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Vector composition for indicating active state of Wnt signal in cell by using BiFC (Bimolecular Fluorescence Complementation) and application of vector composition

An active state and signal technology, which is applied in the direction of using vectors to introduce foreign genetic material, cells modified by introducing foreign genetic material, hybrid peptides, etc., can solve the problem of not reporting the interaction of β-catenin protein

Inactive Publication Date: 2014-08-27
TSINGHUA UNIV
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  • Abstract
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  • Claims
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Problems solved by technology

[0006] At present, there is no report using BiFC technology to display the level of β-catenin and the protein interaction at the downstream level

Method used

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  • Vector composition for indicating active state of Wnt signal in cell by using BiFC (Bimolecular Fluorescence Complementation) and application of vector composition
  • Vector composition for indicating active state of Wnt signal in cell by using BiFC (Bimolecular Fluorescence Complementation) and application of vector composition
  • Vector composition for indicating active state of Wnt signal in cell by using BiFC (Bimolecular Fluorescence Complementation) and application of vector composition

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Experimental program
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Embodiment 1

[0067] Embodiment 1, fusion protein composition and BiFC system constructed therefrom

[0068] Based on the crystal structures of β-catenin and TCF3-CBD, it can be known that the C-terminus of β-catenin (the 667th amino acid, excluding the unknown structure of the C-terminus) is relatively close to the N-terminus of TCF3 in space. Two fragments of fluorescent protein were designed to be fused to the N-terminus of TCF3 and the C-terminus of β-catenin, respectively. Since the C-terminal structure of β-catenin is unknown and does not participate in the interaction between TCFs, at the same time, β-cateninΔC (amino acid residues of β-catenin from the 2nd to 667th amino acid residues at the N-terminal, the β-cateninΔC fragment is hereinafter referred to as β-cat ) can also prevent apoptosis induced by excessive intracellular Wnt signal to a certain extent, and protect cells. Therefore, β-cateninΔC and TCF3 are used as target proteins to construct BiFC system.

[0069] The genbank ...

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Abstract

The invention discloses a system for indicating the active state of a Wnt signal in a cell by using BiFC (Bimolecular Fluorescence Complementation) and application of the vector composition. The invention provides a fusion protein group which consists of fusion protein A and fusion protein B, wherein the fusion protein A comprises a core effector beta-catenin fragment and a fluorescent protein fragment A located at a fragment C of the core effector beta-catenin fragment; the fusion protein B comprises a transcription factor (TCF) and a fluorescent protein fragment B located at a fragment N of the TCF. Proved by experiments, a complex formed by the core effector beta-catenin and the TCF in a Wnt signal channel is visualized by using a strategy on the basis of BiFC, so that the active state of Wnt in the cell is directly displayed; a BiFC system is used for instantaneously transfecting cells or constructing a stable expression cell line, so that specific signals formed in a cell nucleus can be observed and can specifically display and reflect the active state of the Wnt signal channel in a living cell and variation thereof.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a carrier composition and application for indicating the active state of Wnt signal in cells by using BiFC. Background technique [0002] The classic Wnt signaling pathway is a conserved and important signal transduction pathway, which is closely related to embryonic development, adult tissue homeostasis, and carcinogenesis. In a variety of cancers, such as colorectal cancer, breast cancer, lung cancer, skin cancer, etc., abnormal activation of Wnt signaling has been detected due to various reasons such as mutations in pathway members. Therefore, the research on the Wnt signaling pathway and the screening of drugs targeting Wnt signaling are of great significance. [0003] Regarding the working mode of canonical Wnt signaling, it is currently believed that when Wnt signaling is activated, the function of the degradation complex is inhibited, allowing β-catenin to accumul...

Claims

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Application Information

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IPC IPC(8): C07K19/00G01N33/68C12N15/62C12N15/63C12N5/10
Inventor 吴畏丁雅洁汤玮欣
Owner TSINGHUA UNIV
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