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Novel fusion protein for human HMGB1 A box and acidic tail and use thereof

A fusion protein and acidic technology, applied in the field of genetic engineering, can solve the problems of failing to improve the survival rate of patients, and achieve the effects of avoiding inflammatory effects, strong anti-inflammatory activity, and inhibiting growth

Inactive Publication Date: 2010-04-07
ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Inflammatory mediators play an important role in the pathogenesis of systemic inflammatory response syndrome, although some early inflammatory mediators (cytokines secreted 6 hours after inflammatory effector cells are stimulated) such as TNF-α, IL-1, and macrophage migration are inhibited. Animal experiments on systemic inflammation such as inhibitory factor (MIF) have been successful, but clinical trials have failed to improve the survival rate of such patients. It is particularly important to explore inflammatory mediators with a wide therapeutic window and their inhibitory mechanisms

Method used

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  • Novel fusion protein for human HMGB1 A box and acidic tail and use thereof
  • Novel fusion protein for human HMGB1 A box and acidic tail and use thereof
  • Novel fusion protein for human HMGB1 A box and acidic tail and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] 1. Cloning of human HMGB1 cDNA

[0080] 1. Isolation of Human Peripheral Blood Mononuclear Cells

[0081] Take 15ml of human peripheral anticoagulant blood (purchased from the Blood Transfusion Department of Southwest Hospital), add sterile PBS to dilute and mix evenly; draw the diluted blood, and carefully add an equal volume of human lymphocyte separation solution along the tube wall. Centrifuge horizontally at 2000r / min for 15 minutes. Carefully suck out the liquid containing human peripheral blood mononuclear cells in the white mist in the middle layer, wash with sterile PBS twice, and centrifuge at 1000r / min for 15 minutes each time.

[0082] 2. Extraction of total RNA from human peripheral blood mononuclear cells

[0083] Use the Tripure kit to extract total cellular RNA, and follow the instructions: 1ml / 5~10×10 6 Add an appropriate amount of Tripure to each cell, invert the Ep tube to fully mix the Tripure and the cells, and place at room temperature for 5 min...

Embodiment 2

[0104] 1. Cloning of human HMGB1 A box and acidic tail flexible junction coding sequence

[0105] 1. Schematic diagram of the fusion protein structure of human HMGB1 A box and acidic tail flexible linker figure 2 .

[0106] 2. One-step inverse PCR mutagenesis strategy to amplify human HMGB1 A box and acidic tail rigid junction and flexible junction coding sequence respectively

[0107] (1) The basic principle of one-step reverse PCR mutagenesis strategy: design a pair of primers with adjacent 5' ends and opposite directions at the 3' ends to introduce mutation points; in order to ensure the correctness of the amplified product sequence, use high-fidelity DNA polymerase Pyrobest DNA Polymerase was used to amplify; in the same reaction system, the end of the PCR product was smoothed and the 5' end was phosphorylated; the product was then subjected to self-ligation reaction; finally, plasmid DNA transformation and mutant screening were performed. (For specific principles, see ...

Embodiment 3

[0188] 1. The target protein removes LPS

[0189] 1. Detoxi-Gel TM Endotoxin Removing Gel removes LPS in the target protein (refer to Pierce company instructions)

[0190] (1) Detoxi-Gel placed at 4°C TM Endotoxin Removing Gel column was left at room temperature for 30 minutes.

[0191] (2) Remove the cap from the bottom of the column, and add 5 times column volume of 1% sodium deoxycholate to regenerate Detoxi-Gel.

[0192] (3) Add 5 times the column volume of sterile pyrogen-free water to wash the column.

[0193] (4) Add 5 column volumes of sterile pyrogen-free water to equilibrate Detoxi-Gel.

[0194] (5) Cover the cap at the bottom of the column, and each protein of interest is passed through Ni 2+ -NTA column purification collected Elution Buffer and dialyzed liquid respectively Detoxi-Gel TM Endotoxin Removing Gel column, at room temperature for 1 hour.

[0195] (6) Remove the cap at the bottom of the column, wash the column with sterile pyrogen-free water, co...

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Abstract

The invention relates to a recombinant plasmid for coding a novel fusion protein for a human HMGB1 A box and an acidic tail. An HMGB1 B box is deleted between the HMGB1 A box and the acidic tail; and the HMGB1A box is connected with the acidic tail through a flexible connexon. The invention also provides a preparation method of the recombinant plasmid and the novel fusion protein secreted and expressed by the recombinant plasmid. The fusion protein deletes an inflammatory functional domain B box, fully uses two beneficial functions of the antibacterial activity of HMGB1 and the obvious anti-inflammatory effects of the A box, and effectively avoids the side effect of inflammatory effect at the same time. The fusion protein can effectively resist the release of various inflammatory cytokine induced by the HMGB1, has stronger anti-inflammatory activity than the single A box does, and can inhibit growth of bacteria at the same time.

Description

technical field [0001] The invention belongs to the field of genetic engineering and relates to a novel fusion protein of human HMGB1 A box and acidic tail and application thereof. Background technique [0002] High-mobility group box-1 (HMGB1) was discovered in calf thymus tissue in the 1970s. Named for its high mobility during electrophoresis, it mainly includes three structural domains: the A box at the N-terminal rich in lysine; the B box at the middle of the molecule; the acidic tail with a highly conserved amino acid sequence at the C-terminus . HMGB1 can exist in multiple parts such as the nucleus, cytoplasm and extracellular of eukaryotes. It was originally recognized as a nuclear protein, which can non-specifically bind to the minor groove of DNA molecules and stabilize nucleosomes. It also participates in life activities such as cell differentiation, DNA replication, transcription, recombination and repair. HMGB1 can also exist in the cytoplasm and extracellular...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/66C12N15/70C12N15/74C12N1/21C07K14/435A61K38/17A61P29/00A61P31/04C12R1/19
Inventor 何凤田龚薇
Owner ARMY MEDICAL UNIV
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