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CYP2C9 gene segment comprising 637A> G 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 637A> G mutation, encoded protein segment and application thereof
  • CYP2C9 gene segment comprising 637A> G mutation, encoded protein segment and application thereof
  • CYP2C9 gene segment comprising 637A> G 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, the blood samples of patients who are clinically used propofol are collected, the genomic DNA in the blood is extracted, sequencing primers are designed to amplify and sequence the 9 exons of the CYP2C9 gene, and analyze whether there are mutations in the CYP2C9 gene point.

[0043] 1) DNA extraction:

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

[0045] 2) PCR amplification:

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

[0047] Use 50μl PCR reaction system, including: 1×PCR buffer, 1.5mM MgCl 2 , 100-150 ng of genom...

Embodiment 2

[0061] Embodiment 2: the expression of target gene

[0062] Using the plasmid vector with the open reading frame of wild-type CYP2C9*1 (gifted by Professor Zhou Shufeng from the University of South Florida, USA) as a template, CYP2C9*2 (430C>T), CYP2C9*3 (1075A >C) and the open reading frame of the I213V 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 complete this step based on the determined template and target.

[0063] Then the ORFs of the CYP2C9*1 gene and the three mutant genes for site-directed mutagenesis were cloned into the vector pFastBac-dual connected with cytochrome P450 oxidoreductase (OR), so that the CYP2C9 gene and OR were placed in the pH and p10 promoters, respectively. After that, a double expression vector expressing both OR and CYP2C9 (or its mutants) was constructed. The structure diagram of pFastBac-dual vector and the insertion site of CYP2C9 gene ...

Embodiment 3

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

[0069] 1) Chromatography and mass spectrometry conditions: analysis was carried out by liquid chromatography-mass spectrometry. The chromatographic column is a Waters Acquity UPLCBEH C18 reverse-phase chromatographic column (2.1*50mm, 1.7μm, Waters Corp, USA); the mobile phase A is 0.1% formic acid; the mobile phase B is acetonitrile; the column temperature is 40°C, and the flow rate is 0.4ml / min, carry out gradient elution: 0-1.4min, A (60%-10%), 1.4-2.6min, A (10%-60%). Electrospray ionization source (ESI): Positive ion mode is selected for scanning; 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 table below:

[0070]

[0071]

[0072] 2) Incubation conditions:

[0073] The total reaction volume was 200 μL, which included...

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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 637th site of SEQ ID NO.2, ofCYP2C9 gene, the site is mutated from wild type A into 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 disclsoed. 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 specifically, the present invention relates to a 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 CYP enzymes in human liver microsomes. About 10-16% of commonly used clinical drugs are oxidatively metabolized by CYP2C9, mainly including tolbutamide, S-warfarin, phenytoin, glipizide, glibenclamide, toratimide, losartan, urethane Medications 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 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 ind...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/02C12Q1/6888C12N15/11
CPCC12N9/0071C12Y114/14001C12Q1/6888C12Q2600/106C12Q2600/156
Inventor 蔡剑平周晓阳左明章赵思文
Owner BEIJING HOSPITAL
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