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Application of macrophage regulating protein MIRF in preparing anti-inflammatory medicament

An anti-inflammatory drug and protein regulation technology, applied in the field of MIRF protein, can solve the problems of less than 20% sequence homology, unknown structure and function, etc., and achieve the effect of being suitable for popularization and application and low cost

Inactive Publication Date: 2009-12-30
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Protein sequence comparison found that the protein contains a conserved domain of glycohydrolases 18 (Glyco_18), but the sequence homology with the same family protein is less than 20%, and it belongs to a protein whose structure and function are completely unknown

Method used

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  • Application of macrophage regulating protein MIRF in preparing anti-inflammatory medicament
  • Application of macrophage regulating protein MIRF in preparing anti-inflammatory medicament
  • Application of macrophage regulating protein MIRF in preparing anti-inflammatory medicament

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Cloning, expression and purification of MIRF protein

[0024] PCR with 5'-ggggacaagtttgtacaaaaaagcaggcttaATGACCAAGGCCCGGCTGTTCCGG and 5'-ggggaccactttgtacaagaaagctgggtcTCAGTCTCGGAGGAGGTTTTCGGG from the IMAGE library (The IMAGEConsortium) (predenaturation: 94°C, 5 minutes, 1 minute, extension 94°C, 50°C, annealing) : 72°C, 90 seconds; after 30 cycles of denaturation to extension, 72°C, 10 minutes) to obtain cDNA encoding MIRF protein, insert the cDNA encoding MIRF protein into the prokaryotic expression vector pET21DEST (Invitrogen) by Gateway cloning technology, The vector has a histone (His 6 )label. Or insert the cDNA encoding the MIRF protein into the prokaryotic expression vector pGEX4T-1 (GE Company), which has a glutamyl transpeptidase protein (GST) tag at its N-terminus.

[0025] The expression vector was transformed into Escherichia coli BL21(DE3) strain. The transformed bacterial solution was spread on a plate medium (1% sodium chloride, 1% peptone, 0.5% y...

Embodiment 2

[0030] Example 2 Protein crystallization and structure determination

[0031] The purified MIRF protein was concentrated to 10mg / ml, and then the crystals were screened using the meteorological diffusion method (Protein Crystallography.T.L Blundell). The result was 100mM Bis-Tris pH 6.5, 2M NH 4 SO 4 , 10mM MPD, 100mM MgCl 2 Under the conditions to obtain crystals. The crystal diffraction data was collected in the Synchrotron Radiation Laboratory of Stanford University using MAR 325 CCD detector. Using 20% ​​glycerin as the antifreeze, the crystals are diffracted at a temperature of 100K at a rotation angle of 1°. The collected data is processed through Mosflm and CCP4 procedures. The crystallographic parameters and data collection statistics are given in Table 1.

[0032] Table 1. Collection and statistics of MIRF crystallographic parameters

[0033]

[0034] * The value in brackets is the value under high resolution

[0035] The standard equations are used to define different ...

Embodiment 3

[0037] Example 3 Detection of MIRF binding to carbohydrates and lipopolysaccharides

[0038]Use BIAcore 3000 (Amersham Phramacia Biotech) to detect the binding specificity of MIRF with different monosaccharides including glucosamine, galactosamine, acetylglucosamine, acetylgalactosamine, mannose, glucose and ribose. The purified MIRF (200μg / ml) and the control in a 10mM sodium acetate (pH 5.0) solution were fixed on the surface of the sensor chip CM5 (Amersham Phramacia Biotech) that had been activated by primary amine. Excess hydroxysuccinyl The iminoester group is blocked by 1M aminoethanol hydrochloride. Different monosaccharides and LPS dissolved in HBS buffer (10mM HEPES with 150mM NaCl, 3mM EDTA, and 0.05% Tween-20) were injected into the surface of the chip at a speed of 30μl / min, and the binding curve was observed in real time. HBS-ET buffer was used to stop the reaction. The kinetic data was calculated using BIA evaluation software 4.1 (Pharmacia Biosensor AB).

[0039] T...

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Abstract

The invention discloses medical application of MIRF in preparing an anti-inflammatory medicament. Simultaneously, the invention provides the anti-inflammatory medicament which contains the MIRF in effective dose. The experiment shows that the MIRF can inhibit the expression of inflammatory factors induced by LPS, PGN and the like and caused by autoimmune diseases, so the MIRF can be used as an effective inhibitor for immunological diseases of certain inflammations such as asthma, rheumatoid arthritis and the like and has potential clinical application value. In addition, by gene engineering technology, large amount of stable and active recombinant MIRF protein can be obtained with low cost, and the MIRF protein is suitable for popularization and application.

Description

Technical field [0001] The invention relates to the field of biomedicine, in particular to the use of MIRF (Macrophage Inflammation Related Factor) protein in medicine. Background technique [0002] Macrophages are a type of human phagocytic cells, which are distributed in tissues and have the functions of immune information transmission, coordination and phagocytosis and processing of antigens. Monocytes ooze out of blood vessels, enter tissues and organs, and can further differentiate and develop into macrophages, becoming the most phagocytic cells in the body. Macrophages can be immobile or move in an amoeba-like movement. Fixed and migratory macrophages are different stages of the same cell, and the two can change each other, and their morphology also changes with their functional status and location. Macrophages have different names in different tissues: they are called "pulmonary macrophages" in the lungs; they are called "microglia" in the nervous system; they are called "...

Claims

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Application Information

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IPC IPC(8): A61K38/17A61P29/00A61P37/00
Inventor 郑晓峰孟赓罗明赵艳梅白效耘
Owner PEKING UNIV
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