CN103045615B - Comprise the CYP2C9 gene fragment of 146A > G sudden change, coded protein fragments and application thereof - Google Patents
Comprise the CYP2C9 gene fragment of 146A > G sudden change, coded protein fragments and application thereof Download PDFInfo
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Abstract
The invention belongs to field of biology, relate to single base mutation.More specifically, the present invention relates to the mutational site of the 146th that CYP2C9 gene corresponds to SEQ ID NO.2, described site sports G by the A of wild-type, comprises the nucleic acid fragment in this mutational site, its protein fragments of encoding and application thereof.Present invention also offers the allele specific oligonucleotide, test kit and the detection method that detect described mutational site.
Description
Technical field
The invention belongs to field of biology, relate to single base mutation.More specifically, the present invention relates to CYP2C9 gene relative to the 1001st of SEQ ID NO.1 or the mutational site of the 146th of SEQ ID NO.2, comprise 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 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 tolbutamide, S-warfarin, Phenytoin Sodium Salt, Glipizide, U26452, holder draw the medicines (see 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).
CYP2C9 gene has height polymorphism.So far, named allelotrope has 35 kinds (http://www.cypalleles.ki.se) in the world, removing wild-type (CYP2C9*1) outward, having 34 kinds of mutation types can cause CYP2C9 Argine Monohydrochloride composition to change, and also has multiple newfound sudden change not yet to be named in addition.Study more and the mutation type that clinical meaning is larger mainly comprises following 10 kinds: CYP2C9
*2,
*3,
*5,
*6,
*8,
*11,
*12,
*13,
*14,
*16, wherein the widest, that most study, Chinese population research data are the relatively the abundantest saltant type of ethnic group distribution is CYP2C9*2 (430C > T), CYP2C9*3 (1075A > C) (see reference 6-17,20).
According to current clinical studies show, this polymorphism of CYP2C9 gene is the major cause causing CYP2C9 enzymic activity greatly different between individuals, therefore carrying the huge 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, study CYP2C9 gene pleiomorphism and will provide important scientific basis (see reference 18,19,21,22) to clinical rational drug use to the impact of curative effect of medication.
Summary of the invention
The object of this invention is to provide the new single base mutation site of CYP2C9 gene, comprise the nucleic acid fragment in this mutational site, its protein fragments of encoding 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 SEQ IDNO.1, and be at least 10 continuous nucleotides in the nucleotide sequence shown in SEQ ID NO.1, wherein the Nucleotide of the 1001st is G; Or described nucleic acid fragment comprises the mutational site of the 146th corresponding to SEQ ID NO.2, and is at least 10 continuous nucleotides in the nucleotide sequence shown in SEQ ID NO.2, and wherein the Nucleotide of the 146th is G; Or be the complementary sequence of above-mentioned nucleic acid fragment.
Second aspect of the present invention be to provide with containing corresponding to the 1001st of SEQ ID NO.1 or correspond to the allelotrope fragment in the mutational site of the 146th or the allele specific oligonucleotide of all or part of hybridization of its complementary sequence of SEQ ID NO.2, wherein the Nucleotide in the mutational site of the 1001st of SEQ ID NO.1 or the 146th of SEQ ID NO.2 is G; Described allelotrope fragment is at least 10 continuous nucleotides in SEQID NO.1 or the nucleotide sequence shown in SEQ ID NO.2 or its complementary sequence.
3rd aspect of the present invention is to provide for detecting 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 SEQ IDNO.4 and/or SEQ ID NO.5 and/or the sequence fragment shown in SEQ ID NO.16.
4th aspect of the present invention is to provide nucleic acid fragment of the present invention or oligonucleotide is detecting the application in CYP2C9 transgenation, and wherein said nucleic acid fragment or oligonucleotide are used as probe or primer.
5th aspect of the present invention is to provide a kind of medication guide, comprises the single base mutation corresponding to the 1001st of SEQ ID NO.1 or the 146th of SEQ ID NO.2 detecting CYP2C9 gene in testing sample; According to the sudden change detected, adjust by the dosage of the medicine of CYP2C9 metabolism.
6th aspect of the present invention is to provide the method for analysis of nucleic acids, described method comprises in the nucleic acid comprising corresponding to the sequence of SEQ ID NO.1 analyzed in testing sample the Nucleotide corresponding to the 1001st, or analyzes in the nucleic acid comprising corresponding to the sequence of SEQ ID NO.2 in testing sample the Nucleotide corresponding to the 146th.
7th aspect of the present invention is to provide CYP2C9 albumen or its fragment or varient, and described protein sequence is the sequence shown in SEQ ID NO.3; Described fragment or varient comprise the glycine of the 49th corresponding to SEQ ID NO.3, and are at least 10 continuous amino acids of the aminoacid sequence shown in SEQ ID NO.3.
The invention provides the CYP2C9 gene and encoding sequence that comprise new single base mutation.The 146th Nucleotide that this gene is corresponding to SEQ ID NO.2 sports G (146A > G) by A, thus the amino acid causing it to encode is glycine by Aspartic acid mutations, namely correspond to the glycine of the 49th of SEQ ID NO.3.The CYP2C9 albumen (called after D49G) of this sudden change declines to drug metabolism activity.The medication of this single base mutation to the individuality carrying this mutational site has directive significance.
Accompanying drawing explanation
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, the present invention is described, but content of the present invention is not limited thereto.
As illustrated without other, " nucleic acid fragment " of the present invention is made up of Nucleotide or its analogue, can be the fragment of DNA, RNA or its analogue; Can be strand or double-strand; Can be natural (as genomic) or synthesis.
In the present invention, " sudden change " refers to the gene detected, and 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 146th in sequence shown in the 1001st of sequence shown in SEQ ID NO.1 or SEQ ID NO.2.
In the present invention, " allele-specific " refers to hybridize with allelotrope specifically, as hybridized under high stringency conditions, makes to identify that the 146th Nucleotide corresponding to sequence shown in the 1001st of sequence shown in SEQ ID NO.1 or SEQ IDNO.2 is G.
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 are known, and described mutational site both can show in genomic dna, also can performance in encoding sequence (i.e. CDS, corresponding to mRNA sequence).Those skilled in the art, according to detected sample, can detect this mutational site on genomic dna or mRNA level in-site.In the application, SEQ ID NO.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 SEQ ID NO.1 is the mutational site that the present invention relates to.SEQ ID NO.2 is the encoding sequence of the CYP2C9 gene with described mutational site, and wherein the 146th is the mutational site that the present invention relates to.Those skilled in the art are known, and in this article, the 146th site corresponding to SEQID NO.2 and the 1001st the site synonym corresponding to SEQ ID NO.1 are used mutually.
In the present invention, Nucleotide and amino acid whose abbreviation adopt abbreviation mode well known in the art, and as in Nucleotide, A represents VITAMIN B4, G represents guanine, and C represents cytosine(Cyt), and T represents thymus pyrimidine.In 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 the new single base mutation site based on CYP2C9 gene.Described mutational site is the coding region being positioned at CYP2C9 gene, and corresponding to the 146th of SEQ ID NO.2, this site sports G (146A > G) by the A of wild-type; In addition, be glycine (D49G) by the 49th of albumen of the CYP2C9 genes encoding of this sudden change by Aspartic acid mutations.
In first, the invention provides nucleic acid fragment, described nucleic acid fragment comprises the mutational site of the 1001st corresponding to SEQ IDNO.1, and is at least 10 continuous nucleotides in the nucleotide sequence shown in SEQ ID NO.1, and wherein the Nucleotide of the 1001st is G; Or described nucleic acid fragment comprises the mutational site of the 146th corresponding to SEQ ID NO.2, and is at least 10 continuous nucleotides in the nucleotide sequence shown in SEQ ID NO.2, and wherein the Nucleotide of the 146th is G; Or be the complementary sequence of above-mentioned nucleic acid fragment.
In one embodiment, the length of described nucleic acid fragment can be as 10-100,100-200,200-500,500-1000 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 can be positioned at any position of described nucleic acid fragment.
In another embodiment, described nucleic acid fragment is the sequence shown in SEQ ID NO.1.
In another embodiment, described nucleic acid fragment is the sequence shown in SEQ ID NO.2.
In other embodiments, shown nucleic acid fragment can be the sequence shown in SEQ ID NO.24-31.
Second aspect of the present invention be to provide with containing corresponding to the 1001st of SEQ ID NO.1 or correspond to the allelotrope fragment in the mutational site of the 146th or the allele specific oligonucleotide of all or part of hybridization of its complementary sequence of SEQ ID NO.2, wherein the Nucleotide in the mutational site of the 1001st of SEQ ID NO.1 or the 146th of SEQ ID NO.2 is G; Described allelotrope fragment is at least 10 continuous nucleotides in SEQID NO.1 or the nucleotide sequence shown in SEQ ID NO.2 or its complementary sequence.
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 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 Nucleotide, 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 a preferred embodiment, be that mutational site appears at 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 does not need and template complete complementary, but should synthesize to instruct DNA with template Complementary hybridization.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 oligonucleotide is the sequence as shown in SEQ ID NO.32-38.
Based on this, 3rd aspect of the present invention is to provide for detecting 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 SEQ ID NO.4 and/or SEQ ID NO.5 and/or the sequence fragment shown in SEQ ID NO.16.
4th aspect of the present invention is to provide nucleic acid fragment of the present invention or oligonucleotide for detecting the application of CYP2C9 transgenation, and wherein said nucleic acid fragment or oligonucleotide are used as probe or primer.
5th aspect of the present invention is to provide medication guide, comprises the single base mutation corresponding to the 1001st of SEQ ID NO.1 or the 146th of SEQ ID NO.2 detecting CYP2C9 gene in testing sample.When the CYP2C9 gene detected is when the site corresponding to the 1001st of SEQ ID NO.1 or the 146th of SEQ ID NO.2 is G, adjust the dosage of the medicine through CYP2C9 metabolism accordingly.In the particular embodiment, when CYP2C9 gene is when the site of the 146th of SEQ ID NO.2 is G, the CYP2C9 protease activity of this genes encoding declines, therefore needs 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, Acenocoumarol, anticonvulsive drug or mephenytoin; Antidiabetic drug, as tolbutamide, nateglinide, pioglitazone or rosiglitazone; Antiepileptic drug, as Phenytoin Sodium Salt or zonisamide; Antimalarial drug/antiparasitic, as amodiaquine, Tirian 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, pyramidon, quinizine, celecoxib, flurbiprofen, Ibuprofen BP/EP, indomethacin, lornoxicam, mefenamic acid, Naproxen Base, piroxicam or tenoxicam; Anodyne, as, Loperamide, methadone or morphine; Proton pump inhibitor, as lansoprazole or omeprazole; Tranquilizer, as, clobazam, Mephogarbital or Zopiclone.
6th aspect of the present invention is to provide the method for analysis of nucleic acids, and described method comprises in the nucleic acid comprising corresponding to the sequence of SEQ ID NO.1 analyzed in testing sample the Nucleotide that corresponds to the 1001st or analyzes in the nucleic acid comprising corresponding to the sequence of SEQ ID NO.2 in testing sample the Nucleotide corresponding to the 146th.
In one embodiment, described method can be sequencing, comprise and being separated and the nucleotide sequence measured from genomic dna or RNA, analyze and wherein comprise corresponding to corresponding to the Nucleotide of the 1001st or comprising corresponding to corresponding to whether the Nucleotide of the 146th is G in the nucleic acid of the sequence of SEQ ID NO.2 in the nucleic acid of the sequence of SEQ ID NO.1.Sequencing can be any available sequence measurement known in the art.Sequencing primer can design according to the general knowledge of those skilled in the art, as the upstream and downstream appropriate position design primer in site to be detected, with the fragment of expanding packet containing this site to be measured, thus judges the Nucleotide in this site.Also oligonucleotide of the present invention can be adopted as primer sequence.
In another embodiment, described method is the method utilizing probe hybridization, and whether the Nucleotide comprised in identification and detection sample specifically corresponding to corresponding to the 146th in the Nucleotide corresponding to the 1001st in the nucleic acid of the sequence of SEQ ID NO.1 or the nucleic acid comprising corresponding to the sequence of SEQ ID NO.2 is G; The probe adopted in described method is oligonucleotide of the present invention.Such as, from testing sample, isolate nucleic acid, under the condition allowing probe and the specific target sequence that may exist in nucleic acid to hybridize, probe is contacted with nucleic acid; The hybridization that can be detected can be realized by the probe that detectable reagent is labeled by using; Such as, form the enzyme that can detect product with radio isotope, fluorescence dye or energy catalysis and carry out label probe.Label probe, to detect in sample with label probe the method that whether there is target sequence be all well-known to those skilled in the art.
In a kind of concrete embodiment, the method detecting the Nucleotide of the 1001st corresponding to SEQ ID NO.1 is provided, comprises with Taqman probe SNP detection method:
1) design primer to be used for specific amplification and to comprise the PCR primer of the 1001st corresponding to SEQ ID NO.1, design two Taqman-MGB probes, respectively for A and G (as shown in the SEQ ID NO.37 sequence) allelotrope of the 1001st corresponding to SEQ ID NO.1 simultaneously.
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 more than 4 or 4 and match continuously, avoid primer self to form pili annulati card structure;
(3) primer length is at 18 to 24 Nucleotide;
(4) Tm value at 55-65 DEG C, GC content at 40%-60%;
(5) the Tm value difference between primer avoids exceeding 2 DEG C;
(6) 3 ' end of primer is avoided using base A, and 3 ' of primer holds the base avoiding appearance more than 3 or 3 consecutive identical;
(7) pcr amplified fragment length is at 50bp-150bp;
(8) last 5 Nucleotide of prime end can not have G and C more than 2.
Taqman MGB probe design principle is:
(1) 5 ' end of probe is avoided occurring G;
(2) Tm value should be 65-67 DEG C;
(3) shorten Taqman MGB probe, but probe length is no less than 13bp as far as possible;
(4) avoid the base duplicated, especially G base as far as possible, avoid the G of appearance more than 4 or 4 to repeat;
(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 mark two allelotrope.
2) utilize above-mentioned primer and probe, real-time quantitative PCR is carried out to sample to be tested.
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 detects fluorescent signal).
3) data analysis.
According to the power of sample two kinds of fluorescence, analysis design mothod result, judges whether sample to be tested CYP2C9 gene exists 146A > G and suddenly change.
In the present invention, described sample can be any sample comprising 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 with DNA or RNA for target compound.Those skilled in the art are known, when taking DNA as detection target compound, analyze in the nucleic acid comprising corresponding to the sequence of SEQ ID NO.1 in testing sample the Nucleotide corresponding to the 1001st, the probe used or primer are according to the sequences Design of SEQ ID NO.1; When being when detecting target compound with RNA, analyze in the nucleic acid comprising corresponding to the sequence of SEQID NO.2 in testing sample the Nucleotide corresponding to the 146th, the probe used or primer are according to the sequences Design of SEQ ID NO.2.
7th aspect of the present invention is to provide CYP2C9 albumen or its fragment or varient, and described protein sequence is the sequence shown in SEQ ID NO.3; Described fragment or varient comprise the glycine of the 49th corresponding to SEQ ID NO.3, and are at least 10 continuous amino acids of the aminoacid sequence shown in SEQ ID NO.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 exons, the mutational site of screening CYP2C9 gene
1) DNA is extracted:
5ml vein EDTA anticoagulated blood sample is taked from every measured; Then according to common salting-out process and/or adopt special DNA extraction kit (DNA extraction kit of purchased from American 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 obtained.Described amplimer to sequence in table 1.
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 are 0.2 μM, dNTP is the LATaq archaeal dna polymerase 1.5U of 0.4mM, TaKaRa company.Pcr amplification loop parameter is as follows: 94 DEG C of denaturations 5 minutes, and 94 DEG C of sex change 30 seconds, anneal 30 seconds, 72 DEG C extend 2 points 30 seconds, extend 5 minutes again after 30 circulations.Annealing temperature is relevant to primer length, and actual temp is in table 1.
Use the GeneAmp PCR System 9700 amplification instrument amplification of American AB I company.
Table 1: sequencing primer to and annealing temperature
3) purifying amplified production:
Get 50 μ l pcr amplification products and carry out agarose gel electrophoresis separation, blade cuts object band.Reclaim test kit (Omega company) according to E.Z.N.A. gel and require that the DNA carrying out object band reclaims purifying.
4) check order:
With the product after recovery for template, use sequencing primer according to CEQ
tMdTCS-Quick Start Kit sequencing kit (Beckman company of the U.S.) requires that carrying out order-checking PCR reacts, reaction terminates and after purifying, carries out being separated 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) |
| 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.
By compare of analysis, find that the Nucleotide of the 146th of CYP2C9 gene coding region becomes G (as shown in Figure 1) from A in a sample, this sudden change is positioned at the 1st exon of CYP2C9 gene.Infer in the protein of this CYP2C9 genes encoding accordingly, the 49th amino acids sports glycine (G) by aspartic acid (D).This international P450 NK of the 146th new sudden change (146A > G) Yi Bei confirms called after neomorph CYP2C9*37.
The method in qualification new mutant site is exemplarily given in the present embodiment.Those skilled in the art clearly can learn the method detecting specifically and comprise the 1001st Nucleotide corresponding to SEQ ID NO.1 in testing sample according to foregoing: the nucleic acid in sample separation, carry out amplified reaction under experiment condition corresponding in the present embodiment, primer uses primer pair SEQ ID NO.4 and 5; With sequencing primer SEQ IDNO.16, the product of amplification is checked order; By sequencing result and wild-type results comparison, analyze the Nucleotide in the 1001st site corresponding to SEQ ID NO.1.
Embodiment 2: enzymes metabolism activation analysis
According to existing result of study, wild-type is all higher to the metabolic activity of various medicine, and
*the metabolic activity of 2 types has obvious decline than the metabolic activity of wild-type,
*the metabolic activity ratio of 3 types
*2 types lower (see reference 18,19,21,22).Therefore, existing one is like this known together in the art: the enzyme expressed by same genotype can represent the metabolic activity to other substrate medicine to the metabolic activity of specific substrate.Thus, enzyme expressed by a certain genotype can analogize the metabolic activity (e.g., the metabolic activity of the enzyme metabolic activity of enzyme this genotype expressed by and wild-type expressed by can be compared) of the enzyme expressed by this genotype to other substrate medicine to specific substrate metabolic activity data.
In the present embodiment, according to above-mentioned mutational site, with wild-type CYP2C9 (
*1) gene is template, the rite-directed mutagenesis Nucleotide (becoming G from A) of the 146th of CYP2C9 gene coding region, construction of expression vector expresses saltant type CYP2C9 albumen (called after D49G), utilize the CYP2C9 activity detection kit of Promega company (containing CYP2C9 specific substrate) to detect the enzymic activity of this saltant type CYP2C9, with judge with wild-type CYP2C9 (
*1) compare, whether its enzymatic metabolism activity changes.
1) expression of goal gene
With wild-type CYP2C9 (
*1) plasmid vector (being presented by professor Zhou Shufeng of American South University of Florida) is template, 146th Nucleotide of rite-directed mutagenesis CYP2C9 gene coding region, namely the ORF region sequence comprising the Nucleotide shown in SEQ ID NO.2 is obtained, by this goal gene and reference gene Gluc (a kind of secretor type luciferase, its translation product can be secreted in substratum, and fluorescent signal detected by particular agent box; Its skeleton carrier is pIRES pGluc-Basic, purchased from NEB company, article No. N8082S) be connected to double gene expression vector pIRES respectively (purchased from Clontech company, article No. 631605) A, B site in, under making two genes be positioned at the control of same CMV promoter, final acquisition double gene expression vector pIRES-Gluc-2C9.By 8*10
4the COS-7 cell (this cell strain has been widely used in the analysis of CYP2C9 external activity, refers to reference 18,21) of individual African green monkey kidney epithelial cell origin is laid on 24 orifice plates; After incubated overnight, utilize liposome lip2000 transfection 500ng plasmid vector pIRES-Gluc-2C9, to express target protein CYP2C9 (D49G) and internal reference Protein G luc.
2) Enzyme assay
Continue cultivation after 48 hours, add 200 μ l fresh DMEM medium (containing 10%FBS), and the operation instruction of reference Promega company CYP2C9 detection kit (article No.: V8791), add the substrate Luciferin-H of 4 μ l CYP2C9, continue cultivation after mixing and get 50 μ l substratum after 8 hours and add isopyknic Promega test kit (article No.: V8791) and detect damping fluid.Containing Photinus pyralis LUC in this detection damping fluid; Fluorescence can be produced after the product D-luciferin of substrate Luciferin-H after CYP2C9 metabolism and Photinus pyralis LUC react.The GLOMAX 20/20Luminometer of Promega company (GLOMAX 20/20 luminometer) is utilized to detect fluorescent signal.Get 7 μ l substratum again and detect kit (NEB company, article No.: E3300) operation instruction with reference to Gluc, add isopyknic detection damping fluid, utilize GLOMAX 20/20Luminometer to detect Gluc fluorescent signal.Using the fluorescent signal directly related with CYP2C9/internal reference Gluc fluorescent signal value as final experimental data, with wild-type (
*1 type) CYP2C9 Data Comparison, the change of the metabolic activity of mutant to be measured can be analyzed.Often kind of experimental subjects carries out three parallel laboratory tests, and experimental result is in table 3.
Introduction about Luciferin-H:
The exemplary metabolites activity of CYP2C9 can carry out hydroxylation (see reference 23,24, associated viscera is bonded to herein by way of reference) to aromatic ring structure.The detection substrate Luciferin-H applied in the present embodiment is containing aromatic ring structure, CYP2C9 can the 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 react with the Photinus pyralis LUC in detection reagent 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 effectively can reflect the metabolic characteristics of CYP2C9 to Common drugs, mainly comprise CYP2C9 probe medicament warfarin, tolbutamide, diclofenac, Phenytoin Sodium Salt, Ibuprofen BP/EP; Also comprise (see reference 22,24,25) such as part Common drugs sulfaphenazole, troglitazone, Azamulin, piroxicam, sulfaphenazole, miconazole, fluvoxamines simultaneously.Therefore the data of this experimental program generally can be applicable to other medicine through CYP2C9 metabolism, the medicine listed before comprising 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 have obvious decline (the nearly minimizing of nearly 60%),
*3 types decline maximum (approximately declining 93%), this numerical value and trend with have been reported consistent (see reference 18,19,21,22).
This vitro detection system is utilized to find out: D49G metabolic activity reduces about 87% than wild-type, and namely this sudden change can cause the metabolic activity of expressed enzyme obviously to reduce.Therefore, in practice, need to consider suitably to regulate on dosage carrying this genotypic individuality, as reduced the usage quantity of medicine and avoiding the generation of adverse drug reaction.This adjustment of the medicine by gene targeting is even more important for the medicine (as warfarin, Phenytoin Sodium Salt etc.) that treatment window is narrow.
Sequence:
SEQ ID NO.1: genomic dna sequence
AACTAATGTTTGGAAGTTTGTTTTATTTTGCTAAAATTGTTCTCAAGGCAGCTCTGGTGTAAGAGATAATACACCACGATGGGCATCAGAAGACCTCAGCTCAAATCCCAGTTCTGCCAGCTATGAGCTGTGTGGCACCAACAGGTGTCCTGTTCTCCCAGGGTCTCCCTTTTCCCATTTGAAAAATAAAAAATAACAATTCCTGCCTTCAGGAATTTTTTTTAGGGGGTTTAATGGTAAAGGTGTTTATATCTGCTAAGGTAATTTACTTGATATATGTTTGGTTATTTAAGATATATGAGTTATGTTAGCTATTTCATGTTTAGGCTGCTGTATTTTTAGTAGGCTATATTAAATATTTGAAAGGATTTCATTATAAAGAACAAAGTCTCCTAATCTTTGATATAGCATTGACATACTTTTTAAATATACAAGGCATAGAATATGGCCATTTCTGTTAAATCATATATTCCCAACTGGTTATTAATCTAAGAATTCAGAATTTTGAGTAATTGCTTTTGCATCAGATTATTTACTTCAGTGCTCTCAATTATGATGGTGCATTAGAACCATCTGGGTTAACATTTGTTTTTTATTACCAATACCTAGGCTCCAACCAAGTACAGTGAAACTGGAATGTACAGAGTGGACAATGGAACGAAGGAGAACAAGACCAAAGGACATTTTATTTTTATCTGTATCAGTGGGTCAAAGTCCTTTCAGAAGGAGCATATAGTGGACCTAGGTGATTGGTCAATTTATCCATCAAAGAGGCACACACCGAATTAGCATGGAGTGTTATAAAAGGCTTGGAGTGCAAGCTCATGGTTGTCTTAACAAGAAGAGAAGGCTTCAATGGATTCTCTTGTGGTCCTTGTGCTCTGTCTCTCATGTTTGCTTCTCCTTTCACTCTGGAGACAGAGCTCTGGGAGAGGAAAACTCCCTCCTGGCCCCACTCCTCTCCCAGTGATTGGAAATATCCTACAGATAGGTATTAAGGGCATCAGCAAATCCTTAACCAATGTAAGTATGCTCCTTCAGTGGCTTGCAAAAGGTAAGTAAATTCACCTGTATTTTTTAAATAAAGTGTATCCCTAGAGGTACATGTTACAAGAGGTAATGGTAAAGTAAAATACTTTGAAAGGCTTTTGTTGCCTTTTCCAGTCTGTCAGTGTCAGAAATAGTGGAATGAAACCATGTATTTTGTGAGTAGAGAAAGATTTGGGTCTTTGCATGTTAGATTCAAAATAACAAGTGTCAATAGTTTGAAAAGCTGTGTTCCTTCTTCATTTCATAACCATTTGCTATAATTTTTGGCTGAAGGTAAATGGTAAGGTATTGTGGGATCTGGTCAGCAGCCCACAAAGCAACTGGGCTCTCTCTTTTTTCCCAGGTGGATCGGCAGGTTGAGAAATAATAGACACACAAGATAGTGAAAGCTGGGTCCAGGGGGGTCACCGCCTTCTGGTCCCATGGTGCCAAGAATGCACTGGATATACCAGCATTTATTATTAAGTTTAGTGAGGGCAGGGGTAGGTTAGTGAGGGATTTAGGGTCATTTGATTATGAGGTGAGATGGTCACATGGGGATGAAGTAATTCTTTAACATAACATTTGTATGTAGAAGTACAGTACATTTGTATGTAGAAGTACAGTATACGGAGATAAGAATTTACAATATAGTGTGTGCGTCAGTAATTTCTAACAGAGCCTTAAAACAGAAACACAATCTTTCCATAACCTATGATTAGCAAGATATTAATCAGCAGTAACAATTGCAACAAAAGCTGGTTACAAACAATCCATGGAAATAGGATGTGAAGCTAGACAACCAGTTAGACCAGAAATTCTCAGAAGGGAGTATGCCTTAACCCTAAAGAGGCCTAGAAGAGCCATGGCAAGATGAGGGCATTTATAGCCCTATCTTATCCATATGGACAGGTGCCCCCCATGCGTCCATTTATAGGTTCTCCACAAAGGTCGCATTCCATTCCCAGAGCT
SEQ ID NO.2: encoding sequence
ATGGATTCTCTTGTGGTCCTTGTGCTCTGTCTCTCATGTTTGCTTCTCCTTTCACTCTGGAGACAGAGCTCTGGGAGAGGAAAACTCCCTCCTGGCCCCACTCCTCTCCCAGTGATTGGAAATATCCTACAGATAGGTATTAAGGGCATCAGCAAATCCTTAACCAATCTCTCAAAGGTCTATGGCCCTGTGTTCACTCTGTATTTTGGCCTGAAACCCATAGTGGTGCTGCATGGATATGAAGCAGTGAAGGAAGCCCTGATTGATCTTGGAGAGGAGTTTTCTGGAAGAGGCATTTTCCCACTGGCTGAAAGAGCTAACAGAGGATTTGGAATTGTTTTCAGCAATGGAAAGAAATGGAAGGAGATCCGGCGTTTCTCCCTCATGACGCTGCGGAATTTTGGGATGGGGAAGAGGAGCATTGAGGACCGTGTTCAAGAGGAAGCCCGCTGCCTTGTGGAGGAGTTGAGAAAAACCAAGGCCTCACCCTGTGATCCCACTTTCATCCTGGGCTGTGCTCCCTGCAATGTGATCTGCTCCATTATTTTCCATAAACGTTTTGATTATAAAGATCAGCAATTTCTTAACTTAATGGAAAAGTTGAATGAAAACATCAAGATTTTGAGCAGCCCCTGGATCCAGATCTGCAATAATTTTTCTCCTATCATTGATTACTTCCCGGGAACTCACAACAAATTACTTAAAAACGTTGCTTTTATGAAAAGTTATATTTTGGAAAAAGTAAAAGAACACCAAGAATCAATGGACATGAACAACCCTCAGGACTTTATTGATTGCTTCCTGATGAAAATGGAGAAGGAAAAGCACAACCAACCATCTGAATTTACTATTGAAAGCTTGGAAAACACTGCAGTTGACTTGTTTGGAGCTGGGACAGAGACGACAAGCACAACCCTGAGATATGCTCTCCTTCTCCTGCTGAAGCACCCAGAGGTCACAGCTAAAGTCCAGGAAGAGATTGAACGTGTGATTGGCAGAAACCGGAGCCCCTGCATGCAAGACAGGAGCCACATGCCCTACACAGATGCTGTGGTGCACGAGGTCCAGAGATACATTGACCTTCTCCCCACCAGCCTGCCCCATGCAGTGACCTGTGACATTAAATTCAGAAACTATCTCATTCCCAAGGGCACAACCATATTAATTTCCCTGACTTCTGTGCTACATGACAACAAAGAATTTCCCAACCCAGAGATGTTTGACCCTCATCACTTTCTGGATGAAGGTGGCAATTTTAAGAAAAGTAAATACTTCATGCCTTTCTCAGCAGGAAAACGGATTTGTGTGGGAGAAGCCCTGGCCGGCATGGAGCTGTTTTTATTCCTGACCTCCATTTTACAGAACTTTAACCTGAAATCTCTGGTTGACCCAAAGAACCTTGACACCACTCCAGTTGTCAATGGATTTGCCTCTGTGCCGCCCTTCTACCAGCTGTGCTTCATTCCTGTCTGA
SEQ ID NO.3: protein sequence
MDSLVVLVLCLSCLLLLSLWRQSSGRGKLPPGPTPLPVIGNILQIGIKGISKSLTNLSKVYGPVFTLYFGLKPIVVLHGYEAVKEALIDLGEEFSGRGIFPLAERANRGFGIVFSNGKKWKEIRRFSLMTLRNFGMGKRSIEDRVQEEARCLVEELRKTKASPCDPTFILGCAPCNVICSIIFHKRFDYKDQQFLNLMEKLNENIKILSSPWIQICNNFSPIIDYFPGTHNKLLKNVAFMKSYILEKVKEHQESMDMNNPQDFIDCFLMKMEKEKHNQPSEFTIESLENTAVDLFGAGTETTSTTLRYALLLLLKHPEVTAKVQEEIERVIGRNRSPCMQDRSHMPYTDAVVHEVQRYIDLLPTSLPHAVTCDIKFRNYLIPKGTTILISLTSVLHDNKEFPNPEMFDPHHFLDEGGNFKKSKYFMPFSAGKRICVGEALAGMELFLFLTSILQNFNLKSLVDPKNLDTTPVVNGFASVPPFYQLCFIPV
SEQ ID NO.24: nucleic acid fragment
AGATAGGTATTAAGGGCATCAGCAAATCCTT
SEQ ID NO.25: nucleic acid fragment
CCTACAGATAGGTATTAAGGGCATCAGCAAATCCTTAACCA
SEQ ID NO.26: nucleic acid fragment
AATATCCTACAGATAGGTATTAAGGGCATCAGCAAATCCTTAACCAATGTA
SEQ ID NO.27: nucleic acid fragment
TTGGAAATATCCTACAGATAGGTATTAAGGGCATCAGCAAATCCTTAACCAATGTAAGTAT
SEQ ID NO.28: nucleic acid fragment
AGATAGGTATTAAGGGCATCAGCAAATCCTT
SEQ ID NO.29: nucleic acid fragment
CCTACAGATAGGTATTAAGGGCATCAGCAAATCCTTAACCA
SEQ ID NO.30: nucleic acid fragment
AATATCCTACAGATAGGTATTAAGGGCATCAGCAAATCCTTAACCAATCTC
SEQ ID NO.31: nucleic acid fragment
TTGGAAATATCCTACAGATAGGTATTAAGGGCATCAGCAAATCCTTAACCAATCTCTCAAA
SEQ ID NO.32: oligonucleotide sequence
AGGATTTGCTGATGC
SEQ ID NO.33: oligonucleotide sequence
GGTTAAGGATTTGCTGATGC
SEQ ID NO.34: oligonucleotide sequence
ACATTGGTTAAGGATTTGCTGATGC
SEQ ID NO.35: oligonucleotide sequence
TACTTACATTGGTTAAGGATTTGCTGATGC
SEQ ID NO.36: oligonucleotide sequence
GAGCATACTTACATTGGTTAAGGATTTGCTGATGC
SEQ ID NO.37: oligonucleotide sequence
ATGCCCTTAATACCT
SEQ ID NO.38: oligonucleotide sequence
TGCTGATGCCCTTAATACC
Reference:
1.Aquilante CA.Sulfonylurea pharmacogenomics in Type 2diabetes：theinfluence of drug target and diabetes risk polymorphisms.Expert RevCardiovasc Ther.2010；8(3)：359-372.
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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.Zhou Sh.F，Liu J.P.Chowbay B.Polymorphism of human cytochromeP450enzymes and its clinical impact.Drug Metab Rev.2009；41(2)：89-295.
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8.Zhang YN，Cui W，Han M et al：[Gene polymorphism of CYP450 2C9and VKORC1 in Chinese population and their relationships to the maintainingdosage of warfarin.].Zhonghua Liu Xing Bing Xue Za Zhi 2010；31：218-222.
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10.Yu BN，Luo CH，Wang D et al：CYP2C9 allele variants in Chinesehypertension patients and healthy controls.Clin Chim Acta 2004；348：57-61.
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12.Wang SL，Huang J，Lai MD，Tsai JJ：Detection of CYP2C9polymorphism based on the polymerase chain reaction in Chinese.Pharmacogenetics 1995；5：37-42.
13. horse Jingjings, 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, the .Pyrosequencing such as Ding Yuanyuan, Yang Fan detect foundation and the reliability consideration thereof of CYP2C9*3 gene pleiomorphism method. Chinese Clinical pharmacology and therapeutics 2009; 14 (7): 799-803.
15. Ma Xinchao, Yang Jian, Huang Chenrong 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. the research of Xinjiang Uygur healthy population cytochrome P450 gene polymorphism. Chinese medicine and clinical 2007; 7 (2): 91-94.
How 17. shake space, Sun Limin, Li Yueqin etc. Guangdong crowd CYP2C9 allelotrope and genotype distribution frequency. and Guangdong medical science 2006; 27 (8): 1131-1132.
18.Rokitta D，Fuhr U.Comparison of enzyme kinetic parametersobtained in vitro for reactions mediated by human CYP2C enzymes includingmajor CYP2C9 variants.Curr Drug Metab.2010；11(2)：153-161.
19.Van Booven D，Marsh S，McLeod H et al：Cytochrome P4502C9-CYP2C9.Pharmacogenet Genomics 2010；20：277-281.
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Claims (7)
1. nucleic acid fragment, described nucleic acid fragment comprises the mutational site of the 1001st corresponding to SEQ ID NO.1, and is at least 10 continuous nucleotides in the nucleotide sequence shown in SEQ ID NO.1 or its complementary sequence, and wherein the Nucleotide of the 1001st is G; Or described nucleic acid fragment comprises the mutational site of the 146th corresponding to SEQ IDNO.2, and is at least 10 continuous nucleotides in the nucleotide sequence shown in SEQ ID NO.2 or its complementary sequence, and wherein the Nucleotide of the 146th is G.
2. nucleic acid fragment according to claim 1, is characterized 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 2, is characterized 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, is characterized in that, described nucleic acid fragment be SEQID NO.1,2, the sequence shown in 24-31.
5., for detecting and/or the test kit of analysis list base mutation, comprise the nucleic acid fragment described in any one of claim 1-4; Or comprise the fragment of sequence shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.16.
6. the nucleic acid fragment described in any one of claim 1-4 detects the application in the preparation of CYP2C9 transgenation in preparation.
7.CYP2C9 albumen or its fragment or varient, described protein sequence is the sequence shown in SEQ ID NO.3; Described fragment or varient comprise the glycine of the 49th corresponding to SEQ ID NO.3, and are at least 10 continuous amino acids of the aminoacid sequence shown in SEQ ID NO.3.
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Non-Patent Citations (4)
| Title |
|---|
| A systematic genetic polymorphism analysis of the CYP2C9 gene in four different geographical Han populations in mainland China;Xiong et al;《Genomics》;20101129;第97卷(第5期);摘要、材料与方法及表6 * |
| CYP2C9 allele variants in Chinese hypertension patients and healthy controls;Yu et al;《Clinica Chimica Acta》;20041031;第348卷(第1－2期);全文 * |
| CYP2C9 haplotype structure in European American warfarin patients and association with clinical outcomes;Veenstra et al;《CLINICAL PHARMACOLOGY and THERAPEUTICS》;20050531;第77卷(第5期);摘要、讨论部分最后一段及表1 * |
| 细胞色素P450 2C9新等位基因的发现及其对药物代谢的影响;郭颖杰;《中国博士学位论文全文数据库 医药卫生科技辑》;20051015(第6期);全文 * |
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