CN103031318B - Comprise the CYP2C9 genetic fragment of 445G > A sudden change, coded protein fragments and application thereof - Google Patents

Abstract

The invention belongs to field of biology, relate to single base mutation. More specifically, the present invention relates to CYP2C9 gene corresponding to SEQ? ID? the mutational site of the 445th of NO.2, described site sports A by the G of wild type, the nucleic acid fragment that comprises this mutational site, the protein fragments of its coding and application thereof. The present invention also provides the allele specific oligonucleotide, kit and the detection method that detect described mutational site.

Description

Comprise the CYP2C9 genetic fragment of 445G > 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 1001st or mutational site, the nucleic acid fragment that comprise this mutational site and the protein fragments of corresponding encoded thereof of 445th of SEQIDNO.2 of CYP2C9 gene with respect to SEQIDNO.1. The invention still further relates to reagent and the detection method in the described mutational site of qualification, and identify the application of this site in direction of medication usage.

Background technology

CYP2C9 is most important a member in cytochrome P 450 enzymes extended familys CYP2C subfamily, accounts for 20% of people's hepatomicrosome CYP enzyme total amount. There are about 10～16% clinical commonly used drugs via CYP2C9 oxidative metabolism, wherein mainly comprise that orinase, S-warfarin, phenytoinum naticum, Glipizide, glibenclamide, holder draw the medicines (referring to bibliography 1-5) such as thiophene miaow, Losartan, Irbesartan and many non-steroidal anti-inflammatory drugs (as: brufen, Lornoxicam, Diclofenac and naproxen).

CYP2C9 gene has height polymorphism. So far, named allele has 35 kinds (http://www.cypalleles.ki.se) in the world, remove outside 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 saltant type be CYP2C9*2 (430C > T), CYP2C9*3 (1075A > C) (referring to bibliography 6-17,20).

According to current clinical studies show, this polymorphism of CYP2C9 gene is to cause CYP2C9 enzymatic activity very big different main 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 bibliography 18,19,21,22) to the impact of curative effect of medication to clinical rational drug use.

Summary of the invention

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

First aspect of the present 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 nucleotides of the 1001st is A; Or described nucleic acid fragment comprises the mutational site of the 445th corresponding to SEQIDNO.2, and is at least 10 continuous nucleotides in the nucleotide sequence shown in SEQIDNO.2, and wherein the nucleotides of the 445th is A; Or be the complementary series of above-mentioned nucleic acid fragment.

Second aspect of the present invention is to provide and contains corresponding to the 1001st of SEQIDNO.1 or corresponding to the allele fragment in the mutational site of the 445th or the allele specific oligonucleotide of all or part of hybridization of its complementary series of SEQIDNO.2, and wherein the nucleotides in the mutational site of the 445th of the 1001st of SEQIDNO.1 the or SEQIDNO.2 is A; Described allele fragment is at least 10 continuous nucleotides or its complementary series in the nucleotide sequence shown in SEQIDNO.1 or SEQIDNO.2.

The 3rd aspect of the present invention be to provide for detection of and/or the kit of analysis list base mutation, described kit comprises nucleic acid fragment of the present invention or allele specific oligonucleotide, or comprises the sequence fragment shown in SEQIDNO.6 and/or SEQIDNO.7 and/or SEQIDNO.17.

The 4th aspect of the present invention is to provide nucleic acid fragment of the present invention or the application of oligonucleotides in detection CYP2C9 gene mutation, and wherein said nucleic acid fragment or oligonucleotides are as probe or primer.

The 5th aspect of the present invention is to provide a kind of medication guide, comprises the 1001st or the single base mutation of the 445th of SEQIDNO.2 corresponding to the SEQIDNO.1 that detect CYP2C9 gene in testing sample; According to the sudden change detecting, adjust the dosage by the medicine of CYP2C9 metabolism.

The 6th aspect of the present invention is to provide the method for analysis of nucleic acids, described method comprise analyze in testing sample comprise corresponding in the nucleic acid of the sequence of SEQIDNO.1 corresponding to the nucleotides of the 1001st or analyze comprising corresponding to the nucleotides corresponding to the 445th in the nucleic acid of the sequence of SEQIDNO.2 in testing sample.

The 7th aspect of the present 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 threonine of the 149th corresponding to SEQIDNO.3, and are at least 10 continuous amino acids of the amino acid sequence shown in SEQIDNO.3.

The invention provides the CYP2C9 gene and the coded sequence that comprise new single base mutation. This gene sports A (445G > A) at the 445th nucleotides corresponding to SEQIDNO.2 by G, thereby the amino acid that causes its coding is threonine by alanine mutation, corresponding to the threonine of the 149th of SEQIDNO.3. The CYP2C9 albumen (called after A149T) of this sudden change is lower than wild type to the metabolic activity of medicine. This single base mutation has directive significance to the individual medication of carrying this mutational site.

Brief description of the drawings

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

Detailed description of the invention

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

As illustrated without other, " nucleic acid fragment " of the present invention is made up of nucleotides or its analog, can be the fragment of DNA, RNA or its analog; Can be strand or two strands; It can be natural (as genomic) or synthetic.

In the present invention, " sudden change " refers at the gene detecting, in CYP2C9 gene, has the nucleotide site different from wild type CYP2C9 gene order. " mutational site " refers to the position that base is undergone mutation. In the present invention, described mutational site is corresponding to the 445th in sequence shown in the 1001st of sequence shown in SEQIDNO.1 or SEQIDNO.2.

In the present invention, " allele-specific " refer to specifically and allele hybridization, as hybridized under rigorous condition, makes to identify that corresponding to the 445th nucleotides of sequence shown in the 1001st of sequence shown in SEQIDNO.1 or SEQIDNO.2 be A.

Content of the present invention relates to the nonsynonymous mutation of CYP2C9 gene. Because this mutational site is arranged in the coded sequence of gene, therefore, those skilled in the art are known, and described mutational site both can show in genomic DNA, also can performance in coded sequence (being CDS, corresponding to mRNA sequence). Those skilled in the art, according to detected sample, can detect this mutational site in genomic DNA or mRNA level. In the application, SEQIDNO.1 be centered by the application's mutational site, the genomic dna sequence of the each 1kb in front and back, the 1001st of SEQIDNO.1 the is the mutational site the present invention relates to. SEQIDNO.2 is the coded sequence with the CYP2C9 gene in described mutational site, and wherein the 445th is the mutational site the present invention relates to. Those skilled in the art are known, in this article, and corresponding to the 445th site of SEQIDNO.2 with use mutually corresponding to the 1001st the site synonym of SEQIDNO.1.

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

Content of the present invention is the new single base mutation site based on CYP2C9 gene. Described mutational site is the code area that is positioned at CYP2C9 gene, and corresponding to the 445th of SEQIDNO.2, this site sports A (445G > A) by the G of wild type; In addition, the 149th by the albumen of the CYP2C9 gene code of this sudden change is threonine (A149T) by alanine mutation.

Aspect first, the invention provides 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, and wherein the nucleotides of the 1001st is A; Or described nucleic acid fragment comprises the mutational site of the 445th corresponding to SEQIDNO.2, and is at least 10 continuous nucleotides in the nucleotide sequence shown in SEQIDNO.2, and wherein the nucleotides of the 445th is A; Or be the complementary series of above-mentioned nucleic acid fragment.

In one embodiment, the length of described nucleic acid fragment can be as 10-100,100-200,200-500, a 500-1000 nucleotides. 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 nucleotides.

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 SEQIDNO.1.

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

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

Second aspect of the present invention is to provide and contains corresponding to the 1001st of SEQIDNO.1 or corresponding to the allele fragment in the mutational site of the 445th or the allele specific oligonucleotide of all or part of hybridization of its complementary series of SEQIDNO.2, and wherein the nucleotides in the mutational site of the 445th of the 1001st of SEQIDNO.1 the or SEQIDNO.2 is A; Described allele fragment is at least 10 continuous nucleotides or its complementary series in the nucleotide sequence shown in SEQIDNO.1 or SEQIDNO.2.

In one embodiment, described oligonucleotides is as probe. Described probe can be under rigorous condition and the target sequence specific hybrid that comprises mutational site. It is known to those skilled in the art that described probe does not need and target sequence complete complementary, if 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 nucleotides, 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 nucleotides. Described mutational site can appear at any position of probe. In a preferred embodiment, be center or the about center that mutational site appears at probe sequence.

In another embodiment, described oligonucleotides is with coaching the synthetic primer of DNA, sequencing primer or synthetic primer etc. as known in the art. Described primer does not need and template complete complementary, but should be with the complementary hybridization of template to instruct DNA synthetic. 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 nucleotides. 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 oligonucleotides be the sequence as shown in SEQIDNO.28-33.

Based on this, the 3rd aspect of the present invention be to provide for detection of and/or the kit of analysis list base mutation, described kit comprises nucleic acid fragment of the present invention or allele specific oligonucleotide, or comprises the sequence fragment shown in SEQIDNO.6 and/or SEQIDNO.7 and/or SEQIDNO.17.

The 4th aspect of the present invention is to provide the application for detection of CYP2C9 gene mutation of nucleic acid fragment of the present invention or oligonucleotides, and wherein said nucleic acid fragment or oligonucleotides are as probe or primer.

The 5th aspect of the present invention is to provide medication guide, comprises the 1001st or the single base mutation of the 445th of SEQIDNO.2 corresponding to the SEQIDNO.1 that detect CYP2C9 gene in testing sample. When the CYP2C9 gene detecting is in the time being A corresponding to the site of the 445th of the 1001st of SEQIDNO.1 or SEQIDNO.2, adjust accordingly the dosage through the medicine of CYP2C9 metabolism. In specific embodiment, in the time that CYP2C9 gene is A in the site of the 445th of SEQIDNO.2, the CYP2C9 proteinase activity of this gene code declines, therefore need to reduce the dosage through the medicine of CYP2C9 metabolism.

The medicine through CYP2C9 metabolism described in the present invention comprises: cancer therapy drug, as endoxan, ifosfamide or taxol; Anticoagulant, as warfarin, acenocoumarin, anticonvulsive drug or mephenytoin; Antidiabetic drug, as orinase, Nateglinide, Pioglitazone or Rosiglitazone; Antiepileptic, as phenytoinum naticum or Zonisamide; Antimalarial/antiparasitic agent, as amodiaquine, chloroguanide hydrochloride or quinine; Antipsychotic drug, as amitriptyline, Citalopram, imipramine, Perospirone, Sertraline, thioridazine or Venlafaxine; Depressor, as Losartan, Irbesartan or Valsartan; Non-steroidal anti-inflammatory drug, as Diclofenac, aminopyrine, antipyrine, Sai-Mi-Xi-Bu, Flurbiprofen, brufen, Indomethacin, Lornoxicam, mefenamic acid, naproxen, piroxicam or tenoxicam; Antalgesic, as, Loperamide, methadone or morphine; Proton pump inhibitor, as Lansoprazole or Omeprazole; Sedative, as, Clobazam, methylphenobarbital or zopiclone.

The 6th aspect of the present invention is to provide the method for analysis of nucleic acids, described method comprise analyze in testing sample comprise corresponding in the nucleic acid of the sequence of SEQIDNO.1 corresponding to the nucleotides of the 1001st or analyze comprising corresponding to the nucleotides corresponding to the 445th in the nucleic acid of the sequence of SEQIDNO.2 in testing sample.

In one embodiment, described method can be PCR sequencing PCR, comprise the nucleotide sequence that separates and measure from genomic DNA or RNA, analyze wherein comprise corresponding in the nucleic acid of the sequence of SEQIDNO.1 corresponding to the nucleotides of the 1001st or whether comprise corresponding to the nucleotides corresponding to the 445th in the nucleic acid of the sequence of SEQIDNO.2 be A. PCR sequencing PCR 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 the upstream and downstream appropriate position design primer in site to be detected, contains the fragment in this site to be measured, thereby judge the nucleotides in this site with expanding packet. Also can adopt oligonucleotides of the present invention as primer sequence.

In another embodiment, described method is to utilize the method for Probe Hybridization, identify specifically detect comprise in sample corresponding in the nucleic acid of the sequence of SEQIDNO.1 corresponding to the nucleotides of the 1001st or whether comprise in the nucleic acid corresponding to the sequence of SEQIDNO.2 be A corresponding to the nucleotides of the 445th; The probe adopting in described method is oligonucleotides of the present invention. For example, from testing sample, isolate nucleic acid, under the condition that allows probe and the specific target sequence hybridization that may exist in nucleic acid, probe is contacted with nucleic acid; The hybridization that can be detected can be by using the probe of being crossed by detectable reagent mark to realize; For example,, with radio isotope, fluorescent dye or can catalysis form the enzyme that can detect product and carry out label probe. Label probe, detect with label probe whether to have the method for target sequence in sample be all well-known to those skilled in the art.

In a kind of concrete embodiment, provide with Taqman probe SNP detection method and detect the method corresponding to the nucleotides of the 1001st of SEQIDNO.1, comprising:

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

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 the above pairing continuously of 4 or 4, avoid primer self to form pili annulati card structure;

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

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

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

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

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

(8) last 5 nucleotides of primer end can not have the G and the C that exceed 2.

TaqmanMGB probe design principle is:

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

(2) Tm value should be 65-67 DEG C;

(3) shorten TaqmanMGB 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 occurring that 4 or 4 above G repeat;

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

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

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

PCR condition: 95 DEG C of denaturations enter 30 amplification cycles after 10 minutes: 92 DEG C of sex change 12 seconds, 60 DEG C of annealing and extend 1 minute (this stage is detected fluorescence 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 445G > A sudden change.

In the present invention, described sample can be any sample that comprises nucleic acid, as blood; Preferred described sample comes from 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 taking DNA or RNA as object. Those skilled in the art are known, when taking DNA as detecting when object, analyze comprising corresponding to the nucleotides corresponding to the 1001st in the nucleic acid of the sequence of SEQIDNO.1 in testing sample, and the probe using or primer are according to the sequences Design of SEQIDNO.1; When taking RNA as detecting when object, analyze comprising corresponding to the nucleotides corresponding to the 445th in the nucleic acid of the sequence of SEQIDNO.2 in testing sample, the probe using or primer are according to the sequences Design of SEQIDNO.2.

The 7th aspect of the present 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 threonine of the 149th corresponding to SEQIDNO.3, and are at least 10 continuous amino acids of the amino acid sequence shown in SEQIDNO.3, as 10-20,20-50 or 50-100 amino acid.

To further illustrate the present invention by specific embodiment below, but following specific embodiment is only for exemplary object.

Embodiment

Embodiment 1: the qualification 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 blood, design sequencing primer carries out sequence amplification, order-checking to 9 of CYP2C9 gene extrons, 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 method and/or adopt special DNA to extract kit (DNA purchased from Omega company of the U.S. extracts kit) 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 obtaining. Described amplimer to sequence in table 1.

Adopt 50 μ lPCR reaction systems, comprising: 1 × PCR buffer solution, 1.5mMMgCl₂, the genomic DNA of 100～150ng, upstream and downstream primer be the LATaqDNA polymerase 1.5U that 0.2 μ M, dNTP are 0.4mM, TaKaRa company. Pcr amplification loop parameter is as follows: 94 DEG C of denaturations 5 minutes, 94 DEG C of sex change 30 seconds, anneal 30 seconds, 72 DEG C extend 2 points 30 seconds, after 30 circulations, extend again 5 minutes. 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) purifying amplified production:

Get 50 μ lPCR amplified productions and carry out agarose gel electrophoresis separation, blade cuts object band. Reclaiming kit (Omega company) according to E.Z.N.A. gel requires the DNA that carries out object band to reclaim purifying.

4) order-checking:

Taking reclaim after product as template, use sequencing primer according to CEQ^TMDTCS-QuickStartKit sequencing kit (Beckman company of the U.S.) requires to check order PCR reaction, reaction finish and purifying after, separate the sequence with 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)
		Extron 4	TTGCTGTTAAGGGAATTTGTAGGTAAGATA(SEQ ID NO.18)
Extron 5	TAGTGGTCTATTTTGTTATTCATTCAT(SEQ ID NO.19)
		Exon 6	TTCCAGTTTCTATGTTG(SEQ ID NO.20)
Exon 7	ACCCGGTGATGGTAGAGGTT(SEQ ID NO.21)
		Extron 8	ACGGGATTTCCTCATCTG(SEQ ID NO.22)
Extron 9	CGATACACTGAACAGTTATTGC(SEQ ID NO.23)

5) data analysis:

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

By compare of analysis, in a duplicate samples, find that the nucleotides of the 445th of CYP2C9 gene coding region becomes A (as shown in Figure 1) from G, this sudden change is positioned at the 3rd extron of CYP2C9 gene. Infer accordingly in the protein of this CYP2C9 gene code, the 149th amino acids sports threonine (T) by alanine (A). The international P450 of this new 445 sudden changes (445G > A) Yi Bei NK confirms called after neomorph CYP2C9*46.

In the present embodiment, exemplarily provide the method in qualification new mutation site. Those skilled in the art can clearly learn to detect specifically according to foregoing and in testing sample, comprise the method corresponding to the 1001st nucleotides of SEQIDNO.1: the nucleic acid in sample separation, under corresponding experiment condition, carry out amplified reaction in the present embodiment, primer uses primer pair SEQIDNO.6 and 7; With sequencing primer SEQIDNO.17 to amplification product check order; Sequencing result and wild type result are compared, analyzed the nucleotides corresponding to the 1001st site of 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 has obvious decline than the metabolic activity of wild type, the metabolic activity of * 3 types is than * 2 types lower (referring to bibliography 18,19,21,22). Therefore, existing one is like this known together in the art: the expressed enzyme of same genotype can represent the 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 metabolic activity of the expressed enzyme of this genotype to other substrate medicine (as, the metabolic activity of the metabolic activity of enzyme expressed this genotype and the expressed enzyme of wild type can be compared).

In the present embodiment, according to above-mentioned mutational site, taking wild type CYP2C9 (* 1) gene as template, rite-directed mutagenesis the nucleotides (becoming A from G) of the 445th of CYP2C9 gene coding region, construction of expression vector is expressed saltant type CYP2C9 albumen (called after A149T), utilize the active detection kit of CYP2C9 (containing CYP2C9 specific substrate) of Promega company to detect the enzymatic activity of this saltant type CYP2C9, with judgement, compared with wild type CYP2C9 (* 1), whether its enzymatic metabolic activity changes.

1) expression of genes of interest

Taking wild type CYP2C9 (* 1) plasmid vector (professor Zhou Shufeng by American South University of Florida presents) as template, the 445th nucleotides of rite-directed mutagenesis CYP2C9 gene coding region, obtain the ORF region sequence that comprises the nucleotides 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) be connected to respectively double gene expression vector pIRES (purchased from Clontech company, article No. 631605) A, B site in, two genes are positioned under the control of same CMV promoter, finally obtain double gene expression vector pIRES-Gluc-2C9. By 8*10⁴The COS-7 cell (this cell line has been widely used in the analysis of CYP2C9 external activity, refers to bibliography 18,21) in individual cercopithecus aethiops renal epithelial cell source is laid on 24 orifice plates; After incubated overnight, utilize liposome lip2000 transfection 500ng plasmid vector pIRES-Gluc-2C9, to express destination protein CYP2C9 (A149T) and internal reference Protein G luc.

2) enzymatic activity detects

Continue to cultivate after 48 hours, add the fresh DMEM culture medium of 200 μ l (containing 10%FBS), and with reference to the operation instruction of the CYP2C9 of Promega company detection kit (article No.: V8791), add the substrate Luciferin-H of 4 μ lCYP2C9, mix rear continuation and cultivate and after 8 hours, get 50 μ l culture mediums and add isopyknic Promega kit (article No.: V8791) to detect buffer solution. In this detection buffer solution, contain firefly luciferase; After reacting with firefly luciferase, the product D-luciferin of substrate Luciferin-H after CYP2C9 metabolism can produce fluorescence. Utilize the GLOMAX20/20Luminometer (the luminous detector of GLOMAX20/20) of Promega company to detect fluorescence signal. Get again 7 μ l culture mediums and detect kit (NEB company, article No.: E3300) operation instruction with reference to Gluc, add isopyknic detection buffer solution, utilize GLOMAX20/20Luminometer to detect Gluc fluorescence signal. Using the fluorescence signal/internal reference Gluc fluorescence signal value directly related with CYP2C9 as final experimental data, with wild type (* 1 type) CYP2C9 Data Comparison, can analyze the variation of the metabolic activity of mutant to be measured. Every kind of experimental subjects is carried out three parallel laboratory tests, and experimental result is in table 3.

Introduction about Luciferin-H:

The typical metabolic activity of CYP2C9 is to carry out hydroxylation (referring to bibliography 23,24, related content mode is by reference bonded to herein) to aromatic ring structure. The detection substrate Luciferin-H applying in the present embodiment 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 be luminous with detecting that firefly luciferase in reagent react, the power of signal is directly related with the amount of product D-luciferin, and the latter has been reflected the enzymatic activity of CYP2C9 albumen to be checked. There are some researches show, utilize this detection method can effectively reflect the metabolic characteristics of CYP2C9 to Common drugs, mainly comprise CYP2C9 probe medicament warfarin, orinase, Diclofenac, phenytoinum naticum, brufen; Also comprise (referring to bibliography 22,24,25) such as part Common drugs sulfaphenazolum, troglitazone, Azamulin, piroxicam, sulfaphenazolum, Miconazole, Fluvoxamines simultaneously. Therefore the data of this experimental program can generally be applicable to other medicine through CYP2C9 metabolism, comprise the medicine of listing before in 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 decline maximum (approximately declining 93%), this numerical value and trend with have been reported consistent (referring to bibliography 18,19,21,22).

Utilize this vitro detection system to find out: the metabolic activity of A149T is low more about 78% than wild type, 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 on dosage carrying this genotypic individuality, as reduced the use amount and the generation of avoiding adverse drug reaction of medicine. This medicine adjustment by gene targeting is even more important for the narrow medicine (as warfarin, phenytoinum naticum etc.) for the treatment of window.

Sequence:

SEQIDNO.1: genomic dna sequence

TTACTCTGTAGCTCAGGCTGGAGTGCAGTGGTACAATCTTGGCTCACTGCAACCTCCATCTCCCAGGTCCCCATTCAAGAAATTCTCCTGCCTCAGTCCCCCAAGTAGCTAGCATTACAGGCATGCACCACCATGCTCAGCTAATTTTTGTATTTTTAGTAGAGACGTGGTATCACCTTGTTGGCCAGGCTGGTCTTGAACTCCTGACCTTGTGATCCACCTGCCTTGGCCTCCCAAAGTGTTGGGATTACAGGCAGGAGCCACCACACCTGGCCGTTTGTTTAAAATAGAGTAAATAGACCTGCTGAATATGTTGATGTGAGTATTAATTGTAATCTGCATAGCAATTGTCTGACCATTGCCTTGAACATCACAGGCCATCTGAGTGGCAAGTATAATCATCATCATGTTTCTATTTAAAATTCAGAAATATTTGAAGCCTGTGTGGCTGAATAAAAGCATACAAATACAATGAAAATATCATGCTAAATCAGGCTTAGCAAATGGACAAAATAGTAACTTCGTTTGCTGTTATCTCTGTCTACTTTCCTAGCTCTCAAAGGTCTATGGCCCTGTGTTCACTCTGTATTTTGGCCTGAAACCCATAGTGGTGCTGCATGGATATGAAGCAGTGAAGGAAGCCCTGATTGATCTTGGAGAGGAGTTTTCTGGAAGAGGCATTTTCCCACTGGCTGAAAGAGCTAACAGAGGATTTGGTAGGTGTGCATGTGCCTGTTTCAGCATCTGTCTTGGGGATGGGGAGGATGGAAAACAGAGACTTACAGAGCTCCTCGGGCAGAGCTTGGCCCATCCACATGGCTGCCCAGTGTCAGCTTCCTCTTTCTTGCCTGGGATCTCCCTCCTAGTTTCGTTTCTCTTCCTGTTAGGAATTGTTTTCAGCAATGGAAAGAAATGGAAGGAGATCCGGCGTTTCTCCCTCATGACGCTGCGGAATTTTGGGATGGGGAAGAGGAGCATTGAGGACCGTGTTCAAGAGGAAACCCGCTGCCTTGTGGAGGAGTTGAGAAAAACCAAGGGTGGGTGACCCTACTCCATATCACTGACCTTACTGGACTACTATCTTCTCTACTGACATTCTTGGAAACATTTCAGGGGTGGCCATATCTTTCATTATGAGTCCTGGTTGTTAGCTCATGTGAAGCGGGGGTTTGAAGCTGAGAGCCAAGGGAATTTGCACATATTTGTGCTGTGTGTGTACAGGCATGATTGTGCGTACAGTGTGGGTATAAAAGGTTCATTTAATCCCATGTTCTCCTGAACTTTGCTTTTTTGCTTTCAAATAAGAAATGATGAATATAGATTTTGAGTTCATTTTTTGAAAGAGTTAAAGAGCAGTGTTTTTCCCATTACCTATTCCAGAACATGTCACCAGAGAATACTTGACAAGTCAACATGGTGGGAATGGCCCTATCATACCCATATGGAGCATGAACCAAATGGCATGTGCTTTTATTTAATTGGACTGTGTTTGTATGGTCAGCCTCACTGACTTCTCTGGGGTTTCTTTTAGGCCCGTGCTTGCCATTCTGGCCAGTAATGACATTCTACAGTTTTTATTGCTTAGGCATATCTTAGTGCAGTTCTCATCAATTATTATTTCTCTGTAAACACAGCATTATTTTAAAAATAGTATTAATTATTTCTTGTTACTGTATTGATTTATATATTTTCAGTAAATACATCCTGTAGCATAATTCTGTGAAATACCCAAATGTCAATTTATAAAATGATTTATTTAACAAGATTTTACTTATTAGTAATAACTCTGTAATCTGCATTCCCTATGTATGATTTGGCTCTGTTTCAGTTTTGCTTATCTCTTTCCAACCATATTTATGAAATTTTGGCTTAGAAATTTATGTTAATTATTTTTTTTCCATGGCCAACTCTACTCATCTATGAAGTTTTACAATGAATCTGTTTATCAGCTTGGATACCAAATTACCTTGTTTTTAAATTCTGTTTTCCCAATGAAGTTAA

SEQIDNO.2: coded sequence

ATGGATTCTCTTGTGGTCCTTGTGCTCTGTCTCTCATGTTTGCTTCTCCTTTCACTCTGGAGACAGAGCTCTGGGAGAGGAAAACTCCCTCCTGGCCCCACTCCTCTCCCAGTGATTGGAAATATCCTACAGATAGGTATTAAGGACATCAGCAAATCCTTAACCAATCTCTCAAAGGTCTATGGCCCTGTGTTCACTCTGTATTTTGGCCTGAAACCCATAGTGGTGCTGCATGGATATGAAGCAGTGAAGGAAGCCCTGATTGATCTTGGAGAGGAGTTTTCTGGAAGAGGCATTTTCCCACTGGCTGAAAGAGCTAACAGAGGATTTGGAATTGTTTTCAGCAATGGAAAGAAATGGAAGGAGATCCGGCGTTTCTCCCTCATGACGCTGCGGAATTTTGGGATGGGGAAGAGGAGCATTGAGGACCGTGTTCAAGAGGAAACCCGCTGCCTTGTGGAGGAGTTGAGAAAAACCAAGGCCTCACCCTGTGATCCCACTTTCATCCTGGGCTGTGCTCCCTGCAATGTGATCTGCTCCATTATTTTCCATAAACGTTTTGATTATAAAGATCAGCAATTTCTTAACTTAATGGAAAAGTTGAATGAAAACATCAAGATTTTGAGCAGCCCCTGGATCCAGATCTGCAATAATTTTTCTCCTATCATTGATTACTTCCCGGGAACTCACAACAAATTACTTAAAAACGTTGCTTTTATGAAAAGTTATATTTTGGAAAAAGTAAAAGAACACCAAGAATCAATGGACATGAACAACCCTCAGGACTTTATTGATTGCTTCCTGATGAAAATGGAGAAGGAAAAGCACAACCAACCATCTGAATTTACTATTGAAAGCTTGGAAAACACTGCAGTTGACTTGTTTGGAGCTGGGACAGAGACGACAAGCACAACCCTGAGATATGCTCTCCTTCTCCTGCTGAAGCACCCAGAGGTCACAGCTAAAGTCCAGGAAGAGATTGAACGTGTGATTGGCAGAAACCGGAGCCCCTGCATGCAAGACAGGAGCCACATGCCCTACACAGATGCTGTGGTGCACGAGGTCCAGAGATACATTGACCTTCTCCCCACCAGCCTGCCCCATGCAGTGACCTGTGACATTAAATTCAGAAACTATCTCATTCCCAAGGGCACAACCATATTAATTTCCCTGACTTCTGTGCTACATGACAACAAAGAATTTCCCAACCCAGAGATGTTTGACCCTCATCACTTTCTGGATGAAGGTGGCAATTTTAAGAAAAGTAAATACTTCATGCCTTTCTCAGCAGGAAAACGGATTTGTGTGGGAGAAGCCCTGGCCGGCATGGAGCTGTTTTTATTCCTGACCTCCATTTTACAGAACTTTAACCTGAAATCTCTGGTTGACCCAAAGAACCTTGACACCACTCCAGTTGTCAATGGATTTGCCTCTGTGCCGCCCTTCTACCAGCTGTGCTTCATTCCTGTCT

SEQIDNo.3: protein sequence

MDSLVVLVLCLSCLLLLSLWRQSSGRGKLPPGPTPLPVIGNILQIGIKDISKSLTNLSKVYGPVFTLYFGLKPIVVLHGYEAVKEALIDLGEEFSGRGIFPLAERANRGFGIVFSNGKKWKEIRRFSLMTLRNFGMGKRSIEDRVQEETRCLVEELRKTKASPCDPTFILGCAPCNVICSIIFHKRFDYKDQQFLNLMEKLNENIKILSSPWIQICNNFSPIIDYFPGTHNKLLKNVAFMKSYILEKVKEHQESMDMNNPQDFIDCFLMKMEKEKHNQPSEFTIESLENTAVDLFGAGTETTSTTLRYALLLLLKHPEVTAKVQEEIERVIGRNRSPCMQDRSHMPYTDAVVHEVQRYIDLLPTSLPHAVTCDIKFRNYLIPKGTTILISLTSVLHDNKEFPNPEMFDPHHFLDEGGNFKKSKYFMPFSAGKRICVGEALAGMELFLFLTSILQNFNLKSLVDPKNLDTTPVVNGFASVPPFYQLCFIPV

SEQIDNO.24 nucleic acid fragment

CGTGTTCAAGAGGAAACCCGCTGCCTTGTGG

SEQIDNO.25 nucleic acid fragment

AGGACCGTGTTCAAGAGGAAACCCGCTGCCTTGTGGAGGAG

SEQIDNO.26 nucleic acid fragment

CATTGAGGACCGTGTTCAAGAGGAAACCCGCTGCCTTGTGGAGGAGTTGAG

SEQIDNO.27 nucleic acid fragment

AGGAGCATTGAGGACCGTGTTCAAGAGGAAACCCGCTGCCTTGTGGAGGAGTTGAGAAAAA

SEQIDNO.28 oligonucleotide sequence

CAAGGCAGCGGGTTTC

SEQIDNO.29 oligonucleotide sequence

CTCCACAAGGCAGCGGGTTTC

SEQIDNo.30 oligonucleotide sequence

ACTCCTCCACAAGGCAGCGGGTTTC

SEQIDNO.31 oligonucleotide sequence

TCTCAACTCCTCCACAAGGCAGCGGGTTTC

SEQIDNO.32 oligonucleotide sequence

CGGGTTTCCTCTTGAAC

SEQIDNO.33 oligonucleotide sequence

GCAGCGGGTTTCCTCTTGA

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Claims (7)

1. nucleic acid fragment, described nucleic acid fragment comprises prominent corresponding to the 1001st of SEQIDNO.1Displacement point, and be at least 10 continuous nucleotides in the nucleotide sequence shown in SEQIDNO.1 or itsComplementary series, wherein the nucleotides of the 1001st is A; Or described nucleic acid fragment comprises corresponding to SEQIDThe mutational site of the 445th of NO.2, and be in the nucleotide sequence shown in SEQIDNO.2 at least10 continuous nucleotides or its complementary series, wherein the nucleotides of the 445th is A.

2. nucleic acid fragment according to claim 1, is characterized in that, the length of described nucleic acid fragmentIt is 10-100,100-200, a 200-500 or 500-1000 nucleotides.

3. nucleic acid fragment according to claim 2, is characterized in that, the length of described nucleic acid fragmentIt is 10-20,20-30,30-40,40-50,50-60, a 60-100 or 100-300 nucleotides.

4. nucleic acid fragment according to claim 1, is characterized in that, described nucleic acid fragment is SEQSequence shown in IDNO.1,2,24-27.

For detection of and/or the kit of analysis list base mutation, comprise claim 1-4 any one instituteThe nucleic acid fragment of stating.

6. the nucleic acid fragment described in claim 1-4 any one detects CYP2C9 gene mutation in preparationApplication in preparation.

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