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TAT-WNK102 transmembrane protein and application thereof

A TAT-WNK102, transmembrane protein technology, applied in the fields of peptide/protein components, recombinant DNA technology, medical preparations containing active ingredients, etc., can solve problems such as unclear downstream signal transduction pathways

Inactive Publication Date: 2012-03-21
SHANGHAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But its downstream signal transduction pathway is not clear

Method used

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  • TAT-WNK102 transmembrane protein and application thereof
  • TAT-WNK102 transmembrane protein and application thereof
  • TAT-WNK102 transmembrane protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Embodiment 1: Preparation of TAT-WNK102 fusion protein

[0018] The WNK102 protein corresponds to the amino acid sequence 308-409 of the human WNK-1 molecule, and is derived from a subclone using the full-length structure pEGFP-N1-hWNK-1 as a template. The production of the fusion protein TAT-WNK102 is to correctly insert the smallest translocation domain sequence (MRGSHHHHHHHGMARGYGRKKRRQRRRPQ) in the HIV1 Tat protein into the N-terminus of the corresponding WNK1 cDNA. The fusion protein was expressed by BL21 system in vitro and purified by nickel-affinity chromatography column according to the classical method.

Embodiment 2

[0019] Example 2: Culture of SD rat oligodendrocytes

[0020] The brains of SD neonatal rats were taken, and the bilateral cerebral cortex was separated, D. Wash with Hanks solution, pipette to make cell suspension, filter with 200-mesh cell mesh, centrifuge at 180×g for 5 min, discard supernatant, add high-glucose DMEM culture medium containing 10% fetal bovine serum to resuspend cells, dilute to (1.0 -2.0) *106 density planted in 0.025% poly-lysine pretreated 75c㎡ culture flask and cultured in 5% CO2 for 8-11 days, after the cells were confluent and formed obvious stratification, OPCs shaking separation and purification began (the culture flask Place on a constant temperature air bath vibrating shaker, 37 degrees, 150r / min, shake for 2h to remove microglial cells, etc. D. Wash with Hanks solution, change the liquid; 5% CO2 incubator balance for 2h; 230r / min, 16-18h , 280r / min, 1-2h; collect the cell suspension, centrifuge at 400×g for 5 min, resuspend the cells in high-gluc...

Embodiment 3

[0021] Embodiment 3: The SD rat oligodendrocytes purified in Example 2 were inoculated on a 35mM culture plate coated with GST-Nogo66 protein or GST protein in advance, and replaced with fresh Neurobasal containing 2% B27 after three hours -A medium (Gibco), . After cultured for 5 days, the cells were immunohistochemically performed with anti-MBP antibody and observed and photographed under a confocal microscope. See figure 1 and figure 2 , using Nogo66 protein as a substrate to culture primary cultured rat OPC cells will indeed inhibit the differentiation and maturation of OPC cells in its differentiation medium. The number of OPC differentiated cells grown on Nogo66 matrix was significantly reduced. figure 2 for figure 1 Statistical analysis of the proportion of differentiated cells. Data are from three independent experiments and expressed as mean ± standard error. Comparison by Student's-t-test, p<0.001.

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Abstract

The invention relates to a TAT-WNK102 transmembrane protein and application thereof. The protein has an amino acid sequence shown in SEQIDNO1. The TAT-WNK102 transmembrane protein can inhibit the combination of LINGO-1 and WNK1; and after the interaction between the LINGO-1 and the WNK1 is inhibited by the peptide, the differentiation and maturation of cultured oligodendrocytes can be promoted, thereby obtaining the TAT-WNK102 transmembrane protein. The TAT-WNK102 transmembrane protein can be used for the preparation of medicaments for treating demyelinating diseases of the central nervous system.

Description

technical field [0001] The invention relates to a TAT-WNK102 transmembrane protein and its application. Background technique [0002] In the pathogenesis of central nervous system demyelinating diseases such as multiple sclerosis, it is accompanied by the differentiation, maturation and remyelination of OPC cells. At present, the research on the molecular mechanism of how to promote the process of remyelination in patients with multiple sclerosis is a hot spot both at home and abroad. During the pathogenesis of multiple sclerosis, many inhibitory factors are produced in the disintegrated axon myelin sheath. The receptor for these inhibitors, LINGO-1, is expressed on nascent oligodendrocytes and inhibits their maturation and remyelination. The discovery of an antibody that antagonizes the function of Nogo receptor-interacting protein 1 (LINGO-1) with LRR structure and immunoglobulin domain can promote partial recovery of function in experimentally demyelinated rats. But it...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C12N15/70A61K38/45A61P25/28C12R1/19
Inventor 徐晓辉张照环黄海
Owner SHANGHAI UNIV
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