CYP3A4 gene fragment including 337T>A mutation, encoded protein fragment and application thereof

A fragment and gene technology, applied in the biological field, can solve the problems of drug side effects, insufficient treatment, and differences in drug efficacy

Active Publication Date: 2017-04-26
WENZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] According to the current clinical research, the polymorphism of CYP3A4 gene is the main reason for the great difference in CYP3A4 enzyme activity among individuals, so it can cause great differences in the efficacy of drugs among individuals carrying different CYP3A4 genotypes, and even cause severe Drug toxicity or inadequate treatment of

Method used

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  • CYP3A4 gene fragment including 337T>A mutation, encoded protein fragment and application thereof
  • CYP3A4 gene fragment including 337T>A mutation, encoded protein fragment and application thereof
  • CYP3A4 gene fragment including 337T>A mutation, encoded protein fragment and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: Identification of new mutation sites in human CYP3A4 gene

[0046] In this example, 1114 blood samples from healthy volunteers were collected, genomic DNA in the blood was extracted, sequencing primers were designed to amplify and sequence the 13 exons of the CYP3A4 gene, and to analyze whether there were mutation sites in the CYP3A4 gene .

[0047] 1. Extract DNA:

[0048] 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).

[0049] 2. PCR amplification:

[0050] Amplification primers were designed to amplify the 13 exon sequences of the CYP3A4 gene in the obtained genomic DNA sample. The sequences of the amplification primer pairs are shown in Table 1.

[0051] Use 50μL PCR reaction system, including: 1×PCR buffer, 1.5mM MgCl 2 , 100-150ng of genomic DNA, both u...

Embodiment 2

[0067] Embodiment 2: the expression of target gene

[0068]Using the plasmid vector (gifted by Professor Zhou Shufeng from the University of South Florida) connected with the open reading frame of wild-type CYP3A4*1 as a template, CYP3A4*4, CYP3A4*5, CYP3A4*18 and the present invention were respectively obtained by site-directed mutagenesis. The open reading frame (ORF) of the F113I 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.

[0069] Then the ORFs of the CYP3A4*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 CYP3A4 gene and OR were placed in the pH and p10 promoters respectively. After that, a double expression vector expressing both OR and CYP3A4 (or its mutants) was constructed. The structure diagram o...

Embodiment 3

[0074] Example 3: In vitro analysis of the metabolic properties of testosterone using the obtained insect microsomes

[0075] 1. Chromatographic conditions: the chromatographic column is a ZORBAX SB-C18 column (2.1*150mm, 5-Micron, Agilent, USA); the mobile phase is 0.05% TFA: acetonitrile=55:45; the column temperature is 35°C; the detection wavelength is: 240nm.

[0076] 2. Incubation conditions:

[0077] The total reaction volume is 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, 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, *4, *5, *18 types of CYP3A4 and F113I of the present invention respectively) were added to start the reaction. After incubation at 37° C. for 20 min, 50 μL of 0.1 M HCl and 20 μL of 25 ng / μL internal standard desipramine (purchas...

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Abstract

The invention discloses a CYP3A4 gene fragment including a 337T>A mutation, an encoded protein fragment and application thereof and belongs to the technical field of biology. The nucleic acid fragment includes a mutation site corresponding to 1012th site in a sequence table SEQ ID NO:1, and the nucleotide at the 1012th site in at least ten continuous nucleotides in a nucleotide sequence shown by the sequence table SEQ ID NO:1 is A or is a complementary sequence for the nucleic acid fragment. The invention provides a method for rapidly amplifying a target gene through an oligonucleotide fragment. An allele of a biological sample is rapidly analyzed, and whether variation exists or not is prompted. Activity study is conducted on an existing produced altered protein, and adjustment of the drug usage amount can be recommended according to experimental data.

Description

technical field [0001] The invention belongs to the field of biotechnology, and includes the CYP3A4 gene fragment with 337T>A mutation, the encoded protein fragment and application thereof. Background technique [0002] CYP3A4 is the most important member of the CYP3A subfamily of the cytochrome P450 enzyme family, accounting for about 60% of the total CYP enzymes in human liver microsomes and 70% of the total intestinal microsomes. About 40-50% of commonly used clinical drugs are oxidatively metabolized by CYP3A4, which mainly include macrolide antibiotics, antifungal drugs, H1 receptor antagonists, HMG-CoA reductase inhibitors, benzodiazepines, protons, etc. Pump inhibitors, calcium channel blockers, anticancer drugs, antipsychotics and other drugs. [0003] The CYP3A4 gene is highly polymorphic. So far, there are 26 types of alleles named in the world (http: / / www.cypalleles.ki.se), except for the wild type (CYP3A4*1), there are 25 types of mutations that can cause ch...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/02C12Q1/68C12N15/11
CPCC12N9/0077C12Q1/6883C12Q2600/106C12Q2600/156
Inventor 胡国新蔡剑平戴大鹏黄象鑫
Owner WENZHOU MEDICAL UNIV
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