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Anti-inflammatory inhibitor screening model taking MyD88TIR (myeloid differentiation primary response protein 88 Toll/interleukin-1 receptor) dimerization as target point and application thereof

A technology for inhibitor screening and dimerization, applied in recombinant DNA technology, microbial determination/testing, biochemical equipment and methods, etc.

Inactive Publication Date: 2014-04-02
NORTHEAST NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, many studies have designed and synthesized TIR structural analogs based on the amino acid sequence of the MyD88 TIR domain and introduced them into mammalian cells. Pro-inflammatory effects of 18

Method used

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  • Anti-inflammatory inhibitor screening model taking MyD88TIR (myeloid differentiation primary response protein 88 Toll/interleukin-1 receptor) dimerization as target point and application thereof
  • Anti-inflammatory inhibitor screening model taking MyD88TIR (myeloid differentiation primary response protein 88 Toll/interleukin-1 receptor) dimerization as target point and application thereof
  • Anti-inflammatory inhibitor screening model taking MyD88TIR (myeloid differentiation primary response protein 88 Toll/interleukin-1 receptor) dimerization as target point and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: GFP-MyD88 TIR and RFP-MyD88 TIR transfected into mammalian cells can undergo fluorescence resonance energy transfer

[0043] we press figure 1 The combination shown in, the constructed GFP-MyD88 TIR, RFP-MyD88 TIR, GFP, RFP plasmids were co-transfected into HeLa cells. Under 488nm excitation light, cells co-transfected with GFP-MyD88TIR and RFP-MyD88TIR fusion proteins (left photo) emit dim green fluorescence, and some cells even emit orange light. It was proved that the green fluorescence emitted by GFP should be the dimerization of MyD88TIR to transfer the energy to RFP, thus realizing FRET. While the control group was co-transfected with GFP-MyD88 TIR fusion protein and plasmid empty of RFP (middle photo), or conversely, co-transfected with plasmid empty of GFP and RFP-MyD88 TIR fusion protein (right photo), 488nm excitation light Under observation, the cells showed bright green fluorescence. Note: Because the cells co-transfected with GFP-MyD88 TIR and...

Embodiment 2

[0045] Example 2: His-MyD88 TIR recombinant protein can interact with GST-MD88 TIR recombinant protein in vitro

[0046] There is still a problem with the live cell in situ fluorescence model described in Example 1 above: when the FRET phenomenon in cultured cells is blocked by an added compound, we cannot conclude that this compound directly blocks MyD88 TIR The dimerization of MyD88 TIR, or this compound triggers some kind of intracellular signaling cascade that indirectly affects the dimerization of MyD88 TIR. To address this issue, we can use a His-MyD88 TIR and GST-MyD88 TIR recombinant protein interaction assay in vitro to verify.

[0047] To this end, we constructed plasmids for prokaryotic expression of fusion proteins GST-MyD88 TIR and His-MyD88 TIR, and transformed E. coli to induce protein expression (recombinant fusion proteins can be specifically recognized by MyD88 antibody immunoblotting, proving the correct expression of MyD88 TIR) . like Figure 4 As shown,...

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Abstract

The invention relates to an anti-inflammatory inhibitor screening model taking MyD88TIR (myeloid differentiation primary response protein 88 Toll / interleukin-1 receptor) dimerization as a target point and application thereof. The anti-inflammatory inhibitor screening model taking the MyD88TIR dimerization as the target point is characterized in that: an MyD88TIR builds fusion protein plasmids together with protein genes GFP / RFP (Green fluorescent protein / Red fluorescent protein) of a fluorescence donor and a fluorescent receptor, and the fusion protein plasmids are transfected into mammalian cells to build dual-positive expression cell strains; the FRET (fluorescence resonance energy transfer) phenomenon can be detected; when an MyD88TIR dimerization inhibitor exists in a culture medium, the cell strains which depend on dual-positive to express GFP-MyD88-TIR and RFP-MyD88-TIR are suggested, and whether the inhibitor directly blocks the interaction of the TIRs or not can be further determined according to in vitro binding analysis; and combined with the fluorescent FRET blocking results of eukaryotic cells and prokaryotic expression recombinant protein interaction analysis, the MyD88TIR dimerization inhibitor can be determined. The model can be used for widely screening commercialized small molecule libraries, self-prepared natural product components, or various chemical compounds, and modifiers, from which MyD88 dimerization inhibitory compounds are obtained to participate in the drug screening of MyD88 signal pathway-dependent chronic inflammation and autoimmune diseases.

Description

technical field [0001] The invention relates to an anti-inflammatory inhibitor screening model targeting MyD88 TIR dimerization and its application, so as to realize the high-throughput screening of MyD88 dimerization inhibitors in live cells in situ and based on changes in fluorescent signals. Background technique [0002] IL-1R receptors and Toll-like receptors (TLRs) belong to the same superfamily, and the IL-1R subfamily includes receptors and other members of IL-1β and IL-18, which mediate acute and chronic inflammatory responses. The Toll-like receptor subfamily has 10 members that recognize pathogenic microorganisms or their metabolites (such as endotoxin LPS) and mediate innate immunity. When IL-1R receptors and Toll-like receptors bind to ligands, their intracellular TIR (Toll / IL-1receptor homologous region) domains undergo conformational changes, recruiting cytoplasmic receptors that also contain TIR structures domain of adaptor protein molecules, a process critic...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10C12N15/79C12Q1/02G01N21/64
Inventor 巴雪青曾宪录
Owner NORTHEAST NORMAL UNIVERSITY
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