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Analogs of long-acting exendin 4

An analog, long-acting technology, applied in the field of peptides of Exendin4 analogs, can solve the problems of high price and the need for Exendin4, and achieve the effect of reducing cost and reducing immunogenicity

Active Publication Date: 2016-04-13
吴晓琰 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to solve the problem that the current market needs Exendin4 and the price is very expensive. This description provides a 32-amino acid polypeptide derivative of Exendin4

Method used

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  • Analogs of long-acting exendin 4
  • Analogs of long-acting exendin 4
  • Analogs of long-acting exendin 4

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Produce EW1 with genetic engineering method

[0025] material

[0026] EW1

[0027] EcoRIBglIIArgHisGlyGluGlyThrTyrThrSerAspLeuSerSerGlnMetGlu

[0028] CGTCACGGCGAAGGCACCTACACCAGCGATCTGAGCAGCCAGATGGAA

[0029] GluGluAlaValLysLeuPheIleGluTrpLeuValAsnGlyGlyProArg

[0030] GAAGAAGCGGTTAAACTGTTCATCGAATGGCTGGTTAACGGCGGCCCGCGTGGATCCTAG

[0031] BamHlSall

[0032] (1) According to EW1 amino acid sequence and corresponding DNA (positive strand) sequence, synthesize DNA fragment

[0033] 5′AATTCCAGATCTCGTCACGGCGAAGGCACCTACACCAGCGATCTGAGCAGCCAGATGG

[0034] AAGAAGAAGCGGTTAAACTGTTCATCGAATGGCTGGTTAACGGCGGCCCGCGTGGATC

[0035] CTAGTCGAC3′

[0036] Synthetic DNA fragments

[0037] 1) 5'AATTCCAGATCTCGTCACGGCGAAGGCACCTACACCAGCGATCTGAGCAGCCAGATGG3'

[0038] 2) 5'ATTCGATGAACAGTTTAACCGCTTCTTCTTCCATCTGGCTGCTCAGATCGCTGGTGTAG3'

[0039] 3) 5'AAGAAGAAGCGGTTAAACTGTTCATCGAATGGCTGGTTAACGGCGGCCCGCGTGGATCCTAG3'

[0040] 4) 5'CTAGGATCCACGCGGGCCGCCGTTAACCAGCC3'

[0041] 5) 5'GTGCCTTCGCCGTGACGAGATCTGG3'

[0042] 6) 5'tcg...

Embodiment 2

[0062] EW2

[0063] EcoRIBglIIAsnGlyArgHisGlyGluGlyThrTyrThrSerAspLeuSerSerGlnMetGlu

[0064] AACGGCCGTCACGGCGAAGGCACCTACACCAGCGATCTGAGCAGCCAGATGGAA

[0065] GluGluAlaValLysLeuPheIleGluTrpLeuValGlnGlyGlyProArg

[0066] GAAGAAGCGGTTAAACTGTTCATCGAATGGCTGGTTCAGGGCGGCCCGCGTGGATCCTAG

[0067] BamHISaII

[0068] Using EW1 DNA as template, synthesize two DNA fragments

[0069] 487f: 5'ccaatgAATTCCAGATCTAACGGCCGTCACGGCGAAG3'

[0070] 487r: 5'aattGTCGACTAGGATCCACGCGGGCCGCCCTGAACCAGCCAT3'

[0071] Carry out PCR amplification, determine the DNA sequence, and construct EW2 genetically engineered bacteria.

[0072] gAAtTC C AGATCT AACGGCCGTCACGGCGAAGGCACCTACACCAGCGATCTGAGCAGCCAGATGGAAGAA

[0073] GAAGCGGTTAAACTGTTCATCGAATGGCTGGTTCAGGGCGGCCCGCGT G

[0074] GATCT AACGGCCGTCACGGCGAAGGCACCTACACCAGCGATCTGAGCAGCCAGATGGAAGAA

[0075] GAAGCGGTTAAACTGTTCATCGAATGGCTGGTTCAGGGCGGCCCGCGT G

[0076] GATCT AACGGCCGTCACGGCGAAGGCACCTACACCAGCGATCTGAGCAGCCAGATGGAAGAA

[0077] GAAGCGGTTAAACTGTTCATCGAATGGCTGGTTCAGGGCGGCCCGC...

Embodiment 3

[0089] Preparation of EW1 and EW2

[0090] (A) Fermentation of genetically engineered bacteria: Ew1 genetically engineered bacteria were inoculated into 300ml×2 bottles of LB medium, and Ampicillin was added to make the final concentration 50μg / ml, 37°C, and cultured overnight (150rpm) on a shaker. On the second day, the culture was transferred to a 20L New Brunswik fermenter. The composition of the culture medium was M9 medium, with glucose concentration of 1%, Ampicillin concentration of 50μg / ml, ventilation rate of 20L / min, and dissolved oxygen maintained above 20%. The anti-foaming agent is a domestic foam enemy, and the pH is maintained at 7 to 8, adjusted with ammonia. When the cell concentration reaches the center line of the log curve, add IPTG to a concentration of 0.5 mM, continue fermentation for 4-6 hours, and collect the cell by centrifugation to weigh 30-50 g / L.

[0091] (B) Cell disruption: The collected bacteria were homogenized with pH7.0 Tris buffer 20mM, then 1g...

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Abstract

Exendin provided? 4 analog polypeptides. The polypeptide of the present invention and wild Exendin? 4 compared to 7 amino acids shorter at the C-terminus. The length of the peptide chain is similar to that of human GLP-1, and the structure of the peptide contains only one Lys20 residue, so fatty acid acylation occurs exclusively on the -NH2 of Lys20 to form fatty acid acylated derivatives. Also provided are pharmaceutical compositions containing such polypeptides and methods of producing said polypeptides by chemical synthesis or recombinant DNA techniques.

Description

Technical field [0001] The present invention relates to a polypeptide of Exendin4 analogue, which has the effect of lowering blood sugar and can be used for the treatment of type 2 diabetes. The present invention also provides methods for producing these polypeptides through chemical synthesis and recombinant DNA production technology. Background technique [0002] The number of diabetic patients in countries around the world is increasing year by year. Now there are 50 million in China, 60 million in India, 20 million in the United States, 6 million in Japan, and 220 million globally. Diabetic patients are divided into two types, one is insulin-dependent diabetes (type 1 diabetes) and non-insulin-dependent diabetes (type 2 diabetes). Among them, type 2 diabetes accounts for more than 90% of diabetic patients. [0003] Type 2 diabetes patients showed many characteristics, such as decreased β-cells, insufficient postprandial insulin secretion, delayed insulin secretion, increased ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/575A61K38/22A61P3/10
CPCA61K38/26A61P3/10C07K14/57563
Inventor 吴晓琰龚铁军孙玉琨
Owner 吴晓琰