Preparation method of p75NTR-ED-Fc fusion protein and application of p75NTR-ED-Fc fusion protein in regeneration and functional recovery of injured central nerve

A p75ntr-ed-fc, 1.p75ntr-ed-fc technology, applied in the preparation method of peptides, nervous system diseases, peptide/protein components, etc. Purification and regeneration effect

Inactive Publication Date: 2012-07-18
INST OF FIELD OPERATION SURGERY NO 3 MILITARY MEDICL UNIV PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the expressed protein is difficult to fold correctly and has low biological activity, the pET prok

Method used

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  • Preparation method of p75NTR-ED-Fc fusion protein and application of p75NTR-ED-Fc fusion protein in regeneration and functional recovery of injured central nerve
  • Preparation method of p75NTR-ED-Fc fusion protein and application of p75NTR-ED-Fc fusion protein in regeneration and functional recovery of injured central nerve
  • Preparation method of p75NTR-ED-Fc fusion protein and application of p75NTR-ED-Fc fusion protein in regeneration and functional recovery of injured central nerve

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Implementation 1: The preparation method of p75NTR-ED-Fc fusion protein is realized by the following steps:

[0042] (1) Construction and identification of p75NTR-ED-Fc expression vector

[0043] In order to obtain a large number of naturally expressed p75NTR-ED-Fc fusion proteins, the present invention uses the eukaryotic expression vector pcDNA-p75NTR-ED-Fc plasmid (preserved in our laboratory) as a template, and is based on the p75NTR nucleoside numbered NM_002507.1 in Genebank The acid sequence and the nucleotide sequence of the Fc segment of IgG numbered XM_002348257.1 are designed to design specific p75NTR forward primers and semi-specific p75NTR / Fc reverse primers, and introduce Kpn I and Xho I digestion at both ends of the primers. The site and corresponding protective bases were amplified by PCR to obtain the p75NTR-ED-Fc fragment. The primer sequence is as follows:

[0044] Forward primer: 5'CGG GGTACC ATGGGGGCAGGTGCCACC 3'

[0045] KpnI restriction site

[0046] Rev...

Embodiment 2

[0069] Example 2: In vitro observation of p75NTR-ED-Fc against NgR signal to promote the growth of neuronal processes

[0070] Isolate and culture the Dorsal root ganglia (DRG) neurons of newborn SD rats by conventional methods. 6 Cell density per ml was seeded in a 24-well culture plate coated with polylysine. Adjust the concentration of MAG, p75NTR-ED-Fc fusion protein and bovine serum albumin (BSA) to 100ng / ml with PBS in advance. After the above-mentioned cultured DRG neurons adhered to the wall, the fluid was changed, and they were randomly divided into 3 groups, and each group was repeated twice. The first group: add only 1ml MAG; the second group: add 1ml each of MAG and p75NTR-ED-Fc fusion protein; the third group: normal control, only add 1ml BSA. The cells in the above groups were fixed with 4% paraformaldehyde after 24 hours of culture, and the projection length after 24 hours of culture was measured by Image-Pro Plus 5.0 software, and neurite outgrowth assay was perf...

Embodiment 3

[0072] Example 3: In vivo detection of p75NTR-ED-Fc on the repair of injured spinal cord

[0073] The dorsal column of the spinal cord of adult SD rats was transected (1mm deep) with ophthalmic scissors to establish a rat spinal cord injury model. Immediately after injury, p75NTR-ED-Fc fusion protein (2mm each) was injected on both sides of the transversal point ( A total of 25μl, 0.4mg / ml), with spinal cord injury (SCI) as a negative control, while sham operation and normal rats as a positive control, retrograde tracing after 1, 2, 4, and 6w The technique detects the regeneration of axons. The detailed operation process is as follows: After spinal cord injury, each group of animals was injected with nuclear yellow (NY) into the sciatic nerve 16 to 18 hours before perfusion. The number of neurons indicates the regeneration of axons. At the same time, behavioral (BBB score, footprint test) and neuro-electrophysiological methods were used to detect the functional recovery of the i...

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Abstract

The invention, belonging to the field of medical science, particularly relates to a preparation and application of p75NTR-ED-Fc fusion protein. The preparation method of the p75NTR-ED-Fc fusion protein can improve the expression efficiency and biological activity of the p75NTR-ED-Fc fusion protein. The p75NTR-ED-Fc fusion protein can be applied for promoting the regeneration and functional recovery of the injured central nerve.

Description

technical field [0001] The invention belongs to the field of biomedicine, in particular to the preparation and application of p75NTR-ED-Fc fusion protein. Background technique [0002] Central nervous system (Central nervous system, CNS) injury and repair has always been a hot spot in neuroscience research. Developmental neurobiology and a large number of transplant repair experiments have confirmed that adult mammalian CNS can not only regenerate after injury, but also is determined by the inherent characteristics of neurons (Intrinsic properties) and the microenvironment (Environment cues) in which it is located, which is different from the peripheral nervous system after injury. Compared with successful regeneration, the main reason for CNS regeneration failure is due to the presence of multiple regeneration inhibitory molecules in the damaged microenvironment, such as Nogo-A, MAG (Myelin-associated glycoprotein) and OMgp (Oligodendrocyte-myelin glycoprotein). All of the...

Claims

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Application Information

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IPC IPC(8): C12N15/70C07K19/00C07K1/36C07K1/22C07K1/18A61K38/17A61K47/48A61P25/00
Inventor 王永堂鲁秀敏朱枫黄鹏伍亚民龙在云
Owner INST OF FIELD OPERATION SURGERY NO 3 MILITARY MEDICL UNIV PLA
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