CN103031320B - Comprise the CYP2C9 gene fragment of 329T > C sudden change, coded protein fragments and application thereof - Google Patents
Comprise the CYP2C9 gene fragment of 329T > C 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 CYP2C9 gene and correspond to SEQ? ID? the mutational site of the 329th of NO.2, described site sports C by the T 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 SEQIDNO.1 or the mutational site of the 329th of SEQIDNO.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 SEQIDNO.1, and be at least 10 continuous nucleotides in the nucleotide sequence shown in SEQIDNO.1, wherein the Nucleotide of the 1001st is C; Or described nucleic acid fragment comprises the mutational site of the 329th 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 329th is C; 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 SEQIDNO.1 or correspond to the allelotrope fragment in the mutational site of the 329th or the allele specific oligonucleotide of all or part of hybridization of its complementary sequence of SEQIDNO.2, wherein the Nucleotide in the mutational site of the 1001st of SEQIDNO.1 or the 329th of SEQIDNO.2 is C; Described allelotrope fragment is at least 10 continuous nucleotides in the nucleotide sequence shown in SEQIDNO.1 or SEQIDNO.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 the sequence fragment shown in SEQIDNO.6 and/or SEQIDNO.7 and/or SEQIDNO.17.
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 SEQIDNO.1 or the 329th of SEQIDNO.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, and described method comprises in the nucleic acid comprising corresponding to the sequence of SEQIDNO.1 analyzed in testing sample the Nucleotide that corresponds to the 1001st or analyzes in the nucleic acid comprising corresponding to the sequence of SEQIDNO.2 in testing sample the Nucleotide corresponding to the 329th.
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 SEQIDNO.3; Described fragment or varient comprise the Serine of the 110th corresponding to SEQIDNO.3, and are at least 10 continuous amino acids of the aminoacid sequence shown in SEQIDNO.3.
The invention provides the CYP2C9 gene and encoding sequence that comprise new single base mutation.The 329th Nucleotide that this gene is corresponding to SEQIDNO.2 sports C (329T > C) by T, thus the amino acid causing it to encode sports Serine by phenylalanine, namely correspond to the Serine of the 110th of SEQIDNO.3.The CYP2C9 albumen (called after F110S) of this sudden change is higher than wild-type to the metabolic activity of medicine.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 SEQIDNO.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 329th 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 allelotrope specifically, as hybridized under high stringency conditions, makes to identify that the 329th Nucleotide corresponding to sequence shown in the 1001st of sequence shown in SEQIDNO.1 or SEQIDNO.2 is C.
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, 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 encoding sequence of the CYP2C9 gene with described mutational site, and wherein the 329th is the mutational site that the present invention relates to.Those skilled in the art are known, and in this article, the 329th 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 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 329th of SEQIDNO.2, this site sports C (329T > C) by the T of wild-type; In addition, Serine (F110S) is sported by the 110th of albumen of the CYP2C9 genes encoding of this sudden change by phenylalanine.
In 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 Nucleotide of the 1001st is C; Or described nucleic acid fragment comprises the mutational site of the 329th 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 329th is C; 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 SEQIDNO.1.
In another embodiment, described nucleic acid fragment is the sequence shown in SEQIDNO.2.
In other embodiments, shown nucleic acid fragment can be the sequence shown in SEQIDNO.24-31.
Second aspect of the present invention be to provide with containing corresponding to the 1001st of SEQIDNO.1 or correspond to the allelotrope fragment in the mutational site of the 329th or the allele specific oligonucleotide of all or part of hybridization of its complementary sequence of SEQIDNO.2, wherein the Nucleotide in the mutational site of the 1001st of SEQIDNO.1 or the 329th of SEQIDNO.2 is C; Described allelotrope fragment is at least 10 continuous nucleotides in the nucleotide sequence shown in SEQIDNO.1 or SEQIDNO.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 SEQIDNO.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 the sequence fragment shown in SEQIDNO.6 and/or SEQIDNO.7 and/or SEQIDNO.17.
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 SEQIDNO.1 or the 329th of SEQIDNO.2 detecting CYP2C9 gene in testing sample.When the CYP2C9 gene detected is when the site corresponding to the 1001st of SEQIDNO.1 or the 329th of SEQIDNO.2 is C, adjust the dosage of the medicine through CYP2C9 metabolism accordingly.In the particular embodiment, when CYP2C9 gene is when the site of the 329th of SEQIDNO.2 is C, the CYP2C9 protease activity of this genes encoding raises, therefore needs to increase 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 SEQIDNO.1 analyzed in testing sample the Nucleotide that corresponds to the 1001st or analyzes in the nucleic acid comprising corresponding to the sequence of SEQIDNO.2 in testing sample the Nucleotide corresponding to the 329th.
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 329th is C in the nucleic acid of the sequence of SEQIDNO.2 in the nucleic acid of the sequence of SEQIDNO.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 329th in the Nucleotide corresponding to the 1001st in the nucleic acid of the sequence of SEQIDNO.1 or the nucleic acid comprising corresponding to the sequence of SEQIDNO.2 is C; 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 SEQIDNO.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 SEQIDNO.1, design two Taqman-MGB probes, respectively for T and C (as shown in the SEQIDNO.37 sequence) allelotrope of the 1001st corresponding to SEQIDNO.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.
TaqmanMGB probe design principle is:
(1) 5 ' end of probe is avoided 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 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 329T > C 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, and when being when detecting target compound with DNA, analyze in the nucleic acid comprising corresponding to the sequence of SEQIDNO.1 in testing sample the Nucleotide corresponding to the 1001st, the probe used or primer are according to the sequences Design of SEQIDNO.1; When being when detecting target compound with RNA, analyze in the nucleic acid comprising corresponding to the sequence of SEQIDNO.2 in testing sample the Nucleotide corresponding to the 329th, the probe used or primer are according to the sequences Design of SEQIDNO.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 SEQIDNO.3; Described fragment or varient comprise the Serine of the 110th corresponding to SEQIDNO.3, and are at least 10 continuous amino acids of the aminoacid 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 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 lPCR reaction systems, comprising: 1 × PCR damping fluid, 1.5mMMgCl
2, the genomic dna of 100 ~ 150ng, upstream and downstream primer are 0.2 μM, dNTP is the LATaqDNA polysaccharase 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 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.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-QuickStartKit sequencing kit (Beckman company of the U.S.) requires that carrying out order-checking PCR reacts, and 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 329th of CYP2C9 gene coding region becomes C (as shown in Figure 1) from T in a sample, this sudden change is positioned at the 2nd exon of CYP2C9 gene.Infer in the protein of this CYP2C9 genes encoding accordingly, the 110th amino acids sports Serine (S) by phenylalanine (F).The international P450 NK of this new 329 sudden changes (329T > C) Yi Bei confirms called after neomorph CYP2C9*40.
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 SEQIDNO.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 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 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 of * 3 types is lower than * 2 types (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 for template, the rite-directed mutagenesis Nucleotide (becoming C from T) of the 329th of CYP2C9 gene coding region, construction of expression vector expresses saltant type CYP2C9 albumen (called after F110S), the CYP2C9 activity detection kit of Promega company (containing CYP2C9 specific substrate) is utilized to detect the enzymic activity of this saltant type CYP2C9, to judge compared with wild-type CYP2C9 (* 1), 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) for template, 329th Nucleotide of rite-directed mutagenesis CYP2C9 gene coding region, namely the ORF region sequence comprising the Nucleotide shown in SEQIDNO.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 pIRESpGluc-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.The COS-7 cell (this cell strain has been widely used in the analysis of CYP2C9 external activity, refers to reference 18,21) of 8*104 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 (F110S) 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 lCYP2C9, 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 GLOMAX20/20Luminometer of Promega company (GLOMAX20/20 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 GLOMAX20/20Luminometer 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-thiazolecarboxylicacid), 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 has 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).
Utilize this vitro detection system to find out: the metabolic activity of F110S is higher than wild-type by 108%, namely this sudden change can cause the metabolic activity of expressed enzyme obviously to raise.Therefore, in practice, need to consider suitably to regulate on dosage carrying this genotypic individuality, as increased 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:
SEQIDNO.1: genomic dna sequence
TTTGGGAGTCCTAGGTGGAGTCCATAGAGAGATATTTTTAGTATAAATGCTATGATGCATGCTAATCATAAAAGGCTATTAAACCAGGAAATTGTTAGTTCTTGAAGCTGGGTATTGGTCTTAAGGTAGACTCGAAAGTGCTGATAAATTTCTCAAGCATCAGTGTTTGAATAAGCGGAGTTTCAAATTTTGGTCTGCTGTACATTAGCTGTGAGACACTGAAAATGAATTTGTCATTCTCTGAGCTCAGTTTTTTTTTTTTTTTTTTTTTTTTTTGAGACAGAGTCTTACTCTGTAGCTCAGGCTGGAGTGCAGTGGTACAATCTTGGCTCACTGCAACCTCCATCTCCCAGGTCCCCATTCAAGAAATTCTCCTGCCTCAGTCCCCCAAGTAGCTAGCATTACAGGCATGCACCACCATGCTCAGCTAATTTTTGTATTTTTAGTAGAGACGTGGTATCACCTTGTTGGCCAGGCTGGTCTTGAACTCCTGACCTTGTGATCCACCTGCCTTGGCCTCCCAAAGTGTTGGGATTACAGGCAGGAGCCACCACACCTGGCCGTTTGTTTAAAATAGAGTAAATAGACCTGCTGAATATGTTGATGTGAGTATTAATTGTAATCTGCATAGCAATTGTCTGACCATTGCCTTGAACATCACAGGCCATCTGAGTGGCAAGTATAATCATCATCATGTTTCTATTTAAAATTCAGAAATATTTGAAGCCTGTGTGGCTGAATAAAAGCATACAAATACAATGAAAATATCATGCTAAATCAGGCTTAGCAAATGGACAAAATAGTAACTTCGTTTGCTGTTATCTCTGTCTACTTTCCTAGCTCTCAAAGGTCTATGGCCCTGTGTTCACTCTGTATTTTGGCCTGAAACCCATAGTGGTGCTGCATGGATATGAAGCAGTGAAGGAAGCCCTGATTGATCTTGGAGAGGAGTTTTCTGGAAGAGGCATTTTCCCACTGGCTGAAAGAGCTAACAGAGGATCTGGTAGGTGTGCATGTGCCTGTTTCAGCATCTGTCTTGGGGATGGGGAGGATGGAAAACAGAGACTTACAGAGCTCCTCGGGCAGAGCTTGGCCCATCCACATGGCTGCCCAGTGTCAGCTTCCTCTTTCTTGCCTGGGATCTCCCTCCTAGTTTCGTTTCTCTTCCTGTTAGGAATTGTTTTCAGCAATGGAAAGAAATGGAAGGAGATCCGGCGTTTCTCCCTCATGACGCTGCGGAATTTTGGGATGGGGAAGAGGAGCATTGAGGACCGTGTTCAAGAGGAAGCCCGCTGCCTTGTGGAGGAGTTGAGAAAAACCAAGGGTGGGTGACCCTACTCCATATCACTGACCTTACTGGACTACTATCTTCTCTACTGACATTCTTGGAAACATTTCAGGGGTGGCCATATCTTTCATTATGAGTCCTGGTTGTTAGCTCATGTGAAGCGGGGGTTTGAAGCTGAGAGCCAAGGGAATTTGCACATATTTGTGCTGTGTGTGTACAGGCATGATTGTGCGTACAGTGTGGGTATAAAAGGTTCATTTAATCCCATGTTCTCCTGAACTTTGCTTTTTTGCTTTCAAATAAGAAATGATGAATATAGATTTTGAGTTCATTTTTTGAAAGAGTTAAAGAGCAGTGTTTTTCCCATTACCTATTCCAGAACATGTCACCAGAGAATACTTGACAAGTCAACATGGTGGGAATGGCCCTATCATACCCATATGGAGCATGAACCAAATGGCATGTGCTTTTATTTAATTGGACTGTGTTTGTATGGTCAGCCTCACTGACTTCTCTGGGGTTTCTTTTAGGCCCGTGCTTGCCATTCTGGCCAGTAATGACATTCTACAGTTTTTATTGCTTAGGCATATCTTAGTGCAGTTCTCATCAATTATTATTTCTCTGTAAACACAGCATTATTTTAAAAATAGTATTAATTATTTCTTGTTACTGTATTGATTTATATATTTTCAGTAAATACATCCTGTAGCATA
SEQIDNO.2: encoding sequence
ATGGATTCTCTTGTGGTCCTTGTGCTCTGTCTCTCATGTTTGCTTCTCCTTTCACTCTGGAGACAGAGCTCTGGGAGAGGAAAACTCCCTCCTGGCCCCACTCCTCTCCCAGTGATTGGAAATATCCTACAGATAGGTATTAAGGACATCAGCAAATCCTTAACCAATCTCTCAAAGGTCTATGGCCCTGTGTTCACTCTGTATTTTGGCCTGAAACCCATAGTGGTGCTGCATGGATATGAAGCAGTGAAGGAAGCCCTGATTGATCTTGGAGAGGAGTTTTCTGGAAGAGGCATTTTCCCACTGGCTGAAAGAGCTAACAGAGGATCTGGAATTGTTTTCAGCAATGGAAAGAAATGGAAGGAGATCCGGCGTTTCTCCCTCATGACGCTGCGGAATTTTGGGATGGGGAAGAGGAGCATTGAGGACCGTGTTCAAGAGGAAGCCCGCTGCCTTGTGGAGGAGTTGAGAAAAACCAAGGCCTCACCCTGTGATCCCACTTTCATCCTGGGCTGTGCTCCCTGCAATGTGATCTGCTCCATTATTTTCCATAAACGTTTTGATTATAAAGATCAGCAATTTCTTAACTTAATGGAAAAGTTGAATGAAAACATCAAGATTTTGAGCAGCCCCTGGATCCAGATCTGCAATAATTTTTCTCCTATCATTGATTACTTCCCGGGAACTCACAACAAATTACTTAAAAACGTTGCTTTTATGAAAAGTTATATTTTGGAAAAAGTAAAAGAACACCAAGAATCAATGGACATGAACAACCCTCAGGACTTTATTGATTGCTTCCTGATGAAAATGGAGAAGGAAAAGCACAACCAACCATCTGAATTTACTATTGAAAGCTTGGAAAACACTGCAGTTGACTTGTTTGGAGCTGGGACAGAGACGACAAGCACAACCCTGAGATATGCTCTCCTTCTCCTGCTGAAGCACCCAGAGGTCACAGCTAAAGTCCAGGAAGAGATTGAACGTGTGATTGGCAGAAACCGGAGCCCCTGCATGCAAGACAGGAGCCACATGCCCTACACAGATGCTGTGGTGCACGAGGTCCAGAGATACATTGACCTTCTCCCCACCAGCCTGCCCCATGCAGTGACCTGTGACATTAAATTCAGAAACTATCTCATTCCCAAGGGCACAACCATATTAATTTCCCTGACTTCTGTGCTACATGACAACAAAGAATTTCCCAACCCAGAGATGTTTGACCCTCATCACTTTCTGGATGAAGGTGGCAATTTTAAGAAAAGTAAATACTTCATGCCTTTCTCAGCAGGAAAACGGATTTGTGTGGGAGAAGCCCTGGCCGGCATGGAGCTGTTTTTATTCCTGACCTCCATTTTACAGAACTTTAACCTGAAATCTCTGGTTGACCCAAAGAACCTTGACACCACTCCAGTTGTCAATGGATTTGCCTCTGTGCCGCCCTTCTACCAGCTGTGCTTCATTCCTGTCT
SEQIDNO.3: protein sequence
MDSLVVLVLCLSCLLLLSLWRQSSGRGKLPPGPTPLPVIGNILQIGIKDISKSLTNLSKVYGPVFTLYFGLKPIVVLHGYEAVKEALIDLGEEFSGRGIFPLAERANRGSGIVFSNGKKWKEIRRFSLMTLRNFGMGKRSIEDRVQEEARCLVEELRKTKASPCDPTFILGCAPCNVICSIIFHKRFDYKDQQFLNLMEKLNENIKILSSPWIQICNNFSPIIDYFPGTHNKLLKNVAFMKSYILEKVKEHQESMDMNNPQDFIDCFLMKMEKEKHNQPSEFTIESLENTAVDLFGAGTETTSTTLRYALLLLLKHPEVTAKVQEEIERVIGRNRSPCMQDRSHMPYTDAVVHEVQRYIDLLPTSLPHAVTCDIKFRNYLIPKGTTILISLTSVLHDNKEFPNPEMFDPHHFLDEGGNFKKSKYFMPFSAGKRICVGEALAGMELFLFITSILQNFNLKSLVDPKNLDTTPVVNGFASVPPFYQLCFIPV
SEQIDNO.24: nucleic acid fragment
GAGCTAACAGAGGATCTGGTAGGTGTGCATG
SEQIDNO.25: nucleic acid fragment
TGAAAGAGCTAACAGAGGATCTGGTAGGTGTGCATGTGCCT
SEQIDNO.26: nucleic acid fragment
CTGGCTGAAAGAGCTAACAGAGGATCTGGTAGGTGTGCATGTGCCTGTTTC
SEQIDNO.27: nucleic acid fragment
TCCCACTGGCTGAAAGAGCTAACAGAGGATCTGGTAGGTGTGCATGTGCCTGTTT
CAGCAT
SEQIDNO.28: nucleic acid fragment
GAGCTAACAGAGGATCTGGAATTGTTTTCAG
SEQIDNO.29: nucleic acid fragment
TGAAAGAGCTAACAGAGGATCTGGAATTGTTTTCAGCAATG
SEQIDNO.30: nucleic acid fragment
CTGGCTGAAAGAGCTAACAGAGGATCTGGAATTGTTTTCAGCAATGGAAAG
SEQIDNO.31: nucleic acid fragment
TCCCACTGGCTGAAAGAGCTAACAGAGGATCTGGAATTGTTTTCAGCAATGGAAAGAAATG
SEQIDNO.32: oligonucleotide sequence
TGCACACCTACCAGA
SEQIDNO.33: oligonucleotide sequence
GCACATGCACACCTACCAGA
SEQIDNO.34: oligonucleotide sequence
AACAGGCACATGCACACCTACCAGA
SEQIDNO.35: oligonucleotide sequence
GCTGAAACAGGCACATGCACACCTACCAGA
SEQIDNO.36: oligonucleotide sequence
CAGATGCTGAAACAGGCACATGCACACCTACCAGA
SEQIDNO.37: oligonucleotide sequence
CCAGATCCTCTGTTAG
SEQIDNO.38: oligonucleotide sequence
CACCTACCAGATCCTCTGTT
Reference:
1.AquilanteCA.SulfonylureapharmacogenomicsinType2diabetes：theinfluenceofdrugtargetanddiabetesriskpolymorphisms.ExpertRevCardiovascTher.2010；8(3)：359-372.
2.XuHM，MurrayM，MclachlanAJ.Influenceofgeneticpolymorphismsonthepharmacokineticsandpharmacodynamicsofsulfonylureadrugs.CurrentDrugMetabolism.2009；10(6)：643-658.
3.WangB，WangJ，HuangSQ，etal.GeneticpolymorphismofthehumancytochromeP4502C9geneanditsclinicalsignificance.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.PolymorphismofhumancytochromeP450enzymesanditsclinicalimpact.DrugMetabRev.2009；41(2)：89-295.
6.XiongY，WangM，FangKetal：AsystematicgeneticpolymorphismanalysisoftheCYP2C9geneinfourdifferentgeographicalHanpopulationsinmainlandChina.Genomics2011；97：277-281.
7.ZhuJ，ZhangW，LiY，WangH，ZhengW，WangC：ARMStestfordiagnosisofCYP2C9andVKORC1mutationinpatientswithpulmonaryembolisminHanChinese.Pharmacogenomics2010；11：113-119.
8.ZhangYN，CuiW，HanMetal：[GenepolymorphismofCYP4502C9andVKORC1inChinesepopulationandtheirrelationshipstothemaintainingdosageofwarfarin.].ZhonghuaLiuXingBingXueZaZhi2010；31：218-222.
9.LiZ，WangG，WangLSetal：EffectsoftheCYP2C9*13alleleonthepharmacokineticsoflosartaninhealthymalesubjects.Xenobiotica2009；39：788-793.
10.YuBN，LuoCH，WangDetal：CYP2C9allelevariantsinChinesehypertensionpatientsandhealthycontrols.ClinChimActa2004；348：57-61.
11.YangJQ，MorinS，VerstuyftCetal：FrequencyofcytochromeP4502C9allelicvariantsintheChineseandFrenchpopulations.FundamClinPharmacol2003；17：373-376.
12.WangSL，HuangJ，LaiMD，TsaiJJ：DetectionofCYP2C9polymorphismbasedonthepolymerasechainreactioninChinese.Pharmacogenetics1995；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.RokittaD，FuhrU.ComparisonofenzymekineticparametersobtainedinvitroforreactionsmediatedbyhumanCYP2CenzymesincludingmajorCYP2C9variants.CurrDrugMetab.2010；11(2)：153-161.
19.VanBoovenD，MarshS，McLeodHetal；CytochromeP4502C9-CYP2C9.PharmacogenetGenomics2010；20：277-281.
20.SiD，GuoY，ZhangY，YangL，ZhouH，ZhongD.IdentificationofanovelvariantCYP2C9alleleinChinese.Pharmacogenetics.2004；14(7)：465-469.
21.HiratsukaM：InvitroassessmentoftheallelicvariantsofcytochromeP450.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-activityrelationshipsofhumanCytochromeP4502C9andimplicationsindrugdevelopment.CurrMedChem2009；16：3480-3675.
24.CaliJJ，MaD，SobolMetal：LuminogeniccytochromeP450assays.ExpertOpinDrugMetabToxicol2006；2：629-645.
25.AnzenbacherovaE，VeinlichovaA，MasekV，AnzenbacherP：Comparisonof″highthroughput″micromethodsfordeterminationofcytochromeP450activitieswithclassicalmethodsusingHPLCforproductidentification.BiomedPapMedFacUnivPalackyOlomoucCzechRepub2005；149：353-355.
Sequence table
<110> Cai Jian puts down
<120> comprises the CYP2C9 gene fragment of 329T>C sudden change, coded protein fragments and application thereof
<130>DP1F111455ZL/CNCJP/RR
<140>201110430181.8
<141>2011-12-19
<160>38
<170>PatentInversion3.5
<210>1
<211>2001
<212>DNA
<213> people
<400>1
tttgggagtcctaggtggagtccatagagagatatttttagtataaatgctatgatgcat60
gctaatcataaaaggctattaaaccaggaaattgttagttcttgaagctgggtattggtc120
ttaaggtagactcgaaagtgctgataaatttctcaagcatcagtgtttgaataagcggag180
tttcaaattttggtctgctgtacattagctgtgagacactgaaaatgaatttgtcattct240
ctgagctcagttttttttttttttttttttttttttgagacagagtcttactctgtagct300
caggctggagtgcagtggtacaatcttggctcactgcaacctccatctcccaggtcccca360
ttcaagaaattctcctgcctcagtcccccaagtagctagcattacaggcatgcaccacca420
tgctcagctaatttttgtatttttagtagagacgtggtatcaccttgttggccaggctgg480
tcttgaactcctgaccttgtgatccacctgccttggcctcccaaagtgttgggattacag540
gcaggagccaccacacctggccgtttgtttaaaatagagtaaatagacctgctgaatatg600
ttgatgtgagtattaattgtaatctgcatagcaattgtctgaccattgccttgaacatca660
caggccatctgagtggcaagtataatcatcatcatgtttctatttaaaattcagaaatat720
ttgaagcctgtgtggctgaataaaagcatacaaatacaatgaaaatatcatgctaaatca780
ggcttagcaaatggacaaaatagtaacttcgtttgctgttatctctgtctactttcctag840
ctctcaaaggtctatggccctgtgttcactctgtattttggcctgaaacccatagtggtg900
ctgcatggatatgaagcagtgaaggaagccctgattgatcttggagaggagttttctgga960
agaggcattttcccactggctgaaagagctaacagaggatctggtaggtgtgcatgtgcc1020
tgtttcagcatctgtcttggggatggggaggatggaaaacagagacttacagagctcctc1080
gggcagagcttggcccatccacatggctgcccagtgtcagcttcctctttcttgcctggg1140
atctccctcctagtttcgtttctcttcctgttaggaattgttttcagcaatggaaagaaa1200
tggaaggagatccggcgtttctccctcatgacgctgcggaattttgggatggggaagagg1260
agcattgaggaccgtgttcaagaggaagcccgctgccttgtggaggagttgagaaaaacc1320
aagggtgggtgaccctactccatatcactgaccttactggactactatcttctctactga1380
cattcttggaaacatttcaggggtggccatatctttcattatgagtcctggttgttagct1440
catgtgaagcgggggtttgaagctgagagccaagggaatttgcacatatttgtgctgtgt1500
gtgtacaggcatgattgtgcgtacagtgtgggtataaaaggttcatttaatcccatgttc1560
tcctgaactttgcttttttgctttcaaataagaaatgatgaatatagattttgagttcat1620
tttttgaaagagttaaagagcagtgtttttcccattacctattccagaacatgtcaccag1680
agaatacttgacaagtcaacatggtgggaatggccctatcatacccatatggagcatgaa1740
ccaaatggcatgtgcttttatttaattggactgtgtttgtatggtcagcctcactgactt1800
ctctggggtttcttttaggcccgtgcttgccattctggccagtaatgacattctacagtt1860
tttattgcttaggcatatcttagtgcagttctcatcaattattatttctctgtaaacaca1920
gcattattttaaaaatagtattaattatttcttgttactgtattgatttatatattttca1980
gtaaatacatcctgtagcata2001
<210>2
<211>1471
<212>DNA
<213> people
<400>2
atggattctcttgtggtccttgtgctctgtctctcatgtttgcttctcctttcactctgg60
agacagagctctgggagaggaaaactccctcctggccccactcctctcccagtgattgga120
aatatcctacagataggtattaaggacatcagcaaatccttaaccaatctctcaaaggtc180
tatggccctgtgttcactctgtattttggcctgaaacccatagtggtgctgcatggatat240
gaagcagtgaaggaagccctgattgatcttggagaggagttttctggaagaggcattttc300
ccactggctgaaagagctaacagaggatctggaattgttttcagcaatggaaagaaatgg360
aaggagatccggcgtttctccctcatgacgctgcggaattttgggatggggaagaggagc420
attgaggaccgtgttcaagaggaagcccgctgccttgtggaggagttgagaaaaaccaag480
gcctcaccctgtgatcccactttcatcctgggctgtgctccctgcaatgtgatctgctcc540
attattttccataaacgttttgattataaagatcagcaatttcttaacttaatggaaaag600
ttgaatgaaaacatcaagattttgagcagcccctggatccagatctgcaataatttttct660
cctatcattgattacttcccgggaactcacaacaaattacttaaaaacgttgcttttatg720
aaaagttatattttggaaaaagtaaaagaacaccaagaatcaatggacatgaacaaccct780
caggactttattgattgcttcctgatgaaaatggagaaggaaaagcacaaccaaccatct840
gaatttactattgaaagcttggaaaacactgcagttgacttgtttggagctgggacagag900
acgacaagcacaaccctgagatatgctctccttctcctgctgaagcacccagaggtcaca960
gctaaagtccaggaagagattgaacgtgtgattggcagaaaccggagcccctgcatgcaa1020
gacaggagccacatgccctacacagatgctgtggtgcacgaggtccagagatacattgac1080
cttctccccaccagcctgccccatgcagtgacctgtgacattaaattcagaaactatctc1140
attcccaagggcacaaccatattaatttccctgacttctgtgctacatgacaacaaagaa1200
tttcccaacccagagatgtttgaccctcatcactttctggatgaaggtggcaattttaag1260
aaaagtaaatacttcatgcctttctcagcaggaaaacggatttgtgtgggagaagccctg1320
gccggcatggagctgtttttattcctgacctccattttacagaactttaacctgaaatct1380
ctggttgacccaaagaaccttgacaccactccagttgtcaatggatttgcctctgtgccg1440
cccttctaccagctgtgcttcattcctgtct1471
<210>3
<211>490
<212>PRT
<213> people
<400>3
MetAspSerLeuValValLeuValLeuCysLeuSerCysLeuLeuLeu
151015
LeuSerLeuTrpArgGlnSerSerGlyArgGlyLysLeuProProGly
202530
ProThrProLeuProValIleGlyAsnIleLeuGlnIleGlyIleLys
354045
AspIleSerLysSerLeuThrAsnLeuSerLysValTyrGlyProVal
505560
PheThrLeuTyrPheGlyLeuLysProIleValValLeuHisGlyTyr
65707580
GluAlaValLysGluAlaLeuIleAspLeuGlyGluGluPheSerGly
859095
ArgGlyIlePheProLeuAlaGluArgAlaAsnArgGlySerGlyIle
100105110
ValPheSerAsnGlyLysLysTrpLysGluIleArgArgPheSerLeu
115120125
MetThrLeuArgAsnPheGlyMetGlyLysArgSerIleGluAspArg
130135140
ValGlnGluGluAlaArgCysLeuValGluGluLeuArgLysThrLys
145150155160
AlaSerProCysAspProThrPheIleLeuGlyCysAlaProCysAsn
165170175
ValIleCysSerIleIlePheHisLysArgPheAspTyrLysAspGln
180185190
GlnPheLeuAsnLeuMetGluLysLeuAsnGluAsnIleLysIleLeu
195200205
SerSerProTrpIleGlnIleCysAsnAsnPheSerProIleIleAsp
210215220
TyrPheProGlyThrHisAsnLysLeuLeuLysAsnValAlaPheMet
225230235240
LysSerTyrIleLeuGluLysValLysGluHisGlnGluSerMetAsp
245250255
MetAsnAsnProGlnAspPheIleAspCysPheLeuMetLysMetGlu
260265270
LysGluLysHisAsnGlnProSerGluPheThrIleGluSerLeuGlu
275280285
AsnThrAlaValAspLeuPheGlyAlaGlyThrGluThrThrSerThr
290295300
ThrLeuArgTyrAlaLeuLeuLeuLeuLeuLysHisProGluValThr
305310315320
AlaLysValGlnGluGluIleGluArgValIleGlyArgAsnArgSer
325330335
ProCysMetGlnAspArgSerHisMetProTyrThrAspAlaValVal
340345350
HisGluValGlnArgTyrIleAspLeuLeuProThrSerLeuProHis
355360365
AlaValThrCysAspIleLysPheArgAsnTyrLeuIleProLysGly
370375380
ThrThrIleLeuIleSerLeuThrSerValLeuHisAspAsnLysGlu
385390395400
PheProAsnProGluMetPheAspProHisHisPheLeuAspGluGly
405410415
GlyAsnPheLysLysSerLysTyrPheMetProPheSerAlaGlyLys
420425430
ArgIleCysValGlyGluAlaLeuAlaGlyMetGluLeuPheLeuPhe
435440445
LeuThrSerIleLeuGlnAsnPheAsnLeuLysSerLeuValAspPro
450455460
LysAsnLeuAspThrThrProValValAsnGlyPheAlaSerValPro
465470475480
ProPheTyrGlnLeuCysPheIleProVal
485490
<210>4
<211>34
<212>DNA
<213> artificial sequence
<400>4
gacaatggaacgaaggagaacaagaccaaaggac34
<210>5
<211>30
<212>DNA
<213> artificial sequence
<400>5
ggtttcattccactatttctgacactgaca30
<210>6
<211>24
<212>DNA
<213> artificial sequence
<400>6
tacaaatacaatgaaaatatcatg24
<210>7
<211>21
<212>DNA
<213> artificial sequence
<400>7
ctaacaaccaggactcataat21
<210>8
<211>31
<212>DNA
<213> artificial sequence
<400>8
ctattcttgccctttccatctcagtgccttg31
<210>9
<211>30
<212>DNA
<213> artificial sequence
<400>9
cttgttattggtctattcagggatttgact30
<210>10
<211>27
<212>DNA
<213> artificial sequence
<400>10
taggcaagcatggaataagggagtagg27
<210>11
<211>30
<212>DNA
<213> artificial sequence
<400>11
aatcaccattagtttgaaacagattacagc30
<210>12
<211>21
<212>DNA
<213> artificial sequence
<400>12
cccctgaattgctacaacaaa21
<210>13
<211>20
<212>DNA
<213> artificial sequence
<400>13
acccggtgatggtagaggtt20
<210>14
<211>29
<212>DNA
<213> artificial sequence
<400>14
cttctttggaacgggatttcctcatctgc29
<210>15
<211>29
<212>DNA
<213> artificial sequence
<400>15
tctgtccttatcattttgagaaccagcat29
<210>16
<211>17
<212>DNA
<213> artificial sequence
<400>16
tacctctagggatacac17
<210>17
<211>21
<212>DNA
<213> artificial sequence
<400>17
ctaacaaccaggactcataat21
<210>18
<211>30
<212>DNA
<213> artificial sequence
<400>18
ttgctgttaagggaatttgtaggtaagata30
<210>19
<211>27
<212>DNA
<213> artificial sequence
<400>19
tagtggtctattttgttattcattcat27
<210>20
<211>17
<212>DNA
<213> artificial sequence
<400>20
ttccagtttctatgttg17
<210>21
<211>20
<212>DNA
<213> artificial sequence
<400>21
acccggtgatggtagaggtt20
<210>22
<211>18
<212>DNA
<213> artificial sequence
<400>22
acgggatttcctcatctg18
<210>23
<211>22
<212>DNA
<213> artificial sequence
<400>23
cgatacactgaacagttattgc22
<210>24
<211>31
<212>DNA
<213> artificial sequence
<400>24
gagctaacagaggatctggtaggtgtgcatg31
<210>25
<211>41
<212>DNA
<213> artificial sequence
<400>25
tgaaagagctaacagaggatctggtaggtgtgcatgtgcct41
<210>26
<211>51
<212>DNA
<213> artificial sequence
<400>26
ctggctgaaagagctaacagaggatctggtaggtgtgcatgtgcctgtttc51
<210>27
<211>61
<212>DNA
<213> artificial sequence
<400>27
tcccactggctgaaagagctaacagaggatctggtaggtgtgcatgtgcctgtttcagca60
t61
<210>28
<211>31
<212>DNA
<213> artificial sequence
<400>28
gagctaacagaggatctggaattgttttcag31
<210>29
<211>41
<212>DNA
<213> artificial sequence
<400>29
tgaaagagctaacagaggatctggaattgttttcagcaatg41
<210>30
<211>51
<212>DNA
<213> artificial sequence
<400>30
ctggctgaaagagctaacagaggatctggaattgttttcagcaatggaaag51
<210>31
<211>61
<212>DNA
<213> artificial sequence
<400>31
tcccactggctgaaagagctaacagaggatctggaattgttttcagcaatggaaagaaat60
g61
<210>32
<211>15
<212>DNA
<213> artificial sequence
<400>32
tgcacacctaccaga15
<210>33
<211>20
<212>DNA
<213> artificial sequence
<400>33
gcacatgcacacctaccaga20
<210>34
<211>25
<212>DNA
<213> artificial sequence
<400>34
aacaggcacatgcacacctaccaga25
<210>35
<211>30
<212>DNA
<213> artificial sequence
<400>35
gctgaaacaggcacatgcacacctaccaga30
<210>36
<211>35
<212>DNA
<213> artificial sequence
<400>36
cagatgctgaaacaggcacatgcacacctaccaga35
<210>37
<211>16
<212>DNA
<213> artificial sequence
<400>37
ccagatcctctgttag16
<210>38
<211>20
<212>DNA
<213> artificial sequence
<400>38
cacctaccagatcctctgtt20
Claims (7)
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 sequence, and wherein the Nucleotide of the 1001st is C; Or described nucleic acid fragment comprises the mutational site of the 329th corresponding to SEQIDNO.2, and is at least 10 continuous nucleotides in the nucleotide sequence shown in SEQIDNO.2 or its complementary sequence, and wherein the Nucleotide of the 329th is C.
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 SEQIDNO.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.
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, described protein sequence is the sequence shown in SEQIDNO.3.
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| CN201110430181.8A CN103031320B (en) | 2011-12-19 | 2011-12-19 | Comprise the CYP2C9 gene fragment of 329T > C sudden change, coded protein fragments and application thereof |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101434994A (en) * | 2008-12-25 | 2009-05-20 | 上海交通大学 | Method for detecting CYP2C9 gene exon 9 mononucleotide polymorphism |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101434994A (en) * | 2008-12-25 | 2009-05-20 | 上海交通大学 | Method for detecting CYP2C9 gene exon 9 mononucleotide polymorphism |
Non-Patent Citations (4)
| Title |
|---|
| CYP2C9基因多态性及其功能意义研究进展;李智等;《中国临床药理学与治疗学》;20080630;第13卷(第6期);601-609 * |
| Identification of a novel variant CYP2C9 allele in Chinese;Si D等;《Pharmacogenetics》;20041231;第14卷(第7期);全文 * |
| NM_000771.3;GenBank;《GenBank》;20111211;1-5 * |
| 中国汉族人群CYP2C9常见多态性位点的检测及其与国外其他人群的比较研究;徐仁爱等;《医学研究杂志》;20110630;第40卷(第6期);全文 * |
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