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Macaca fascicularis P450 2B6 medical metabolic enzyme and co-expression recombinant carrier with macaca fascicularis P450 oxidoreductase

A P4502B6, recombinant vector 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

Co-expression of cynomolgus P450 2B6 with cynomolgus P450 oxidoreductase genes has not been reported so far

Method used

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  • Macaca fascicularis P450 2B6 medical metabolic enzyme and co-expression recombinant carrier with macaca fascicularis P450 oxidoreductase
  • Macaca fascicularis P450 2B6 medical metabolic enzyme and co-expression recombinant carrier with macaca fascicularis P450 oxidoreductase
  • Macaca fascicularis P450 2B6 medical metabolic enzyme and co-expression recombinant carrier with macaca fascicularis P450 oxidoreductase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1. Cloning of cynomolgus monkey drug metabolizing enzyme P450 2B6 gene (cyp 2B6)

[0019] According to the published human P450 2B6 gene sequence (Genebank, Accession NO.: NM_000767), the following primers were designed:

[0020] Primer 1: 5'-ATGGAGCTCAGCGTCCTC-3'

[0021] Primer 2: 5'-CCTTCAGCGGGGCAGG-3'

[0022] Using cDNA reverse transcribed from cynomolgus monkey liver RNA as a template, the coding region sequence (cyp 2B6) of cynomolgus monkey P450 2B6 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-cynoP2B6 plasmid. Among them, the designed primers do not contain the coding region sequence (cyp 2B6) of cynomolgus monkey P450 2B6 except the start codon (ATG). After sequencing, the obtained coding region sequence has a full length of 1482 base pairs, encoding 494 amino acids. figure 1 It is the RT-PCR result map of cynomolgus monkey P450 2B6 gene (cyp 2B...

Embodiment 2

[0023] Example 2. Construction and expression of cynomolgus monkey drug metabolizing enzyme P450 2B6 insect expression system

[0024] Using the above cloned cynomolgus monkey P450 2B6 gene (cyp 2B6) as a template, design primers comprising RsrII and HindIII restriction endonuclease sites:

[0025] Primer 3: 5'-ATACGGTCCGATGGAGCTCAGCGTCCTC-3'

[0026] Primer 4: 5'-AGAAAGCTTCAGCGGGGCAGGAAGCGG-3'

[0027]The coding region sequence of cynomolgus monkey P450 2B6 (cyp 2B6) was amplified by PCR and inserted between the RsrII and HindIII restriction sites of the insect expression vector pFastBac-Dual to obtain the pFB-cynoP2B6 expression vector. Sequencing to confirm that the gene is correctly inserted into the vector. The cynomolgus P450 2B6 protein was expressed in sf-9 insect cells according to the baculovirus expression manual. Briefly, the pFB-cynoP2B6 plasmid constructed above was used to transfect DH10Bac competent cells, positive clones were identified by PCR, recombinant ...

Embodiment 3

[0028] Example 3. Cloning of cynomolgus monkey NADPH-cytochrome P450 oxidoreductase gene

[0029] 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:

[0030] Primer 5: 5'-ATGATCAACATGGGAGACTCCC-3'

[0031] Primer 6: 5'-CTAGCTCCACACGTCCAGGG-3'

[0032] Using the cDNA reverse transcribed from cynomolgus monkey liver RNA as a template, the coding region sequence of the cynomolgus monkey NADPH-cytochrome P450 oxidoreductase gene was amplified by polymerase chain reaction (PCR). The resulting PCR product was ligated with the pMD 18-T vector to obtain the 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, 750b...

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Abstract

The invention discloses cynomolgus monkey P450 2B6 drug metabolizing enzyme and a co-expression recombinant vector of the cynomolgus monkey P450 2B6 drug metabolizing enzyme and cynomolgus monkey P450 oxidoreductase. The cynomolgus monkey P450 2B6 drug metabolizing enzyme can catalyze hydroxylation of bupropion hydrochloride and N end demethylation of 3-methyl benzene phenytoin, a gene sequence of the cynomolgus monkey P450 2B6 drug metabolizing enzyme or a complementary sequence of the cynomolgus monkey P450 2B6 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 2B6 drug metabolizing enzyme and the cynomolgus monkey P450 oxidoreductase comprises an open reading frame of the sequence of the cynomolgus monkey P450 2B6 drug metabolizing enzyme and the open reading frame of 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 2B6 hypotype and the system is closet 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 cynomolgus monkey (Cynomolgus Monkey, Macacafascicularis) P450 2B6 drug metabolizing enzyme (Cytochrome P450 2B6) and its co-production with cynomolgus monkey P450 oxidoreductase (NADPH-cytochrome P450 oxidoreductae) Expression of recombinant vectors. 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 2B subfamily is the main member of the cytochrome P450 gene family, mainly including 2B6 and so on. Cytochrome P450 2B6 can metabolize various drugs such as bupropion hydrochloride (Bupropion), 3-methylphenytoin ((s)-(+)-Mephenytoin). 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 c...

Claims

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Application Information

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IPC IPC(8): C12N9/00C12N15/52C12N15/53C12N15/62C12N15/866
Inventor 戴仁科佘妙琴王海涛温鼎声白杨
Owner GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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