Three functional mutants of human phase II metabolic enzyme, construction and use thereof

A functional and metabolic enzyme technology, applied in the field of genetic engineering, can solve the problems of enhanced and weakened glucuronidation activity, complicated drug interactions, etc., to simplify the operation, save time, and improve the efficiency of recombination.

Inactive Publication Date: 2008-12-17
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The above studies show that the presence of UGT2B7 gene polymorphisms may potentially complicate the drug interaction situation, because patients with allele mutations may show increased or decreased glucuronidation activity. Will exhibit different pharmacokinetic / pharmacokinetic (PK / PD) behavior [7，8]

Method used

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  • Three functional mutants of human phase II metabolic enzyme, construction and use thereof
  • Three functional mutants of human phase II metabolic enzyme, construction and use thereof
  • Three functional mutants of human phase II metabolic enzyme, construction and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1 produces the plasmid of mutant recombinase: the construction of pGEM-UGT2B7*71S, pGEM-UGT2B7*3 and pGEM-UGT2B7*5

[0038] 1. Materials and methods

[0039] (1) Experimental materials

[0040] Escherichia coli strain E.coli DH5α was preserved in this experiment. PCR primers, Shanghai Sangong. The cloning plasmid pGEM-T is a Promega product.

[0041] Takara MutanBEST kit was purchased from Takara Company, restriction endonucleases, PCR product recovery kits, plasmid extraction kits, and agarose gel recovery kits were purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd. Synthesis was performed by Sangon Bioengineering Technology Service Co., Ltd., and the sequencing service was completed by Shanghai Sangon Bioengineering Technology Service Company.

[0042] (2) Experimental method

[0043] 1. Cloning construction of human UGT2B7 cDNA

[0044] Extract the mRNA of human liver tissue, take about 1 mg, add 1 ml of TRIzol reagent, and ho...

Embodiment 2

[0077] Example 2 Construction of pFastBac-UGT2B7*71S, pFastBac-UGT2B7*2 and pFastBac-UGT2B7*5

[0078] The correctly sequenced pGEM-UGT2B7*71S, pGEM-UGT2B7*2 and pGEM-UGT2B7*5 were digested with SalI / XhoI, and pFastBac TM 1-UGT2B7*1 was also digested with SalI / XhoI, the mutant target gene and vector were ligated with T4DNA ligase, transformed into E.coli DH 5α competent cells, and positive colonies were picked to extract pFastBac-UGT2B7*71S, pFastBac -UGT2B7*2 and pFastBac-UGT2B7*5 recombinant plasmids, simultaneous enzyme digestion identification, see Figure 5 . Figure 5 Middle: Lanes 1 and 10: DNA marker; Lane 2: Plasmid pFastBac-UGT2B7*1; Lane 3: Plasmid pFastBac-UGT2B7*1 digested with SalI / XhoI; Lane 4: Plasmid pFastBac-UGT2B7*71S; Lane 5: pFastBac-UGT2B7*71S plasmid was digested by SalI / XhoI; lane 6: pFastBac-UGT2B7*2 plasmid; lane 7: pFastBac-UGT2B7*2 plasmid was digested by SalI / XhoI; lane 8: pFastBac-UGT2B7*5 plasmid ; Lane 9: pFastBac-UGT2B7*5 plasmid was digeste...

Embodiment 3

[0079] Example 3 Preparation of Bacmid-UGT2B7*71S, Bacmid-UGT2B7*2 and Bacmid-UGT2B7*5

[0080] The pFastBac-UGT2B7*71S, pFastBac-UGT2B7*2 and pFastBac-UGT2B7*5 recombinant plasmids identified by restriction enzyme digestion were transformed into E.coli DH 10Bac, and the pFastBac empty plasmid was operated in parallel. After the white colonies were confirmed by blue-white screening, the transposed Bacmid-UGT2B7*71S, Bacmid-UGT2B7*2 and Bacmid-UGT2B7*5 were extracted by alkaline lysis. Extract the non-transposed Bacmid from E.coliDH 10Bac bacteria in the same way, and use M13(+) / M13(-) as primers for PCR verification. The three PCR products should have bands at about 3900, 2300, and 300bp respectively. See Image 6 , Figure 7 . Image 6 Middle: Swimming lane 1. DNA marker; Swimming lane 2. PCR result of pFastBac-UGT1A9 cosmid after transfection (3900bp); Swimming lane 3. PCR result of cosmid transfection pFastBac (2300bp); Swimming lane 4. Not transfected The PCR result of ...

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Abstract

The invention provides three functional mutant recombinases of human phase II metabolic enzyme, namely, UGT2B771S, UGT2B72 and UGT2B75, wherein, a mutant site is led in a primer; a wild type gene plasmid with human UGT2B7 is taken as a template; after PCR reaction, phosphorylation, self-ligation and conversion, three functional mutant recombinase gene plasmids with the UGT2B7 are respectively synthesized. The functional mutant recombinases provided by the invention have the advantages of high level expression in insect cells, comparatively short period, exact host range of baculovirus, no human pathogenicity, safe operation, increased recombination efficiency, time saving, simple and effective construction and expression method and being easy to be amplified for mass production. The functional mutant recombinases can be applied to the screening of medicines, forecast drug side effect likely to be caused by individualized medication, thus instructing reasonable medication; the functional mutant recombinases can also be applied to the research of structure-activity relationship between enzymes and substrate drugs at molecular level.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and more specifically relates to obtaining three functional mutant genes of human two-phase metabolic enzyme UGT2B7 by using a baculovirus / insect cell expression system, and a recombinant cosmid containing the mutant genes. The vector was transfected into insect cells to obtain the mutant recombinant enzyme. Background technique [0002] Uridine diphosphate glucuronosyltransferases (UGTs) are glycoproteins located on the endoplasmic reticulum membrane that catalyze a series of endogenous and exogenous lipophilic glycoside substrates (such as bilirubin, steroid hormones, drugs, Insecticides, etc.) to glucuronidate them. According to the homology of amino acid sequence, UGTs can be divided into two families: UGT1A and UGT2B. UGT2B7 belongs to the UGT2B subfamily, and its gene contains 6 exons with a total length of 16kb, of which the first exon and the second exon encode the substrate...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/866C12N15/70C12N15/81C12Q1/48
Inventor 曾苏袁玲敏陈静
Owner ZHEJIANG UNIV
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