A kind of human-derived secreted fndc5 protein and its preparation method and application

A FNDC5-R, secretory technology, applied in the field of genetic engineering, to inhibit the formation of atherosclerotic plaques, increase the volume and strength of cortical bone, and have a simple structure

Active Publication Date: 2021-08-03
THE SECOND HOSPITAL OF SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] FNDC5 is the precursor protein of irisin, and there are few studies on it at present. The new subtype of human-derived FNDC5 protein that does not contain a transmembrane domain and can be directly secreted and its application in various diseases have not yet been investigated. been reported

Method used

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  • A kind of human-derived secreted fndc5 protein and its preparation method and application
  • A kind of human-derived secreted fndc5 protein and its preparation method and application
  • A kind of human-derived secreted fndc5 protein and its preparation method and application

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Experimental program
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Effect test

Embodiment 1

[0043] A human-derived secreted FNDC5 protein does not contain a transmembrane domain and can be directly secreted and released into the blood. The amino acid sequence of the human-derived secreted FNDC5 protein is SED ID NO: 1:

[0044] IHPGSPSAWPPRARAALRLWLGCVCFALVQADSPSAPAVNVTVRHLKANSAVVSWDVLEDEVVIGFAISQQKKDVRMLRFIQEVNTTTRSCALWGLHILPSHPGFHESYPGSSCPSLEPEWCEVTGLSLHCPFLV;

Embodiment 2

[0046] The preparation method of the human-derived secreted FNDC5 protein described in embodiment 1, comprises the following steps:

[0047]1. Design primers from different sites at the C-terminus of the FNDC5 gene, perform PCR, run electrophoresis, and detect FNDC5 subtypes that are different from the C-terminals of the three known FNDC5 isoforms reported in Pubmed (that is, new FNDC5 isoforms body), specifically:

[0048] The primers are:

[0049] Upstream primer FNDC5-F: TGAGGCCGAGAAGATGGCCTCTAA;

[0050] Downstream primer FNDC5-R: TAGTGACAATGGCTGCTCTCTGCC;

[0051] Extract RNA from Blox5 cells, reverse transcribe into cDNA, and use the above primers for PCR;

[0052] PCR reaction system:

[0053] h 2 O: 36.75 μl

[0054] Go Taq DNA Polymerase: 0.25 μl (Promega)

[0055] 5x Go Taq Buffer: 10 μl (Promega)

[0056] FNDC5-F: 1 μl

[0057] FNDC5-R: 1 μl

[0058] cDNA: 1 μl

[0059] Total: 50μl

[0060] 94°C for 5 minutes;

[0061] 94°C for 30 seconds, 56°C for 30 s...

Embodiment 3

[0154] Application of human-derived secreted FNDC5 protein in the preparation of drugs for treating obesity-related metabolic disorders

[0155] Establishment of obesity model: 6-week-old C57BL / 6 male mice were randomly divided into 3 groups, 6 mice in each group:

[0156] The first group: normal diet mice-normal saline group: negative control group, given normal diet all the time, and intraperitoneal injection of normal saline for 2 weeks after 12 weeks;

[0157] The second group: obese mice-normal saline group: as the positive control group, a high-fat diet was given to make obese mouse models, and after 12 weeks, normal saline was injected intraperitoneally for 2 weeks;

[0158] The third group: obese mice-secreted FNDC5 group: as the experimental group, high-fat diet was given to make obese mouse models, and secreted FDNC5 protein was injected intraperitoneally for 2 weeks after 12 weeks.

[0159] Administration method and administration concentration: The secreted FNDC5 ...

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Abstract

The invention discloses a human-derived secreted FNDC5 protein and its preparation method and application, and relates to the technical field of genetic engineering; wherein the human-derived secreted FNDC5 protein does not contain a transmembrane domain, and can be directly secreted and released into blood; The human-derived secreted FNDC5 protein of the invention can be used for preparing medicines for treating atherosclerosis, promoting bone formation and treating aplastic anemia, and can prepare medicines for proliferation of endothelial cells. The human-derived secreted FNDC5 protein of the present invention can effectively reduce the body weight of obese mice after fasting injection to obese mouse models fed with high fat, indicating that the protein can significantly improve body fat metabolism of obese patients, significantly reduce plasma cholesterol, The levels of free fatty acids and triglycerides indicate that the protein can significantly improve lipid metabolism disorders in obese patients.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a human-derived secreted FNDC5 protein and its preparation method and application. Background technique [0002] Irisin (irisin) is a protein containing 112 amino acid residues discovered in 2012. It is the product of an unknown protease that cleaves type III fibronectin domain-containing protein 5 (FNDC5). Irisin is a part of FNDC5. cutting product. The three FNDC5 subtypes found so far all contain a transmembrane domain (exon5), which anchors the protein on the cell membrane. The sequence of irisin is highly conserved in mammals. Human, mouse and rabbit irisin The amino acid sequence is exactly the same; however, the C-terminal base sequence of the FNDC5 gene of human and mouse and rabbit (animal source) is different. If the animal-derived FNDC5 protein is directly injected into the human body, rejection may occur due to species issues. In addition to having all t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/78C12N15/81A61K38/39A61P3/04A61P9/10A61P19/08A61P7/06C12R1/84
CPCA61K38/00A61P3/04A61P7/06A61P9/10A61P19/08C07K14/78C12N15/815
Inventor 唐东起李世武李慧
Owner THE SECOND HOSPITAL OF SHANDONG UNIV
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