PirB extracellular polypeptide and application

A sequence and drug technology, applied in the field of bioengineering protein recombination, can solve the problem of no effective therapeutic drugs and achieve the effect of promoting axon growth

Inactive Publication Date: 2013-05-22
FOURTH MILITARY MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no effective treatment for neurological dysfunction caused by central nervous system (central nervous system, CNS) injury

Method used

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  • PirB extracellular polypeptide and application
  • PirB extracellular polypeptide and application
  • PirB extracellular polypeptide and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: Principles and Ideas of Recombining PEP

[0024] In 2008, Atwal et al. first reported that PirB is another functional receptor of myelin inhibitory factor in "Science". Later, some scholars published a review in "Neuron" and others, and believed that the discovery of PirB as a myelin receptor is a clinically promoted axis. Synaptic regeneration and neurological recovery provide a new therapeutic target. PirB is a type 1 transmembrane glycoprotein consisting of six extracellular immunoglobulin-like domains and intracellular polypeptides containing four immunoreceptor tyrosine-dependent inhibitory motifs (ITIMs) ( figure 1) . Relying on library screening technology, Atwal et al. found that PirB is a functional receptor of myelin protein, which can bind with MAG, Nogo-66, Omgp with high affinity ( figure 2) . Compared with NgR1, PirB appears to be more important in myelin inhibition, on the basis that genetic knockout of PirB causes more axonal regeneration...

Embodiment 2

[0025] Example 2: PEP expression, identification and purification

[0026] 1. Reagents and materials

[0027] (1). Plasmids, strains

[0028] Strain: BL21(DE3)

[0029] Plasmid: pET32a

[0030] (2).Main reagents

[0031] Amp (ampicillin)

[0032] TPTG (isopropyl-β-D-thiogalactoside)

[0033] PBS (phosphate buffered saline)

[0034] SDS (Sodium Dodecyl Sulfate)

[0035] LB Borth Medium

[0036] LB Ager Medium

[0037] 2. According to the molecular structure of PirB ( figure 1) And bioinformatics, relying on library screening technology to determine the amino acid sequence of PirB extracellular polypeptide.

[0038] The amino acid sequence of PirB extracellular polypeptide PEP is:

[0039] GSLPKPILRVQPDSVVSRRTKVTFLCEETIGANEYRLYKDGKLYKTVTKNKQKPENKAEFSFSNVDLSNAGQYRCSYSTQYKSSGYSDLLELVVTGHYWTPSLLAQASPVVTSGGYVTLQCESWHNDHKFILTVEGPQKLSWTQDSQYNYSTRKYHALFSVGPVTPNQRWICRCYSYDRNRPYVWSPPSESVELLVSGNLQKPTIKAEPGSVITSKRAMTIWCQGNLDAEVYFLHNEKSQKTQSTQTLQEPGNKGKFFIPSVTLQHAGQYRCYCYGSAGWSQ...

Embodiment 3

[0055] Embodiment three: PEP efficacy test

[0056] 1. Effects of different concentrations of PEP on the viability of primary neurons

[0057] (1). The hippocampal neurons were cultured until the fifth day, and different concentrations of purified fusion protein PEP were added, the concentrations were 62, 5 μg / L, 125 μg / L, and 250 μg / L;

[0058] (2). Normal culture, cell viability analysis (MTT method) at 12h, 24h, 48h after adding protein;

[0059] (3). The results showed that different concentrations of PEP protein had no effect on the viability of primary neurons, indicating that PEP had no toxicity to normal primary neurons.

[0060] 2. PEP promotes the growth of primary neuron axons and their branches and the formation of skeleton proteins

[0061] (1). Coating: Coat Nogo-66, MAG, OMgp(Sigma) (10mg / well) respectively in 96-well plates, incubate at 4°C for 24h, dry and wash with PBS (pH7.3 ) rinse 3 times;

[0062] (2). Blocking: add 2.5% calf serum (BSA) containing 0....

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PUM

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Abstract

The invention provides PirB extracellular polypeptide which potentially treats central lesion. The polypeptide has an extracellular amino acid sequence of a paired immunoglobin-like receptor. The polypeptide segment can be massively expressed in escherichia coli and combined with myelin inhibiting factors MAG, Nogo-66 and OMgp in high affinity, so as to antagonize the bioactivity and function of PirB. The PEP provided by the invention can be effectively combined with myelin inhibiting factors MAG, Nogo-66 and OMgp so as to promote axon growth. The PEP provided by the invention can be massively prepared, is low in cost and high in activity, can be used for preparing medicines for treating various CNS (Central Nervous System) damages such as cerebral ischemia and anoxia, cerebral hemorrhage, cerebral trauma and spinal cord injury, and can effectively promote the regeneration of nervous tissues and neural functional recovery.

Description

technical field [0001] The invention belongs to the field of bioengineering protein recombination, and specifically relates to a PirB extracellular polypeptide (PirB extracellular peptides, PEP) capable of treating central system damage and an application thereof. Background technique [0002] Trauma, stroke, cerebral hemorrhage, and spinal cord injury are characterized by high mortality and disability. How to reduce the degree of neurological damage and how to better improve the function of damaged neurons and improve the quality of life of patients is of great significance to patients, their families and society, and is a worldwide problem. Currently, there is no effective treatment for neurological dysfunction caused by central nervous system (central nervous system, CNS) injury. Therefore, finding new and effective drugs for the treatment of brain CNS injury is a research hotspot. [0003] In the past, it was generally believed that the main reason for the difficulty i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/705C12N15/12A61K38/17A61P25/00
Inventor 王强苟兴春郭钒邓斌姜凤良熊利泽
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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