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Mimic polypeptides of myeloid differentiation protein-2 and application thereof

A myeloid, protein technology, applied in the field of biopharmaceuticals

Inactive Publication Date: 2010-06-09
THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although TLR4 plays a key role in the recognition and activation of LPS effects, TLR4 alone is not sufficient to trigger the inflammatory response of LPS in MD-2-deficient mouse Ba / F3 cells
At present, there is no report on the research and development of antagonistic endotoxin preparations derived from MD-2 protein itself

Method used

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  • Mimic polypeptides of myeloid differentiation protein-2 and application thereof
  • Mimic polypeptides of myeloid differentiation protein-2 and application thereof
  • Mimic polypeptides of myeloid differentiation protein-2 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 MDMP has the effect of neutralizing LPS activity in vitro

[0035] 1.1 MDMP inhibits LPS-induced LAL activation

[0036] The ability of MDMP to neutralize LPS was assessed using commercially available Chromogenic LAL assays. Method: Take 25ul of different concentrations of MDMP / MDSP (1-50ug / ml) and 1EU / ml of LPS, mix well and bathe in water at 37°C for 30min, so that the combination of the two reaches equilibrium. Then 50ul LAL was added and incubated for 10min, and the absorbance value (OD) at 405nm was measured 10min and 16min after adding 100ul chromogenic substrate, respectively. Calculate the 10min and 16min OD value difference of the control sample, and subtract the control sample value from the OD value difference of the experimental sample to obtain the measured value.

[0037] The results are attached figure 1 As shown, MDMP inhibited LPS-induced LAL activation in a dose-dependent manner (1-50ug / ml), while MDSP had no such effect.

[0038] 1.2 MDM...

Embodiment 2

[0043] Example 2 MDMP inhibits the response of RAW264.7 cells to LPS stimulation

[0044] 2.1 MDMP down-regulates the expression of TLR4-MD-2 complex on the surface of RAW264.7 cells

[0045] Expression of the TLR4-MD-2 complex on the surface of RAW264.7 cells was assessed using phycoerythrin (PE)-labeled monoclonal antibody MTS510 against the murine TLR4-MD-2 complex after LPS treatment of RAW264.7 cells for 6 hours . After blocking anti-FcrR, the cells were co-incubated with fluorescently labeled monoclonal antibodies for 30 min at 4°C, washed twice with PBS, and then the fluorescence intensity on the cell surface was analyzed by flow cytometry.

[0046] Since MTS510 can specifically react with the murine TLR4-MD-2 complex on the cell surface, it can be used to recognize the complex. attached Figure 4 It was shown that MDMP treatment alone could down-regulate the expression of TLR4-MD-2 complex, LPS alone treatment had a similar effect, and MDMP pretreatment could furthe...

Embodiment 3

[0053] Example 3 Effect of MDMP on LPS-induced acute lung injury in mice

[0054] 3.1 Establishment and detection method of acute lung injury model in mice

[0055] According to the method of Moon et al. (Moon., 2009), the LPS-induced acute lung injury model in mice was established. Mice were anesthetized by intraperitoneal injection of a mixture of ketamine (45 mg / kg) and xylazine (8 mg / kg), and 50 μl of LPS (1.0 mg / kg, dissolved in sterile saline) was placed behind the throat to be inhaled into the lungs , with sterile saline as the control. MDMP or MDSP (5 mg / kg) was injected intraperitoneally 30 minutes before LPS treatment. The degree of lung injury was assessed 24 hours after LPS treatment.

[0056] Bronchoalveolar lavage fluid (BALF) acquisition method: wash the airway with 3ml of normal saline without Ca2+ / Mg2+ ions through the tracheal cannula to obtain BALF three times, mix well and centrifuge at 1500g for 10min at 4°C. The collected supernatant was analyzed by B...

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Abstract

The invention provides a group of novel polypeptides derived from myeloid differentiation protein-2 (MD-2), which comprises an amino acid sequence of SEQ1 (SEQ1: CHGHDDDYSFCFSFEGILFPKGHYR) or a variant or a fragment thereof. The actual detection results show that the polypeptides can inhibit a TLR4 signal path which is triggered by LPS from being activated, inhibit excessive inflammatory response induced by in vitro-in vivo LPS, and improve survival rate of mouse damaged by endotoxin, thus being capable to be used for preparing a curative drug for endotoxin related inflammation including pyaemia.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and in particular relates to a group of polypeptides simulating myeloid differentiation protein-2 (MD-2), and the polypeptides are used to prepare and treat Gram-negative bacterial infectious diseases and endotoxin-related inflammatory diseases. medicine. Background technique [0002] Endotoxemia and subsequent sepsis is a severe systemic inflammatory response syndrome caused by infection, with an annual incidence of about 18 million worldwide. Although various advanced medical technologies and new drugs are continuously applied clinically, the mortality rate is still more than 60%. This lethal syndrome is initially caused by lipopolysaccharide (LPS) in the cell wall components of Gram-negative bacilli. LPS can activate host immune cells by triggering Toll like receptor 4 (TLR4) signaling pathway, and secrete pro-inflammatory mediators such as TNF-α, IL-6, etc. However, excessive and unbalanc...

Claims

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Application Information

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IPC IPC(8): C07K14/47A61K38/17A61P31/04A61P29/00A61P7/00
Inventor 段光杰刘友生朱江邓军许成燕
Owner THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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