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Human glucagon-like peptide-1 analogue

A technology for glucagon and related peptides, applied in the field of genetic engineering, can solve the problems of many steps, low yield and high cost of chemical synthesis method

Inactive Publication Date: 2012-11-28
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the chemical synthesis method has many steps, low yield, and high cost; the genetic engineering method also has many difficulties in production, such as peptide mRNA and expressed protein with a length between 20 and 80 amino acids are unstable in the host cell, The expression efficiency is very low; high-efficiency expression can be obtained by fusion

Method used

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  • Human glucagon-like peptide-1 analogue
  • Human glucagon-like peptide-1 analogue
  • Human glucagon-like peptide-1 analogue

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] M1: 5'-ATATGGATCCGCCGCACGGTGAAGGCACCTTTACCTCTGACGTG TCTTCT TACCTG-3', containing BamH I restriction site

[0067] M2: 5'-GACGTGTCTTTCTTACCTGGAAGGCCAGGCTGCTAAAGAATTTATTGCTTGG-3'

[0068] M3: 5'-GGCCAAGCTTAATCACGACCTTTCACCAGCCAAGCAATAAATTCTTT-3', containing HindIII restriction site

[0069] M4: 5'-GGCCAAGCTTAATCACGACCTTTCACCAG-3'

[0070]Synthesized by Shanghai GenScript Biotechnology Co., Ltd. The PCR reaction was carried out on a Bernie PCR amplifier. Among them, M1, M2, and M3 were mixed according to the concentration ratio of 10:1:10, at 94°C for 30s; at 54°C for 60s; at 72°C for 90s; a total of 30 cycles. The PCR products were identified by 1.8% agarose gel electrophoresis, and the target fragments were recovered with a gel recovery kit and stored at -20°C.

[0071] The PCR target fragment and the carrier plasmid pED recovered by the gel were digested by BamH I and HindIII respectively, the target fragment was recovered by agarose gel electrophoresis, ligated ove...

Embodiment 2

[0080] Shake the recombinant expression bacteria in liquid LB medium overnight at 37 degrees, transfer to corn steep liquor fermentation medium with 3% inoculum, culture at 37 degrees for 4 hours, add final concentration of 5 mmol / L lactose to induce expression, collect and ferment after 6-8 hours bacteria. Reserve samples were analyzed by 15% SDS-PAGE. An obvious protein band appeared at the molecular weight of about 17700Da. The fusion protein reached a stable maximum expression level 6h after induction, and BandScan analysis showed that the fusion protein accounted for more than 40% of the total bacterial protein, and formed inclusion bodies in the cell.

[0081] Example 3 Fusion Protein Insertion of His Tag

Embodiment 3

[0082] According to the preferred codon and design requirements of Escherichia coli, 6 histidines were inserted upstream of the fusion protein to form a histidine tag. The His-tag was inserted by PCR, and the upstream and downstream primers were designed:

[0083] Nco I His Up: 5'-TATACCATGGATCATCATCATCATCATACGCCATTCG-3', containing Nco I restriction site

[0084] HindIII down1: 5'-GGCCAAGCTTAATCACGACCTTTCACCAG-3', containing HindIII restriction site

[0085] The plasmid pED-PP-h[Gly8]GLP-1(7-36)D was used as a template, and Nco I His Up and HindIII down1 were used as upstream and downstream primers for PCR amplification. The reaction conditions were the same as in Example 1. The PCR product was digested with Nco I and BamH I, and the plasmid pED-PP-h[Gly8]GLP-1(7-36)D was digested with Nco I and HindIII to remove AnsB-C-PP-h[Gly8] GLP-1(7-36)D gene fragment, the restriction products of the two were ligated to construct the recombinant plasmid pET-His-AnsB-C-pro-pro-h[Gly8]GL...

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Abstract

The invention relates to the field of gene engineering, and discloses a construction and an expression for two engineering bacteria of two human glucagon-like peptide-1 analogues, and a preparation method for the objective peptides. The objective peptides are formed by inserting objective sequence after double digestions, adding His labels at the front ends of fusion proteins, obtaining objective peptides by cutting the fusion proteins with acid via gaining the fusion proteins, and after an isoelectric precipitation, obtaining the objective peptides again through an affinity chromatography of a Ni agarose gel after an isoelectric precipitation. Freeze-dried objective peptides are dissolved in a PBS buffer solution, oral glucose tolerance test and determination of serum insulin concentration are carried out on normal ICR mice. The two analogues are modified peptides of human glucagon-like peptide-1. Experiments demonstrate that both the two peptides have effects for reducing blood glucose and can be used for treating type 2 diabetes mellitus. Furthermore, the invention also provides the preparation method for the peptides.

Description

technical field [0001] The invention belongs to the field of genetic engineering and discloses two human glucagon-related peptide-1 analogs pro-pro-h[Gly8]GLP-1(7-36)D peptide and pro-pro-h[Gly8, Glu22] Construction of GLP-1(7-35)PPSRD peptide engineering bacteria, preparation method of polypeptide and detection of hypoglycemic activity. Using the new fusion expression preparation technology platform for active polypeptides with a length of 30-70aa constructed in our laboratory, we constructed a high-efficiency fusion expression of human glucagon-related peptide-1 analogues in E. coli cells. [0002] pro-pro-h[Gly8]GLP-1(7-36)D genetically engineered bacteria, the N-terminal alanine (Ala) residue of the human glucagon-related peptide-1 analog is replaced by Gly, and the C-terminal Add D amino acid residues. pro-pro-h [Gly8, Glu22] GLP-1 (7-35) PPSRD genetically engineered bacteria, the alanine (Ala) residue at the N-terminal of the human glucagon-related peptide-1 analog is ...

Claims

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Application Information

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IPC IPC(8): C07K14/605C12N15/16C12N15/63C12N1/15C12N1/19C12N1/21A61K38/26A61P3/10
Inventor 李泰明马艳红刘景晶徐辰刘涛谷春娇
Owner CHINA PHARM UNIV
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