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CYP2C9 gene segment containing 373C>T mutation, encoded protein segment and application of CYP2C9 gene segment

A technology of nucleic acid fragments and mutation sites, applied in the field of biology, can solve problems such as differences in drug efficacy, insufficiency, drug toxicity and side effects, etc.

Inactive Publication Date: 2021-03-09
杨杰孚 +1
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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 containing 373C>T mutation, encoded protein segment and application of CYP2C9 gene segment
  • CYP2C9 gene segment containing 373C>T mutation, encoded protein segment and application of CYP2C9 gene segment
  • CYP2C9 gene segment containing 373C>T mutation, encoded protein segment and application of CYP2C9 gene segment

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Identification of new mutation sites in human CYP2C9 gene

[0041] In this example, the blood samples of patients with clinically low warfarin dosage were collected, the genomic DNA in the blood was extracted, and sequencing primers were designed to amplify and sequence the 9 exons of the CYP2C9 gene to analyze whether the CYP2C9 gene There are mutation sites.

[0042] 1) DNA extraction:

[0043] 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).

[0044] 2) PCR amplification:

[0045] 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 primer pairs are shown in Table 1.

[0046] Use 50μl PCR reaction system, including: 1×PCR buffer, 1.5mM MgCl 2 , 100-150ng ...

Embodiment 2

[0060] Embodiment 2: the expression of target gene

[0061] The open reading frame (ORF) of the R125C mutant of the present invention was respectively obtained by site-directed mutagenesis using the plasmid vector (gifted by Professor Zhou Shufeng from the University of South Florida) connected with the open reading frame of wild-type CYP2C9*1 as a template. 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.

[0062] Then clone the ORF of the CYP2C9*1 gene and the mutant gene for site-directed mutagenesis into the vector pFastBac-dual connected with cytochrome P450 oxidoreductase (OR), so that the CYP2C9 gene and OR are respectively placed behind the PH and p10 promoters , to construct a dual expression vector expressing OR and CYP2C9 (or its mutants) simultaneously. The structure of pFastBac-dual vector and the insertion site of CYP2C9 ge...

Embodiment 3

[0066] Example 3: In vitro analysis of the metabolic properties of diclofenac using the obtained insect microsomes:

[0067] 1) Chromatographic conditions: CORTECST UPLC C18 column (1.6μm, 2.1×50mm); phase A acetonitrile, phase B 0.1% formic acid-water, flow rate 0.4ml / min; 0-0.5min, 20%A; 0.5-1min , 20%-95%A; 1-2min, 95%A; 2-2.3min, 95%-20%A, running time 3min. The column temperature is 40°C.

[0068] 2) Mass spectrometry detection conditions:

[0069] The ion pair of 4-hydroxydiclofenac is 311.94>265.95, the cone voltage is 20v, and the collision energy is 18v; the internal standard diazepam ion pair is 285.1>193.1, the cone voltage is 35v, and the collision energy is 30v. Use the MRM model. The detection wavelength is: 280nm.

[0070] 3) Incubation conditions:

[0071]The total volume of the reaction was 200 μL, including: 100 mM Tris-HCl (pH 7.4), 1×NADPH coenzyme generation system (Promega, USA), 2 pmol cytochrome b5 and diclofenac (purchased from Sigma, USA, the fin...

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Abstract

The invention discloses a CYP2C9 gene segment containing the 373C>T mutation, a coded protein segment and an application of the CYP2C9 gene segment, belongs to the field of biology, and relates to single base mutations. More specifically, the invention relates to a mutation site, corresponding to a 373th site of SEQ ID NO.2, of a CYP2C9 gene, a nucleic acid fragment containing the mutation site, aprotein fragment coded by the nucleic acid fragment, and an application of the nucleic acid fragment, wherein the site is mutated from wild type C to T. The mutant CYP2C9 protein, namely R125C, caused by the single base mutation has lower metabolic activity of drugs compared than wild type CYP2C9 protein, and therefore the guiding significance is provided for the medication of individuals carrying the mutation site.

Description

technical field [0001] The invention belongs to the field of biology and relates to a single base mutation; more specifically, the 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 individual...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/6883C12Q2600/106C12Q2600/156
Inventor 杨杰孚陈浩
Owner 杨杰孚
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