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CYP2C9 gene segment comprising 419G> A mutation, encoded protein segment and application thereof

A technology of fragments and nucleic acid fragments, applied in the field of biology, can solve the problems of drug toxicity and side effects, insufficient treatment, and differences in drug efficacy.

Active Publication Date: 2020-04-14
BEIJING HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

According to current clinical research, this polymorphism of CYP2C9 gene is the main reason for the great difference in CYP2C9 enzyme activity among individuals, so it can cause great differences in drug efficacy among individuals carrying different CYP2C9 genotypes, Even severe drug side effects or inadequate treatment

Method used

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  • CYP2C9 gene segment comprising 419G> A mutation, encoded protein segment and application thereof
  • CYP2C9 gene segment comprising 419G> A mutation, encoded protein segment and application thereof
  • CYP2C9 gene segment comprising 419G> A mutation, encoded protein segment and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: Identification of new mutation sites in human CYP2C9 gene

[0042] In this example, blood samples were collected from patients using propofol clinically, genomic DNA in the blood was extracted, and sequencing primers were designed to amplify and sequence the 9 exons of the CYP2C9 gene, and analyze whether the CYP2C9 gene has a mutation. point.

[0043] 1) Extract DNA:

[0044] A 5ml intravenous EDTA anticoagulated blood sample was taken from the subject; then the genomic DNA of the blood sample to be tested was extracted according to the common salting-out method and / or using a special DNA extraction kit (DNA extraction kit purchased from Omega, USA).

[0045] 2) PCR amplification:

[0046] The amplification primers were designed to amplify the 9 exon sequences of the CYP2C9 gene in the obtained genomic DNA samples. The sequences of the amplification primer pairs are shown in Table 1.

[0047] A 50 μl PCR reaction system was used, including: 1× PCR buffer...

Embodiment 2

[0061] Example 2: Expression of target genes

[0062] Using the plasmid vector linked with the open reading frame of wild-type CYP2C9*1 (gifted by Professor Zhou Shufeng of the University of South Florida) as a template, CYP2C9*2 (430C>T) and CYP2C9*3 (1075A) were obtained by site-directed mutagenesis. >C) and the open reading frame of the S140N mutant of the invention. The technique of site-directed mutagenesis is well-known in the art, and those skilled in the art can undoubtedly know how to accomplish this step according to the determined template and target.

[0063] Then the ORFs of the CYP2C9*1 gene and the three mutant genes of site-directed mutagenesis were cloned into the vector pFastBac-dual linked with cytochrome P450 oxidoreductase (OR), so that the CYP2C9 gene and OR were placed in the PH and p10 promoters, respectively Then, construct a dual expression vector expressing OR and CYP2C9 (or its mutants) at the same time. For the structure of the pFastBac-dual vect...

Embodiment 3

[0068] Example 3: In vitro analysis of the metabolic properties of tolbutamide using the obtained insect microsomes:

[0069] 1) Chromatography and mass spectrometry conditions: LC-MS was used for analysis. The chromatographic column is Waters Acquity UPLCBEH C18 reversed-phase column (2.1*50mm, 1.7μm, Waters Corp, USA); mobile phase A is 0.1% formic acid; mobile phase B is acetonitrile; column temperature is 40°C, flow rate is 0.4ml / min, carry out gradient elution: 0-1.4 min, A (60%-10%), 1.4-2.6 min, A (10%-60%). Electrospray ion source (ESI): Select positive ion mode to scan; the ion source temperature is 500°C, the signal acquisition method is multi-stage reaction monitoring, and the parameters of metabolites and internal standards are shown in the following table:

[0070]

[0071]

[0072] 2) Incubation conditions:

[0073] The total reaction volume was 200 μL, including: 100 mM Tris-HCl (pH 7.4), 1×NADPH coenzyme generation system, 2 pmol cytochrome b5 and tolbut...

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Abstract

The invention belongs to the field of biology, and relates to single base mutation, and more specifically, the invention relates to a mutation site, corresponding to the 419th site of SEQ ID NO.2, ofCYP2C9 gene; the site is mutated into A from wild-type G, a nucleic acid fragment containing the mutation site, a protein fragment coded by the nucleic acid fragment and application of the nucleic acid fragment are disclosed. The invention also provides allele-specific oligonucleotides, kits and detection methods for detecting the mutation site.

Description

technical field [0001] The invention belongs to the field of biology, and relates to single-base mutation; more particularly, the invention relates to single-base mutation of CYP2C9 gene. Background technique [0002] CYP2C9 is the most important member of the CYP2C subfamily of the cytochrome P450 enzyme family, accounting for about 20% of the total amount of human liver microsomal CYP enzymes. About 10-16% of commonly used clinical drugs are metabolized by CYP2C9 oxidation, mainly including tolbutamide, S-warfarin, phenytoin, glipizide, glyburide, tolatimide, losartan, and Drugs such as besartan and many NSAIDs (eg: ibuprofen, lornoxicam, diclofenac, and naproxen) (see references 1-5). [0003] The CYP2C9 gene is highly polymorphic. According to the current clinical research, this polymorphism of the CYP2C9 gene is the main reason for the great difference in CYP2C9 enzyme activity among individuals, so it can cause great differences in drug efficacy among individuals wit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N15/11C12Q1/6883
CPCC12N9/0081C12Q1/6883C12Y114/15006C12Q2600/106C12Q2600/156
Inventor 蔡剑平周晓阳左明章赵思文
Owner BEIJING HOSPITAL
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