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MG53 (mitsugumin53) mutant as well as preparation method and application thereof

A mutant and site mutation technology, applied in the field of MG53 mutants, can solve the problems of unsolved channels, coexistence of positive and negative effects, etc.

Active Publication Date: 2017-10-20
MUDANJIANG YOUBO PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This is a problem that the industry is concerned about and does not want its positive and negative effects to coexist. So far, there is no solution

Method used

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  • MG53 (mitsugumin53) mutant as well as preparation method and application thereof
  • MG53 (mitsugumin53) mutant as well as preparation method and application thereof
  • MG53 (mitsugumin53) mutant as well as preparation method and application thereof

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Experimental program
Comparison scheme
Effect test

specific Embodiment approach

[0092] The coiled-coil domain of MG53 protein is a well-studied domain that mediates the interaction between proteins. It presents a unique repeating unit in sequence and is widely distributed. It participates in membrane fusion, membrane vesicle transport and many other important life process. Functionally, the coiled coil domain provides sufficient space for the head and tail structures of the protein during vesicle transport. In addition, the coiled coil domain can also serve as a backbone to recruit proteins to form complexes. Transfer the cell proliferation, differentiation and apoptosis signals into the MAP kinase module to form a signaling pathway.

[0093] The applicant believes that the coiled coil domain plays an important role in the vesicle transport and signal pathway transduction of MG53 protein. Therefore, the applicant chose the coiled coil domain of MG53 protein as the research object. Moreover, the applicant found that in the regulation of insulin signalin...

Embodiment 1

[0219] Embodiment 1: Construction of MG53 mutant plasmid MG53 S189A

[0220] 1. MG53 gene optimization, synthesis and MG53 S189A subcloning and plasmid preparation:

[0221] The following are the reagents and their sources used in the construction of the MG53 S189A plasmid:

[0222] Reagent: T4DNA Ligase Source: NEB Lot Number: 00309971,

[0223] PET-22b plasmid, source: Novagen,

[0224] Competent cell source: Tiangen Biochemical Technology (Beijing) Co., Ltd. (CB105-02),

[0225] Experimental water source: ddH2O Batch number: 10 / 13 / 2015,

[0226] Endonuclease NdeI Source: NEB Cat. No.: R0111S,

[0227] Source of endonuclease XhoI: NEB Cat. No.: R5146S.

[0228] In addition, the equipment used in the construction of the MG53 S189A plasmid and its sources are listed in Table 1

[0229] Table 1, equipment used in constructing MG53 S189A plasmid

[0230] equipment name

model

manufacturers

centrifuge

MEGAFUGE 8R

Thermo

Electrophores...

Embodiment 2

[0291] Example 2: Expression and purification of MG53 mutant plasmid MG53 S189A

[0292] 2.1 Expression of MG53 mutant plasmid MG53 S189A

[0293] The medium used for expression is as follows:

[0294] 1) Shaker medium (400ml): LB medium (tryptone 2%; yeast powder 1%; sodium chloride 2%; 100 μg / ml Amp 1%);

[0295] 2) Fermentation medium (5 L): (tryptone 12%; yeast powder 24%; glycerol 4ml / L; dipotassium hydrogen phosphate 16.4%; potassium dihydrogen phosphate 2.32%; 100 μg / ml Amp 1%).

[0296] 3) Feed medium: 50% glycerol. The feed medium is used to supplement the carbon source and energy needed by the bacteria in the late stage of fermentation.

[0297] In the middle and late stages of the fermentation process, after the carbon source and energy in the fermentation medium are exhausted, the feed medium is used to supplement the carbon source and energy needed by the bacteria.

[0298] Express MG53 S189A according to the following steps:

[0299] 1) Activation of MG53 S189...

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Abstract

The invention provides an MG53 (mitsugumin53) mutant and a preparation method thereof. The mutant is a serine-site mutant of MG53 protein in a Coiled-coil structural domain, and the mutant results in inactivation of MG53 phosphorylation. Meanwhile, the MG53 protein mutant can be used for preventing or treating diseases related to cellular membrane damage, such as diseases related to myocardial cell injury, diseases related to ulcer, trauma with a wound, particularly a difficult-to-heal wound, intestinal leakage, kidney injury and the like. The diseases related to myocardial cell injury can include one or more diseases related to following symptoms: myocardial ischemia, heart ischemia, heart ischemia / reperfusion injury, myocardial infarction, cardiac failure, arrhythmia and cardiac rupture. The diseases related to ulcer include one or more of following diseases: chronic ulcer, peptic ulcer, diabetic foot ulcer, diabetic foot gangrene and chronic gastric ulcer. Besides, the invention further provides a dry powder preparation, a spray preparation, a gel preparation and an emulsion containing MG53 or MG53 mutant as well as preparation methods of the preparations.

Description

field of invention [0001] The present invention relates to a MG53 mutant (also referred to as MG53 protein mutant), and the use of the MG53 mutant in preventing or treating diseases related to cell membrane damage, such as diseases related to myocardial cell damage, diseases related to ulcers, diseases with Wounds, especially hard-to-heal wounds, intestinal leaks, and kidney damage. Diseases associated with cardiomyocyte injury may include one or more selected from the group consisting of: myocardial ischemia, cardiac ischemia / reperfusion injury, myocardial infarction, heart failure, cardiac arrhythmia, and cardiac rupture. And ulcer-related diseases include one or more selected from the following: chronic ulcer, peptic ulcer, diabetic foot ulcer, diabetic foot gangrene, and chronic gastric ulcer. While protecting the heart and treating heart diseases caused by cell death, the MG53 mutant avoids side effects such as insulin resistance, obesity, diabetes and metabolic syndrome...

Claims

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Application Information

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IPC IPC(8): C07K14/47A61K38/17A61P9/10A61P9/04A61P9/06A61P1/04A61K9/19A61K9/08A61K9/12A61K9/06A61K9/107
CPCA61K9/0019A61K9/06A61K9/08A61K9/107A61K9/12A61K9/145A61K9/19A61K38/00A61K47/36C07K14/4702
Inventor 慎东
Owner MUDANJIANG YOUBO PHARMA CO LTD
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