Irisin recombinant protein and synthetic method thereof

A recombinant protein and protein technology, which is applied in the field of genetic engineering, can solve the problems such as the localization and expression analysis and identification of Irisin protein that cannot be used, and achieve the effect of accurate research and localization analysis.

Inactive Publication Date: 2016-11-30
THE WEST CHINA SECOND UNIV HOSPITAL OF SICHUAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The above-mentioned prior art realizes the expression of Irisin protein, but the antibody against Irisin has certain specificity problems, and the receptor of Irisin has not been purified and identified so far, and the specific analysis and identification of the localized expression of Irisin protein cannot be carried out

Method used

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  • Irisin recombinant protein and synthetic method thereof
  • Irisin recombinant protein and synthetic method thereof
  • Irisin recombinant protein and synthetic method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0052] An Irisin recombinant protein, the N-terminal of the Irisin recombinant protein has a His-tag.

Embodiment 2

[0054] A kind of synthetic method of the DNA sequence of Irisin recombinant protein, comprises the following steps:

[0055] (1) Design and sequence acquisition of Irisin prokaryotic expression plasmid.

[0056] (2) Obtain the cohesive Irisin sequence.

[0057] (3) Obtain the pET28a(+) linearly linked plasmid with cohesive ends.

[0058] (4) Ligate the pET28a(+) plasmid and the Irisin sequence product.

Embodiment 3

[0060] The design and sequence acquisition of Irisin prokaryotic expression plasmids include the following steps:

[0061] (1) Design of sequences and primers:

[0062] A. Check the FNDC5 amino acid sequence and FNDC5 cDNA sequence from NCBI and Ensembl websites. The FNDC5 amino acid sequence includes the Irisin amino acid sequence, and the FNDC5 cDNA sequence includes the Irisin cDNA sequence.

[0063] The sequence of Irisin is highly conserved, and the amino acid sequences of human, rat, and mouse are completely consistent. In the present invention, the sequence of mouse is selected for recombination and synthesis, and the mRNA containing FNDC5 sequence obtained from the mRNA of mouse preadipocyte strain 3T3-L1 full sequence.

[0064] B. Design primers according to NdeI and XhoI enzyme digestion. The 5' end of the primer has a sequence of enzyme cutting points, and the rest are sequences located at both ends of Irisin. The codons of amino acids after connection are consiste...

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Abstract

The invention discloses Irisin recombinant protein and a synthetic method thereof. His-tag is arranged at an N end of the Irisin recombinantprotein, and the His-tag is a mouse monoclonal antibody after affinity purification, and is used for detecting the sequence of labels on an expression vector for analyzing the expression content and the function of target protein. The His-tag contains 6 histidine which can be idiosyncratically combined with nickel ions, and the recombinant protein with histidine labels can be adsorbed by nickel columns, so that the purification purpose can be achieved. If Irisin is directly used, the expression of the protein cannot be analyzed and identified, and identification on biological functions indicates that the Irisin recombinant protein with the His-tag can perform idiosyncratic analysis and identification for the positioning expression of the protein, so that the expression content and the function of the Irisin recombinant protein are analyzed.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to an Irisin recombinant protein and a synthesis method thereof. Background technique [0002] Irisin is a new myokine reported by Spiegelman's laboratory in Nature in early 2012. It is a secretable polypeptide fragment formed after proteolytic enzymes cleave type III fibronectin module containing protein 5 (FNDC5). There is a signal sequence at the N-terminus of human FNDC5, a type III fibronectin domain (FND) in the middle, followed by a hydrophobic amino acid region and the C-terminus. Irisin is the main part of the fibronectin domain in FNDC5. It is an N-glycosylated protein hormone composed of 111 amino acid residues. Its sequence is highly conserved among species. The amino acid sequence homology of human and mouse Irisin is 100%. [0003] The discoverer named Irisin after the messenger goddess Iris in Greek mythology, implying that like Iris, it acts as a mess...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/78C12N15/12C12N15/70C12N1/21
CPCC07K14/78C12N15/70C12N2800/101
Inventor 谯小勇许良智马亚仙胡颖聂颖程冉曾如君
Owner THE WEST CHINA SECOND UNIV HOSPITAL OF SICHUAN
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