Two series major vault protein of glucagon-like peptide-2 of people and preparation method thereof

A glucagon and protein technology, applied in the direction of glucagon, peptide/protein components, animal/human proteins, etc., can solve the problems of unclear molecular mechanism, tumor-promoting effect on GLP-2 and its analogs, etc. , to achieve the effect of promoting proliferation, convenient preparation, and prolonging action time.

Active Publication Date: 2013-06-19
FOURTH MILITARY MEDICAL UNIVERSITY
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Problems solved by technology

However, the molecular mechanism of GLP-2 is still unclear, which is also the focus and difficulty of current research
In addition, the controversy about the role of GLP-2 in promoting cancer will also affect the clinical application of GLP-2 and its analogues

Method used

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  • Two series major vault protein of glucagon-like peptide-2 of people and preparation method thereof
  • Two series major vault protein of glucagon-like peptide-2 of people and preparation method thereof
  • Two series major vault protein of glucagon-like peptide-2 of people and preparation method thereof

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Embodiment Construction

[0070] The present invention will be described in detail through specific examples below.

[0071] According to the technical scheme adopted in the present invention, the human glucagon-like peptide-2 dyad gene is synthesized by using the preferred codons of Escherichia coli, and the human glucagon-like peptide-2 dyad gene expression vector pET-22b is constructed. (+)-GLP-2②, the recombinant plasmid was transformed into Escherichia coli BL21 competent cells, and after IPTG induced expression, the recombinant protein was purified by salting out and ion exchange chromatography, and finally a high-purity human glucagon-like peptide was obtained- 2 dimer protein, and use Western blot and HPLC to identify it. The biological activity was determined by Caco-2 cell proliferation assay. The results showed that human glucagon-like peptide-2 dimer protein can promote the proliferation of Caco-2 cells, and its ability to promote the proliferation of Caco-2 cells is higher than that of ch...

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Abstract

The invention belongs to the field of medical biotechnology, and relates to two series major vault protein of glucagon-like peptide-2 (GLP-2) of people and a preparation method of the two series major vault protein. A second place of an amino acid fragment of the glucagon-like peptide-2 of people is mutated, two segments are connected in series through connecting peptide, a dimmer structure of the GLP-2 is simulated, encoding gene of the two series major vault protein of the GLP-2 is obtained by a synthetic method, the encoding gene is cloned to a pET22b(+) pronucleus so as to express a vector, Escherichia coli BL21 is transformed, the encoding gene is expressed in the Escherichia coli efficiently, the purified reconstructed purpose protein is obtained through salt fractionation of ammonium sulfate in fractional and chromatography of anion exchange columns. The reconstructed purpose protein can stimulate a Caco-2 cell, multiplication of the Caco-2 cell can be promoted notably, the two series major vault protein of GLP-2 of people has natural biological activities, and therefore a foundation is established for further study and extensive use of the GLP-2 of people.

Description

technical field [0001] The present invention belongs to the field of medical biotechnology, and in particular relates to a double-string protein of human glucagon-like peptide-2 (GLP-2), its construction method, preparation method, and the expression, purification and processing of the double-string protein. Identification, in vitro biological activity detection, etc. Background technique [0002] Glucagon-like peptide-2 (GLP-2) is one of proglucagon (PG)-derived peptides (PGDP), which is processed by PG transcription and post-translation The polypeptide containing 33 amino acids has the sequence of amino acid residues: HADGSFSDEMNTILDNLAARDFINWLIQTKITD. PG is expressed in pancreatic A cells and intestinal L cells, but its expression products are different due to tissue-specific processing modifications. Glucagon is mainly produced in the pancreas, and PGDP (1-160 amino acids) is mainly produced in the intestine. PGDP mainly includes GLP-1, GLP-2, glucagon, oxyntomodulin, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/605C12N15/16C12N15/63C12N5/10C12N1/15C12N1/19C12N1/21C12N7/01A61K38/26A61P1/00A23L1/29A23L1/305A23L33/00
Inventor 薛晓畅刘烁王增禄张伟张英起
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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