Cytochrome P450 enzyme and NADPH-cytochrome P450 oxidation-reduction enzyme of crab-eating macaque

A cytochrome and cynomolgus monkey technology, applied in the field of genetic engineering, can solve problems such as no reports of cynomolgus monkey clones

Inactive Publication Date: 2010-06-23
GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, the P450 oxidoreductase genes of humans and rats have been cloned, but there is no report on the cloning of P450 oxidoreductase genes in cynomolgus monkeys

Method used

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  • Cytochrome P450 enzyme and NADPH-cytochrome P450 oxidation-reduction enzyme of crab-eating macaque
  • Cytochrome P450 enzyme and NADPH-cytochrome P450 oxidation-reduction enzyme of crab-eating macaque
  • Cytochrome P450 enzyme and NADPH-cytochrome P450 oxidation-reduction enzyme of crab-eating macaque

Examples

Experimental program
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Effect test

Embodiment 1

[0033]Microsomes of cynomolgus monkey P450 3A5 recombinantly expressed in sf-9 insect cells were prepared as in Example 1;

[0034] Put 0.36 mL of the mixture of microsomes, testosterone at a fixed concentration and serially diluted ketoconazole into a reaction tube, and place in a water bath at 37° C. for 5 minutes to incubate. Add 40 μL of 10 mM NADPH solution to start the reaction. After 10 minutes of reaction, add 0.8mL reaction termination solution containing internal standard to terminate the reaction, mix well and place in ice water;

[0035] The above-mentioned reaction tubes were centrifuged at 2500g for 30 minutes, and 100 μL of the supernatant was taken and added to a 96-well plate for sample injection. LC / MS / MS quantitative analysis and data processing were performed, and the activity of ketoconazole to inhibit the metabolism of testosterone by cynomolgus monkey P450 3A5 was calculated.

[0036] Cytochrome P450 enzymes and NADPH-cytochrome P450 oxidoreductase in c...

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Abstract

The invention relates to genetic engineering technical field, in particular to macaca fascicularis cytochromeP4502 3A5 gene, macaca fascicularis NADPH-cytochrome P450 oxidoreductase gene, a recombinant vector which contains the two genes, a host transformed by the vector and the recombined virus produced by the transformant. The invention includes the production of gene expression products whichhave the enzyme activity and the application of the gene expression products in medicine screening.

Description

[technical field] [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a gene of cytochrome P4503A5, a recombinant vector containing the gene, a host transformed with the vector, an expression product of the gene produced by a transformant and an application thereof. [Background technique] [0002] Cytochrome P450 is an important drug metabolism phase I enzyme, and more than 80% of drugs on the market are metabolized by cytochrome P450. The 3A subfamily is the main member of the cytochrome P450 gene family, mainly including several members of 3A4, 3A5 and 3A7. Cytochrome P450 3A5 has a metabolic profile similar to 3A4, and can metabolize a variety of drugs such as midazolam, nifedipine, testosterone, progesterone, etc. [0003] Cytochrome P450 gene sequences of various animals including humans, dogs, rats, and mice have been cloned, and various cytochrome P450s have also been expressed in hosts such as Escherichia coli, yea...

Claims

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Application Information

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Patent Type & AuthorityPatents(China)
IPC IPC(8): C12N9/02C12N15/53C12N15/866C12N15/12
Inventor戴仁科吴志聪陈彦郭宾
OwnerGUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI