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.
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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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