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Recombined membrane-penetrating peptide with WNK (with no lysine (K)) kinase inhibiting effect

A kinase inhibition, membrane-penetrating peptide technology, applied in recombinant DNA technology, medical preparations containing active ingredients, peptides, etc., can solve problems such as poisoning and death

Inactive Publication Date: 2012-04-11
SHANGHAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

; It acts on the M-type and N-type Ach receptors in the central nervous system to produce analgesic effects, but there is also a great risk of poisoning death

Method used

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  • Recombined membrane-penetrating peptide with WNK (with no lysine (K)) kinase inhibiting effect
  • Recombined membrane-penetrating peptide with WNK (with no lysine (K)) kinase inhibiting effect
  • Recombined membrane-penetrating peptide with WNK (with no lysine (K)) kinase inhibiting effect

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Example 1: Preparation of TAT-LIC peptide

[0017] The LIC peptide (LINGO-1, intracellular domain) corresponds to the amino acid sequence 580-620 of the human LINGO-1 molecule, and is derived from a subclone using the full-length structure pEGFP-N1-hLINGO-1 as a template. The production of the fusion protein TAT-LIC peptide is to correctly insert the smallest translocation domain sequence (MRGSHHHHHHGMARGYGRKKRRQRRRPQ) in the HIV1 Tat protein into the N-terminus of the corresponding LIC cDNA. The fusion protein was expressed by BL21 system in vitro and purified by nickel-affinity chromatography column according to the classical method.

[0018]

Embodiment 2

[0019] Example 2: Culture of dorsal root ganglion neurons in SD rat spinal cord

[0020] Extraction and culture of dorsal root ganglion of the spinal cord Aseptically remove embryos from E16 pregnant mice after ether anesthesia, put them into 4 ℃ PBS solution, expose the spinal canal and intervertebral foramen of SD fetal mice under a dissecting microscope, and use forceps to remove the embryos of the spinal cord Take out the DRGs on both sides and put them into PBS solution, remove the DRGs one by one with a microscope, and peel off the fascia on the surface of the ganglion as much as possible, put it into a 35mm culture dish, cut the ganglion into pieces with ophthalmic scissors, and transfer it into 2mL 0.25% In the centrifuge tube of trypsin digestion solution, digest in a CO2 incubator at 37 ℃ for 20 minutes, suck off the digestion solution, directly add DMEM medium containing 10% FBS to terminate the digestion reaction, discard the supernatant after centrifugation, and re...

Embodiment 3

[0021] Example 3: Treating primary cultured spinal cord dorsal root ganglion neurons of SD rats with TAT-LIC peptide can significantly inhibit the phosphorylation of WNK kinase, that is, inhibit the activity of WNK kinase. (See figure 1 )

[0022] The specific experimental process is as follows: the primary cultured spinal cord dorsal root ganglion neurons of SD rats obtained in Example 2 were treated with TAT-LIC peptide (final concentration: 100nM) or control peptide (final concentration: 100nM) for 3 hours . Wash twice with PBS and add RIPAI tissue lysate (20mM Tris-HCl, pH 7.5, 137mM NaCI, 50mM NaF, 0.02%NaN3, 1%NP-40, 2mM EDTA, 2mM benzamidine, 1mM PMSF, 2pg / ml pepstatin A, 2pg / ml leupeptin, 4 pg / ml antipain, 2pg / ml aprotinin, 2mM DTT) and phosphatase inhibitors. The cells were manually homogenized on ice, and the homogenate was centrifuged at 14,000 rpm for 10 min at 4°C, and the supernatant was collected. Co-immunoprecipitation and Western blot detection: Take 500 μ...

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Abstract

The invention relates to a recombined membrane-penetrating peptide with a WNK (with no lysine (K)) kinase inhibiting effect. The gene has an amino acid sequence shown in SEQID NO.1. Serving as a specific inhibitor of WNK serine / threonine kinase kinase, the recombined membrane-penetrating peptide can be used for reducing the activity of NKCC1 activated by the WNK kinase after being combined with the WNK kinase, thus specifically reducing the Cl<-> in neuron cells of spinal cord dorsal root ganglion (DRG) and trigeminal ganglion, inhibiting the excitability action of GABA (gamma-amino butyric acid) in the DRG and inhibiting the transfer of pain sense.

Description

technical field [0001] The invention relates to a recombinant penetrating peptide with inhibitory effect on WNK (silk threonine protein kinase without lysine in the kinase domain) kinase and its application. Background technique [0002] Pain is a complex physiological and psychological activity, and it is one of the most common clinical symptoms. It includes the pain sensation caused by noxious stimuli acting on the body, and the body's pain response to noxious stimuli (body motor response and / or visceral vegetative response, often accompanied by strong emotional color). Pain can be used as a warning that the body is injured, causing a series of defensive protective reactions of the body. But on the other hand, pain also has its limitations as an alarm (such as when pain occurs in cancer, it is too late). And some long-term severe pain has become a kind of unbearable torture to the body. Therefore, analgesia is an important task faced by medical workers. [0003] Pain ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70A61K38/16A61P25/04A61P29/00
Inventor 徐晓辉张照环黄海
Owner SHANGHAI UNIV
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