 31; 1532 (1-2): 97-104), change has taken place in these lipid acid when fat, cause the unusual of PPAR γ signal conduction, and PPAR γ, PPAR α, PPAR δ also can promote high expression level (the Buechler C of ADFP, Ritter M, Duong CO etal.Adipophilin is a sensitive marker for lipid loading in human blood monocytesBiochim Biophys Acta.2001 May 31; 1532 (1-2): 97-104; Gupta RA, Brockman JA, Sarraf P, et al.Target genes of peroxisome proliferator-activated receptor gamma incolorectal cancer cells.J Biol Chem.2001 Aug 10; 276 (32): 29681-7).Free fatty acid increases also can suppress resistin gene (resistin), promote the leptin expression of gene, thereby caused insulin resistant (Juan CC, Au LC, Fang VS, Kang SF, Ko YH, Kuo SF, Hsu YP, Kwok CF, HoLT.Suppressed Gene Expression of Adipocyte Resistin in an Insulin-Resistant RatModel Probably bv Elevated Free Fatty Acids.Biochem Biophys Res Commun 2001Dec 21; 289 (5): 1328-1333).The activity that stops PKC in addition, to cause the high expression level of ADFP and cell lactones to drip and increase, become big, when using medicine that ADFP genetic expression is descended, to make fat drip (the Chen JS that melts, disappears, Greenberg AS, Tseng YZ, Wang SM.Possible involvement of proteinkinase C in the induction of adipose differentiation-related protein by Sterol ester inRAW 264.7 macrophages.J Cell Biochem 2001; 83 (2): 187-99).

As the protein-bonded Adipophilin of lipid within endothelial cells droplet, (Perilipin) is closely related with the Peri gene.Intracellular lipid droplet or wrap up by adipophilin or by Perilipin that is to say to have the relation that suppresses mutually between them.Experimental study shows that Perilipin can regulate the hydrolysis of triglyceride, Peri ^-/-With Peri ^{+ /+}Mouse is compared, and obviously rising of Leptin concentration in the blood plasma, impaired glucose tolerance, hyperinsulinemia, insulin resistant, adipolysis strengthen, and body fat reduces and can reach about 30%.Peri ^-/-Whether a series of pathologic-physiological reactions of mouse relevant with Adipophilin still awaits further confirmation.

Two sudden changes of the ADFP that institute of the present invention finds also are heterozygotes though a normal people is arranged in 118 familys, and this may be not show as diabetes because she less at age, further make achievement in research can follow up a case by regular visits to by tracking.Two missense mutation Ser252Pro and Thr397Ala cause hydroxyl polare Aminosaeren to become nonpolar amino acid, Threonine (Thr) and Serine (Ser) are hydroxyl polare Aminosaeren, and L-Ala (Ala) and proline(Pro) (Pro) are nonpolar amino acids.Research prompting in the past, hydroxyl amino acid and phosphorylation have closely gets in touch, and phosphorylation is relevant with the differentiation of cell.Test-results prompting of the present invention, two sudden changes of ADFP gene may be respectively the pathogenic sites of two diabetes B familys.The early stage index that ADFP breaks up as adipocyte, and closely related with the metabolism of lipid acid, therefore, there are certain dependency in ADFP and diabetes.

Embodiment 2

Diabetes B susceptibility detection kit

Prepare a test kit, it contains:

Title sequence (5 ' → 3 ') numbering concentration

Forward primer CCTCAACTGGCTGGTAGGTCC SEQ ID NO:2 dry powder 20D

Reverse primer TCACACCAGAGCAGACACCAGT SEQ ID NO:3 dry powder 20D

The PCR reaction solution contains Taq enzyme dNTP magnesium ion PCR reaction buffer

Extract the blood 3ml of object to be detected, use ordinary method (or using specific test kit) from blood, to extract DNA.PCR primer in the diabetes B detection kit is diluted to 1 μ mol/ μ l, is that template is carried out the PCR reaction with the primer that is provided with the DNA that is extracted.Behind the PCR product purification, use ABI-PRISM ^TMThe 377DNA sequenator carries out the two-way order-checking of the terminal cessation method of fluorescent mark, and the interpretation and the SNP that carry out sequence with Polyphred software confirm.

As a result, the 697th is in the detected object of G in SEQ ID NO:1, and its diabetes B susceptibility is higher than normal population.

All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

Sequence table

＜110〉Shanghai Endocrine-Metabolic Diseases Inst.

＜120〉dependency of white gene of lipocyte differentiation associated protein and diabetes B

<130>050056

<160>23

<170>PatentIn version 3.1

<210>1

<211>12814

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>1

ttttttcttt tctttttttt tttttagaca gggtctcagt cactcaggct ggggtgcagt 60

ggtacagtgg cgcgatctcg actcattgca gcctcgacca cctgggctca aaagattctt 120

tcacctcagc cacccacccg cctgccccca accccgctaa acagccggga ctagctggga 180

ctacaggcgc gcgccaccac gctgggctgc tttctttgta ttttgagaga gacggggttt 240

cgccatgttg gccaggctgg tgattcgctc gcctcggcct cccaaagtgc taggattaca 300

ggcgtgagcc accgcgccca gccgccacaa aacttttaaa agttattact tcgctaaaaa 360

taacagcgta gaagccattg tgcagcgcca cgcgatctcg gctcactgca gcctcgacct 420

cccaggctca agcgatcctc ccatcttagc ctcgcaggta gctggggcca caggcgcacg 480

acaccacgct gggctatttt ttaaaataaa ataaaaatct tcgccaggct ggtctcgaac 540

tcctaggctc gacagatcct ctcgcctcag catcccaaag tgttgtgatt acaggcgtga 600

gccaccgctt ccatttttag agttctattc tgaggactaa cgccgttcaa caagttggtg 660

tgaatttgtt tgcatttttc aaaaccccaa agccggcctg ggcgccatcc ccatttgccc 720

cgggcctctc aggaccccga ccacaggcag gaccccgacc acaggcagcg ccccgactcc 780

aggcagcgcc ccgactccag gcagggctac ctccaactcc cagcctcccc aggccgggcc 840

cccgcccggc tcccgacagg ctcctctccc cgtggcgggg cccctcgccc aggcagcaca 900

ccctctcaac ccgaccgggc ccccaccact ctaggagggc cggcgttggg ggagggcgcc 960

gggacgtgcc cgagggtgac actcgggctt gggacagggc gtgctgccgc gggtcacgtg 1020

ctgcggaggc ttggggaggg gcggcgaggc ggggtttata gcccgggcgc ccgcgggccc 1080

cacgctttga ccgggtcgtg gcagccggag tcgtcttcgg gacgcgcctg ctcttcgcct 1140

ttcgctgcag tccgtcggtg agtgcgcgcc cagtgggtgc cccagcgaac cggtcccggg 1200

cccccagcgg cgcccagttg gggtgggggg ctccccgggg ccttgggacc gctgctctgc 1260

tccccgcggg gctcaaaggt ggacgggacg cgggggtgat ggtccgagac cccccgctcg 1320

cctcgcggct tccaggtcct ggctgggtag gggcggtctg ctgaacccgc gctttcgacc 1380

cttgcctttg tgcgttgagg cttctttcct gccaacagat ttagggcgat tgctatcgaa 1440

tgtcccttgg cctgcttggg tggttgtggg aaaacaatat ttgtcgaagg gacattatcg 1500

aaagggaaac gcgttagaaa accgcatttt ttttttgacg gagtctcgct ctgtcgccca 1560

ggctggagtg cagtgacgcc atctaggctc actgcaacct ccgcctcccg ggttcaagcg 1620

attctcctgc ctcagcctcc agagcagctg ggataacagg cgcccgccac cacgcccggc 1680

taatttttgt atttttagaa gagacggggt ttcaccacgt tggtcaggct ggtcttgaac 1740

tcctgacatc aagcagtcca cccccctcgg cctcccaaag tgctgggatt acaggcgtga 1800

gccaccgcgt ccggcttggc tagcattttt gttaacagat actcggagaa acaagagaag 1860

tatgtcaaca actttacttc tccctcctga gtattgcaag tttacacata agctctgaaa 1920

ctttgcctca aaagagggag tggggttgtg tgggtgtggg tctttcagga gggggacctg 1980

gagagctgga gagaggaaag aagtgcagaa gactacaggg agctcaccct gcatttttct 2040

ctacttgtaa tcagcatttg cttgggttgg ggaatgaatt tgggagagtc ttatcccggt 2100

gttctgaaat aagtctcttt tctgcagatt tctttctcca ggaagaaaaa tggcatccgt 2160

tgcagttgat ccacaaccgg tatgaatttt gagtagcctt tgtctacttt ctctcctttt 2220

gtgttttgag agattaataa gcataccctc cctactttga ttgcagagtg tggtgactcg 2280

ggtggtcaac ctgcccttgg tgagctccac gtatgacctc atgtcctcag cctatctcag 2340

tacaaaggac cagtatccct acctgaagtc tgtgtgtgag atggcagaga acggtgtgaa 2400

gaccatcacc tccgtggcca tgaccagtgc tctgcccatc atccagaagc tagagccgca 2460

aagtgagttc tgatttttca agatgcaagt caagggactg gtcagtggcg agcccctgag 2520

ctcacagcag taacatctgg aactcctcag cccttcctgg ggcatacact gcctttacct 2580

agaaactttc aggtgaccct cattaccgta agaggactgt ttcttttctt ttctttcctt 2640

tttttttttt tttgagatgg agtctcactc tatcactcag gctggagttc agtggcgcag 2700

tcttggctca ctgcaacctc tgcctgctgg gttcaagtga ttctcctgcc tcagcctccc 2760

gagtagctgg gactacaggc tcccaccacc atgcccagct aatttttgta tttttagtag 2820

agacgcggtt tcacaatatt ggccaggctt aactcctgac tttgtgatcc acccacctct 2880

gcctcccaaa gtgctgggat tacaggcgtg agccaccgcg cctgtccaca gaaggcctgt 2940

ttcatagctt tggcagatgt ttatgatcct ttaattaaaa atcccccaga ggatgggcac 3000

ggtggcctgc acctgtaatc ctagcacttt gggaggtcta gatgggagga tcgcttgagg 3060

cctggaattt gagactgctc actgcagtct ctgcctctcg ggctcaggtg attgtcctgc 3120

ctcagctccc tgagtagttg ggattacagg catacaccac catgcccagc taattttgta 3180

tttttagtag agacagggtt ttgccatgtt ggccaggctg gtcaggaact cctgacccca 3240

ggtgatccgc ccgcctcggc ctcccaaagt gctaggacta caggtgtgag ccaccatgcc 3300

tggccatgaa tgagttttag aaatggatag tggtgatggt tgtataatgt tgtaaatgga 3360

cttaatgcca ctgaattgca cagttaaaaa tggttgaaat ggtgaaattt gttatgtata 3420

ttttaccgaa ataaagaata gaaatgtcat actccatgag aaatctcata ctctcatact 3480

tagtagtcac tccccatttc ctaacaacca ccctgaggca gctactcatc tactttgtct 3540

ttatagattt gcctatgctg gatatttctt ataaatgaaa tcataaaatg tgtgggtttt 3600

tttgtgattg gcttctttcg ttaagcataa tgttttcaaa gttaatctgt cttgtcacat 3660

catatcagta cttcatctct ttttattgtt gaatagtaat ttactgtcta gacagacaat 3720

atttattcat tcttcaactg atggatgttt gggttcgttc tacttttgga gattatgtat 3780

aatgcttctg tgaacatttg tatacaagtt tttgtgtgga catgttttca ggtttttttg 3840

gtatataccc aggagtggaa ttgctgggtt atataataac tgtttaagct tttgaggagc 3900

tgccagactg ttttccaaat cagctgtacc attttacatt cccaccaaca ctgtatggag 3960

gctccaattt ctccacaaca tgaactgctt ttgaataaag atttaaatca ttctttcaaa 4020

tggttaagcc agggactttt aggattataa ggagccttta gagaccagcc attccaactc 4080

tgctacttcc taaatgaatt tggtactagt acttggtttg gtattcttag tgggaagctc 4140

acactaaaat ctcccttgtt agggagatcc agggtggtct tggcatactc tttcctcctg 4200

aggaccccaa atacactgac ctgtgctgaa gtaagcgtag gtagggatta aactagcatg 4260

ggaatgccga ttttgagaga gtttccagat accttattct ctgaatgaca gttcagggaa 4320

agcccggcta ctcttggcat taacattagg ggccagttga taaggaagaa gcttcagagc 4380

cagcttcctg tttggcctgt cactgcctct gcctgctcct ccctcacacc cccatatttc 4440

cttaccttta gaagagagtt gccagcaact tgccaggcct cttcctgtca gaactagata 4500

aaccaggcaa tgccttctgt tgtataagat caacattctt gccttttttt ttttttttga 4560

gatggagtct tgctctgtca cccaggctgg agtgcaatga tgcatcttgg ctcactggaa 4620

cccctgcttc ctgagttcaa gcgattctcc tgcctcagcc tcctgagtag ctgggactac 4680

aggcgcacac catcacgccc agctaatttt tgtattttta gtagagacgg ggtttcacca 4740

tgttggccag gatggtctcg atctcttgac ctcgtgatcc gcctgcctca gcctcccaaa 4800

gtgctgggat tacaggcgtg agccaccgtg cccagcccat tattgccatt ttttataagg 4860

cctatggtaa tgtgtgttca ttctatacct atagtactac attaacattt tcccatttgt 4920

gattctagtt gcagttgcca atacctatgc ctgtaagggg ctagacagga ttgaggagag 4980

actgcctatt ctgaatcagc catcaactca ggtgagcttg ggacaaaagt gctgagactt 5040

gacacttcac atgttttcat cttctatctg ttaaaacaag ctgtacatat cacaaacagg 5100

caaatacttg ttttcccaac ctggatctgg atgtatgttt gaaaaataat ggccacttcc 5160

ttacttcctg aactgttgtc aagttgtatc tacgaagcag agaagaacta gcctcctttg 5220

gattagaaaa cttttagtgt gttgtctatc agtttgtgtc tttgctccta taaatgaaat 5280

tgctgtctca agtaactgat tgtcttgtgg tattaaataa atgcagacgt gatatatata 5340

accttttctc acctggcttg tacaatgctg ccaggaacta ccatccatat gggatagcta 5400

gccagtgttg acaaaaatga taggcttgtc tgtcctggtg atcctttggt tcagataatg 5460

tcctaaaatt ttgtttcaaa ttaagaatac agaatggcac aaaaggatac cagttaggga 5520

aaaatatagc tctgtgtctt ttcataatct aaatacagtc atctgtcact tagtgatggg 5580

aatatatttt gagaaatgca cattaggtga ttttgccatt gtgtgaacat cacagagtat 5640

actcacacca acctagatgg tagagcccag tggtccccaa gctttatggc accaggaact 5700

ggttccatgg aagacagttt ttccatggac cagcaggggg tgggtggtgt ttttgggatg 5760

attcaagtgc actatattta tcattagatt ctcataagga gcatgcacgt gcacagttca 5820

caagagggtt agggttaggt ctcctatgag aatctaatgc cgcctctgac tggtaatgga 5880

ctggtaatgg actggtaatg gtccatggcc cgggggttag ggacttgtgg tgtagcctgc 5940

tatataccta ggctatatgg tgtatagcct gctatatacc taggctatat ggtgtatagc 6000

ctgctatata cctaggctat atggtgtata gcctgctata tacctaggct atatggtgta 6060

tagcctgcta tatacctagg ctatatggta tatagcctgt tgcttgcttc taggctacaa 6120

acctgcacag catgactaca ctgactactg taacacaatg ataagtattc gtgtatctaa 6180

actagaaaag gtacagtgaa aaaaaaaagt atacctgtta tagggcagtt cattataatc 6240

ttatgggact atcattgaat agtggtccat cgctgaccaa aatattatgt ggtacacaac 6300

tataggtagg tttagtgtat agcctttaga atgcttgtgc aaatctttta aatttttcac 6360

ttacattgct tgagactgtg ctaatggtga gagttatttt ccatttcgga tattttcttt 6420

aaatgttggt aacttgtgtt cttttttttt tttttttttt tttttttgag acagagtctt 6480

gctctgtcac ccaggctgga gtgcagtggc acaatctcgg ctcactgctg tctcttcctc 6540

ctgggttcaa gcaattgtcc tgcttcagcc tccccagtag ctgggttcac aggcacgccc 6600

cgccacaccc agctaatttt ttttgtcatt gtttgtttgt ttttgagaca gagttttgct 6660

cttgttgccc aggctgtagt gtaatggtgc aattttggct cactgcaacc tccacctctg 6720

gggttcaagt gattcttctg cctcagtctc ccaagtagct ggggttacag gcatgtgcca 6780

ccacgcccaa ctaatttttt gtatttagta gagacggggt ttcaccatgt tggtgaggct 6840

ggtctcgaac tcctaacctc aggtgatcca accaactcag ctttgcaaag tgctggcatt 6900

acaggcatga gccaccatgc cctgccatgt ataataactg catactaata acaccccgag 6960

ggccaggtat agtttgatct tcagtttcct ctctggattt gtgtgacttt ggaataagct 7020

atataactta ccttcttttc ttttcttttt ttttcttttc tcttctcttt tttttttttt 7080

ttttttgaga cagggtcttg ctctgtcacc caggctgagt gcagtggcac aatcacagct 7140

cactgcagcc ttggcaaaca gacaaatttc atatctgttt acttcatttg ctcctcctgc 7200

aattcccaag gattggcagg tagggcagat atacttgata gggaaattca ggctcatcaa 7260

ctcttagtga cttgcccaag acctaaagct agagtgagag gactagattc cttctttaac 7320

tcttcagctg tgctgcctta gttaagacct taggtatgtc cttgtgctca tttttctcac 7380

cagccagggg atctgctttt ctacttacag attgttgcca atgccaaagg cgctgtgact 7440

ggggcaaaag atgctgtgac gactactgtg actggggcca aggattctgt ggccagcacg 7500

atcacagggg tgatggacaa gaccaaaggg gcagtgactg gcagtgtgga gaagaccaag 7560

tctgtggtca gtggcagcat taacacagtc ttggggagtc ggatgatgca gctcgtgagc 7620

agtggcgtag aaaatgcact caccaaatca gagctgttgg tagaacagta cctccctctc 7680

actgaggaag aactaggtaa ctggaatttt atcttgaaat actgttttat attaaatctt 7740

tcttaaacag cttcattgac atatactctc atcttgagtc taaatttaca cagttgtgca 7800

agcatcactg caatccagtt tcagaacact tctgctaccc caaaagatcc tcatgcctgt 7860

ttgcagtcag tcctttttaa tttaatttaa ttttatagag atgggatctc actatgttgc 7920

ccaggctgat ctcgaactcc tgggctcaaa cgatccacct gccttgacct cccaaagttc 7980

tgggattaca gatgtgagca accactaatc tttttgcctc tatagatttt ccttcctaaa 8040

gcaatcataa agttcacatt gaatcgtacg tagtaagttt tgaaacctta gcaaaaattg 8100

gccttgaaaa atattttctg taaatggata gtctttaatg gattgcttta taaaatcctc 8160

cttacaaaga atcatcatta ggtaatgtgt gccaggtacc atgccgctca agttcactct 8220

aggctgggag caatggtgca tgcatgccta caatcccagc actttgggag gctaagacag 8280

gcagatcgct tgagttcagg agtttgagat tagcctgggc aacatggcga aaccctaaaa 8340

aacacaaaaa ttagctgggc atggtggctt gttcctagag tcctagctac tcgggaggct 8400

gaggtgagag gatcagttga gctcgggagg ttgaggctgc agtgagccaa aattgtgcca 8460

ctgtactcca gcctgggcaa caaagcaaga cctgtttcca aaaaaaaaaa agcagtttac 8520

tctagtggga gaaacgtgta gatgcatgca gtgtgtatca gatacagtaa tatgttcagg 8580

gtagtcaagt ccagcagcag acgtgagcag tcttggaaaa atgactgcat gattgctaag 8640

tgattgggta gaagtaagaa agtcaagacc caggttagag tccaggcctt atcacttgtc 8700

actgtgatcc cttaagtttt tttgtctctt atttctaact tcagaaaaag aagcaaaaaa 8760

agttgaagga tttgatctgg ttcagaagcc aagttattat gttagactgg gatccctgtc 8820

taccaagctt cactcccgtg cctaccagca ggctctcagc agggttaaag aagctaagca 8880

aaaaagccaa cagaccattt ctcagctcca ttctactgtt cacctggtga gtaaaacttt 8940

attttaaggt gtctgaagtg ggttcattta tctctaggcc caagaattaa atgtaatcac 9000

aaaatccaag aatttcattc ttagttgaca aacaacccaa tagaaaactg gacaaagaat 9060

atggagacag ctcacagaaa aaggaagtat aactagttga aatataggaa gcaatgctaa 9120

tctcgctctg aggggtcaaa tactgacatc tctcttgtcc tgccacacgg gaaatgtggc 9180

tgttggcaac ggtgtagaga aagaaccaca cttgtgcagc tcttgctcta agggactgat 9240

tcaagagaca gttttagtaa agttacaagc atacctacac ttggagctgt tttacttttt 9300

atcttacaga tgtgtatatt tgtttaaagt tgcaagagac tataacaatg cacatccact 9360

aacaggacta attaaattga tatatcacaa tagactactg tgtagccatt aaaggtttcc 9420

aagttaaaga acagtatgta ccattctgta ctgccatgag aaaaccattt cttcttagaa 9480

ggggaaatag tggttggcaa cagggaagag gaaggtcaaa ttcctttctg ttttgtaatg 9540

ctaacttatc ctttactcat gtacacatta aattttagtt gctggctggg cgtggtggct 9600

catgcctata atcccagcac tttaggaggg caaggtggtt ggatcacctg aggtcaggag 9660

ttcgagacca gtctggccaa caaggtgaaa tcctgtctct actaaaaata caaaaattag 9720

ctgggcatgg tggcgtatgc ctgtaatccc agctactcgg taggctaagg cacgagaagg 9780

aggttcagtg agctgtgatc gcgccactgt acttcagcct gggtgacaga gtgacactca 9840

aaaaaaattt tttgtagttt tttagttgct ttacctgtgc taaactggaa gtaaatgtgg 9900

agtcagtgat ttaatagtta aatagaggca gtaaagagct actactgctt ggttaattta 9960

cacatattga taataaataa taatgatagc aggaatagat cttattcaac tccaggaaat 10020

tcttagtttt tatgttgcct gatttacatc atacatctgc aaacaggcta acgtgaatat 10080

ctgacttgtg tgttcctttg tcttgtttca atgtgtctag attgaatttg ccaggaagaa 10140

tgtgtatagt gccaatcaga aaattcagga tgctcaggat aagctctacc tctcatgggt 10200

agagtggaaa aggagcattg gatatgatga tactgatgag tcccactgtg ctgaggtaag 10260

atgactggga cggtcatttg ttggttgctg aagaagttct tatcagactt ttcatttcaa 10320

tttttccata ctatgtctta gaatactctt ctctatcaaa aggttgaaca ataccatgta 10380

cataggtggc tagttatgtg gcagaaaagc aaaactaggc agcgaataat tttatctaac 10440

aacctatgag tatacatatg ccccaagtga gatacaacta aatgagaaac acaggccttg 10500

ccctcataat gggtacagac taacagggga tacagccagc catgctgtcc tattttgtgc 10560

ctttttaatg ctccttgcag aagagggcta tctcataggc agtacactca gagtagccaa 10620

gtccggttgt tgtttaacgg cataaataca ggtactattg gctgggcgcg atggctcacg 10680

cctgtaatcc caacactttg ggaggccgag gcgggtagat cacgaggtca ggagatcgag 10740

accatcctgg ccaacatggt gaaaccctgt ctctactaaa aatacaaaaa atgagccagg 10800

catggtggca cgtgcctgta gtcccagcta ctcgggcagc tgaggcagga gaatagcttg 10860

aacctgggag gcagaggttg ccatgagccg agatcatgct actgcactcc agcctgggca 10920

acagagtaag actctgtctc aaaaaaaaaa aaaagaaaac atatgggaag actatctcag 10980

gcagccttgg agtgggggta gttgagtcac attgctttct ggaagagatg aatggtaagc 11040

tgagaactaa tgggtgaata aactaggtga aagggcaata aggtgaccaa ggttccagag 11100

ggaaggcata gcatcttggc tgggtgcaat ggctcacacc tgtaatctca gcattttggg 11160

aggccaaggc aggcagatca cttgagccca ggaattcagg accagcctgg gcaactgtga 11220

aaccccatct ctacaaaaaa tacaaaaatt agcagggtgt ggtggtgtgc acctgtagtc 11280

ctagttgctc gagaggctga ggtgggagga tcacctaagc ccgagaggtc gggtccgcag 11340

tgagccatga ttgtgccagt gccctccagc ctgggcaaca gactgagacc ctgtctcaga 11400

aagaaagaaa gaaaagaaaa gaaagaaaga agaaagaaag aaaaaggaag ggaagcaggg 11460

aggaaggaag ggaaaagaaa tatcctctta tagtcctaga aagtggttct ttgtgtccca 11520

aaagctctga agtcaatatg aactgtttgt tcatgtcttg tcattttatt tgaggaggga 11580

atatatacat tacactacta acatgaagga tatttccatt cttttgggtc tcccctacct 11640

aatatgtttc ttcagtttaa gatgatgaac tttggagact caggcactag ttcgaatccc 11700

tacccagctg ttttccagtt gttaactgaa gccatctact taaactctaa gcttatattc 11760

atatctatga aatgtgaata atactgctgc actcggcatt gtaatgagga tttttaaaag 11820

ccaaagaatc cacatatgtg gtggacacat aaccagccta ctgtcatcga actaatttat 11880

acatagcact gttggatcca ctgctaattt taagttttat ttttagcaca ttgagtcacg 11940

tactcttgca attgcccgca acctgactca gcagctccag accacgtgcc acaccctcct 12000

gtccaacatc caaggtgtac cacagaacat ccaagatcaa gccaagcaca tgggggtgat 12060

ggcaggcgac atctactcag tgttccgcaa tgctgcctcc tttaaagaag tgtctgacag 12120

cctcctcact tctagcaagg ggcagctgca gaaaatgaag gaatctttag atgacgtgat 12180

ggattatctt gttaacaaca cgcccctcaa ctggctggta ggtccctttt atcctcagct 12240

gactgagtct cagaatgctc aggaccaagg tgcagagatg gacaagagca gccaggagac 12300

ccagcgatct gagcataaaa ctcattaaac ctgcccctat cactagtgca tgctgtggcc 12360

agacagatga caccttttgt tatgttgaaa ttaacttgct aggcaaccct aaattgggaa 12420

gcaagtagct agtataaagg ccctcaattg tagttgtttc cagctgaatt aagagcttta 12480

aagtttctgg cattagcaga tgatttctgt tcacctggta agaaaagaat gataggcttg 12540

tcagagccta tagccagaac tcagaaaaaa ttcaaatgca cttatgttct cattctatgg 12600

ccattgtgtt gcctctgtta ctgtttgtat tgaataaaaa catcttcatg tgggctgggg 12660

tagaaactgg tgtctgctct ggtgtgatct gaaaaggcgt cttcactgct ttatctcatg 12720

atgcttgctt gtaaaacttg attttagttt ttcatttctc aaataggaat actacctttg 12780

aattcaataa aattcactgc aggatagacc agtt 12814

<210>2

<211>25

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>2

agttctattc tgaggactaa cgccg 25

<210>3

<211>18

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>3

aaaggcgaag agcaggcg 18

<210>4

<211>19

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>4

tgttggccag gctggtgat 19

<210>5

<211>19

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>5

tgtggtcggg gtcctgaga 19

<210>6

<211>20

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>6

ctggcgcaac ttgtctgctc 20

<210>7

<211>19

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>7

aagttttgtg gcggctggg 19

<210>8

<211>19

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>8

cgaggcgggg tttatagcc 19

<210>9

<211>21

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>9

atgcggtttt ctaacgcgtt t 21

<210>10

<211>25

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>10

atgaatttgg gagagtctta tcccg 25

<210>11

<211>19

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>11

gctcgccact gaccagtcc 19

<210>12

<211>20

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>12

cccagcccat tattgccatt 20

<210>13

<211>23

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>13

caggaagtaa ggaagtggcc att 23

<210>14

<211>26

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>14

gctgtgctgc cttagttaag acctta 26

<210>15

<211>21

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>15

ctggattgca gtgatgcttg c 21

<210>16

<211>25

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>16

gactgcatga ttgctaagtg attgg 25

<210>17

<211>23

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>17

agtatttgac ccctcagagc gag 23

<210>18

<211>22

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>18

catctgcaaa caggctaacg tg 22

<210>19

<211>22

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>19

tgtacccatt atgagggcaa gg 22

<210>20

<211>21

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>20

gtggtggaca cataaccagc c 21

<210>21

<211>22

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>21

cccaatttag ggttgcctag ca 22

<210>22

<211>21

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>22

cctcaactgg ctggtaggtc c 21

<210>23

<211>22

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>23

tcacaccaga gcagacacca gt 22

Claims (10)

1. method that the diabetes B susceptibility of individuality is diagnosed is characterized in that it comprises step:

Detect this individual ADRP gene, transcript and/or albumen, and compare with normal ADRP gene, transcript and/or albumen,

The possibility that there are differences with regard to showing this individuality trouble diabetes B is higher than normal population.

2. the method for claim 1 is characterized in that, detection be gene or the transcript of ADRP, and with normal ADRP nucleotide sequence comparing difference.

3. the method for claim 1 is characterized in that, described difference is the single nucleotide polymorphism that is selected from down group:

697 A → G;

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

4. whether a test sample exists the method for the single nucleotide polymorphism of ADRP gene, it is characterized in that, comprises step:

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

(b) detect whether there is the single nucleotide polymorphism that is selected from down group in the amplified production:

697 A → G;

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

5. method as claimed in claim 4 is characterized in that, described single nucleotide polymorphism is selected from down group: 697 A → G.

6. the test kit of a complementary detection diabetes B is characterized in that, it comprises the primer and the specification sheets of specific amplification ADRP gene or transcript.

7. test kit as claimed in claim 6 is characterized in that, it also contains the reagent that is selected from down group:

(a) with mutable site bonded probe;

(b) restriction enzyme in identification mutational site.

8. test kit as claimed in claim 7 is characterized in that, described sudden change is the single nucleotide polymorphism that is selected from down group:

697 A → G;

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

9 test kits as claimed in claim 6 is characterized in that, described primer is the primer of sequence shown in SEQ IDNO:2 and 3.

10. an ADRP gene and proteic purposes is characterized in that, are used to prepare the reagent of complementary detection diabetes B.

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