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BiFC (Bimolecular Fluorescence Complementation) intracellular detection method and system for indicating NrF2 (Nuclear factor erythroid 2-related factor)-Keap1 (Kelch-like ECH associated protein 1) interaction

A detection system, pegfp-c1 technology, applied in the field of BiFC intracellular detection methods and systems, can solve the problems of expensive detection instruments, inability to respond to the interaction between Nrf2 and Keap1 functional proteins, and the inability to monitor the interaction between Nrf2 and Keap1 in real time, to achieve Intuitive effect of detection system

Inactive Publication Date: 2017-10-10
NORTHEAST FORESTRY UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

These analyzes either cannot monitor the interaction of Nrf2 and Keap1 in cells in real time, or cannot reflect the interaction of intact Nrf2 and Keap1 functional proteins, or require expensive and complex detection instruments

Method used

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  • BiFC (Bimolecular Fluorescence Complementation) intracellular detection method and system for indicating NrF2 (Nuclear factor erythroid 2-related factor)-Keap1 (Kelch-like ECH associated protein 1) interaction
  • BiFC (Bimolecular Fluorescence Complementation) intracellular detection method and system for indicating NrF2 (Nuclear factor erythroid 2-related factor)-Keap1 (Kelch-like ECH associated protein 1) interaction
  • BiFC (Bimolecular Fluorescence Complementation) intracellular detection method and system for indicating NrF2 (Nuclear factor erythroid 2-related factor)-Keap1 (Kelch-like ECH associated protein 1) interaction

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Experimental program
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Embodiment

[0045] Fusion fragments: A.Nrf2+linker+N158; B.N158+linker+Nrf2; C.Keap1+linker+C159; D.C159+linker+Keap1

[0046] Design the following primers (Table 1), using the vector pEGFP-C1 containing eGFP, the vector pET28a-Nrf2 containing Nrf2, and the vector pET28a-Keap1 containing Keap1 as templates, add primers (1) and (2) to synthesize eN158 plus Fragment of linker sequence; Add primers (3), (4) PCR to synthesize fragments of Linker and Nrf2; Add primers (5), (6) PCR to synthesize eC159 fragments with linker sequence; Add primers (7), (8) PCR synthesis of fragments of Linker and Keap1; addition of primers (9), (10) PCR synthesis of Nrf2 fragments with linker sequences; addition of primers (11), (12) PCR synthesis of fragments of Linker and eN158; addition of primers (13), (14) Synthesize Keap1 fragments with linker sequences added by PCR; add primers (15) and (16) PCR synthesize fragments of Linker and eC159.

[0047] System 50μl

Dosage

Template (30-50ng)

...

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Abstract

The invention discloses a BiFC (Bimolecular Fluorescence Complementation) intracellular detection method and system for indicating NrF2 (Nuclear factor erythroid 2-related factor)-Keap1 (Kelch-like ECH associated protein 1) interaction. A recombinational expression vector constructed by Nrf2 protein which is fused with eGFP N-end 158-locus amino acid and a Keap1 protein coding gene which is fused with eGFP C-end 159-locus amino acid is provided; meanwhile after cells are transfected, a complete fluorescent protein is reconstructed through fused N-end and C-end of fluorescent protein eGFP under the expressed NrF2-Keap1 interaction. By adopting the BIFC system, cells are transfected instantly or constructed into a stable expression cell line, a specific signal generated in a cell nucleus can be observed, the signal can be displayed specifically and reflect the activity state and change of a Nrf2-Keap1 signal path in a living cell, and a foundation is laid for screening of small molecule compounds capable of influencing Nrf2-Keap1 interaction and antitumor medicaments by detecting BiFC fluorescence change.

Description

technical field [0001] The invention relates to the biological field, in particular to a BiFC intracellular detection method and system for indicating Nrf2-Keap1 interaction. Background technique [0002] Chemotherapy is a conventional therapy for tumor treatment, but chemotherapy resistance is still a difficult point in clinical cancer treatment, which will lead to tumor recurrence and metastasis, and seriously affect the quality of life of patients. Nuclear factor erythroid 2-related factor (Nrf2) can regulate antioxidant response and resist cell damage caused by reactive oxygen species and electrophiles. Kelch-like ECH associated protein 1 (Keap1), a specific substrate adapter protein of (Cullin-3)-dependent E3 ligase complex, negatively regulates the activity of Nrf2. The Keap1-Nrf2 pathway in tumor cells is often overactivated, and the expression of Nrf2 is up-regulated to resist the attack of high levels of intracellular ROS and chemotherapeutic drugs. However, the me...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/85
CPCC12N15/85C12Q1/6897
Inventor 周波
Owner NORTHEAST FORESTRY UNIVERSITY
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