CN103031319A

CN103031319A - CYP2C9 gene segment comprising 371G>A, coded protein segment and application thereof

Info

Publication number: CN103031319A
Application number: CN2011104301184A
Authority: CN
Inventors: 蔡剑平; 戴大鹏; 徐仁爱; 胡国新; 杨丽萍
Original assignee: 蔡剑平
Current assignee: Beijing Hospital
Priority date: 2011-12-19
Filing date: 2011-12-19
Publication date: 2013-04-10
Anticipated expiration: 2031-12-19
Also published as: CN103031319B

Abstract

The invention belongs to the field of biology, and relates to single base mutation. More particularly, the invention relates to a mutation site of a 371th site of SEQ ID NO.2 corresponding to a CYP2C9 gene, wherein the mutation site is mutated into A from wild type G. The invention also relates to a nucleic acid segment, a coded protein segment and application thereof. The invention also provides an allele-specific oligonucleotide, kit and detection method for detecting the mutation site.

Description

Comprise the 371G＞CYP2C9 gene fragment of A sudden change, coded protein fragments and application thereof

Technical field

The invention belongs to field of biology, relate to single base mutation.More specifically, the present invention relates to the CYP2C9 gene with respect to the 1001st or the 371st the mutational site of SEQ ID NO.2 of SEQ ID NO.1, comprise the nucleic acid fragment in this mutational site and the protein fragments of corresponding encoded thereof.The invention still further relates to reagent and the detection method of identifying described mutational site, and identify the application of this site in direction of medication usage.

Background technology

CYP2C9 is most important a member in the cytochrome P 450 enzymes extended familys CYP2C subfamily, accounts for 20% of people's hepatomicrosome CYP enzyme total amount.About 10～16% clinical commonly used drugs are arranged via the CYP2C9 oxidative metabolism, comprise mainly that wherein tolbutamide, S-warfarin, Phenytoin Sodium Salt, Glipizide, U26452, holder draw the medicines (referring to reference 1-5) such as thiophene miaow, losartan, irbesartan and many non-steroidal anti-inflammatory drugs (as: Ibuprofen BP/EP, lornoxicam, diclofenac and Naproxen Base).

The CYP2C9 gene has the height polymorphism.So far, named allelotrope has 35 kinds (http://www.cypalleles.ki.se) in the world, remove outside the wild-type (CYP2C9*1), have 34 kinds of mutation types and can cause that CYP2C9 Argine Monohydrochloride composition changes, and also has multiple newfound sudden change not yet to be named in addition.Study mutation type more and that clinical meaning is larger and mainly comprise following 10 kinds: CYP2C9*2, * 3, * 5, * 6, * 8, * 11, * 12, * 13, * 14, * 16, wherein ethnic group distribute the widest, most study, Chinese population research data relatively the abundantest mutant be CYP2C9*2 (430C＞T), CYP2C9*3 (1075A＞C) (referring to reference 6-17,20).

According to present clinical studies show, this polymorphism of CYP2C9 gene is to cause CYP2C9 enzymic activity very big different major cause between individuality, therefore carrying the very big difference that can cause curative effect of medication between the genotypic individuality of different CYP2C9, even producing serious poisonous side effect of medicine or treat insufficient.Therefore, research CYP2C9 gene pleiomorphism will provide important scientific basis (referring to reference 18,19,21,22) to clinical rational drug use to the impact of curative effect of medication.

Summary of the invention

The new single base mutation site that the purpose of this invention is to provide the CYP2C9 gene, comprise this mutational site nucleic acid fragment, its coding protein fragments and identify the application of this mutational site in medication guide.

First aspect of the present invention provides nucleic acid fragment, described nucleic acid fragment comprises the 1001st mutational site corresponding to SEQ ID NO.1, and be at least 10 continuous nucleotides in the nucleotide sequence shown in the SEQ ID NO.1, wherein the 1001st Nucleotide is A; Perhaps described nucleic acid fragment comprises the 371st mutational site corresponding to SEQ ID NO.2, and is at least 10 continuous nucleotides in the nucleotide sequence shown in the SEQ ID NO.2, and wherein the 371st Nucleotide is A; It perhaps is the complementary sequence of above-mentioned nucleic acid fragment.

Second aspect of the present invention provides and contains corresponding to the 1001st of SEQ ID NO.1 or corresponding to the allelotrope fragment in the 371st the mutational site of SEQ ID NO.2 or the allele specific oligonucleotide of all or part of hybridization of its complementary sequence, and wherein the Nucleotide in the 371st the mutational site of the 1001st of SEQ ID NO.1 the or SEQ ID NO.2 is A; Described allelotrope fragment is at least 10 continuous nucleotides or its complementary sequence in the nucleotide sequence shown in SEQ ID NO.1 or the SEQ ID NO.2.

The 3rd aspect of the present invention provide for detection of and/or the test kit of analysis list base mutation, described test kit comprises nucleic acid fragment of the present invention or allele specific oligonucleotide, or comprises the sequence fragment shown in SEQ ID NO.6 and/or SEQ ID NO.7 and/or the SEQ ID NO.17.

The 4th aspect of the present invention provides nucleic acid fragment of the present invention or the application of oligonucleotide in detecting the CYP2C9 transgenation, and wherein said nucleic acid fragment or oligonucleotide are as probe or primer.

The 5th aspect of the present invention provides a kind of medication guide, comprises the 1001st or the 371st the single base mutation of SEQ ID NO.2 corresponding to the SEQ ID NO.1 that detect CYP2C9 gene in the testing sample; According to the sudden change that detects, adjust the dosage by the medicine of CYP2C9 metabolism.

The 6th aspect of the present invention provides the method for analysis of nucleic acids, described method comprise analyze in the testing sample comprise corresponding in the nucleic acid of the sequence of SEQ ID NO.1 corresponding to the 1001st Nucleotide or analyze in the testing sample comprise corresponding in the nucleic acid of the sequence of SEQ ID NO.2 corresponding to the 371st Nucleotide.

The 7th aspect of the present invention provides CYP2C9 albumen or its fragment or varient, and described protein sequence is the sequence shown in the SEQ ID NO.3; Described fragment or varient comprise the 124th glutamine corresponding to SEQ ID NO.3, and are at least 10 continuous amino acids of the aminoacid sequence shown in the SEQ ID NO.3.

The invention provides the CYP2C9 gene and the encoding sequence that comprise new single base mutation.This gene corresponding to the 371st Nucleotide of SEQ ID NO.2 by G sport A (371G＞A), thus the amino acid that causes its coding sports glutamine by arginine, namely corresponding to the 124th the glutamine of SEQ ID NO.3.The CYP2C9 albumen of this sudden change (called after R124Q) to medicine almost without metabolic activity.This single base mutation has directive significance to the medication of the individuality that carries this mutational site.

Description of drawings

Fig. 1 is the 1001st the nucleotide sequencing collection of illustrative plates of SEQ ID NO.1 of the present invention.

Embodiment

By following embodiment explanation the present invention, but content of the present invention is not limited to this.

As without other explanation, " nucleic acid fragment " of the present invention is comprised of Nucleotide or its analogue, can be the fragment of DNA, RNA or its analogue; Can be strand or two strands; It can be natural (as genomic) or synthetic.

Among the present invention, " sudden change " refers at the gene that detects, namely has the nucleotide site different from wild-type CYP2C9 gene order in the CYP2C9 gene." mutational site " refers to the position that base is undergone mutation.In the present invention, described mutational site is corresponding to the 371st in the sequence shown in the 1001st of sequence shown in the SEQ ID NO.1 or the SEQ ID NO.2.

In the present invention, " allele-specific " refer to specifically and allelotrope hybridization, as hybridizing under rigorous condition, so that identify that the 371st Nucleotide corresponding to sequence shown in the 1001st of sequence shown in the SEQ ID NO.1 or the SEQ ID NO.2 is A.

Content of the present invention relates to the nonsynonymous mutation of CYP2C9 gene.Because this mutational site is arranged in the encoding sequence of gene, therefore, those skilled in the art as can be known, described mutational site both can show in genomic dna, also can performance in encoding sequence (being CDS, corresponding to the mRNA sequence).Those skilled in the art can detect this mutational site on genomic dna or mRNA level according to detected sample.Among the application, SEQ ID NO.1 be centered by the application's mutational site, the genomic dna sequence of each 1kb of front and back, namely the 1001st of SEQ ID NO.1 the is the mutational site that the present invention relates to.SEQ ID NO.2 is the encoding sequence with the CYP2C9 gene in described mutational site, and wherein the 371st is the mutational site that the present invention relates to.Those skilled in the art in this article, use corresponding to the 371st site of SEQ ID NO.2 with corresponding to the 1001st the site synonym of SEQ ID NO.1 as can be known mutually.

Among the present invention, abbreviation mode well known in the art is adopted in Nucleotide and amino acid whose abbreviation, represents VITAMIN B4 such as A in the Nucleotide, and G represents guanine, and C represents cytosine(Cyt), and T represents thymus pyrimidine.In the amino acid, A represents L-Ala, and R represents arginine, and N represents l-asparagine, D represents aspartic acid, and C represents halfcystine, and Q represents glutamine, and E represents L-glutamic acid, G represents glycine, and H represents Histidine, and I represents Isoleucine, and L represents leucine, K represents Methionin, and M represents methionine(Met), and F represents phenylalanine, P represents proline(Pro), and S represents Serine, and T represents Threonine, W represents tryptophane, and Y represents tyrosine, and V represents α-amino-isovaleric acid.

Content of the present invention is based on the new single base mutation site of CYP2C9 gene.Described mutational site is the coding region that is positioned at the CYP2C9 gene, and corresponding to the 371st of SEQ ID NO.2, this site sports A (371G＞A) by the G of wild-type; In addition, sport glutamine (R124Q) by the 124th of the albumen of the CYP2C9 genes encoding of this sudden change by arginine.

Aspect first, the invention provides nucleic acid fragment, described nucleic acid fragment comprises the 1001st mutational site corresponding to SEQ IDNO.1, and is at least 10 continuous nucleotides in the nucleotide sequence shown in the SEQ ID NO.1, and wherein the 1001st Nucleotide is A; Perhaps described nucleic acid fragment comprises the 371st mutational site corresponding to SEQ ID NO.2, and is at least 10 continuous nucleotides in the nucleotide sequence shown in the SEQ ID NO.2, and wherein the 371st Nucleotide is A; It perhaps is the complementary sequence of above-mentioned nucleic acid fragment.

In one embodiment, the length of described nucleic acid fragment can be such as 10-100,100-200,200-500, a 500-1000 Nucleotide.Preferably, the length of described nucleic acid fragment is 10-20,20-30,30-40,40-50,50-60, a 60-100 or 100-300 Nucleotide.

Described mutational site can be positioned at any position of described nucleic acid fragment.

In another embodiment, described nucleic acid fragment is the sequence shown in the SEQ ID NO.1.

In another embodiment, described nucleic acid fragment is the sequence shown in the SEQ ID NO.2.

In other embodiments, nucleic acid fragment shown in can be the sequence shown in the SEQ ID NO.24-27.

Second aspect of the present invention provides and contains corresponding to the 1001st of SEQ ID NO.1 or corresponding to the allelotrope fragment in the 371st the mutational site of SEQ ID NO.2 or the allele specific oligonucleotide of all or part of hybridization of its complementary sequence, and wherein the Nucleotide in the 371st the mutational site of the 1001st of SEQ ID NO.1 the or SEQ ID NO.2 is A; Described allelotrope fragment is at least 10 continuous nucleotides or its complementary sequence in the nucleotide sequence shown in SEQID NO.1 or the SEQID NO.2.

In one embodiment, described oligonucleotide is as probe.Described probe can be under rigorous condition and the target sequence specific hybrid that comprises the mutational site.It is known to those skilled in the art that described probe does not need and the target sequence complete complementary, if can with the target sequence specific hybridization.In preferred embodiments, described hybridization conditions can satisfy make probe only with the target sequence specific hybrid.The length of described probe can be 5-100 Nucleotide, such as 5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,40,50,60,70,80,90 or 100 Nucleotide.Described mutational site can appear at any position of probe.In preferred embodiment, be center or the about center that the mutational site appears at probe sequence.

In another embodiment, the primer that described oligonucleotide synthesizes with the DNA that coaches, as known in the art sequencing primer or synthetic primer etc.Described primer does not need and the template complete complementary, but should hybridize to instruct DNA synthetic with template is complementary.The length of described primer can be 15-40 length of nucleotides, is preferably 18,19,20,21,22,23,24,25,26,27,28,29 or 30 Nucleotide.Described mutational site can appear at any position of described primer; Preferably, described mutational site appears at 3 ' end of described primer.

Some preferred embodiment in, shown in oligonucleotide be sequence shown in SEQ ID NO.28-33.

Based on this, the 3rd aspect of the present invention provide for detection of and/or the test kit of analysis list base mutation, described test kit comprises nucleic acid fragment of the present invention or allele specific oligonucleotide, or comprises the sequence fragment shown in SEQ ID NO.6 and/or SEQ ID NO.7 and/or the SEQ ID NO.17.

The 4th aspect of the present invention provides nucleic acid fragment of the present invention or oligonucleotide for detection of the application of CYP2C9 transgenation, and wherein said nucleic acid fragment or oligonucleotide are as probe or primer.

The 5th aspect of the present invention provides medication guide, comprises the 1001st or the 371st the single base mutation of SEQ ID NO.2 corresponding to the SEQ ID NO.1 that detect CYP2C9 gene in the testing sample.When the CYP2C9 gene that detects is A in the 371st site corresponding to the 1001st of SEQ ID NO.1 or SEQ ID NO.2, adjust accordingly the dosage through the medicine of CYP2C9 metabolism.In specific embodiment, when the CYP2C9 gene was A in the 371st the site of SEQ ID NO.2, the CYP2C9 protease activity of this genes encoding descended, so need to reduce the dosage through the medicine of CYP2C9 metabolism.

The medicine through the CYP2C9 metabolism described in the present invention comprises: cancer therapy drug, such as endoxan, ifosfamide or taxol; Anticoagulant is such as warfarin, Acenocoumarol, anticonvulsive drug or mephenytoin; Antidiabetic drug is such as tolbutamide, nateglinide, pioglitazone or rosiglitazone; Antiepileptic drug is such as Phenytoin Sodium Salt or zonisamide; Antimalarial drug/antiparasitic is such as amodiaquine, Tirian or quinine; Antipsychotic drug is such as amitriptyline, citalopram, imipramine, Perospirone, Sertraline, thioridazine or Venlafaxine; Depressor is such as losartan, irbesartan or valsartan; Non-steroidal anti-inflammatory drug is such as diclofenac, pyramidon, quinizine, celecoxib, flurbiprofen, Ibuprofen BP/EP, indomethacin, lornoxicam, mefenamic acid, Naproxen Base, piroxicam or tenoxicam; Anodyne is such as, Loperamide, methadone or morphine; Proton pump inhibitor draws azoles such as lansoprazole or difficult to understand closing; Tranquilizer is such as, clobazam, Mephogarbital or Zopiclone.

In one embodiment, described method can be sequencing, comprise and separate and measure nucleotide sequence from genomic dna or RNA, analyze wherein comprise corresponding in the nucleic acid of the sequence of SEQ ID NO.1 corresponding to the 1001st Nucleotide or whether comprise corresponding to the Nucleotide corresponding to the 371st in the nucleic acid of the sequence of SEQ ID NO.2 be A.Sequencing can be any available sequence measurement known in the art.Sequencing primer can design according to those skilled in the art's general knowledge, as at the upstream and downstream appropriate position in site to be detected design primer, comprises the fragment in this site to be measured with expansion, thereby judges the Nucleotide in this site.Also can adopt oligonucleotide of the present invention as primer sequence.

In another embodiment, described method is to utilize the method for probe hybridization, comprise in the identification and detection sample specifically corresponding in the nucleic acid of the sequence of SEQ ID NO.1 corresponding to the 1001st Nucleotide or whether comprise corresponding to the Nucleotide corresponding to the 371st in the nucleic acid of the sequence of SEQ ID NO.2 be A; The probe that adopts in the described method is oligonucleotide of the present invention.For example, from testing sample, isolate nucleic acid, under the condition of the specific target sequence hybridization that in allowing probe and nucleic acid, may exist probe is contacted with nucleic acid; The hybridization that can be detected can realize by using the probe of being crossed by detectable reagent mark; For example, form the enzyme that can detect product with radio isotope, fluorescence dye or energy catalysis and come label probe.Label probe, all be well-known to those skilled in the art with the method that whether has target sequence in the label probe test sample.

In a kind of concrete embodiment, provide with the method for Taqman probe SNP detection method detection corresponding to the 1001st the Nucleotide of SEQ ID NO.1, comprising:

1) the design primer is used for specific amplification and comprises the 1001st PCR product corresponding to SEQ ID NO.1, design simultaneously two Taqman-MGB probes, respectively for the 1001st G and A (sequence shown in SEQ ID NO.32) allelotrope corresponding to SEQ ID NO.1.

The design of primers principle is:

(1) choose should be at the conservative section of gene for sequence;

(2) avoid primer self or and primer between form continuously pairing more than 4 or 4, avoid primer self to form the pili annulati card structure;

(3) primer length is at 18 to 24 Nucleotide;

(4) the Tm value is at 55-65 ℃, and GC content is at 40%-60%;

(5) the Tm value between the primer differs and avoids above 2 ℃;

(6) 3 ' of primer end avoids using base A, and 3 ' end of primer avoids occurring base consecutive identical more than 3 or 3;

(7) pcr amplified fragment length is at 50bp-150bp;

(8) terminal last 5 Nucleotide of primer can not have G and the C above 2.

Taqman MGB probe design principle is:

(1) 5 ' of probe end avoids occurring G;

(2) the Tm value should be 65-67 ℃;

(3) shorten Taqman MGB probe, but probe length is no less than 13bp as far as possible;

(4) avoid the base, especially the G base that duplicate as far as possible, avoid the G that occurs more than 4 or 4 to repeat;

(5) mutational site with probe is placed on middle 1/3 place as far as possible.

Fluorophor can adopt FAM, VIC etc. to come two allelotrope of mark.

2) utilize above-mentioned primer and probe, sample to be tested is carried out real-time quantitative PCR.

The PCR condition: 95 ℃ of denaturations enter 30 amplification cycles after 10 minutes: 92 ℃ of sex change 12 seconds, 60 ℃ of annealing and extend 1 minute (this stage is detected fluorescent signal).

3) data analysis.

Analyze experimental result, judge according to the power of two kinds of fluorescence of sample whether sample to be tested CYP2C9 gene exists 371G＞A sudden change.

Among the present invention, described sample can be any sample that comprises nucleic acid, such as blood; Preferred described sample comes from the people.Described nucleic acid can be DNA or coding RNA, is preferably genomic dna.The method of analysis of nucleic acids of the present invention can be take DNA or RNA as target compound.Those skilled in the art as can be known, when take DNA as the detection target compound, analyze in the testing sample comprise corresponding in the nucleic acid of the sequence of SEQ ID NO.1 corresponding to the 1001st Nucleotide, employed probe or primer are according to the sequences Design of SEQ ID NO.1; When take RNA when detecting target compound, analyze in the testing sample comprise corresponding in the nucleic acid of the sequence of SEQ ID NO.2 corresponding to the 371st Nucleotide, employed probe or primer are according to the sequences Design of SEQ ID NO.2.

The 7th aspect of the present invention provides CYP2C9 albumen or its fragment or varient, and described protein sequence is the sequence shown in the SEQ ID NO.3; Described fragment or varient comprise the 124th glutamine corresponding to SEQ ID NO.3, and are at least 10 continuous amino acids of the aminoacid sequence shown in the SEQ ID NO.3, such as 10-20,20-50 or 50-100 amino acid.

The below will further specify the present invention by specific embodiment, but the following specific embodiment purpose of property presented for purpose of illustration only.

Embodiment

Embodiment 1: the evaluation in the mutational site that people CYP2C9 gene is new

In the present embodiment, gather 2127 parts of blood samples, extract the genomic dna in the blood, the design sequencing primer carries out sequence amplification, order-checking to 9 exons of CYP2C9 gene, the mutational site of screening CYP2C9 gene

1) extract DNA:

Take 5ml vein EDTA anticoagulated blood sample from every measured; Then according to common salting-out process and/or adopt special DNA extraction test kit (available from the DNA extraction test kit of U.S. Omega company) to extract the genomic dna of blood sample to be measured.

2) pcr amplification:

Design of amplification primers increases to 9 exon sequences of the CYP2C9 gene in the genome DNA sample that obtains.Described amplimer sees Table 1 to sequence.

Adopt 50 μ l PCR reaction systems, comprising: 1 * PCR damping fluid, 1.5mM MgCl ₂, the genomic dna of 100～150ng, upstream and downstream primer be the LATaq archaeal dna polymerase 1.5U that 0.2 μ M, dNTP are 0.4mM, TaKaRa company.The pcr amplification loop parameter is as follows: 94 ℃ of denaturations 5 minutes, and 94 ℃ of sex change 30 seconds were annealed 30 seconds, and 72 ℃ were extended 2 minutes and 30 seconds, and extended 5 minutes after 30 circulations again.Annealing temperature is relevant with primer length, and actual temp sees Table 1.

Use the GeneAmp PCR System 9700 amplification instruments amplification of American AB I company.

Table 1: sequencing primer is to reaching annealing temperature

3) purifying amplified production:

Get 50 μ l pcr amplification products and carry out the agarose gel electrophoresis separation, blade cuts the purpose band.Reclaiming test kit (Omega company) according to the E.Z.N.A. gel requires the DNA that carries out the purpose band to reclaim purifying.

4) order-checking:

Product after to reclaim uses sequencing primer according to CEQ as template ^TMDTCS-Quick Start Kit sequencing kit (U.S. Beckman company) the PCR reaction that requires to check order, reaction finish and purifying after, separate sequence with the interpretation amplified production with the CEQ8000 type gene sequencer of U.S. Beckman company.Sequencing primer sees Table 2.

Table 2: sequencing primer

The zone	Sequencing primer (5 '-3 ')
		Exons 1	TACCTCTAGGGATACAC(SEQ ID NO.16)
Wai Xianzi2 ﹠3	CTAACAACCAGGACTCATAAT(SEQ ID NO.17)
		Exon 4	TTGCTGTTAAGGGAATTTGTAGGTAAGATA(SEQ ID NO.18)
Exon 5	TAGTGGTCTATTTTGTTATTCATTCAT(SEQ ID NO.19)
		Exon 6	TTCCAGTTTCTATGTTG(SEQ ID NO.20)
Exon 7	ACCCGGTGATGGTAGAGGTT(SEQ ID NO.21)
		Exon 8	ACGGGATTTCCTCATCTG(SEQ ID NO.22)
Exon 9	CGATACACTGAACAGTTATTGC(SEQ ID NO.23)

5) data analysis:

The sequence and the wild-type CYP*1 sequence (GenBank number of registration NM_000771.3) that record are compared.

By compare of analysis, find that in a duplicate samples the 371st Nucleotide of CYP2C9 gene coding region becomes A (as shown in Figure 1) by G, this sudden change is positioned at the 3rd exon of CYP2C9 gene.Infer accordingly in the protein of this CYP2C9 genes encoding that the 124th amino acids sports glutamine (Q) by arginine (R).These 371 new sudden changes (371G＞A) by the international P450 affirmation called after neomorph CYP2C9*43 of NK.

Exemplarily provided the method for identifying the new mutant site in the present embodiment.Those skilled in the art can clearly learn according to foregoing and detect specifically the method that comprises in the testing sample corresponding to the 1001st Nucleotide of SEQ ID NO.1: the nucleic acid in the sample separation, carry out amplified reaction under the corresponding experiment condition in the present embodiment, primer uses primer to SEQ ID NO.6 and 7; With sequencing primer SEQ ID NO.17 the product of amplification is checked order; Sequencing result and wild-type result are compared, analyzed the Nucleotide corresponding to the 1001st site of SEQ ID NO.1.

Embodiment 2: the enzymes metabolism activation analysis

According to existing result of study, wild-type is all higher to the metabolic activity of various medicines, and the metabolic activity of * 2 types has obvious decline than the metabolic activity of wild-type, and the metabolic activity of * 3 types is than * 2 types lower (referring to reference 18,19,21,22).Therefore, existing a kind of like this common recognition in the art: the expressed enzyme of same genotype can represent metabolic activity to other substrate medicine to the metabolic activity of specific substrate.Thereby, according to the expressed enzyme of a certain genotype to specific substrate metabolic activity data can analogize the expressed enzyme of this genotype to the metabolic activity of other substrate medicine (as, the metabolic activity of the enzyme that the metabolic activity of enzyme that can this genotype is expressed and wild-type are expressed compares).

In the present embodiment, according to above-mentioned mutational site, take wild-type CYP2C9 (* 1) gene as template, rite-directed mutagenesis the 371st Nucleotide (becoming A by G) of CYP2C9 gene coding region, construction of expression vector is expressed mutant CYP2C9 albumen (called after R124Q), utilize the active detection kit of CYP2C9 (containing the CYP2C9 specific substrate) of Promega company to detect the enzymic activity of this mutant CYP2C9, compare with wild-type CYP2C9 (* 1) to judge, whether its enzymatic metabolic activity changes.

1) expression of goal gene

Take wild-type CYP2C9 (* 1) plasmid vector (by professor's Zhou Shufeng present of American South University of Florida) as template, the 371st Nucleotide of rite-directed mutagenesis CYP2C9 gene coding region, namely obtain comprising the ORF region sequence of the Nucleotide shown in the SEQ ID NO.2, with this goal gene and reference gene Gluc (a kind of secretor type luciferase, its translation product can be secreted in the substratum, and detects fluorescent signal by the particular agent box; Its skeleton carrier is pIRES pGluc-Basic, available from NEB company, article No. N8082S) is connected to respectively double gene expression vector pIRES (available from Clontech company, article No. 631605) in A, the B site, it is lower to make two genes be positioned at same CMV promotor control, finally obtains double gene expression vector pIRES-Gluc-2C9.With 8*10 ⁴The COS-7 cell (this cell strain has been widely used in the analysis of CYP2C9 external activity, sees reference 18,21 for details) in individual cercopithecus aethiops renal epithelial cell source is laid on 24 orifice plates; After the incubated overnight, utilize liposome lip2000 transfection 500ng plasmid vector pIRES-Gluc-2C9, to express target protein CYP2C9 (R124Q) and confidential reference items Protein G luc.

2) enzymic activity detects

Continue to cultivate after 48 hours, add the fresh DMEM substratum of 200 μ l (containing 10%FBS), and with reference to the CYP2C9 of Promega company detection kit (article No.: operation instruction V8791), the substrate Luciferin-H that adds 4 μ l CYP2C9 continue to cultivate behind the mixing and gets 50 μ l substratum after 8 hours and add isopyknic Promega test kit (article No.: V8791) detect damping fluid.Contain Photinus pyralis LUC in this detection damping fluid; Substrate Luciferin-H can produce fluorescence after the product D-luciferin after the CYP2C9 metabolism and Photinus pyralis LUC reaction.Utilize the GLOMAX 20/20Luminometer (GLOMAX 20/20 luminous detection instrument) of Promega company to detect fluorescent signal.Get again 7 μ l substratum with reference to Gluc detect kit (NEB company, article No.: E3300) operation instruction, add isopyknic detection damping fluid, utilize GLOMAX 20/20Luminometer to detect the Gluc fluorescent signal.Fluorescent signal that will be directly related with CYP2C9/confidential reference items Gluc fluorescent signal value with wild-type (* 1 type) CYP2C9 Data Comparison, can be analyzed the variation of the metabolic activity of mutant to be measured as final experimental data.Every kind of experimental subjects is carried out three parallel laboratory tests, and experimental result sees Table 3.

Introduction about Luciferin-H:

The typical metabolic activity of CYP2C9 is to carry out hydroxylation (referring to reference 23,24, associated viscera mode by reference is bonded to this paper) to aromatic ring structure.The detection substrate Luciferin-H that uses in the present embodiment contains aromatic ring structure, CYP2C9 can specific R1 position hydroxylation to this substrate, and produce new metabolism substrate D-Luciferin ([4S]-4,5-dihydro-2-[6 '-hydroxy-2 '-benzothiazolyl]-4-thiazolecarboxylic acid), the latter can with detection reagent in Photinus pyralis LUC reaction and luminous, the power of signal is directly related with the amount of product D-luciferin, and namely the latter has been reflected the enzymatic activity of CYP2C9 albumen to be checked.There are some researches show, utilize this detection method can reflect effectively that CYP2C9 to the metabolic characteristics of Common drugs, mainly comprises CYP2C9 probe medicament warfarin, tolbutamide, diclofenac, Phenytoin Sodium Salt, Ibuprofen BP/EP; Also comprise simultaneously (referring to reference 22,24,25) such as part Common drugs sulfaphenazole, troglitazone, Azamulin, piroxicam, sulfaphenazole, miconazole, fluvoxamines.Therefore the data of this experimental program can generally be applicable to other through the medicine of CYP2C9 metabolism, comprise the medicine of listing before in the present specification.

Table 3: the experimental result of enzymes metabolism activity

As can be seen from the results, 3 negative controls have all presented expection trend: empty carrier can't detect the signal of 2C9, with respect to wild-type * 1 type, known mutations type * 2 type activity have obvious decline (nearly nearly 60% minimizing), * 3 types descend maximum (approximately descending 93%), this numerical value and trend with have been reported consistent (referring to reference 18,19,21,22).

Utilize this vitro detection system to find out: R124Q is almost without metabolic activity, i.e. this sudden change can cause that the metabolic activity of expressed enzyme obviously reduces.Therefore, in practice, need to consider suitably to regulate at dosage carrying this genotypic individuality, such as the usage quantity that reduces medicine and the generation of avoiding adverse drug reaction.This medicine adjustment by gene targeting is even more important for the narrow medicine (such as warfarin, Phenytoin Sodium Salt etc.) for the treatment of window.

Sequence:

SEQ ID NO.1: genomic dna sequence

AGACACTGAAAATGAATTTGTCATTCTCTGAGCTCAGTTTTTTTTTTTTTTTTTTTTTTTTTTGAGACAGAGTCTTACTCTGTAGCTCAGGCTGGAGTGCAGTGGTACAATCTTGGCTCACTGCAACCTCCATCTCCCAGGTCCCCATTCAAGAAATTCTCCTGCCTCAGTCCCCCAAGTAGCTAGCATTACAGGCATGCACCACCATGCTCAGCTAATTTTTGTATTTTTAGTAGAGACGTGGTATCACCTTGTTGGCCAGGCTGGTCTTGAACTCCTGACCTTGTGATCCACCTGCCTTGGCCTCCCAAAGTGTTGGGATTACAGGCAGGAGCCACCACACCTGGCCGTTTGTTTAAAATAGAGTAAATAGACCTGCTGAATATGTTGATGTGAGTATTAATTGTAATCTGCATAGCAATTGTCTGACCATTGCCTTGAACATCACAGGCCATCTGAGTGGCAAGTATAATCATCATCATGTTTCTATTTAAAATTCAGAAATATTTGAAGCCTGTGTGGCTGAATAAAGCATACAAATACAATGAAAATATCATGCTAAATCAGGCTTAGCAAATGGACAAAATAGTAACTTCGTTTGCTGTTATCTCTGTCTACTTTCCTAGCTCTCAAAGGTCTATGGCCCTGTGTTCACTCTGTATTTTGGCCTGAAACCCATAGTGGTGCTGCATGGATATGAAGCAGTGAAGGAAGCCCTGATTGATCTTGGAGAGGAGTTTTCTGGAAGAGGCATTTTCCCACTGGCTGAAAGAGCTAACAGAGGATTTGGTAGGTGTGCATGTGCCTGTTTCAGCATCTGTCTTGGGGATGGGGAGGATGGAAAACAGAGACTTACAGAGCTCCTCGGGCAGAGCTTGGCCCATCCACATGGCTGCCCAGTGTCAGCTTCCTCTTTCTTGCCTGGGATCTCCCTCCTAGTTTCGTTTCTCTTCCTGTTAGGAATTGTTTTCAGCAATGGAAAGAAATGGAAGGAGATCCAGCGTTTCTCCCTCATGACGCTGCGGAATTTTGGGATGGGGAAGAGGAGCATTGAGGACCGTGTTCAAGAGGAAGCCCGCTGCCTTGTGGAGGAGTTGAGAAAAACCAAGGGTGGGTGACCCTACTCCATATCACTGACCTTACTGGACTACTATCTTCTCTACTGACATTCTTGGAAACATTTCAGGGGTGGCCATATCTTTCATTATGAGTCCTGGTTGTTAGCTCATGTGAAGCGGGGGTTTGAAGCTGAGAGCCAAGGGAATTTGCACATATTTGTGCTGTGTGTGTACAGGCATGATTGTGCGTACAGTGTGGGTATAAAAGGTTCATTTAATCCCATGTTCTCCTGAACTTTGCTTTTTTGCTTTCAAATAAGAAATGATGAATATAGATTTTGAGTTCATTTTTTGAAAGAGTTAAAGAGCAGTGTTTTTCCCATTACCTATTCCAGAACATGTCACCAGAGAATACTTGACAAGTCAACATGGTGGGAATGGCCCTATCATACCCATATGGAGCATGAACCAAATGGCATGTGCTTTTATTTAATTGGACTGTGTTTGTATGGTCAGCCTCACTGACTTCTCTGGGGTTTCTTTTAGGCCCGTGCTTGCCATTCTGGCCAGTAATGACATTCTACAGTTTTTATTGCTTAGGCATATCTTAGTGCAGTTCTCATCAATTATTATTTCTCTGTAAACACAGCATTATTTTAAAAATAGTATTAATTTATTCTTGTTACTGTATTGATTTATATATTTTCAGTAAATACATCCTGTAGCATAATTCTGTGAAATACCCAAATGTCAATTTATAAAATGATTTATTTAACAAGATTTTACTTATTAGTAATAACTCTGTAATCTGCATTCCCTATGTATGATTTGGCTCTGTTTCAGTTTTGCTTATCTCTTTCCAACCATATTTATGAAATTTTGGCTTAGAAATTTATGTTAATTATTTTTTTTCCATGGCCAACTCTACTCATCTATGAAGTT

SEQ ID NO.2 encoding sequence

ATGGATTCTCTTGTGGTCCTTGTGCTCTGTCTCTCATGTTTGCTTCTCCTTTCACTCTGGAGACAGAGCTCTGGGAGAGGAAAACTCCCTCCTGGCCCCACTCCTCTCCCAGTGATTGGAAATATCCTACAGATAGGTATTAAGGACATCAGCAAATCCTTAACCAATCTCTCAAAGGTCTATGGCCCTGTGTTCACTCTGTATTTTGGCCTGAAACCCATAGTGGTGCTGCATGGATATGAAGCAGTGAAGGAAGCCCTGATTGATCTTGGAGAGGAGTTTTCTGGAAGAGGCATTTTCCCACTGGCTGAAAGAGCTAACAGAGGATTTGGAATTGTTTTCAGCAATGGAAAGAAATGGAAGGAGATCCAGCGTTTCTCCCTCATGACGCTGCGGAATTTTGGGATGGGGAAGAGGAGCATTGAGGACCGTGTTCAAGAGGAAGCCCGCTGCCTTGTGGAGGAGTTGAGAAAAACCAAGGCCTCACCCTGTGATCCCACTTTCATCCTGGGCTGTGCTCCCTGCAATGTGATCTGCTCCATTATTTTCCATAAACGTTTTGATTATAAAGATCAGCAATTTCTTAACTTAATGGAAAAGTTGAATGAAAACATCAAGATTTTGAGCAGCCCCTGGATCCAGATCTGCAATAATTTTTCTCCTATCATTGATTACTTCCCGGGAACTCACAACAAATTACTTAAAAACGTTGCTTTTATGAAAAGTTATATTTTGGAAAAAGTAAAAGAACACCAAGAATCAATGGACATGAACAACCCTCAGGACTTTATTGATTGCTTCCTGATGAAAATGGAGAAGGAAAAGCACAACCAACCATCTGAATTTACTATTGAAAGCTTGGAAAACACTGCAGTTGACTTGTTTGGAGCTGGGACAGAGACGACAAGCACAACCCTGAGATATGCTCTCCTTCTCCTGCTGAAGCACCCAGAGGTCACAGCTAAAGTCCAGGAAGAGATTGAACGTGTGATTGGCAGAAACCGGAGCCCCTGCATGCAAGACAGGAGCCACATGCCCTACACAGATGCTGTGGTGCACGAGGTCCAGAGATACATTGACCTTCTCCCCACCAGCCTGCCCCATGCAGTGACCTGTGACATTAAATTCAGAAACTATCTCATTCCCAAGGGCACAACCATATTAATTTCCCTGACTTCTGTGCTACATGACAACAAAGAATTTCCCAACCCAGAGATGTTTGACCCTCATCACTTTCTGGATGAAGGTGGCAATTTTAAGAAAAGTAAATACTTCATGCCTTTCTCAGCAGGAAAACGGATTTGTGTGGGAGAAGCCCTGGCCGGCATGGAGCTGTTTTTATTCCTGACCTCCATTTTACAGAACTTTAACCTGAAATCTCTGGTTGACCCAAAGAACCTTGACACCACTCCAGTTGTCAATGGATTTGCCTCTGTGCCGCCCTTCTACCAGCTGTGCTTCATTCCTGTCT

SEQ ID NO.3 protein sequence

MDSLVVLVLCLSCLLLLSLWRQSSGRGKLPPGPTPLPVIGNILQIGIKDISKSLTNLSKVYGPVFTLYFGLKPIVVLHGYEAVKEALIDLGEEFSGRGIFPLAERANRGFGIVFSNGKKWKEIQRFSLMTLRNFGMGKRSIEDRVQEEARCLVEELRKTKASPCDPTFILGCAPCNVICSIIFHKRFDYKDQQFLNLMEKLNENIKILSSPWIQICNNFSPIIDYFPGTHNKLLKNVAFMKSYILEKVKEHQESMDMNNPQDFIDCFLMKMEKEKHNQPSEFTIESLENTAVDLFGAGTETTSTTLRYALLLLLKHPEVTAKVQEEIERVIGRNRSPCMQDRSHMPYTDAVVHEVQRYIDLLPTSLPHAVTCDIKFRNYLIPKGTTILISLTSVLHDNKEFPNPEMFDPHHFLDEGGNFKKSKYFMPFSAGKRICVGEALAGMELFLFLTSILQNFNLKSLVDPKNLDTTPVVNGFASVPPFYQLCFIPV

SEQ ID NO.24 nucleic acid fragment

AATGGAAGGAGATCCAGCGTTTCTCCCTCAT

SEQ ID NO.25 nucleic acid fragment

AAAGAAATGGAAGGAGATCCAGCGTTTCTCCCTCATGACGC

SEQ ID NO.26 nucleic acid fragment

AATGGAAAGAAATGGAAGGAGATCCAGCGTTTCTCCCTCATGACGCTGCGG

SEQ ID NO.27 nucleic acid fragment

TCAGCAATGGAAAGAAATGGAAGGAGATCCAGCGTTTCTCCCTCATGACGCTGCGGAATTT

SEQ ID NO.28 oligonucleotide sequence

GAGGGAGAATCGCTG

SEQ ID NO.29 oligonucleotide sequence

GTCATGAGGGAGAATCGCTG

SEQ ID NO.30 oligonucleotide sequence

GCAGCGTCATGAGGGAGAATCGCTG

SEQ ID NO.31 oligonucleotide sequence

ATTCCGCAGCGTCATGAGGGAGAATCGCTG

SEQ ID NO.32 oligonucleotide sequence

CGCTGGATCTCCTTCC

SEQ ID NO.33 oligonucleotide sequence

GAGAATCGCTGGATCTCCT

Reference:

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Claims

1. nucleic acid fragment, described nucleic acid fragment comprises the 1001st mutational site corresponding to SEQ ID NO.1, and is at least 10 continuous nucleotides in the nucleotide sequence shown in the SEQ ID NO.1, and wherein the 1001st Nucleotide is A; Perhaps described nucleic acid fragment comprises the 371st mutational site corresponding to SEQ ID NO.2, and is at least 10 continuous nucleotides in the nucleotide sequence shown in the SEQ ID NO.2, and wherein the 371st Nucleotide is A; It perhaps is the complementary sequence of described nucleic acid fragment.

2. nucleic acid fragment according to claim 1 is characterized in that, the length of described nucleic acid fragment is 10-100,100-200, a 200-500 or 500-1000 Nucleotide; Preferably, the length of described nucleic acid fragment is 10-20,20-30,30-40,40-50,50-60, a 60-100 or 100-300 Nucleotide; Further preferably, described nucleic acid fragment is the sequence shown in SEQ ID NO.1,2, the 24-27.

3. allele specific oligonucleotide, described oligonucleotide with contain corresponding to the 1001st of SEQ ID NO.1 or corresponding to allelotrope fragment or all or part of hybridization of its complementary sequence in the 371st the mutational site of SEQ ID NO.2, wherein the Nucleotide in the 371st the mutational site of the 1001st of SEQ ID NO.1 the or SEQ ID NO.2 is A; Described allelotrope fragment is at least 10 continuous nucleotides or its complementary sequence in the nucleotide sequence shown in SEQID NO.1 or the SEQ ID NO.2.

4. allele specific oligonucleotide according to claim 3 is characterized in that, described oligonucleotide is probe or primer; Preferably, when described oligonucleotide was probe, the length of described oligonucleotide was 5-100 Nucleotide; When described oligonucleotide was primer, the length of described oligonucleotide was 15-40 Nucleotide; Preferably, when described oligonucleotide was probe, described mutational site was positioned at center or about center of probe sequence; When described oligonucleotide was primer, described mutational site was positioned at 3 ' end of primer.

5. the allele specific oligonucleotide shown in according to claim 4 is characterized in that described oligonucleotide is the sequence shown in SEQID NO.28-33.

For detection of and/or the test kit of analysis list base mutation, comprise claim 1 or 2 described nucleic acid fragments and/or each described allele specific oligonucleotide of claim 3-5; Perhaps comprise the sequence fragment shown in SEQ ID NO.6 and/or SEQ ID NO.7 and/or the SEQID NO.17.

7. claim 1 or 2 described nucleic acid fragments and/or each described allele specific oligonucleotide of claim 3-5 application in detecting the CYP2C9 transgenation, wherein said nucleic acid fragment or oligonucleotide are as probe or primer.

8. a medication guide comprises the 1001st or the 371st the single base mutation of SEQ ID NO.2 corresponding to the SEQ ID NO.1 that detect CYP2C9 gene in the testing sample; According to the sudden change that detects, adjust the dosage through the medicine of CYP2C9 metabolism; Preferably, described medicine is endoxan, ifosfamide, taxol, warfarin, Acenocoumarol, mephenytoin, tolbutamide, nateglinide, pioglitazone, rosiglitazone, Phenytoin Sodium Salt, zonisamide, amodiaquine, Tirian, quinine, amitriptyline, citalopram, imipramine, Perospirone, Sertraline, thioridazine, Venlafaxine, losartan, irbesartan, valsartan, diclofenac, pyramidon, quinizine, celecoxib, flurbiprofen, Ibuprofen BP/EP, indomethacin, lornoxicam, mefenamic acid, Naproxen Base, piroxicam, tenoxicam, Loperamide, methadone, morphine, lansoprazole, omeprazole, clobazam, Mephogarbital or Zopiclone.

9. the method for an analysis of nucleic acids, comprise analyze in the testing sample comprise corresponding in the nucleic acid of the sequence of SEQ ID NO.1 corresponding to the 1001st Nucleotide or analyze in the testing sample comprise corresponding in the nucleic acid of the sequence of SEQ ID NO.2 corresponding to the 371st Nucleotide; Preferably, described method is sequencing or probe hybridization method.

10.CYP2C9 albumen or its fragment or varient, described protein sequence are the sequence shown in the SEQ ID NO.3; Described fragment or varient comprise the 124th glutamine corresponding to SEQ ID NO.3, and are at least 10 continuous amino acids of the aminoacid sequence shown in the SEQ ID NO.3.