Polypeptide analogue for blocking NR2B signal path and preparation method and medical usage thereof

A technology of analogs and fragments, which can be used in drug combinations, chemical instruments and methods, medical preparations containing active ingredients, etc., and can solve problems such as harmfulness and ineffectiveness of NMDA receptor antagonists.

Inactive Publication Date: 2008-03-05
NANJING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Clinical data also show that NMDA receptor

Method used

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  • Polypeptide analogue for blocking NR2B signal path and preparation method and medical usage thereof
  • Polypeptide analogue for blocking NR2B signal path and preparation method and medical usage thereof
  • Polypeptide analogue for blocking NR2B signal path and preparation method and medical usage thereof

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Experimental program
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Effect test

Embodiment 1

[0059] Design, Synthesis and Cloning of Tat-HA-NR2B9C Polypeptide Gene

[0060] According to the amino acid sequence of the Tat-HA-NR2B9C polypeptide gene, the codon preferred by Escherichia coli was selected, and the complete sequence of the Tat-HA-NR2B9CP polypeptide gene was designed with the aid of a computer. Based on the sequence, four oligonucleotide fragments were designed and chemically synthesized. Using the C-terminal of the L-ansB-C gene of pED as a template, the gene fragment containing the target peptide sequence, that is, the Tat-HA-NR2B9C polypeptide gene, was obtained by adding end PCR, and transformed into Escherichia coli Escherichiacoli BL21 (DE3). The extracted plasmid was named pED-Tat-HA-NR2B9C. The specific method is as follows:

[0061] The 4 oligonucleotides are as follows:

[0062] P1: 5′-TACCATGGATACGCCATTCG-3′

[0063]P2: 5′-CGGCGGCGCTGGCGGCGTTTTTTACGACCGTAACCCGGATCCGCGTACTGGTT-3′

[0064] P3: 5′-TACCAGCAACGTCCGGAACGTCGTACGGGTAACCGCGGCGGCGCTGG...

Embodiment 2

[0069] Improvement of LB medium and formulation of arginine enriched LB medium

[0070] The recombinant plasmid pED-Tat-HA-NR2B9C was transformed into Escherichia coli BL21. When the fusion protein was induced and expressed in ordinary LB medium, the expression level of the fusion protein was very low, as shown in Figure 4A, Lane5. In order to induce a large amount of expression of the fusion protein AnsB-C-Tat-HA-NR2B9C, a certain amount of Arg hydrochloride was added to the LB medium as a source of special amino acids. The strains were inoculated in the LB medium containing Kan (50 μg / ml) at a ratio of 2%, and the LB medium was supplemented with arginine according to the amount of 0, 0.1%, 0.5%, and 1% (W / V) respectively. After 4 hours of incubation at 37°C, the inducer lactose was added, and after 4 hours of induction, samples were taken for SDS-PAGE analysis. The results showed that when the content of arginine in the LB medium was 0.1% (W / V), the expression of the fusio...

Embodiment 3

[0078] Expression of Tat-HA-NR2B9C Polypeptide Gene in Escherichia coli

[0079] Pick a single colony from the plate and inoculate it into arginine-enriched-LB liquid medium, shake it overnight at 37°C, transfer it to a fresh liquid medium at a ratio of 2%, cultivate it at 37°C for 4 hours, add the final IPTG at a concentration of 0.1 mmol / L induced Escherichia coli to express T7 RNA polymerase, thereby expressing the fusion protein. Samples were taken at 1, 2, 3, and 4 hours after the addition of the inducer, and the results of SDS-PAGE electrophoresis showed that the expression of the fusion protein reached a peak and remained at a stable level after the addition of the inducer 3 hours, and there was no increase with the induction time. The changes were obvious, so the cells were collected around 3.5 hours after induction, and the results are shown in Figure 4B.

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Abstract

The present invention provides one kind of fusion protein in C-terminal of recombinant NMDA receptor NR2B subtype and produced through gene engineering process, polynucleotides encoding the fusion protein, and process of preparing and purifying the fusion protein. The fusion protein consists of the PSD95 linking region segment of NMDA receptor NR2B subtype and the transduction region segment of HIV coded Tat protein, and has one hemagglutinin marker segment for influenza virus inserted between the two above said segments. The fusion protein may be expressed effectively in colibacillus, has simple purifying process and is suitable for production in large scale. The Tat-HA-NR2B9C polypeptide of the present invention can block the interaction between NMDA receptor and PSD95, inhibit NMDA receptor mediated excitable neurotoxicity effect and protect nerves. It may be applied in treating cerebral apoplexy and relevant nerve diseases.

Description

(1) Technical field [0001] In the neurobiological field of medicine, peptide blockers targeting receptors are often used to treat neurological diseases. The invention relates to a method for polypeptide analogs, in particular to a molecular design and preparation method for polypeptide analogs that block signal transduction between NMDA receptors and PSD-95 protein. (2) Technical background [0002] N-methyl-D-aspartate (NMDA) receptor is a gated ion channel complex, as one of the excitatory amino acid receptors, mainly distributed in the cerebral cortex, hippocampus, striatum and other parts, is It is an important basis for the transmission of excitatory amino acid transmitters and a key factor for maintaining the normal function of the central nervous system. Under physiological conditions, NMDA receptors not only mediate synaptic plasticity, neuron growth and differentiation, but also regulate synaptic depolarization through postsynaptic signals to maintain certain physi...

Claims

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Application Information

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IPC IPC(8): C07K19/00C07K14/155A61K38/16A61P25/00
Inventor 朱东亚徐进署江君罗春霞朱明媚吴海银
Owner NANJING MEDICAL UNIV
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