CN1519351A - Polymorphism of 7th exon in ITGB2 gene - Google Patents

Abstract

A single nucleotide polymorphism (SNP) for the extron No.7 of ITGB2 gene and a method for analyzing the SNP and activity of said extron No.7 are disclosed. Said SNP is G->A polymorphism at the site No.126 in its sequence shown by SEQ ID No.1 and can cause the change Gly(G)->Glu(E) at the site No.26 of coding protein, so changing protein activity.

Description

No. seven exon polymorphism of ITGB2 gene

Technical field

The present invention relates to the single nucleotide polymorphism of No. seven exon of ITGB2 gene.The invention still further relates to the method for the allelic mutation of analyzing No. seven exon of ITGB2 gene.

Background technology

ITGB2 (Integrin gene beta 2) is No. 2 β subunits of integral protein gene, and its variation is considered to the basic reason that the Canis animals white corpuscle is supported deficiency disease (Canine leukocyte adhesion deficiency).(J?Vet?Intern?Med.2002?Sep-Oct；16(5)：518-23)。Present research also discloses, and ITGB2 genovariation also causes a large amount of crowds to get congenital white corpuscle support deficiency disease.(J?BiomedBiotechnol?2001；1(3)：114-121)

Before the application, also there is not the relevant report of No. seven exon polymorphism of ITGB2 gene.

Summary of the invention

Purpose of the present invention just provides the polymorphism and the detection method thereof of No. seven exon of ITGB2 gene.

In a first aspect of the present invention, the method for the single nucleotide polymorphism of No. seven exon of a kind of people of detection ITGB2 gene is provided, it comprises step:

(a) determine the 126th Nucleotide in sequence shown in the SEQ ID NO:1 of No. seven exon of people ITGB2 gene;

(b) detect whether there is single nucleotide polymorphism in described position.

In a preference, described single nucleotide polymorphism is to have A at the 126th.

In a second aspect of the present invention, a kind of isolating nucleic acid is provided, it has the sequence shown in the SEQ ID NO:1, and the 126th is A.

In a third aspect of the present invention, a kind of allele specific nucleic acid primer is provided, its length is 15-50bp, and hybridizes specifically and amplify shown in the SEQ ID NO:1 that contains No. seven exon of people ITGB2 gene the amplified production of the 126th single nucleotide polymorphism in the sequence.

In a fourth aspect of the present invention, a kind of allele specific oligonucleotide probe is provided, its length is 15-50bp, and hybridizes specifically and detect shown in the SEQ ID NO:1 that contains No. seven exon of people ITGB2 gene the 126th single nucleotide polymorphism in the sequence.

A kind of test kit comprises:

(1) primer of No. seven exon of specific amplification people ITGB2 gene is right,

(2) detect No. seven exon of amplified production and normal ITGB2 gene and compare the reagent that whether exists variation required, the 126th single nucleotide polymorphism in the sequence shown in the described SEQ ID NO:1.

Another kind of diagnostic kit comprises: the oligonucleotide probe that the primer that the present invention is above-mentioned and/or the present invention are above-mentioned.

Embodiment

The inventor is by extensive and deep research, had been found that in the ITGB2 gene protein sequence that No. seven exon is coded the 26th Gly (G)-Glu (E) polymorphic (be last 126 G-of mRNA〉A polymorphism), finished the present invention on this basis.

According to normal people ITGB2 gene sequencing result, found this SNP.Find that by analysis this SNP causes the 26th amino acids by Gly (G)-Glu (E).Because this is the amino acid whose displacement of different properties, therefore, can cause the ITGB2 gene protein-active that No. seven exon is coded to change.

The cDNA sequence of No. seven exon of ITGB2 gene is listed in SEQ ID NO:1, and wherein open reading frame is the 48-203 position, and its coded aminoacid sequence is listed in SEQ ID NO:2.The genome sequence of No. seven exon of ITGB2 gene can obtain from GenBank.

Those skilled in the art will appreciate that a large amount of analytical technologies can be used for detecting whether the site exists single nucleotide polymorphism described in No. seven exon of ITGB2 gene.These technology comprise (but being not limited to): dna sequencing, sequencing by hybridization; Enzymatic mispairing cutting, heteroduple analysis, dot blot, few nuclear former times acid array (DNA chip), Mini-sequencing, Taqman technology, molecular beacon etc., sex change high performance liquid chromatography (DHPLC).

On the other hand, detection method of the present invention is used to assess the individual susceptibility of suffering from No. seven exon relative disease of ITGB2 gene.

Being used for specimen of the present invention and being not particularly limited, for detecting SNP, can be DNA or the mRNA that extracting goes out from samples such as blood, tissue.For detect No. seven exon of ITGB2 gene active for, can anyly contain the sample of No. seven exon of ITGB2 gene, as blood etc.

The primer and the probe that are used for the inventive method or detection kit design according to the cDNA sequence (sequence of SEQ ID NO:1) or the genome sequence of No. seven exon of ITGB2 gene, and the synthetic technology of available routine is synthesized and got final product.

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 normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:ColdSpring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment 1

The extracting of gene and order-checking

Extract DNA with conventional phenol chloroform method from people's blood, concentration correction is used for conventional pcr amplification to 20ng/ul.Primer is:

Adopted primer is arranged: 5 '-ctgggaaact gaggcaggta-3 ' (SEQ ID NO:3)

Antisense primer: 5 '-acttggggct tccagaagtt t-3 ' (SEQ ID NO:4)

The product of the 343bp that amplifies carries out dna sequencing behind the purifying.

Embodiment 2

The acquisition of SNP

The ITGB2 gene is directly checked order.Each sample sequence of measuring is compared, thereby draw the difference of sequence, obtain SNP.

Embodiment 3

Detection kit

Preparation one detects the detection kit of ITGB2 gene-correlation disease susceptibility, and wherein, it is right to contain the following primer that amplifies 126 SNP:

Adopted primer is arranged: 5 '-ctgggaaact gaggcaggta-3 ' (SEQ ID NO:3)

Antisense primer: 5 '-acttggggct tccagaagtt t-3 ' (SEQ ID NO:4)

Amplified production and normal control are carried out stratographic analysis with sex change high performance liquid chromatograph (DHPLC), can detect 126 G-easily A type SNP.

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〉Research Center of Shanghai Human Genome

＜120〉No. seven exon polymorphism of ITGB2

<130>030115

<160>4

<170>PatentIn?version?3.1

<210>1

<211>343

<212>DNA

<213>Homo?sapiens

<220>

<221>CDS

<222>(48)..(203)

<223>

<400>1

ctgggaaact?gaggcaggta?accctgcccc?ccacgccttc?ttctcag?gag?gaa?atc 56

Glu?Glu?Ile

1

ggc?tgg?cgc?aac?gtc?acg?cgg?ctg?ctg?gtg?ttt?gcc?act?gat?gac?ggc 104

Gly?Trp?Arg?Asn?Val?Thr?Arg?Lcu?Leu?Val?Phe?Ala?Thr?Asp?Asp?Gly

5 10 15

ttc?cat?ttc?gcg?ggc?gac?ggg?aag?ctg?ggc?gcc?atc?ctg?acc?ccc?aac 152

Phe?His?Phe?Ala?Gly?Asp?Gly?Lys?Leu?Gly?Ala?Ile?Leu?Thr?Pro?Asn

20 25 30 35

gac?ggc?cgc?tgt?cac?ctg?gag?gac?aac?ttg?tac?aag?agg?agc?aac?gaa 200

Asp?Gly?Arg?Cys?His?Leu?Glu?Asp?Asn?Leu?Tyr?Lys?Arg?Ser?Asn?Glu

40 45 50

ttc?gtaagtcccc?accccaggca?cccaggcacc?gcctggcagg?acaccactga 253

Phe

cggaggagac?aagggtgggg?tctccacctg?acagagcctc?cttctgaccc?aaggagcaga 313

ttcctgagga?aacttctgga?agccccaagt 343

<210>2

<211>52

<212>PRT

<213>Homo?sapiens

<400>2

Glu?Glu?Ile?Gly?Trp?Arg?Asn?Val?Thr?Arg?Leu?Leu?Val?Phe?Ala?Thr

1 5 10 15

Asp?Asp?Gly?Phe?His?Phe?Ala?Gly?Asp?Gly?Lys?Leu?Gly?Ala?Ile?Leu

20 25 30

Thr?Pro?Asn?Asp?Gly?Arg?Cys?His?Leu?Glu?Asp?Asn?Leu?Tyr?Lys?Arg

35 40 45

Ser?Asn?Glu?Phe

50

<210>3

<211>20

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>3

ctgggaaact?gaggcaggta 20

<210>4

<211>21

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>4

acttggggct?tccagaagtt?t 21

Claims (7)

1. method that detects the single nucleotide polymorphism of No. seven exon of people ITGB2 gene is characterized in that it comprises step:

(a) determine the 126th Nucleotide in sequence shown in the SEQ ID NO:1 of No. seven exon of people ITGB2 gene;

(b) detect whether there is single nucleotide polymorphism in described position.

2. the method for claim 1 is characterized in that, described single nucleotide polymorphism is to have A at the 126th.

3. an isolating nucleic acid is characterized in that, it has the sequence shown in the SEQ ID NO:1, and the 126th is A.

4. allele specific nucleic acid primer, it is characterized in that, its length is 15-50bp, and hybridizes specifically and amplify shown in the SEQ ID NO:1 that contains No. seven exon of people ITGB2 gene the amplified production of the 126th single nucleotide polymorphism in the sequence.

5. an allele specific oligonucleotide probe is characterized in that, its length is 15-50bp, and hybridizes specifically and detect shown in the SEQ ID NO:1 that contains No. seven exon of people ITGB2 gene the 126th single nucleotide polymorphism in the sequence.

6. diagnostic kit is characterized in that it comprises:

(1) primer of No. seven exon of specific amplification people ITGB2 gene is right,

(2) detect No. seven exon of amplified production and normal ITGB2 gene and compare the reagent that whether exists variation required, the 126th single nucleotide polymorphism in the sequence shown in the described SEQ ID NO:1.

7. a diagnostic kit is characterized in that it comprises: described primer of claim 4 and/or the described oligonucleotide probe of claim 5.