CYP2C9 gene fragment containing 1300A>T mutation, protein fragment encoded through same and application thereof

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

Active Publication Date: 2013-06-26
BEIJING HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] 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 fragment containing 1300A>T mutation, protein fragment encoded through same and application thereof
  • CYP2C9 gene fragment containing 1300A>T mutation, protein fragment encoded through same and application thereof
  • CYP2C9 gene fragment containing 1300A>T mutation, protein fragment encoded through same and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1: Identification of new mutation sites in human CYP2C9 gene

[0073] 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.

[0074] 1) DNA extraction:

[0075] Take a 5ml 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).

[0076] 2) PCR amplification:

[0077] 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.

[0078] Use 50μl PCR reaction system, including: 1×PCR buffer, 1.5mM MgCl 2 , 100-150ng o...

Embodiment 2

[0093] Embodiment 2: the expression of target gene

[0094] 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, CYP2C9*2, CYP2C9*3, CYP2C9*13 and the present invention were respectively obtained by site-directed mutagenesis. Open reading frame (ORF) of the I434F mutant. 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.

[0095] Then the ORFs of the CYP2C9*1 gene and the four 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, construct a dual expression vector that simultaneously expresses OR and CYP2C9 (or its mutants). The structure of pFas...

Embodiment 3

[0100] Example 3: In vitro analysis of the metabolic properties of diclofenac using the obtained insect microsomes:

[0101] 1) Chromatographic conditions: the chromatographic column is ZORBAX SB-C18 column (2.1*150mm, 5-Micron, Agilent, USA); the mobile phase is 0.1% TFA:water:acetonitrile=20:35:45; the column temperature is 40°C; The detection wavelength is: 280nm.

[0102] 2) Incubation conditions:

[0103] The total reaction volume is 200 μL, including: 100 mM Tris-HCl (pH7.4), 1×NADPH coenzyme generation system (Promega, USA), 2 pmol cytochrome b5 and diclofenac (purchased from Sigma, USA, the final reaction concentration is 1-100 μM ). After pre-incubation at 37°C for 5 min, 2-5 pmol of recombinant microsomes constructed in Example 2 (expressing *1, *2, *3, *13 types of CYP2C9 and I434F of the present invention respectively) were added to start the reaction. After incubation at 37°C for 20 min, 100 μL of 0.1 M HCl and 10 μL of 20 ng / μL internal standard carbamazepine ...

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Abstract

The invention belongs to the field of biology, relates to single-base mutation and particularly relates to a 1300th bit mutation site of a CYP2C9 gene corresponding to SEQ ID NO.2. The 1300th bit mutation site is mutated from wild type A into T. The invention relates to a nucleic acid fragment containing the 1300th bit mutation site, a protein fragment encoded through the same and application thereof. The invention also provides allelomorphic gene specific oligonucleotide for detecting the 1300th bit mutation site, a kit and a detection method.

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 the mutation site of CYP2C9 gene relative to the 1001st position of SEQ ID NO.1 or the 1300th position of SEQ ID NO.2, the nucleic acid fragment comprising the mutation site and the corresponding encoded protein fragment thereof . The invention also relates to a reagent and a detection method for identifying the mutation site, and the application of the identification site in guiding medication. 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 clinically used drugs are oxidatively metabolized by CYP2C9, mainly including tolbutamide, S-warfarin, phenytoin, glipizide, glibenclamide, toratimide, losartan, urethane Medications such as besartan and many ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N9/02C12Q1/68
Inventor 蔡剑平李传保胡国新戴大鹏王双虎耿培武
Owner BEIJING HOSPITAL
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