CN103173443B - Including 1300A > T sudden change CYP2C9 genetic fragment, coded protein fragments and application thereof - Google Patents
Including 1300A > T sudden change CYP2C9 genetic fragment, 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 particularly it relates to CYP2C9 gene is corresponding to the mutational site of the 1300th of SEQ ID NO.2, described site is sported T by the A of wild type, comprises the nucleic acid fragment in this mutational site, the protein fragments of its coding and application thereof.Present invention also offers and detect the allele specific oligonucleotide in described mutational site, test kit and detection method.
Description
Technical field
The invention belongs to field of biology, relate to single base mutation.More particularly it relates to CYP2C9
The sudden change position of relative to the 1001st or SEQ ID NO.2 of SEQ ID NO.1 the 1300th of gene
Point, comprises the nucleic acid fragment in this mutational site and the protein fragments of corresponding encoded thereof.The invention still further relates to mirror
The reagent in fixed described mutational site and detection method, and identify the application in direction of medication usage of this site.
Background technology
CYP2C9 is most important a member in cytochrome P 450 enzymes extended familys CYP2C subfamily, accounts for
The 20% of people's hepatomicrosome CYP enzyme total amount.Have about 10~16% clinical commonly used drug via CYP2C9
Oxidative metabolism, mainly includes tolbutamide, S-warfarin, phenytoin, glipizide, lattice row benzene
Urea, torr draw thiophene miaow, losartan, irbesartan and many non-steroidal anti-inflammatory drugs (such as: ibuprofen, chlorine promise
Former times health, diclofenac and naproxen) etc. medicine (seeing list of references 1-5).
CYP2C9 gene has height polymorphism.So far, the allele the most named has
57 kinds (http://www.cypalleles.ki.se), removes wild type (CYP2C9*1) outward, has 56 kinds
Mutation type can cause CYP2C9 Argine Monohydrochloride composition to change, the most multiple newfound prominent
Become and not yet named.Study more and that clinical meaning is bigger mutation type and mainly include following 10 kinds:
CYP2C9*2, * 3, * 5, * 6, * 8, * 11, * 12, * 13, * 14, * 16, wherein ethnic group is distributed
Extensively, the saltant type that most study, Chinese population research data are the abundantest is CYP2C9*2(430C > T),
CYP2C9*3(1075A > C) and CYP2C9*13(269T > C) (see list of references 6-17,20).
According to current clinical studies show, this polymorphism of CYP2C9 gene is to cause CYP2C9 enzyme
The main cause that activity is the most different, therefore at the individuality carrying different CYP2C9 genotype
Between can cause the huge difference of curative effect of medication, even produce serious poisonous side effect of medicine or treat insufficient.
Therefore, research CYP2C9 gene pleiomorphism will provide important to clinical rational drug use to the impact of curative effect of medication
Scientific basis (seeing list of references 18,19,21,22).
Summary of the invention
It is an object of the invention to provide the new single base mutation site of CYP2C9 gene, comprise this sudden change position
Point nucleic acid fragment, its coding protein fragments and identify the application in medication guide of this mutational site.
The first aspect of the invention is to provide nucleic acid fragment, and described nucleic acid fragment comprises corresponding to SEQ ID
The mutational site of the 1001st of NO.1, and be in the nucleotide sequence shown in SEQ ID NO.1 extremely
Few 10 continuous nucleotides, wherein the nucleotide of the 1001st is T;Or it is right that described nucleic acid fragment comprises
Should in the mutational site of the 1300th of SEQ ID NO.2, and be the nucleoside shown in SEQ ID NO.2
At least 10 continuous nucleotides in acid sequence, wherein the nucleotide of the 1300th is T;Or it is above-mentioned
The complementary series of nucleic acid fragment.
The second aspect of the invention is to provide and the 1001st or right contained corresponding to SEQ ID NO.1
Should be whole in the allele fragment in the mutational site of the 1300th of SEQ ID NO.2 or its complementary series
Or the allele specific oligonucleotide of partial hybridization, wherein the 1001st of SEQ ID NO.1 or
The nucleotide in the mutational site of the 1300th of SEQ ID NO.2 is T;Described allele fragment is SEQ
At least 10 continuous nucleotides in nucleotide sequence shown in ID NO.1 or SEQ ID NO.2 or it is mutual
Complementary series.
The third aspect of the invention is to provide the test kit for detecting and/or analyze single base mutation, institute
State nucleic acid fragment or allele specific oligonucleotide that test kit comprises the present invention, or comprise SEQ ID
Sequence fragment shown in NO.14 and/or SEQ ID NO.15 and/or SEQ ID NO.23.
The fourth aspect of the invention is to provide the nucleic acid fragment of the present invention or oligonucleotide in detection
Application in CYP2C9 gene mutation, wherein said nucleic acid fragment or oligonucleotide are used as probe or primer.
The fifth aspect of the invention is to provide a kind of medication guide, including CYP2C9 in detection testing sample
Single alkali corresponding to the 1300th of the 1001st or SEQ ID NO.2 of SEQ ID NO.1 of gene
Base suddenlys change;According to the sudden change detected, adjust by the dosage of the medicine of CYP2C9 metabolism.
The sixth aspect of the invention is to provide the method analyzing nucleic acid, and described method includes analyzing testing sample
In comprise in the nucleic acid of the sequence corresponding to SEQ ID NO.1 corresponding to the 1001st nucleotide or point
Comprising in the nucleic acid of the sequence corresponding to SEQ ID NO.2 corresponding to the 1300th in analysis testing sample
Nucleotide.
The seventh aspect of the invention is to provide CYP2C9 albumen or its fragment or variant, described albumen sequence
It is classified as the sequence shown in SEQ ID NO.3;Described fragment or variant comprise corresponding to SEQ ID NO.3
The phenylalanine of the 434th, and at least 10 of the aminoacid sequence shown in SEQ ID NO.3
Continuous amino acid.
The invention provides the CYP2C9 gene and coded sequence comprising new single base mutation.This gene exists
The 1300th nucleotide corresponding to SEQ ID NO.2 is sported T(1300A by A > T), thus draw
The aminoacid playing its coding is phenylalanine by isoleucine mutation, i.e. corresponding to the of SEQ ID NO.3
The phenylalanine of 434.The metabolism of medicine is lived by the CYP2C9 albumen (named I434F) of this sudden change
Property than wild type decline.This single base mutation has guidance meaning to the individual medication carrying this mutational site
Justice.
Accompanying drawing explanation
Fig. 1 is the 1001st nucleotide of the SEQ ID NO.1 sequence corresponding to the present invention in embodiment 1
Sequencing chromatogram;
Fig. 2 is insect expression vector pFastBac-dual structure chart;
Fig. 3 is the Western result figure of each microsome expressing protein in embodiment 2;
Fig. 4 is the datagram of each microsomal metabolism diclofenac in embodiment 3;
Fig. 5 is the datagram of each microsomal metabolism tolbutamide in embodiment 4;
Fig. 6 is the datagram of each microsomal metabolism losartan in embodiment 5.
Detailed description of the invention
By following detailed description of the invention, the present invention is described, but present invention is not limited to this.
As illustrated without other, " nucleic acid fragment " of the present invention is made up of nucleotide or its analog, Ke Yishi
DNA, RNA or the fragment of its analog;Can be strand or double-strand;Can be natural (such as gene
Group) or synthesis.
In the present invention, " sudden change " refers to the gene in detection, i.e. exist and wild type in CYP2C9 gene
The nucleotide site that CYP2C9 gene order is different." mutational site " refers to the position that base is undergone mutation.
In the present invention, described mutational site correspond to the 1001st of sequence shown in SEQ ID NO.1 or
In sequence shown in SEQ ID NO.2 the 1300th.
In the present invention, " allele-specific " refers to hybridize, as at high stringency conditions with allele specifically
Under hybridize so that identify corresponding to the 1001st or SEQ ID of sequence shown in SEQ ID NO.1
1300th nucleotide of sequence shown in NO.2 is T.
Present invention relates to the nonsynonymous mutation of CYP2C9 gene.Owing to this mutational site is positioned at gene
In coded sequence, therefore, skilled person will appreciate that, described mutational site both can be at genomic DNA
Middle performance, it is also possible to performance in coded sequence (i.e. CDS, corresponding to mRNA sequence).This area
Technical staff, can be to this mutational site on genomic DNA or mRNA level in-site according to detected sample
Detect.In the application, SEQ ID NO.1 be centered by the mutational site of the application, the most each
The genomic dna sequence of 1kb, i.e. the 1001st of SEQ ID NO.1 is the sudden change position that the present invention relates to
Point.SEQ ID NO.2 is the cDNA sequence of the CYP2C9 gene with described mutational site, Qi Zhong
1300 is the mutational site that the present invention relates to.Skilled person will appreciate that, in this article, correspond to
1300th site of SEQ ID NO.2 and the 1001st the site synonym corresponding to SEQ ID NO.1 are mutual
With.
In the present invention, nucleotide and amino acid whose abbreviation use abbreviation mode well known in the art, such as nucleotide
Middle A represents that adenine, G represent that guanine, C represent that cytosine, T represent thymus pyrimidine.Aminoacid
In, A represents that alanine, R represent that arginine, N represent that agedoite, D represent aspartic acid, C table
Show that cysteine, Q represent that glutamine, E represent that glutamic acid, G represent that glycine, H represent histidine,
I represents that isoleucine, L represent that leucine, K represent that lysine, M represent that methionine, F represent phenylpropyl alcohol
Propylhomoserin, P represents that proline, S represent that serine, T represent that threonine, W represent that tryptophan, Y represent
Tyrosine, V represents valine.
Present disclosure is new single base mutation site based on CYP2C9 gene.Described mutational site
It is in the coding region of CYP2C9 gene, corresponding to the 1300th of SEQ ID NO.2, this site
T(1300A is sported by the A of wild type > T);Additionally, by the CYP2C9 gene code of this sudden change
The 434th of albumen is phenylalanine (I434F) by isoleucine mutation.
At first aspect, the present invention provides nucleic acid fragment, described nucleic acid fragment to comprise corresponding to SEQ ID
The mutational site of the 1001st of NO.1, and be in the nucleotide sequence shown in SEQ ID NO.1 extremely
Few 10 continuous nucleotides, wherein the nucleotide of the 1001st is T;Or it is right that described nucleic acid fragment comprises
Should in the mutational site of the 1300th of SEQ ID NO.2, and be the nucleoside shown in SEQ ID NO.2
At least 10 continuous nucleotides in acid sequence, wherein the nucleotide of the 1300th is T;Or it is above-mentioned
The complementary series of 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 be 10-20,20-30,
30-40,40-50,50-60,60-100 or 100-300 nucleotide.
Described mutational site may be located at any position of described nucleic acid fragment.
In another embodiment, described nucleic acid fragment is the sequence shown in SEQ ID NO.1.
In another embodiment, described nucleic acid fragment is the sequence shown in SEQ ID NO.2.
In other embodiments, described nucleic acid fragment can be the sequence shown in SEQ ID NO.24-31.
The second aspect of the invention is to provide and the 1001st or right contained corresponding to SEQ ID NO.1
Should be whole in the allele fragment in the mutational site of the 1300th of SEQ ID NO.2 or its complementary series
Or the allele specific oligonucleotide of partial hybridization, wherein the 1001st of SEQ ID NO.1 or
The nucleotide in the mutational site of the 1300th of SEQ ID NO.2 is T;Described allele fragment is SEQ
At least 10 continuous nucleotides in nucleotide sequence shown in ID NO.1 or SEQ ID NO.2 or it is mutual
Complementary series.
In one embodiment, described oligonucleotide is used as probe.Described probe can be at high stringency conditions
Down with the target sequence specific hybrid comprising mutational site.It is known to those skilled in the art that described probe is not required to
Will be with target sequence complete complementary, as long as can be with target sequence specific hybridization.In preferred embodiments,
Described hybridization conditions can meet make probe only with target sequence specific hybrid.The length of described probe can be
5-100 nucleotide, such as 5,6,7,8,9,10,11,12,13,14,15,16,17,18,
19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、
40,50,60,70,80,90 or 100 nucleotide.Described mutational site can occur in probe
Any position.In a preferred embodiment, described mutational site occurs in the center or about of probe sequence
Center.
In another embodiment, the described oligonucleotide primer of the DNA synthesis that coaches, such as ability
Sequencing primer or synthetic primer etc. known to territory.Described primer need not and template complete complementary, but it should with
Template Complementary hybridization synthesizes instructing DNA.The length of described primer can be 15-40 length of nucleotides,
It is preferably 18,19,20,21,22,23,24,25,26,27,28,29 or 30 nucleotide.
Described mutational site can occur in any position of described primer;Preferably, described mutational site occurs in
3 ' ends of described primer.
Some preferred embodiment in, described oligonucleotide is the sequence as shown in SEQ ID NO.32-38
Row.
Based on this, the third aspect of the invention is to provide the examination for detecting and/or analyze single base mutation
Agent box, described test kit comprises nucleic acid fragment or the allele specific oligonucleotide of the present invention, or comprises
Sequence fragment shown in SEQ ID NO.14 and/or SEQ ID NO.15 and/or SEQ ID NO.23.
The fourth aspect of the invention is to provide the nucleic acid fragment of the present invention or oligonucleotide for detecting
The application of CYP2C9 gene mutation, wherein said nucleic acid fragment or oligonucleotide are used as probe or primer.
The fifth aspect of the invention is to provide medication guide, including CYP2C9 gene in detection testing sample
The single base corresponding to the 1300th of the 1001st or SEQ ID NO.2 of SEQ ID NO.1 dash forward
Become.When the CYP2C9 gene detected is corresponding to the 1001st or SEQ ID of SEQ ID NO.1
When the site of the 1300th of NO.2 is T, adjust accordingly through CYP2C9 metabolism medicine to
Dose.In the particular embodiment, when CYP2C9 gene is the position of the 1300th of SEQ ID NO.2
When point is for T, the CYP2C9 proteinase activity of this gene code declines, therefore needs to adjust through CYP2C9
The dosage of the medicine of metabolism.
The heretofore described medicine through CYP2C9 metabolism includes: cancer therapy drug, such as cyclophosphamide, different
Cyclophosphamide or paclitaxel;Anticoagulant, such as warfarin, acenocoumarol, anticonvulsant or mephenytoin;
Antidiabetic drug, such as tolbutamide, Nateglinide, pioglitazone or rosiglitazone;Antuepileptic, as benzene is appropriate
English or zonisamide;Antimalarial/antiparasitic, such as amodiaquine, chloroguanide hydrochloride or quinine;Anti-spirit
Sick medicine, as amitriptyline, citalopram, imipramine, cis-N-[4-[4-(1,2-Benzisothiazol-3-yl)-1-piperazinyl, Sertraline, thioridazine or literary composition draw
Method is pungent;Depressor, such as losartan, irbesartan or valsartan;Non-steroidal anti-inflammatory drug, as double chlorine are fragrant
Acid, aminophenazone, phenazone, celecoxib, flurbiprofen, ibuprofen, indomethacin, chlorine promise former times
Health, mefenamic acid, naproxen, piroxicam or tenoxicam;Analgesic, such as, loperamide, U.S. husky
Ketone or morphine;Proton pump inhibitor, such as lansoprazole or omeprazole;Tranquilizer, as, clobazam, first
Benzene is than appropriate or zopiclone.
The sixth aspect of the invention is to provide the method analyzing nucleic acid, and described method includes analyzing testing sample
In comprise in the nucleic acid of the sequence corresponding to SEQ ID NO.1 corresponding to the 1001st nucleotide or point
Comprising in the nucleic acid of the sequence corresponding to SEQ ID NO.2 corresponding to the 1300th in analysis testing sample
Nucleotide.
In one embodiment, described mode can be restriction fragment length polymorphism analysis (RFLP).
Those skilled in the art can be according to present invention contrived experiment to analyze the sequence of SEQ ID NO.1
In the nucleic acid of the nucleotide of the 1001st in nucleic acid or the sequence of SEQ ID NO.2 the 1300th 's
Whether nucleotide is T.
In another embodiment, described method can be sequencing, including separating and measuring from gene
The nucleotide sequence of group DNA or RNA, analyzes the core wherein comprising the sequence corresponding to SEQ ID NO.1
In acid corresponding to the nucleotide of the 1001st or comprise the nucleic acid of the sequence corresponding to SEQ ID NO.2
Whether be T corresponding to the nucleotide of the 1300th.Sequencing can be known in the art any available
Sequence measurement.Sequencing primer can be designed according to the general knowledge of those skilled in the art, as to be detected
The upstream and downstream appropriate position design primer in site, with the expanding packet fragment containing this site to be measured, thus judges
The nucleotide in this site.The oligonucleotide of the present invention can also be used as primer sequence.
In another embodiment, described method is the method utilizing probe to hybridize, and specifically identifies inspection
Test sample product comprise in the nucleic acid of the sequence corresponding to SEQ ID NO.1 corresponding to the nucleoside of the 1001st
Acid or comprise in the nucleic acid of the sequence corresponding to SEQ ID NO.2 corresponding to the 1300th nucleotide whether
For T;The probe used in described method is the oligonucleotide of the present invention.Such as, separate from testing sample
Go out nucleic acid, make under conditions of specific target sequence that may be present hybridization in allowing probe and nucleic acid probe with
Nucleic acid contacts;The hybridization that can be detected can be come real by the probe that detectable reagent is labeled by using
Existing;Such as, maybe can be catalyzed with radiosiotope, fluorescent dye to be formed and can detect the enzyme of product and carry out labelling and visit
Pin.Label probe, it is all art technology with whether label probe detection sample exists the method for target sequence
Known to personnel.
In a kind of specific embodiment, it is provided that with the detection of Taqman probe SNP detection method corresponding to
The method of the nucleotide of the 1001st of SEQ ID NO.1, including:
1) design primer comprises the PCR of corresponding to SEQ ID NO.1 the 1001st for specific amplification
Product, simultaneously two Taqman-MGB probes of design, be respectively directed to the corresponding to SEQ ID NO.1
A and the T allele of 1001.
Design of primers principle is:
(1) choose should be at the conservative section of gene for sequence;
(2) avoid primer self or and primer between form 4 or more than 4 and match continuously, it is to avoid
Primer self forms pili annulati card structure;
(3) primer length is at 18 to 24 nucleotide;
(4) Tm value is at 55-65 DEG C, and G/C content is at 40%-60%;
(5) the Tm value difference between primer avoids exceeding 2 DEG C;
(6) primer 3 ' end avoid use base A, primer 3 ' end avoid the occurrence of 3 or 3 with
Upper consecutive identical base;
(7) pcr amplified fragment length is at 50bp-150bp;
(8) last 5 nucleotide of prime end can not have more than G and C of 2.
Taqman MGB probe design principle is:
(1) 5 ' ends of probe avoid the occurrence of G;
(2) Tm value should be 65-67 DEG C;
(3) shorten Taqman MGB probe as far as possible, but probe length is no less than 13bp;
(4) avoid the occurrence of the base of repetition, especially G base, it is to avoid occur 4 or 4 as far as possible
Individual above G repeats;
(5) mutational site of probe is placed on as far as possible the place of middle 1/3.
Fluorophor can use FAM, VIC etc. to carry out two allele of labelling.
2) utilize above-mentioned primer and probe, sample to be tested is carried out real-time quantitative PCR.
PCR condition: 95 DEG C of denaturations enter 30 amplification cycles after 10 minutes: 92 DEG C of degeneration 12 seconds,
60 DEG C of annealing and extension 1 minute (this stage detection fluorescence signal).
3) data analysis.
Analyzing experimental result, whether strong and weak according to two kinds of fluorescence of sample judges sample to be tested CYP2C9 gene
There is 1300A > T sudden change.
In the present invention, described sample can be any sample comprising nucleic acid, such as blood;The most described sample
Come from people.Described nucleic acid can be DNA or coding RNA, preferably genomic DNA.The present invention
Analyze nucleic acid method can be with DNA or RNA as object.Skilled person will appreciate that, when
During with DNA for detection object, analyze and testing sample comprises the sequence corresponding to SEQ ID NO.1
Nucleic acid in corresponding to the nucleotide of the 1001st, the probe used or primer are according to SEQ ID NO.1
Sequential design;When with RNA for detection object, analyze comprising corresponding to SEQ in testing sample
Corresponding to the nucleotide of the 1300th, the probe used or primer root in the nucleic acid of the sequence of ID NO.2
Sequential design according to SEQ ID NO.2.
The seventh aspect of the invention is to provide CYP2C9 albumen or its fragment or variant, described albumen sequence
It is classified as the sequence shown in SEQ ID NO.3;Described fragment or variant comprise corresponding to SEQ ID NO.3
The phenylalanine of the 434th, and at least 10 companies of the aminoacid sequence shown in SEQ ID NO.3
Continuous aminoacid, such as 10-20,20-50 or 50-100 aminoacid.
The present invention will be further illustrated by specific embodiment below, but embodiment in detail below merely for
Exemplary purpose.
Embodiment
Embodiment 1: the qualification in the mutational site that people's CYP2C9 gene is new
In the present embodiment, gather the clinical obvious patient blood sample on the low side of warfarin medication dose, extract blood
In genomic DNA, design sequencing primer 9 exons of CYP2C9 gene are carried out sequence amplification,
Order-checking, analyzes whether its CYP2C9 gene exists mutational site.
1) DNA is extracted:
5ml vein EDTA anticoagulated blood sample is taked from measured;Then according to common salting out method and/or
Special DNA extraction kit (purchased from the DNA extraction kit of Omega company of the U.S.) is used to carry
Take the genomic DNA of blood sample to be measured.
2) PCR amplification:
Design amplimer, shows outside 9 of the CYP2C9 gene in the genome DNA sample obtained
Subsequence expands.Described amplimer is shown in Table 1 to sequence.
Use 50 μ l PCR reaction systems, including: 1 × PCR buffer, 1.5mM MgCl2, 100~
The genomic DNA of 150ng, upstream and downstream primer be 0.2 μM, dNTP be that 0.4mM, TaKaRa are public
The LATaq archaeal dna polymerase 1.5U of department.PCR amplification cycles parameter is as follows: 94 DEG C of denaturations 5 minutes,
94 DEG C of degeneration 30 seconds, anneal 30 seconds, and 72 DEG C extend 2 points 30 seconds, re-extend 5 minutes after 30 circulations.
Annealing temperature is relevant to primer length, and actual temp is shown in Table 1.
Use the GeneAmp PCR System9700 amplification instrument amplification of American AB I company.
Table 1: sequencing primer to and annealing temperature
3) purification amplified production:
Taking 50 μ l pcr amplification products and carry out agarose gel electrophoresis separation, blade cuts purpose band.Press
Reclaim test kit (Omega company) according to E.Z.N.A. gel and require that the DNA carrying out purpose band reclaims pure
Change.
4) order-checking:
With the product after recovery as template, use sequencing primer according to CEQTM DTCS-Quick Start Kit
Sequencing kit (Beckman company of the U.S.) requires to carry out PCR reaction of checking order, and reaction terminates and purification
After, carry out separating with interpretation amplified production with the CEQ8000 type gene sequencer of Beckman company of the U.S.
Sequence.Sequencing primer is shown 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:
By the sequence recorded and wild type CYP2C9*1 sequence (GenBank number of registration NM_000771.3)
Compare.
Being analyzed by comparison, the nucleotide of the 1300th of discovery CYP2C9 gene coding region is become from A
T(as it is shown in figure 1, wherein W represent A or T), it is the 9th outer aobvious that this sudden change is positioned at CYP2C9 gene
In son.Infer that, in the protein of this CYP2C9 gene code, the 434th amino acids is by isoleucine accordingly
(I) phenylalanine (F) is sported.This sudden change is by the named neomorph of P450 NK
CYP2C9*59, but the most externally announce.
The present embodiment exemplarily gives the method identifying new mutation site.Those skilled in the art according to
Foregoing can clearly be learnt specifically to detect in testing sample and comprise corresponding to SEQ ID NO.1
The method of the 1001st nucleotide: separate the nucleic acid in sample, experiment bar corresponding in the present embodiment
Carrying out amplified reaction under part, primer uses primer to SEQ ID NO.14 and 15;With sequencing primer SEQ ID
The product of amplification is checked order by NO.23;By sequencing result and wild-type results comparison, analyze corresponding to
The nucleotide in the 1001st site of SEQ ID NO.1.
Embodiment 2: the expression of target gene
To connect the plasmid vector of the open reading frame having wild type CYP2C9*1 (by American South Florida
University professor Zhou Shufeng present) be template, utilize side-directed mutagenesis obtain respectively CYP2C9*2,
The open reading frame (ORF) of the I434F mutant of CYP2C9*3, CYP2C9*13 and the present invention.Fixed point
Induced-mutation technique is techniques well known, and those skilled in the art, can milli according to the template determined and target
Free burial ground for the destitute has known how this step undoubtedly.
Then the ORF of CYP2C9*1 gene and four mutant genes of direct mutagenesis is cloned into connection
Have in the carrier pFastBac-dual of cytochrome P450 reductase (OR) so that CYP2C9
After gene and OR are respectively placed in PH and p10 promoter, build simultaneously express OR and CYP2C9(or
Its mutant) dual-expression vector.PFastBac-dual carrier structure figure and CYP2C9 gene and OR
Insertion point see Fig. 2.Using do not comprise CYP2C9 gene, only include the carrier of OR gene as
Negative control vector (pOR).
(purchased from American I nvitrogen company, use according to Bac-to-Bac baculovirus expression system test kit
Mass expressing external genes of interest in insect cell) operation instruction, utilize the dual-expression vector that builds
Pack P1 generation and P2 respectively with control vector for insect viruses, obtained P2 generation virus measure after titre according to
MOI(multiplicity-of-infection) be 4 the amount of infecting infect sf21 insect cell.Infect 72 little
Centrifugal collecting cell time after, uses the Ultrasonic Cell Disruptor (SONIC company of the U.S.) energy according to 40% to surpass
Raw smudge cells, utilizes differential centrifugation to extract insect cell microsome.Each micro-with the detection of Western method
The expression of CYP2C9 and OR in plastochondria;(it is called for short STD, purchased from the U.S. with CYP2C9 standard substance
BD Gentest company) obtained microsome is carried out quantitatively.
Western result is as shown in Figure 3.The first row is shown that CYP2C9 expression, and the second row shows
Be OR expression.As seen from the figure, control vector pOR only expresses OR albumen, 5 dual-expression vectors
All express OR albumen, and express * 1, * 2, * 3, * 13 types CYP2C9 and the I434F of the present invention respectively
Albumen.
Enzymes metabolism activity analysis
According to existing result of study, wild type (* 1 type) is the highest to the metabolic activity of various medicines,
And the metabolic activity of * 2 types is decreased obviously than the metabolic activity of wild type, the metabolic activity of * 3 types is than * 2 types
Lower (seeing list of references 18,19,21,22).Therefore, the most such a is known together:
The metabolic activity of specific substrate can be represented other substrate medicine by the enzyme expressed by same genotype
Metabolic activity.Thus, permissible to specific substrate metabolic activity data according to the enzyme expressed by a certain genotype
Analogizing the enzyme expressed by this genotype (e.g., can be by this genotype institute table to the metabolic activity of other substrate medicine
The metabolic activity of the enzyme reached compares with the metabolic activity of the enzyme expressed by wild type).
Embodiment 3: the Insect Microsomes analyzed in vitro that utilization the obtains metabolic characteristic to diclofenac:
1) chromatographic condition: chromatographic column be ZORBAX SB-C18 post (2.1*150mm, 5-Micron, Agilent,
The U.S.);Flowing is 0.1%TFA: water mutually: acetonitrile=20:35:45;Column temperature is 40 DEG C;Detection wavelength is: 280nm.
2) incubation conditions:
Reaction cumulative volume 200 μ L, including: 100mM Tris-HCl (pH7.4), 1 × NADPH
Coenzyme generates system (Promega company of the U.S.), 2pmol cytochrome b5 and diclofenac (purchased from U.S.
Sigma company of state, reacts final concentration of 1-100 μM).After 37 DEG C of preincubate 5min, add 2-5pmol
Embodiment 2 build restructuring microsome (respectively express * 1, * 2, * 3, * 13 types CYP2C9, this
Bright I434F) to start reaction.After 37 DEG C hatch 20min, add 100 μ L0.1M HCl and 10
μ L20ng/ μ L internal standard carbamazepine (purchased from Sigma Co., USA) vortex concussion 2min.Add
Vortex concussion 2min after 800 μ L glacial acetic acid ethyl esters, at 4 DEG C 10,000 × g is centrifuged 5min.Careful turn
Move organic layer, dry up with Nitrogen evaporator, add 100 μ L flowings and redissolve mutually and take 20 μ L in Waters
E2695 type high performance liquid chromatograph detects.
The Michaelis-Menten data results of the present embodiment is as shown in Figure 4.Carry out medicine further
Dynamic analysis, result is as shown in table 3:
Table 3: the pharmacokinetic analysis result of each microsomal metabolism diclofenac
Wherein, VmaxRepresent maximum reaction rate (numerical value is the biggest, shows that catalytic efficiency is the highest), KmFor rice
Family name's constant (numerical value is the biggest, shows that catalytic efficiency is the lowest), Vmax/KmSpeed removed by the medicine then reflecting entirety
Rate, for comprehensive performance assessment criteria, numerical value is the lowest, shows that the overall the enzyme activity of mutant is the lowest, drug metabolism
Speed is the lowest, and the individuality carrying this saltant type is the lowest to the requirement of medicine, the most easily occurs in medicine
Poison phenomenon.
From Fig. 4 and table 3, the overall the enzyme activity of the I434F of the present invention is well below wild type * 1
Type, also significantly lower than saltant type * 2 type and * 3 types, but higher than saltant type * 13 type.
Embodiment 4: the Insect Microsomes analyzed in vitro that utilization the obtains metabolic characteristic to tolbutamide:
1) chromatographic condition: chromatographic column be ZORBAX SB-C18 post (2.1*150mm, 5-Micron, Agilent,
The U.S.);Flowing is 0.1%TFA: water mutually: acetonitrile=20:40:40;Column temperature is 40 DEG C;Detection wavelength is: 230nm.
2) incubation conditions:
Reaction cumulative volume 200 μ L, including: 100mM Tris-HCl (pH7.4), 1 × NADPH
Coenzyme generates system, and 10pmol cytochrome b5 and tolbutamide are (purchased from Sigma Co., USA, instead
Answer final concentration of 10-1000 μM).After 37 DEG C of preincubate 5min, add the embodiment of 10-20pmol
The 2 restructuring microsomes built start reaction.After 37 DEG C hatch 60min, add 40 μ L0.1M HCl
2min is shaken with 50 μ L20ng/ μ L internal standard chlorpropamide (purchased from Sigma Co., USA) vortex.
Adding vortex concussion 2min after 800 μ L glacial acetic acid ethyl esters, at 4 DEG C 10,000 × g is centrifuged 5min.Little
Heart transfer organic layer, dries up under Nitrogen evaporator, adds 100 μ L flowings and redissolves mutually and take 20 μ L in Waters
E2695 type high performance liquid chromatograph detects.
The Michaelis-Menten data results of the present embodiment is as shown in Figure 5.Carry out medicine further
Dynamic analysis, result is as shown in table 4:
Table 4: the pharmacokinetic analysis result of each microsomal metabolism tolbutamide
From Fig. 5 and table 4, the overall the enzyme activity of the I434F of the present invention is significantly lower than wild type * 1
Type and saltant type * 2 type, also below saltant type * 3 type, but higher than saltant type * 13 type.
Embodiment 5: utilize the Insect Microsomes analyzed in vitro the obtained metabolic characteristic to losartan
1, chromatographic condition: chromatographic column be ZORBAX SB-C18 post (2.1*150mm, 5-Micron, Agilent,
The U.S.);Flowing is 0.1%TFA: water mutually: acetonitrile=20:42:38;Column temperature is 40 DEG C;Detection wavelength is: 230nm.
2, incubation conditions:
Reaction cumulative volume 200 μ L, including: 100mM Tris-HCl (pH7.4), 1 × NADPH
Coenzyme generates system, 10pmol cytochrome b5 and losartan, and (purchased from Sigma Co., USA, reaction is eventually
Concentration is 0.5-25 μM).After 37 DEG C of preincubate 5min, the embodiment 2 adding 10-20pmol builds
Restructuring microsome start reaction.After 37 DEG C hatch 30min, add 40 μ L0.1M HCl and 10 μ L
10ng/ μ L internal standard diazepam (purchased from Sigma Co., USA) vortex concussion 2min.Add 800 μ
Vortex concussion 2min after L glacial acetic acid ethyl ester, at 4 DEG C 10,000 × g is centrifuged 5min.Careful transfer is organic
Layer, dries up under Nitrogen evaporator, adds 100 μ L flowings and redissolves mutually and take 20 μ L in Waters e2695 type
High performance liquid chromatograph detects.
The Michaelis-Menten data results of the present embodiment is as shown in Figure 6.Carry out medicine further
Dynamic analysis, result is as shown in table 5:
Table 5: the pharmacokinetic analysis result of each microsomal metabolism losartan
From Fig. 5 and table 4, the overall the enzyme activity of the I434F of the present invention is far below wild type * 1 type,
Also below saltant type * 2 type and * 3 types, but higher than saltant type * 13 type.
According to above-described embodiment it can be seen that the I434F mutant enzyme metabolic activity of the present invention is well below wild
Type * 1 type, corresponding to saltant type * 2 type, its metabolic activity also ratio is relatively low.Therefore, in practice, need
Consider the individuality carrying this genotype is suitably regulated on dosage, as reduced the usage amount of medicine
With the generation avoiding adverse effect.This narrow for treatment window by the medicine adjustment of gene targeting
Medicine (such as warfarin, phenytoin etc.) is even more important.
Sequence:
SEQ ID NO.1: genomic dna sequence
GGACAGAGCCCTCCTGACTTATTCACTTCCTAAAAGGAGCCATCTCTTTAGTAAT
GTTGCATTGGGATTATGTGTCAACATATGAAATTGGGGGAGGCATATTCAGACCATAGC
ACATTTTTCAATGGAAATATAATGTTGAGGAAACCCAGAGAAGGCAACATTTTCTTGC
TCAAGGAGATGAGAAAGAGGGTAAAAAAGGAGATAAAATTTGACCTATGTCCTGACT
GTGGTAATAGAAAAGTTCATCTTGGCTAAAAGGAGCAGCATGATATAAAATTTGAAAC
CTCATGGTGTGTTGGAGACTGATGATGAGTGGCTATGCCTAGAGTTGACAGTATCGGA
TTTGAAGAGTGTAAGGAGTGATGTGGATCATCAGACTGGAAACAGAATGTGAGGGTC
CAGATCAATCCATTGGGACCTTATCCTATAGGACATACAGGGAAGCCATTTAAAGTTTT
AAAGTGAGAAGGTGACATGTTTAGACATGTGCTCCTGAAAGTACCTAGAGGAAAAAA
ATCTTTGGCTGCATATTGAGCCAGAAATACAAAGGGAAATACAGTATGTTAGCCTCCTC
CTCTAAGCCCTTCTCAGTTCAACCCACTGGACAAGAAATGTATGTTTCTAAAGAAAGA
TTGATGAAGACATTTAAAGTCTCTTGAAAGATTTTAATAAAGTGCTTGGCATGTAGCTG
GTACTCAACAAATATTTGTTGAATACAGGGTGCCTGTTAAGATCTGATATTAGGTGAAG
AGTAAGTATGTCCATTCATTTTTCAGTTGCCTATACATCCATCCATTCATCCATTTATCCA
TCCACTCATCCATCCATTCATTCATGCATGCACCCATCCACCCATCTATCTCTTCATCTCT
TCTACGATACACTGAACAGTTATTGCATATTCTGTTTGTGCCAGTTACAGAGACAGTGT
TTGTCACTGTCACAGTTACGCATGAGGAGTAACTGCTCTCTGTGTTTGCTATTTTCAGG
AAAACGGTTTTGTGTGGGAGAAGCCCTGGCCGGCATGGAGCTGTTTTTATTCCTGACC
TCCATTTTACAGAACTTTAACCTGAAATCTCTGGTTGACCCAAAGAACCTTGACACCA
CTCCAGTTGTCAATGGATTTGCCTCTGTGCCGCCCTTCTACCAGCTGTGCTTCATTCCT
GTCTGAAGAAGAGCAGATGGCCTGGCTGCTGCTGTGCAGTCCCTGCAGCTCTCTTTCC
TCTGGGGCATTATCCATCTTTCACTATCTGTAATGCCTTTTCTCACCTGTCATCTCACAT
TTTCCCTTCCCTGAAGATCTAGTGAACATTCGACCTCCATTACGGAGAGTTTCCTATGT
TTCACTGTGCAAATATATCTGCTATTCTCCATACTCTGTAACAGTTGCATTGACTGTCAC
ATAATGCTCATACTTATCTAATGTTGAGTTATTAATATGTTATTATTAAATAGAGAAATAT
GATTTGTGTATTATAATTCAAAGGCATTTCTTTTCTGCATGTTCTAAATAAAAAGCATTA
TTATTTGCTGAGTCAGTTTATTAGACCTTCCTTCTTTTATGCATAATGTAGGTCAGAAAT
TAAAGAAAATAGAGTTCCAGGAGGCCATGCTGGTTCTCAAAATGATAAGGACAGAAA
GGACAAAGAGGAAGAGGGTAGGGAAGCTATTTTGGGTGAGTGTTAGAGTTACTTGAG
GATTGGATTTGAAAGTGAGAAACTGTGTCCAGGGGCAGCTCTAACCTCTAGGGAAATA
TTCAGAGGATCAGTCAAAGGGTGGAATGGACATTAAATGCTAGAATTCTTATATCCACA
TTGGTGTTCCTTTTTTTTTGAGACAAAGTCTTGCTCTGTCACCCAGGCTGGAGTGCAG
TGGTGTGATCTCAGCTCTCTATAACCTCCGCCTCCCAGGTTCAAGTGATTCTCCTGCCT
CAGCCTCCTGAGTAGCTGGGATTACAGGTGCATGCCACCACACCTGGCTAATTTTTTGT
ATTTTTAG
SEQ ID NO.2: coded sequence
ATGGATTCTCTTGTGGTCCTTGTGCTCTGTCTCTCATGTTTGCTTCTCCTTTCACTC
TGGAGACAGAGCTCTGGGAGAGGAAAACTCCCTCCTGGCCCCACTCCTCTCCCAGTG
ATTGGAAATATCCTACAGATAGGTATTAAGGACATCAGCAAATCCTTAACCAATCTCTC
AAAGGTCTATGGCCCTGTGTTCACTCTGTATTTTGGCCTGAAACCCATAGTGGTGCTGC
ATGGATATGAAGCAGTGAAGGAAGCCCTGATTGATCTTGGAGAGGAGTTTTCTGCAAG
AGGCATTTTCCCACTGGCTGAAAGAGCTAACAGAGGATTTGGAATTGTTTTCAGCAAT
GGAAAGAAATGGAAGGAGATCCGGCGTTTCTCCCTCATGACGCTGCGGAATTTTGGG
ATGGGGAAGAGGAGCATTGAGGACCGTGTTCAAGAGGAAGCCCGCTGCCTTGTGGA
GGAGTTGAGAAAAACCAAGGCCTCACCCTGTGATCCCACTTTCATCCTGGGCTGTGCT
CCCTGCAATGTGATCTGCTCCATTATTTTCCATAAACGTTTTGATTATAAAGATCAGCAA
TTTCTTAACTTAATGGAAAAGTTGAATGAAAACATCAAGATTTTGAGCAGCCCCTGGA
TCCAGATCTGCAATAATTTTTCTCCTATCATTGATTACTTCCCGGGAACTCACAACAAAT
TACTTAAAAACGTTGCTTTTATGAAAAGTTATATTTTGGAAAAAGTAAAAGAACACCA
AGAATCAATGGACATGAACAACCCTCAGGACTTTATTGATTGCTTCCTGATGAAAATG
GAGAAGGAAAAGCACAACCAACCATCTGAATTTACTATTGAAAGCTTGGAAAACACT
GCAGTTGACTTGTTTGGAGCTGGGACAGAGACGACAAGCACAACCCTGAGATATGCT
CTCCTTCTCCTGCTGAAGCACCCAGAGGTCACAGCTAAAGTCCAGGAAGAGATTGAA
CGTGTGATTGGCAGAAACCGGAGCCCCTGCATGCAAGACAGGAGCCACATGCCCTAC
ACAGATGCTGTGGTGCACGAGGTCCAGAGATACATTGACCTTCTCCCCACCAGCCTGC
CCCATGCAGTGACCTGTGACATTAAATTCAGAAACTATCTCATTCCCAAGGGCACAAC
CATATTAATTTCCCTGACTTCTGTGCTACATGACAACAAAGAATTTCCCAACCCAGAGA
TGTTTGACCCTCATCACTTTCTGGATGAAGGTGGCAATTTTAAGAAAAGTAAATACTTC
ATGCCTTTCTCAGCAGGAAAACGGTTTTGTGTGGGAGAAGCCCTGGCCGGCATGGAG
CTGTTTTTATTCCTGACCTCCATTTTACAGAACTTTAACCTGAAATCTCTGGTTGACCC
AAAGAACCTTGACACCACTCCAGTTGTCAATGGATTTGCCTCTGTGCCGCCCTTCTAC
CAGCTGTGCTTCATTCCTGTCTGA
SEQ ID NO.3: protein sequence
MDSLVVLVLCLSCLLLLSLWRQSSGRGKLPPGPTPLPVIGNILQIGIKDISKSLTNLSK
VYGPVFTLYFGLKPIVVLHGYEAVKEALIDLGEEFSARGIFPLAERANRGFGIVFSNGKKW
KEIRRFSLMTLRNFGMGKRSIEDRVQEEARCLVEELRKTKASPCDPTFILGCAPCNVICSII
FHKRFDYKDQQFLNLMEKLNENIKILSSPWIQICNNFSPIIDYFPGTHNKLLKNVAFMKSYI
LEKVKEHQESMDMNNPQDFIDCFLMKMEKEKHNQPSEFTIESLENTAVDLFGAGTETTST
TLRYALLLLLKHPEVTAKVQEEIERVIGRNRSPCMQDRSHMPYTDAVVHEVQRYIDLLPT
SLPHAVTCDIKFRNYLIPKGTTILISLTSVLHDNKEFPNPEMFDPHHFLDEGGNFKKSKYF
MPFSAGKRFCVGEALAGMELFLFLTSILQNFNLKSLVDPKNLDTTPVVNGFASVPPFYQL
CFIPV
SEQ ID NO.24: nucleic acid fragment
ACGGTTTTGTGTGG
SEQ ID NO.25: nucleic acid fragment
CAGGAAAACGGTTTTGTGTGG
SEQ ID NO.26: nucleic acid fragment
AGGAAAACGGTTTTGTGTGGGAGAAGC
SEQ ID NO.27: nucleic acid fragment
CAGGAAAACGGTTTTGTGTGGGAGAAGCCCTG
SEQ ID NO.28: nucleic acid fragment
CTATTTTCAGGAAAACGGTTTTGTGTGGGAGAAGCCCTG
SEQ ID NO.29: nucleic acid fragment
TTGCTATTTTCAGGAAAACGGTTTTGTGTGGGAGAAGCCCTGGC
SEQ ID NO.30: nucleic acid fragment
GTTTGCTATTTTCAGGAAAACGGTTTTGTGTGGGAGAAGCCCTGGCCG
SEQ ID NO.31: nucleic acid fragment
GTTTGCTATTTTCAGGAAAACGGTTTTGTGTGGGAGAAGCCCTGGCCGGCATG
SEQ ID NO.32: oligonucleotide sequence
GCTTCTCCCACACAAAAC
SEQ ID NO.33: oligonucleotide sequence
CAGGGCTTCTCCCACACAAAAC
SEQ ID NO.34: oligonucleotide sequence
GGCCAGGGCTTCTCCCACACAAAAC
SEQ ID NO.35: oligonucleotide sequence
GCCGGCCAGGGCTTCTCCCACACAAAACC
SEQ ID NO.36: oligonucleotide sequence
CATGCCGGCCAGGGCTTCTCCCACACAAAACCG
SEQ ID NO.37: oligonucleotide sequence
CAAAACCGTTTTCCTG
SEQ ID NO.38: oligonucleotide sequence
CACACAAAACCGTTTTCCTG
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CYP2C19 gene pleiomorphism. Chinese Clinical pharmacology and therapeutics 2009;14(9):966-973.
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Claims (7)
1. nucleic acid fragment, described nucleic acid fragment comprises the prominent of the 1001st corresponding to SEQ ID NO.1
Displacement point, and be at least 10 continuous nucleotides in the nucleotide sequence shown in SEQ ID NO.1 or its
Complementary series, wherein the nucleotide of the 1001st is T;Or described nucleic acid fragment comprises corresponding to SEQ ID
The mutational site of the 1300th of NO.2, and be in the nucleotide sequence shown in SEQ ID NO.2 extremely
Few 10 continuous nucleotides or its complementary series, wherein the nucleotide of the 1300th is T.
Nucleic acid fragment the most according to claim 1, it is characterised in that the length of described nucleic acid fragment
It is 10-100,100-200,200-500 or 500-1000 nucleotide.
Nucleic acid fragment the most according to claim 2, it is characterised in that the length of described nucleic acid fragment
It is 10-20,20-30,30-40,40-50,50-60,60-100 or 100-300 nucleotide.
Nucleic acid fragment the most according to claim 1, it is characterised in that described nucleic acid fragment is SEQ
ID NO.1,2, the sequence shown in 24-31.
5. for detecting and/or analyze the test kit of single base mutation, including any one of claim 1-4 institute
The nucleic acid fragment stated.
6. the nucleic acid fragment described in any one of claim 1-4 is in preparation detection CYP2C9 gene mutation
Application in preparation.
7.CYP2C9 albumen, described protein sequence is the sequence shown in SEQ ID NO.3.
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