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Macaca fascicularis P450 2C9 medical metabolic enzyme and co-expression recombinant carrier with macaca fascicularis P450 oxidoreductase

A recombinant vector, cynomolgus monkey technology, applied in the field of genetic engineering research

Inactive Publication Date: 2008-08-27
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

Coexpression of cynomolgus monkey P450 2C9 with cynomolgus monkey P450 oxidoreductase gene has not been reported so far

Method used

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  • Macaca fascicularis P450 2C9 medical metabolic enzyme and co-expression recombinant carrier with macaca fascicularis P450 oxidoreductase
  • Macaca fascicularis P450 2C9 medical metabolic enzyme and co-expression recombinant carrier with macaca fascicularis P450 oxidoreductase
  • Macaca fascicularis P450 2C9 medical metabolic enzyme and co-expression recombinant carrier with macaca fascicularis P450 oxidoreductase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1. Cloning of cynomolgus monkey drug metabolizing enzyme P450 2C9 gene (cyp 2C9)

[0020] According to the published human P450 2C9 gene sequence (Genebank, Accession NO.: NM_000771), design primers:

[0021] Primer 1: 5'-ATGGATTCTCTTGTGGTC-3'

[0022] Primer 2: 5'-CTTCAAACAGGAATGAAGCAC-3'

[0023] Using cDNA reverse transcribed from cynomolgus monkey liver RNA as a template, the coding region sequence (cyp 2C9) of cynomolgus monkey P450 2C9 was amplified by polymerase chain reaction (PCR), and the amplified PCR product was compatible with pMD 18- The T vector was ligated to obtain the pT-cynoP2C9 plasmid. Among them, the designed primers do not contain the coding region sequence (cyp 2C9) of cynomolgus monkey P450 2C9 except the start codon (ATG). After sequencing, the obtained coding region sequence has a full length of 1473 base pairs, encoding 491 amino acids. figure 1 It is the RT-PCR result map of cynomolgus monkey P450 2C9 gene (cyp 2C9), Lane 1: Mark...

Embodiment 2

[0024] Example 2. Construction and expression of cynomolgus monkey drug metabolizing enzyme P450 2C9 insect expression system

[0025] Using the above cloned cynomolgus monkey P450 2C9 gene (cyp 2C9) as a template, design primers comprising RsrII and HindIII restriction endonuclease sites:

[0026] Primer 3: 5'-TGTCGGTCCGATGGATTCTCTTGTGGTC-3'

[0027] Primer 4: 5'-CGCAAGCTTCAAACAGGAATGAAGCAC-3'

[0028]The coding region sequence of cynomolgus monkey P450 2C9 (cyp 2C9) was amplified by PCR and inserted between the RsrII and HindIII restriction sites of the insect expression vector pFastBac-Dual to obtain the pFB-cynoP2C9 expression vector. Sequencing to confirm that the gene is correctly inserted into the vector. Cynomolgus P450 2C9 protein was expressed in sf-9 insect cells according to the Bac-to-Bac Baculovirus Expression System Instruction Manual (provided by Invitrogen). Briefly, the pFB-cynoP2C9 plasmid constructed above was used to transfect DH10Bac competent cells, p...

Embodiment 3

[0029] Example 3. Cloning of cynomolgus monkey NADPH-cytochrome P450 oxidoreductase gene

[0030] According to the published human NADPH-cytochrome P450 oxidoreductase gene sequence (Genebank, Accession NO.: NM_000941), the following primers (primer 5 and primer 6) were designed:

[0031] Primer 5: 5'-ATGATCAACATGGGAGACTCCC-3'

[0032] Primer 6: 5'-CTAGCTCCACACGTCCAGGG-3'

[0033] Using cDNA reverse transcribed from cynomolgus monkey liver RNA as a template, the coding region sequence of NADPH-cytochrome P450 oxidoreductase gene was amplified from cynomolgus monkey by polymerase chain reaction. The obtained PCR product was ligated with pMD 18-T vector to obtain pT-cynoPOR plasmid. After sequencing, the obtained coding region sequence has a full length of 2034 base pairs, encoding 677 amino acids. Figure 4 It is the RT-PCR result map of cynomolgus monkey P450 oxidoreductase, Lane 1: Marker DL2000 (the band sizes from top to bottom are: 2000bp, 1000bp, 750bp, 500bp, 200bp, 1...

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Abstract

The invention discloses cynomolgus monkey P450 2C9 drug metabolizing enzyme and a co-expression recombinant vector of the cynomolgus monkey P450 2C9 drug metabolizing enzyme and cynomolgus monkey P450 oxidoreductase. The cynomolgus monkey P450 2C9 drug metabolizing enzyme can catalyze 4'-hydroxylation of diclofenac, the 4'-hydroxylation of tolbutamide and the 7'-hydroxylation of warfarin and a gene sequence of the cynomolgus monkey P450 2C9 drug metabolizing enzyme or a complementary sequence of the cynomolgus monkey P450 2C9 drug metabolizing enzyme is a sequence in SEQ ID NO:1 with the mutation rate of 0-1%. The co-expression recombinant vector of the cynomolgus monkey P450 2C9 drug metabolizing enzyme and the cynomolgus monkey P450 oxidoreductase comprises an open reading frame of the sequence of the cynomolgus monkey P450 2C9 drug metabolizing enzyme and the sequence of the cynomolgus monkey P450 oxidoreductase. The sequence or the complementary sequence of the cynomolgus monkey P450 oxidoreductase is shown in SEQ ID NO:2. Protein which is expressed by a heterogeneous source of the invention only represents the cynomolgus monkey P450 2C9 and the system is closer to the real situation of metabolism in vivo of cynomolgus monkeys.

Description

technical field [0001] The present invention relates to the field of genetic engineering research, in particular to a P450 2C9 drug metabolizing enzyme (Cytochrome P450 2C9) of cynomolgus Monkey (Macafascicularis) and its co-expression recombination with P450 oxidoreductase (NADPH-cytochrome P450 oxidoreductae) carrier. 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 2C subfamily is the main member of the cytochrome P450 gene family, mainly including several members of 2C8, 2C9 and 2C18. Cytochrome P450 2C9 can metabolize a variety of drugs such as Diclofenac (Diclofenac), Tolbutamide (Tolbutamide), warfarin ((s)-(-)-warfarin) and so on. At present, the 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, ye...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/00C12N15/52C12N15/53C12N15/62C12N15/866
Inventor 戴仁科温鼎声王海涛佘妙琴白杨
Owner GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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