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Expression method and application of fusion protein

A fusion protein and spherical technology, applied in the fields of peptide/protein components, chemical instruments and methods, medical preparations containing active ingredients, etc., can solve the problem that the GLP-1 clearance mechanism cannot be explained, the half-life in vivo has not been correspondingly improved, and in vitro Half-life extension, etc.

Inactive Publication Date: 2011-08-17
ZHEJIANG UNIV
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Problems solved by technology

However, only the participation of dipeptide peptidase IV cannot explain all the clearance mechanisms of GLP-1 in vivo. The half-life of GLP-1 in plasma in vitro is 20 minutes, far exceeding the in vivo half-life of 3 to 11 minutes obtained by many studies.
The in vitro half-life of GLP-1-A obtained by Deacon et al. by substituting glycine for the eighth amino acid alanine of GLP-1 (7-37) was significantly prolonged, but the in vivo half-life was not correspondingly improved, suggesting that there are other mechanisms Involved in the clearance of GLP-1 in vivo

Method used

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  • Expression method and application of fusion protein
  • Expression method and application of fusion protein

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Embodiment 1

[0041] 1. Materials and Instruments

[0042] 1.1 Materials

[0043] E. coli strain Escherichia coli DH5α, BL21(DE3) was provided by the Cancer Research Institute of the Second Affiliated Hospital of Zhejiang University, the prokaryotic expression plasmid PET28a was purchased from Novagen, Germany, and the RNA extract (Trizol) was purchased from Gibcol BRL, USA. Ethyl ester, Oligo(dT) 15 , UNIQ-10 Column Genomic DNA Extraction Kit were purchased from Shanghai Sangon Bioengineering Company, M-MLV Reverse Transcriptase, RNasin, dNTPs, Taq DNA polymerase, agarose, T 4 DNA ligase, restriction endonucleases Nhe I, BamH I, HindIII, and Xba I were all purchased from Promega Company in the United States, and lymphocyte extract was purchased from Shanghai Hengxin Chemical Reagent Co., Ltd., GeneRuler TM DNA Ladder Mix was purchased from Fermentas Company in Germany, DNA Marker DL2000, Isopropyl-beta-D-thiogalactopyranoside (IPTG) was purchased from TaKaRa Company in Japan, SDS polya...

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Abstract

The invention provides an expression method of a human globular adiponectin-glucagon-like peptide-1 analog (gAd-GLP-1-A) peptide fusion protein. The method comprises the following steps of: constructing a prokaryotic expression vector containing human gAd-GLP-1-A fusion genes, converting Escherichia coliBL21 (DE3), obtaining a gAd-GLP-1-A fusion protein containing 6*His tag from an inclusion body by inducible expression, and further digesting the gAd-GLP-1-A fusion protein by using enterokinase to obtain the protein. Animal research shows that the fusion protein has remarkable activity of reducing blood sugar. The fusion protein can promote pancreas islet beta cells to secrete insulin, improve the insulin resistance and remarkably correct the internal metabolic disturbance of type-2 diabetes, and can be applied to preparation of medicaments for treating the type-2 diabetes.

Description

technical field [0001] The present invention relates to the biological field, and relates to a fusion protein expression method, in particular to the expression method and application of human adiponectin globular domain-glucagon-like peptide-1 analogue peptide fusion protein. Background technique [0002] Type 2 diabetes (Diabetes Mellitus, DM) is a disease that seriously endangers human health. The number of patients is increasing rapidly with the improvement of living standards, the aging of the population, and the advancement of diagnostic techniques. Recent studies have shown that insulin resistance and insulin insufficiency are the characteristic pathophysiological changes of type 2 diabetes, and the interaction between the two eventually leads to the occurrence of type 2 diabetes. Not only that, insulin resistance is also involved in the occurrence and development of chronic complications of diabetes, such as cardiovascular complications, diabetic nephropathy, etc., a...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/70A61K38/26A61P3/10A61K38/17
Inventor 赵同峰赵江佩黄晓
Owner ZHEJIANG UNIV
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