CN103031319B

CN103031319B - Including the CYP2C9 genetic fragment of 371G > A sudden change, coded protein fragments and application thereof

Info

Publication number: CN103031319B
Application number: CN201110430118.4A
Authority: CN
Inventors: 蔡剑平; 戴大鹏; 徐仁爱; 胡国新; 杨丽萍
Original assignee: Individual
Current assignee: Beijing Hospital
Priority date: 2011-12-19
Filing date: 2011-12-19
Publication date: 2016-06-29
Anticipated expiration: 2031-12-19
Also published as: CN103031319A

Abstract

The invention belongs to field of biology, relate to single base mutation.More particularly it relates to CYP2C9 gene is corresponding to the mutational site of the 371st of SEQ ID NO.2, described site is sported A by the G of wild type, comprises the nucleic acid fragment in this mutational site, the protein fragments of its coding and application thereof.Present invention also offers and detect the allele specific oligonucleotide in described mutational site, test kit and detection method.

Description

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

Technical field

The invention belongs to field of biology, relate to single base mutation.More particularly it relates to CYP2C9 gene relative to the 1001st of SEQIDNO.1 or the mutational site of the 371st of SEQIDNO.2, comprises the nucleic acid fragment in this mutational site and the protein fragments of corresponding encoded thereof.The invention still further relates to the reagent and detection method of identifying described mutational site, and identify the application in direction of medication usage of this site.

Background technology

CYP2C9 is most important a member in cytochrome P 450 enzymes extended familys CYP2C subfamily, accounts for the 20% of people's hepatomicrosome CYP enzyme total amount.There are about 10～16% clinical commonly used drugs via CYP2C9 oxidative metabolism, wherein mainly include tolbutamide, thiophene miaow is drawn in S-warfarin, phenytoin, glipizide, glibenclamide, holder, the medicine (referring to list of references 1-5) such as losartan, irbesartan and many non-steroidal anti-inflammatory drugs (such as ibuprofen, lornoxicam, diclofenac and naproxen).

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

According to current clinical studies show, this polymorphism of CYP2C9 gene is the main cause causing CYP2C9 enzymatic activity greatly different between individuals, therefore carrying the huge difference that can cause curative effect of medication between the different genotypic individuality of CYP2C9, even produce serious poisonous side effect of medicine or treat insufficient.Therefore, clinical rational drug use will be provided important scientific basis (referring to list of references 18,19,21,22) by the impact of curative effect of medication by research CYP2C9 gene pleiomorphism.

Summary of the invention

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

The first aspect of the invention is to provide nucleic acid fragment, described nucleic acid fragment comprises the mutational site of the 1001st corresponding to SEQIDNO.1, and be at least 10 continuous nucleotides in the nucleotide sequence shown in SEQIDNO.1, wherein the nucleotide of the 1001st is A；Or described nucleic acid fragment comprises the mutational site of the 371st corresponding to SEQIDNO.2, and is at least 10 continuous nucleotides in the nucleotide sequence shown in SEQIDNO.2, and wherein the nucleotide of the 371st is A；Or it is the complementary series of above-mentioned nucleic acid fragment.

The second aspect of the invention is to provide and contains corresponding to the 1001st of SEQIDNO.1 or corresponding to the allele specific oligonucleotide of the allele fragment in the mutational site of the 371st of SEQIDNO.2 or all or part of hybridization of its complementary series, and wherein the nucleotide in the mutational site of the 371st of the 1001st of SEQIDNO.1 or SEQIDNO.2 is A；Described allele fragment is at least 10 continuous nucleotides in the nucleotide sequence shown in SEQIDNO.1 or SEQIDNO.2 or its complementary series.

The third aspect of the invention is to provide for detecting and/or the test kit of analysis list base mutation, described test kit comprises nucleic acid fragment or the allele specific oligonucleotide of the present invention, or comprises the sequence fragment shown in SEQIDNO.6 and/or SEQIDNO.7 and/or SEQIDNO.17.

The fourth aspect of the invention is to provide nucleic acid fragment or the oligonucleotide application in detection CYP2C9 gene mutation of the present invention, and wherein said nucleic acid fragment or oligonucleotide are used as probe or primer.

The fifth aspect of the invention is to provide a kind of medication guide, including the single base mutation of the 371st of the 1001st that corresponds to SEQIDNO.1 or SEQIDNO.2 detecting CYP2C9 gene in testing sample；According to the sudden change detected, adjust by the dosage of the medicine of CYP2C9 metabolism.

The sixth aspect of the invention is to provide the method analyzing nucleic acid, described method include analyzing in testing sample comprise corresponding in the nucleic acid of the sequence of SEQIDNO.1 corresponding to the nucleotide of the 1001st or analyze comprising corresponding to nucleotide corresponding to the 371st in the nucleic acid of the sequence of SEQIDNO.2 in testing sample.

The seventh aspect of the invention is to provide CYP2C9 albumen or its fragment or variant, and described protein sequence is the sequence shown in SEQIDNO.3；Described fragment or variant comprise the glutamine of the 124th corresponding to SEQIDNO.3, and at least 10 continuous amino acids of the aminoacid sequence shown in SEQIDNO.3.

The invention provides the CYP2C9 gene and coded sequence that comprise new single base mutation.This gene is sported A (371G > A) at the 371st nucleotide corresponding to SEQIDNO.2 by G, thus causing the aminoacid of its coding to be sported glutamine by arginine, namely corresponding to the glutamine of the 124th of SEQIDNO.3.The CYP2C9 albumen (called after R124Q) of this sudden change to medicine almost without metabolic activity.The individual medication carrying this mutational site is had directive significance by this single base mutation.

Accompanying drawing explanation

Fig. 1 is the 1001st the nucleotide sequencing collection of illustrative plates of the SEQIDNO.1 of the present invention.

Detailed description of the invention

By following detailed description of the invention, the present invention is described, but present invention is not limited to this.

As illustrated without other, " nucleic acid fragment " of the present invention is made up of nucleotide or its analog, it is possible to be the fragment of DNA, RNA or its analog；Can be strand or double-strand；Can be natural (as genomic) or synthesis.

In the present invention, " sudden change " refers to the gene in detection, namely there is the nucleotide site different from wild type CYP2C9 gene order in CYP2C9 gene." mutational site " refers to the position that base is undergone mutation.In the present invention, described mutational site corresponds to the 371st in sequence shown in the 1001st of sequence shown in SEQIDNO.1 or SEQIDNO.2.

In the present invention, " allele-specific " refers to hybridize with allele specifically, as hybridized under high stringency conditions so that identify that shown in the 1001st corresponding to sequence shown in SEQIDNO.1 or SEQIDNO.2, the 371st nucleotide of sequence is A.

Present invention relates to the nonsynonymous mutation of CYP2C9 gene.Owing to this mutational site is arranged in the coded sequence of gene, therefore, skilled person will appreciate that, described mutational site both can show in genomic DNA, it is also possible to performance in coded sequence (i.e. CDS, corresponding to mRNA sequence).Those skilled in the art are according to detected sample, it is possible on genomic DNA or mRNA level in-site, this mutational site is detected.In the application, SEQIDNO.1 be centered by the mutational site of the application, the genomic dna sequence of each 1kb in front and back, namely the 1001st of SEQIDNO.1 is the mutational site that the present invention relates to.SEQIDNO.2 is the coded sequence of the CYP2C9 gene with described mutational site, and wherein the 371st is the mutational site that the present invention relates to.Skilled person will appreciate that, in this article, the 371st site corresponding to SEQIDNO.2 and the 1001st the site synonym corresponding to SEQIDNO.1 are used mutually.

In the present invention, nucleotide and amino acid whose abbreviation adopt abbreviation mode well known in the art, and in nucleotide, A represents that adenine, G represent that guanine, C represent cytosine, and T represents thymus pyrimidine.In aminoacid, A represents that alanine, R represent arginine, and N represents agedoite, D represents that aspartic acid, C represent that cysteine, Q represent glutamine, and E represents glutamic acid, G represents that glycine, H represent that histidine, I represent isoleucine, and L represents leucine, K represents that lysine, M represent methionine, and F represents phenylalanine, P represents that proline, S represent serine, and T represents threonine, W represents that tryptophan, Y represent tyrosine, and V represents valine.

Present disclosure is based on the new single base mutation site of CYP2C9 gene.Described mutational site is in the coding region of CYP2C9 gene, and corresponding to the 371st of SEQIDNO.2, this site is sported A (371G > A) by the G of wild type；Additionally, sported glutamine (R124Q) by the 124th of the albumen of the CYP2C9 gene code of this sudden change by arginine.

In first, the present invention provides the mutational site that nucleic acid fragment, described nucleic acid fragment comprise corresponding to SEQIDNO.1 the 1001st, and is at least 10 continuous nucleotides in the nucleotide sequence shown in SEQIDNO.1, and wherein the nucleotide of the 1001st is A；Or described nucleic acid fragment comprises the mutational site of the 371st corresponding to SEQIDNO.2, and is at least 10 continuous nucleotides in the nucleotide sequence shown in SEQIDNO.2, and wherein the nucleotide of the 371st is A；Or it is the complementary series of above-mentioned nucleic acid fragment.

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

Described mutational site may be located at any position of described nucleic acid fragment.

In another embodiment, described nucleic acid fragment is the sequence shown in SEQIDNO.1.

In another embodiment, described nucleic acid fragment is the sequence shown in SEQIDNO.2.

In other embodiments, it is shown that nucleic acid fragment can be the sequence shown in SEQIDNO.24-27.

In one embodiment, described oligonucleotide is used as probe.Described probe can under high stringency conditions with comprise the target sequence specific hybrid in mutational site.It is known to those skilled in the art that described probe need not with target sequence complete complementary, as long as can with target sequence specific hybridization.In preferred embodiments, described hybridization conditions can meet make probe only with 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 occur in any position of probe.In a preferred embodiment, it is that mutational site occurs in the center of probe sequence or about center.

In another embodiment, the described oligonucleotide primer of the DNA synthesis that coaches, sequencing primer as known in the art or synthetic primer etc..Described primer need not with template complete complementary, but it should with template Complementary hybridization to instruct DNA to synthesize.The length of described primer can be 15-40 length of nucleotides, it is therefore preferable to 18,19,20,21,22,23,24,25,26,27,28,29 or 30 nucleotide.Described mutational site can occur in any position of described primer；Preferably, described mutational site occurs in 3 ' ends of described primer.

Some preferred embodiment in, it is shown that oligonucleotide is the sequence as shown in SEQIDNO.28-33.

Based on this, the third aspect of the invention is to provide for detecting and/or the test kit of analysis list base mutation, described test kit comprises nucleic acid fragment or the allele specific oligonucleotide of the present invention, or comprises the sequence fragment shown in SEQIDNO.6 and/or SEQIDNO.7 and/or SEQIDNO.17.

The fourth aspect of the invention is to provide the nucleic acid fragment of the present invention or oligonucleotide for detecting the application of CYP2C9 gene mutation, and wherein said nucleic acid fragment or oligonucleotide are used as probe or primer.

The fifth aspect of the invention is to provide medication guide, including the single base mutation of the 371st of the 1001st that corresponds to SEQIDNO.1 or SEQIDNO.2 detecting CYP2C9 gene in testing sample.When the CYP2C9 gene detected is when the site corresponding to the 371st of the 1001st of SEQIDNO.1 or SEQIDNO.2 is A, adjust the dosage of medicine through CYP2C9 metabolism accordingly.In the particular embodiment, when CYP2C9 gene is when the site of the 371st of SEQIDNO.2 is A, the CYP2C9 proteinase activity of this gene code declines, therefore needs to reduce the dosage of the medicine through CYP2C9 metabolism.

The heretofore described medicine through CYP2C9 metabolism includes: cancer therapy drug, such as cyclophosphamide, ifosfamide or paclitaxel；Anticoagulant, such as warfarin, acenocoumarol, anticonvulsant or mephenytoin；Antidiabetic drug, such as tolbutamide, Nateglinide, pioglitazone or rosiglitazone；Antuepileptic, such as phenytoin or zonisamide；Antimalarial/antiparasitic, such as amodiaquine, chloroguanide hydrochloride or quinine；Psychosis, such as amitriptyline, citalopram, imipramine, cis-N-[4-[4-(1,2-Benzisothiazol-3-yl)-1-piperazinyl, Sertraline, thioridazine or venlafaxine；Depressor, such as losartan, irbesartan or valsartan；Non-steroidal anti-inflammatory drug, such as diclofenac, aminophenazone, phenazone, celecoxib, flurbiprofen, ibuprofen, indomethacin, lornoxicam, mefenamic acid, naproxen, piroxicam or tenoxicam；Analgesic, as, loperamide, methadone or morphine；Proton pump inhibitor, as lansoprazole or difficult to understand closing draw azoles；Tranquilizer, as, clobazam, enphenemal or zopiclone.

In one embodiment, described method can be sequencing, including separating and measuring the nucleotide sequence from genomic DNA or RNA, analyze and wherein comprise corresponding to the nucleotide corresponding to the 1001st in the nucleic acid of the sequence of SEQIDNO.1 or comprise corresponding to whether the nucleotide corresponding to the 371st in the nucleic acid of the sequence of SEQIDNO.2 is A.Sequencing can be any available sequence measurement known in the art.Sequencing primer can be designed according to the general knowledge of those skilled in the art, as primer is designed in the upstream and downstream appropriate position in site to be detected, with the expanding packet fragment containing this site to be measured, thus judging the nucleotide in this site.The oligonucleotide of the present invention can also be adopted as primer sequence.

In another embodiment, described method is the method utilizing probe hybridization, comprises specifically corresponding to the nucleotide corresponding to the 1001st in the nucleic acid of the sequence of SEQIDNO.1 or comprise whether the nucleotide corresponding to corresponding to the 371st in the nucleic acid of the sequence of SEQIDNO.2 is A in identification and detection sample；The probe adopted in described method is the oligonucleotide of the present invention.Such as, from testing sample, isolate nucleic acid, make probe contact with nucleic acid when allowing probe specific target sequence that may be present with nucleic acid to hybridize；The hybridization that can be detected can be realized by the probe that detectable reagent is labeled by use；Such as, form, with radiosiotope, fluorescent dye or energy catalysis, the enzyme that product can be detected and carry out label probe.Label probe, detect in sample the method that whether there is target sequence with label probe be all well-known to those skilled in the art.

In a kind of specific embodiment, it is provided that with the Taqman probe SNP detection method detection method corresponding to the nucleotide of the 1001st of SEQIDNO.1, including:

1) design primer comprises the PCR primer of corresponding to SEQIDNO.1 the 1001st for specific amplification, two Taqman-MGB probes of design simultaneously, are respectively directed to G and A (as shown in the SEQIDNO.32 sequence) allele of corresponding to SEQIDNO.1 the 1001st.

Design of primers principle is:

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

(2) avoid primer self or and primer between form 4 or more than 4 continuously pairings, it is to avoid primer self formation pili annulati card structure；

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

(4) Tm value is at 55-65 DEG C, and G/C content is at 40%-60%；

(5) the Tm value difference between primer avoids exceeding 2 DEG C；

(6) 3 ' ends of primer are avoided using base A, and 3 ' ends of primer avoid the occurrence of 3 or more than 3 consecutive identical bases；

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

(8) last 5 nucleotide of prime end can not have more than G and the C of 2.

TaqmanMGB probe design principle is:

(1) 5 ' ends of probe avoid the occurrence of G；

(2) Tm value should be 65-67 DEG C；

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

(4) avoid the occurrence of the base of repetition, especially G base, it is to avoid occur that the G of 4 or more than 4 repeats as far as possible；

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

Fluorophor can adopt FAM, VIC etc. to carry out two allele of labelling.

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

PCR condition: 95 DEG C of denaturations enter 30 amplification cycles after 10 minutes: 92 DEG C of degeneration 12 seconds, 60 DEG C of annealing and extend 1 minute (this stage detection fluorescence signal).

3) data analysis.

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

In the present invention, described sample can be any sample comprising nucleic acid, such as blood；Preferred described sample comes from people.Described nucleic acid can be DNA or coding RNA, it is preferred to genomic DNA.The method analyzing nucleic acid of the present invention can with DNA or RNA for object.Skilled person will appreciate that, when with DNA for detection object, analyze comprising corresponding to nucleotide corresponding to the 1001st in the nucleic acid of the sequence of SEQIDNO.1, the probe used or the primer sequential design according to SEQIDNO.1 in testing sample；When with RNA for detection object, analyze comprising corresponding to nucleotide corresponding to the 371st in the nucleic acid of the sequence of SEQIDNO.2, the probe used or the primer sequential design according to SEQIDNO.2 in testing sample.

The seventh aspect of the invention is to provide CYP2C9 albumen or its fragment or variant, and described protein sequence is the sequence shown in SEQIDNO.3；Described fragment or variant comprise the glutamine of the 124th corresponding to SEQIDNO.3, and at least 10 continuous amino acids of the aminoacid sequence shown in SEQIDNO.3, such as 10-20,20-50 or 50-100 aminoacid.

Further illustrate the present invention following by specific embodiment, but embodiment in detail below is merely for exemplary purpose.

Embodiment

Embodiment 1: the qualification in the mutational site that people's CYP2C9 gene is new

In the present embodiment, gathering 2127 parts of blood samples, extract the genomic DNA in blood, 9 exons of CYP2C9 gene are carried out sequence amplification, order-checking, the mutational site of screening CYP2C9 gene by design sequencing primer

1) DNA is extracted:

5ml vein EDTA anticoagulated blood sample is taked from every measured；Then according to common salting out method and/or adopt the special DNA extraction kit DNA extraction kit of company (purchased from American Omega) to extract the genomic DNA of blood sample to be measured.

2) pcr amplification:

9 exon sequences of the CYP2C9 gene in the genome DNA sample obtained are expanded by design amplimer.Described amplimer to sequence in Table 1.

Adopt 50 μ lPCR reaction systems, including: 1 × PCR buffer, 1.5mMMgCl₂, the genomic DNA of 100～150ng, upstream and downstream primer be 0.2 μM, dNTP be the LATaqDNA polymerase 1.5U of 0.4mM, TaKaRa company.Pcr amplification loop parameter is as follows: 94 DEG C of denaturations 5 minutes, 94 DEG C of degeneration 30 seconds, anneals 30 seconds, and 72 DEG C extend 2 points 30 seconds, re-extend 5 minutes after 30 circulations.Annealing temperature is relevant to primer length, and actual temp is in Table 1.

Use the GeneAmpPCRSystem9700 amplification instrument amplification of American AB I company.

Table 1: sequencing primer to and annealing temperature

3) purification amplified production:

Taking 50 μ lPCR amplified productions and carry out agarose gel electrophoresis separation, blade cuts purpose band.Reclaim test kit (Omega company) according to E.Z.N.A. gel and require that the DNA carrying out purpose band reclaims purification.

4) order-checking:

With the product after recovery for template, use sequencing primer according to CEQ^TMDTCS-QuickStartKit sequencing kit (Beckman company of the U.S.) requires to carry out order-checking PCR reaction, and reaction terminates and after purification, is easily separated with the sequence of interpretation amplified production with the CEQ8000 type gene sequencer of Beckman company of the U.S..Sequencing primer is in Table 2.

Table 2: sequencing primer

Region	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 recorded and wild type CYP*1 sequence (GenBank number of registration NM_000771.3) are compared.

Being analyzed by comparison, in a sample, the nucleotide of the 371st of discovery CYP2C9 gene coding region is become A (as shown in Figure 1) from G, and this sudden change is positioned at the 3rd exon of CYP2C9 gene.Infer that, in the protein of this CYP2C9 gene code, the 124th amino acids is sported glutamine (Q) by arginine (R) accordingly.This new 371 sudden changes (371G > A) Yi Bei world P450 NK confirms called after neomorph CYP2C9*43.

The present embodiment exemplarily gives the method identifying new mutation site.Those skilled in the art can clearly learn according to foregoing and detect the method comprising the 1001st nucleotide corresponding to SEQIDNO.1 in testing sample specifically: separate the nucleic acid in sample, carrying out amplified reaction under experiment condition corresponding in the present embodiment, primer uses primer pair SEQIDNO.6 and 7；With sequencing primer SEQIDNO.17, the product of amplification is checked order；By sequencing result and wild-type results comparison, analyze the nucleotide in the 1001st site corresponding to SEQIDNO.1.

Embodiment 2: enzymes metabolism activity 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 is decreased obviously than the metabolic activity of wild type, and the metabolic activity of * 3 types is lower than * 2 types (referring to list of references 18,19,21,22).Therefore, existing such a is known together in the art: the metabolic activity of specific substrate can be represented the metabolic activity to other substrate medicine by the enzyme expressed by same genotype.Thus, enzyme expressed by a certain genotype specific substrate metabolic activity data can be analogized the enzyme expressed by this genotype to the metabolic activity of other substrate medicine (as, it is possible to the metabolic activity of the metabolic activity of the enzyme expressed by this genotype with the enzyme expressed by wild type is compared).

In the present embodiment, according to above-mentioned mutational site, with wild type CYP2C9 (* 1) gene for template, the nucleotide (being become A from G) of the 371st of rite-directed mutagenesis CYP2C9 gene coding region, construction of expression vector expresses saltant type CYP2C9 albumen (called after R124Q), the CYP2C9 activity detection kit (containing CYP2C9 specific substrate) utilizing Promega company detects the enzymatic activity of this saltant type CYP2C9, to judge compared with wild type CYP2C9 (* 1), whether its enzymatic metabolism activity changes.

1) expression of genes of interest

With wild type CYP2C9 (* 1) plasmid vector (being presented by professor Zhou Shufeng of American South University of Florida) for template, 371st nucleotide of rite-directed mutagenesis CYP2C9 gene coding region, namely obtain comprising the ORF region sequence of the nucleotide shown in SEQIDNO.2, by this genes of interest and reference gene Gluc (a kind of secreting type luciferase, its translation product can be secreted in culture medium, and fluorescence signal detected by particular agent box；Its skeleton carrier is pIRESpGluc-Basic, purchased from NEB company, article No. N8082S) it is respectively connecting to double gene expression vector pIRES (purchased from Clontech company, article No. 631605) A, B site in, two genes are made to be positioned under the control of same CMV promoter, final acquisition double gene expression vector pIRES-Gluc-2C9.By 8*10⁴The COS-7 cell (this cell strain has been widely used in CYP2C9 external activity analysis, refers to list of references 18,21) of individual African green monkey kidney epithelial cell origin is laid on 24 orifice plates；After incubated overnight, liposome lip2000 is utilized to transfect 500ng plasmid vector pIRES-Gluc-2C9, to express destination protein CYP2C9 (R124Q) and internal reference Protein G luc.

2) Enzyme assay

After continuing cultivation 48 hours, add 200 μ l fresh DMEM medium (containing 10%FBS), and with reference to the operation instruction of Promega company CYP2C9 detection kit (article No.: V8791), add the substrate Luciferin-H of 4 μ lCYP2C9, take the 50 μ l culture medium isopyknic Promega test kit of addition (article No.: V8791) after continuing cultivation after mixing 8 hours and detect buffer.Containing LUC Photinus pyralis LUC Photinus pyralis FL in this detection buffer；Substrate Luciferin-H product D-luciferin after CYP2C9 metabolism can produce fluorescence after reacting with LUC Photinus pyralis LUC Photinus pyralis FL.The GLOMAX20/20Luminometer (GLOMAX20/20 luminometer) utilizing Promega company detects fluorescence signal.Take 7 μ l culture medium again and detect kit (NEB company, article No.: E3300) operation instruction with reference to Gluc, add isopyknic detection buffer, utilize GLOMAX20/20Luminometer to detect Gluc fluorescence signal.Using the fluorescence signal directly related with CYP2C9/internal reference Gluc fluorescence signal value as final experimental data, and wild type (* 1 type) CYP2C9 Data Comparison, the change of the metabolic activity of mutant to be measured can be analyzed.Every kind of experimental subject carries out three parallel laboratory tests, and experimental result is in Table 3.

Introduction about Luciferin-H:

The exemplary metabolites activity of CYP2C9 is that aromatic ring structure can carry out hydroxylation (referring to list of references 23,24, related content combines by way of reference to herein).In the present embodiment, the detection substrate Luciferin-H of application is containing aromatic ring structure, CYP2C9 can the specific R1 position hydroxylating to this substrate, and produce new metabolism substrate D-Luciferin ([4S]-4,5-dihydro-2-[6 '-hydroxy-2 '-benzothiazolyl]-4-thiazolecarboxylicacid), the latter can react with the LUC Photinus pyralis LUC Photinus pyralis FL in detectable and luminous, the power of signal is directly related with the amount of product D-luciferin, and namely the latter reflects the enzymatic activity of CYP2C9 albumen to be checked.There are some researches show, utilize this detection method can effectively reflect the CYP2C9 metabolic characteristics to Common drugs, mainly include CYP2C9 probe medicament warfarin, tolbutamide, diclofenac, phenytoin, ibuprofen；Also include (referring to lists of references 22,24,25) such as part Common drugs sulfaphenazole, troglitazone, azamulin, piroxicam, sulfaphenazole, miconazole, fluvoxamines simultaneously.Therefore the data of this experimental program can be generally applicable to other medicine through CYP2C9 metabolism, including the medicine listed before in present specification.

Table 3: the experimental result of enzymes metabolism activity

As can be seen from the results, 3 negative controls all present expection trend: empty carrier can't detect the signal of 2C9, relative to wild type * 1 type, known mutations type * 2 type activity has and is decreased obviously (the nearly minimizing of nearly 60%), * 3 types decline maximum (about declining 93%), this numerical value and trend with have been reported consistent (referring to list of references 18,19,21,22).

Utilizing this vitro detection system it can be seen that R124Q is almost without metabolic activity, namely this sudden change can cause the metabolic activity of expressed enzyme substantially to reduce.Therefore, in practice, it is necessary to consider suitably to regulate on dosage carrying this genotypic individuality, as reduced making consumption and avoiding the generation of adverse effect of medicine.This even more important for the medicine (such as warfarin, phenytoin etc.) that treatment window is narrow by the medicine adjustment of gene targeting.

Sequence:

SEQIDNO.1: genomic dna sequence

AGACACTGAAAATGAATTTGTCATTCTCTGAGCTCAGTTTTTTTTTTTTTTTTTTTTTTTTTTGAGACAGAGTCTTACTCTGTAGCTCAGGCTGGAGTGCAGTGGTACAATCTTGGCTCACTGCAACCTCCATCTCCCAGGTCCCCATTCAAGAAATTCTCCTGCCTCAGTCCCCCAAGTAGCTAGCATTACAGGCATGCACCACCATGCTCAGCTAATTTTTGTATTTTTAGTAGAGACGTGGTATCACCTTGTTGGCCAGGCTGGTCTTGAACTCCTGACCTTGTGATCCACCTGCCTTGGCCTCCCAAAGTGTTGGGATTACAGGCAGGAGCCACCACACCTGGCCGTTTGTTTAAAATAGAGTAAATAGACCTGCTGAATATGTTGATGTGAGTATTAATTGTAATCTGCATAGCAATTGTCTGACCATTGCCTTGAACATCACAGGCCATCTGAGTGGCAAGTATAATCATCATCATGTTTCTATTTAAAATTCAGAAATATTTGAAGCCTGTGTGGCTGAATAAAAGCATACAAATACAATGAAAATATCATGCTAAATCAGGCTTAGCAAATGGACAAAATAGTAACTTCGTTTGCTGTTATCTCTGTCTACTTTCCTAGCTCTCAAAGGTCTATGGCCCTGTGTTCACTCTGTATTTTGGCCTGAAACCCATAGTGGTGCTGCATGGATATGAAGCAGTGAAGGAAGCCCTGATTGATCTTGGAGAGGAGTTTTCTGGAAGAGGCATTTTCCCACTGGCTGAAAGAGCTAACAGAGGATTTGGTAGGTGTGCATGTGCCTGTTTCAGCATCTGTCTTGGGGATGGGGAGGATGGAAAACAGAGACTTACAGAGCTCCTCGGGCAGAGCTTGGCCCATCCACATGGCTGCCCAGTGTCAGCTTCCTCTTTCTTGCCTGGGATCTCCCTCCTAGTTTCGTTTCTCTTCCTGTTAGGAATTGTTTTCAGCAATGGAAAGAAATGGAAGGAGATCCAGCGTTTCTCCCTCATGACGCTGCGGAATTTTGGGATGGGGAAGAGGAGCATTGAGGACCGTGTTCAAGAGGAAGCCCGCTGCCTTGTGGAGGAGTTGAGAAAAACCAAGGGTGGGTGACCCTACTCCATATCACTGACCTTACTGGACTACTATCTTCTCTACTGACATTCTTGGAAACATTTCAGGGGTGGCCATATCTTTCATTATGAGTCCTGGTTGTTAGCTCATGTGAAGCGGGGGTTTGAAGCTGAGAGCCAAGGGAATTTGCACATATTTGTGCTGTGTGTGTACAGGCATGATTGTGCGTACAGTGTGGGTATAAAAGGTTCATTTAATCCCATGTTCTCCTGAACTTTGCTTTTTTGCTTTCAAATAAGAAATGATGAATATAGATTTTGAGTTCATTTTTTGAAAGAGTTAAAGAGCAGTGTTTTTCCCATTACCTATTCCAGAACATGTCACCAGAGAATACTTGACAAGTCAACATGGTGGGAATGGCCCTATCATACCCATATGGAGCATGAACCAAATGGCATGTGCTTTTATTTAATTGGACTGTGTTTGTATGGTCAGCCTCACTGACTTCTCTGGGGTTTCTTTTAGGCCCGTGCTTGCCATTCTGGCCAGTAATGACATTCTACAGTTTTTATTGCTTAGGCATATCTTAGTGCAGTTCTCATCAATTATTATTTCTCTGTAAACACAGCATTATTTTAAAAATAGTATTAATTTATTCTTGTTACTGTATTGATTTATATATTTTCAGTAAATACATCCTGTAGCATAATTCTGTGAAATACCCAAATGTCAATTTATAAAATGATTTATTTAACAAGATTTTACTTATTAGTAATAACTCTGTAATCTGCATTCCCTATGTATGATTTGGCTCTGTTTCAGTTTTGCTTATCTCTTTCCAACCATATTTATGAAATTTTGGCTTAGAAATTTATGTTAATTATTTTTTTTCCATGGCCAACTCTACTCATCTATGAAGTT

SEQIDNO.2 coded sequence

ATGGATTCTCTTGTGGTCCTTGTGCTCTGTCTCTCATGTTTGCTTCTCCTTTCACTCTGGAGACAGAGCTCTGGGAGAGGAAAACTCCCTCCTGGCCCCACTCCTCTCCCAGTGATTGGAAATATCCTACAGATAGGTATTAAGGACATCAGCAAATCCTTAACCAATCTCTCAAAGGTCTATGGCCCTGTGTTCACTCTGTATTTTGGCCTGAAACCCATAGTGGTGCTGCATGGATATGAAGCAGTGAAGGAAGCCCTGATTGATCTTGGAGAGGAGTTTTCTGGAAGAGGCATTTTCCCACTGGCTGAAAGAGCTAACAGAGGATTTGGAATTGTTTTCAGCAATGGAAAGAAATGGAAGGAGATCCAGCGTTTCTCCCTCATGACGCTGCGGAATTTTGGGATGGGGAAGAGGAGCATTGAGGACCGTGTTCAAGAGGAAGCCCGCTGCCTTGTGGAGGAGTTGAGAAAAACCAAGGCCTCACCCTGTGATCCCACTTTCATCCTGGGCTGTGCTCCCTGCAATGTGATCTGCTCCATTATTTTCCATAAACGTTTTGATTATAAAGATCAGCAATTTCTTAACTTAATGGAAAAGTTGAATGAAAACATCAAGATTTTGAGCAGCCCCTGGATCCAGATCTGCAATAATTTTTCTCCTATCATTGATTACTTCCCGGGAACTCACAACAAATTACTTAAAAACGTTGCTTTTATGAAAAGTTATATTTTGGAAAAAGTAAAAGAACACCAAGAATCAATGGACATGAACAACCCTCAGGACTTTATTGATTGCTTCCTGATGAAAATGGAGAAGGAAAAGCACAACCAACCATCTGAATTTACTATTGAAAGCTTGGAAAACACTGCAGTTGACTTGTTTGGAGCTGGGACAGAGACGACAAGCACAACCCTGAGATATGCTCTCCTTCTCCTGCTGAAGCACCCAGAGGTCACAGCTAAAGTCCAGGAAGAGATTGAACGTGTGATTGGCAGAAACCGGAGCCCCTGCATGCAAGACAGGAGCCACATGCCCTACACAGATGCTGTGGTGCACGAGGTCCAGAGATACATTGACCTTCTCCCCACCAGCCTGCCCCATGCAGTGACCTGTGACATTAAATTCAGAAACTATCTCATTCCCAAGGGCACAACCATATTAATTTCCCTGACTTCTGTGCTACATGACAACAAAGAATTTCCCAACCCAGAGATGTTTGACCCTCATCACTTTCTGGATGAAGGTGGCAATTTTAAGAAAAGTAAATACTTCATGCCTTTCTCAGCAGGAAAACGGATTTGTGTGGGAGAAGCCCTGGCCGGCATGGAGCTGTTTTTATTCCTGACCTCCATTTTACAGAACTTTAACCTGAAATCTCTGGTTGACCCAAAGAACCTTGACACCACTCCAGTTGTCAATGGATTTGCCTCTGTGCCGCCCTTCTACCAGCTGTGCTTCATTCCTGTCT

SEQIDNO.3 protein sequence

MDSLVVLVLCLSCLLLLSLWRQSSGRGKLPPGPTPLPVIGNILQIGIKDISKSLTNLSKVYGPVFTLYFGLKPIVVLHGYEAVKEALIDLGEEFSGRGIFPLAERANRGFGIVFSNGKKWKEIQRFSLMTLRNFGMGKRSIEDRVQEEARCLVEELRKTKASPCDPTFILGCAPCNVICSIIFHKRFDYKDQQFLNLMEKLNENIKILSSPWIQICNNFSPIIDYFPGTHNKLLKNVAFMKSYILEKVKEHQESMDMNNPQDFIDCFLMKMEKEKHNQPSEFTIESLENTAVDLFGAGTETTSTTLRYALLLLLKHPEVTAKVQEEIERVIGRNRSPCMQDRSHMPYTDAVVHEVQRYIDLLPTSLPHAVTCDIKFRNYLIPKGTTILISLTSVLHDNKEFPNPEMFDPHHFLDEGGNFKKSKYFMPFSAGKRICVGEALAGMELFLFLTSILQNFNLKSLVDPKNLDTTPVVNGFASVPPFYQLCFIPV

SEQIDNO.24 nucleic acid fragment

AATGGAAGGAGATCCAGCGTTTCTCCCTCAT

SEQIDNO.25 nucleic acid fragment

AAAGAAATGGAAGGAGATCCAGCGTTTCTCCCTCATGACGC

SEQIDNO.26 nucleic acid fragment

AATGGAAAGAAATGGAAGGAGATCCAGCGTTTCTCCCTCATGACGCTGCGG

SEQIDNO.27 nucleic acid fragment

TCAGCAATGGAAAGAAATGGAAGGAGATCCAGCGTTTCTCCCTCATGACGCTGCGGAATTT

SEQIDNO.28 oligonucleotide sequence

GAGGGAGAATCGCTG

SEQIDNO.29 oligonucleotide sequence

GTCATGAGGGAGAATCGCTG

SEQIDNO.30 oligonucleotide sequence

GCAGCGTCATGAGGGAGAATCGCTG

SEQIDNO.31 oligonucleotide sequence

ATTCCGCAGCGTCATGAGGGAGAATCGCTG

SEQIDNO.32 oligonucleotide sequence

CGCTGGATCTCCTTCC

SEQIDNO.33 oligonucleotide sequence

GAGAATCGCTGGATCTCCT

List of references:

1.AquilanteCA.SulfonylureapharmacogenomicsinType2diabete s:theinfluenceofdrugtargetanddiabetesriskpolymorphisms.E xpertRevCardiovascTher.2010；8 (3): 359-372.

2.XuHM, MurrayM, MclachlanAJ.Influenceofgeneticpolymorphismsonthepharmaco kineticsandpharmacodynamicsofsulfonylureadrugs.CurrentDr ugMetabolism.2009；10 (6): 643-658.

3.WangB, WangJ, HuangSQ, etal.GeneticpolymorphismofthehumancytochromeP4502C9genea nditsclinicalsignificance.CurrentDrugMetabolism.2009；10 (7): 781-834.

4. Li Zhi, Wang Guo, Zhou Honghao .CYP2C9 gene pleiomorphism and functional meaning progress thereof. Chinese Clinical pharmacology and therapeutics 2008；13 (6): 601-609.

5.ZhouSh.F, LiuJ.P.ChowbayB.PolymorphismofhumancytochromeP450enzymes anditsclinicalimpact.DrugMetabRev.2009；41 (2): 89-295.

6.XiongY.WangM, FangKetal:AsystematicgeneticpolymorphismanalysisoftheCYP 2C9geneinfourdifferentgeographicalHanpopulationsinmainla ndChina.Genomics2011；97:277-281.

7.ZhuJ, ZhangW, LiY, WangH, ZhengW, WangC:ARMStestfordiagnosisofCYP2C9andVKORC1mutationinpat ientswithpulmonaryembolisminHanChinese.Pharmacogenomics2 010；11:113-119.

8.ZhangYN, CuiW, HanMetal:[GenepolymorphismofCYP4502C9andVKORC1inChinesep opulationandtheirrelationshipstothemaintainingdosageofwa rfarin.] .ZhonghuaLiuXingBingXueZaZhi2010；31:218-222.

9.LiZ, WangG, WangLSetal:EffectsoftheCYP2C9*13alleleonthepharmacokinet icsoflosartaninhealthymalesubjects.Xenobiotica2009；39:788-793.

10.YuBN, LuoCH, WangDetal:CYP2C9allelevariantsinChinesehypertensionpatie ntsandhealthycontrols.ClinChimActa2004；348:57-61.

11.YangJQ, MorinS, VerstuyftCetal:FrequencyofcytochromeP4502C9allelicvarian tsintheChineseandFrenchpopulations.FundamClinPharmacol20 03；17:373-376.

12.WangSL, HuangJ, LaiMD, TsaiJJ:DetectionofCYP2C9polymorphismbasedonthepolymerase chainreactioninChinese.Pharmacogenetics1995；5:37-42.

13. horse Jingjing, Li Jinheng, Cheng Lu. three dimensional gel gene chips Study of China crowd CYP2C9 and CYP2C19 gene pleiomorphism. Chinese Clinical pharmacology and therapeutics 2009；14 (9): 966-973.

14. Zhao Gang great waves, Ding Yuanyuan, the foundation of the .Pyrosequencing detection CYP2C9*3 gene pleiomorphism methods such as Yang Fan and reliability consideration thereof. Chinese Clinical pharmacology and therapeutics 2009；14 (7): 799-803.

15. Cor Equi surpasses, Yang Jian, Huang Rong in morning etc. the gene pleiomorphism of vitamin K epoxide reductase subunit 1 unit 1 cytochrome P450 2C9 in Han population in Jiangsu province. University Of Suzhou's journal (medicine) 2009；29 (2): 279-282.

16. Tang and year, cuckoo Kui, Zhang Zonggang. Xinjiang Uygur healthy population cytochrome P450 gene polymorphism research. China's medicine and clinical 2007；7 (2): 91-94.

17. how to shake space, Sun Limin, Li Yueqin etc. Guangdong crowd's CYP2C9 allele and genotype distribution frequency. Guangdong medical science 2006；27 (8): 1131-1132.

18.RokittaD, FuhrU.Comparisonofenzymekineticparametersobtainedinvitro forreactionsmediatedbyhumanCYP2CenzymesincludingmajorCYP 2C9variants.CurrDrugMetab.2010；11 (2): 153-161.

19.VanBoovenD, MarshS, McLeodHetal:CytochromeP4502C9-CYP2C9.PharmacogenetGenomi cs2010；20:277-281.

20.SiD, GuoY, ZhangY, YangL, ZhouH, ZhongD.IdentificationofanovelvariantCYP2C9alleleinChines e.Pharmacogenetics.2004；14 (7): 465-469.

21.HiratsukaM:Invitroassessmentoftheallelicvariantsofcyt ochromeP450.DrugMetabPharmacokinet2011.

22.GuoY, WangY, SiD, FawcettPJ, ZhongD, ZhouH.CatalyticactivitiesofhumancytochromeP4502C9*1,2C9*3and2C9*13.Xenobiotica2005；35:853-861.

23.ZhouSF, ZhouZW, YangLP, CaiJP:Substrates, inducers, inhibitorsandstructure-activityrelationshipsofhumanCytoc hromeP4502C9andimplicationsindrugdevelopment.CurrMedChem 2009；16:3480-3675.

24.CaliJJ, MaD, SobolMetal:LuminogeniccytochromeP450assays.ExpertOpinDru gMetabToxicol2006；2:629-645.

25.AnzenbacherovaE, VeinlichovaA, MasekV, AnzenbacherP；Comparisonof " highthroughput " micromethodsfordeterminationofcytochromeP450activitieswi thclassicalmethodsusingHPLCforproductidentification.Biom edPapMedFacUnivPalackyOlomoucCzechRepub2005；149:353-355.

Claims

1. nucleic acid fragment, described nucleic acid fragment comprises the mutational site of the 1001st corresponding to SEQIDNO.1, and is at least 10 continuous nucleotides in the nucleotide sequence shown in SEQIDNO.1 or its complementary series, and wherein the nucleotide of the 1001st is A；Or described nucleic acid fragment comprises the mutational site of the 371st corresponding to SEQIDNO.2, and is at least 10 continuous nucleotides in the nucleotide sequence shown in SEQIDNO.2 or its complementary series, and wherein the nucleotide of the 371st is A.

2. nucleic acid fragment according to claim 1, it is characterised in that the length of described nucleic acid fragment is 10-100,100-200,200-500 or 500-1000 nucleotide.

3. nucleic acid fragment according to claim 1, it is characterised in that the length of described nucleic acid fragment is 10-20,20-30,30-40,40-50,50-60,60-100 or 100-300 nucleotide.

4. nucleic acid fragment according to claim 1, it is characterised in that described nucleic acid fragment be SEQIDNO.1,2, the sequence shown in 24-27.

5. for detecting and/or the test kit of analysis list base mutation, including the nucleic acid fragment described in any one of claim 1-4.

6. the application in the preparation of preparation detection CYP2C9 gene mutation of the nucleic acid fragment described in any one of claim 1-4.

7.CYP2C9 albumen, described protein sequence is the sequence shown in SEQIDNO.3.