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Method for preparing human pancreatic glucagons like polypeptide - 1, and fusion proten of human serum albumin, and products

A human serum albumin and fusion protein technology, applied in the field of long-acting fusion protein drugs, can solve the problems of clinical application limitations and difficulty in wide application, and achieve the effect of avoiding the stability of fusion proteins

Inactive Publication Date: 2007-02-21
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although GLP-1 has a good anti-diabetic effect, because its half-life in vivo is only 2 minutes (mainly due to the hydrolysis and renal excretion of DPP-IV), and it is a polypeptide drug injected in vivo, it must be administered continuously to be effective. Clinical application brings great limitations, so far it is difficult to be widely used clinically

Method used

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  • Method for preparing human pancreatic glucagons like polypeptide - 1, and fusion proten of human serum albumin, and products
  • Method for preparing human pancreatic glucagons like polypeptide - 1, and fusion proten of human serum albumin, and products
  • Method for preparing human pancreatic glucagons like polypeptide - 1, and fusion proten of human serum albumin, and products

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: (GLP-1) 2 cDNA cloning

[0027] Synthetic (GLP-1) 2cDNA (completed by Shanghai Sangon Bioengineering Technology Service Co., Ltd.). Artificially synthesized (GLP-1) 2 cDNA as template, amplified by PCR (GLP-1) 2 cDNA, the primers used are as follows:

[0028] PG1: 5'TGC GGATCC CACGGTGAAGGTACTTTCACTTCTGA-3'

[0029] PG2: 5'-ATA GCGGCCGC TTAACGACCCTTAACCAACCAAGC-3'

[0030] The PCR method is as follows: Add to the 50 μl reaction system: 1.5 μl of 10 μmol / L PG1 and PG2 primers, 5 μl of 2 mmol / L dNTP, 1 μl of 10×pfu Buffer, 0.5 μl of 5 U / μl pfu DNA polymerase (dNTP, 10×pfu Buffer and pfu DNA polymerase were purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd., the same below), (GLP-1) 2 cDNA 1μg, add double distilled water to make up 50μl, in PTC-100 of MJ Research TM On the PCR instrument (the same below), the PCR conditions are: denaturation at 95°C for 10 minutes, denaturation at 94°C for 1 minute, annealing at 60°C for 1 min...

Embodiment 2

[0032] Example 2: Cloning of HSA cDNA

[0033] HSA cDNA was amplified from the human fetal liver cDNA library by PCR, and the primers used were:

[0034] PH1: 5'-AG GTC GAC GATGCACACAAGAGTGAGGTTGCTC-3'

[0035] PH2: 5'-GC AAGCTT TTATAAGCCTAAGGCAGCTTGACTT-3'

[0036] The PCR method is as follows: Add to the 50 μl reaction system: 1.5 μl of 10 μmol / L PH1 and PH2 primers, 5 μl of 2 mmol / L dNTP, 1 μl of 10×pfu Buffer, 0.5 μl of 5 U / μl pfu DNA polymerase, human fetal Liver cDNA library 1 μg, add double distilled water to make up 50 μl; PCR conditions are: denaturation at 95°C for 5 minutes, denaturation at 94°C for 1 minute, annealing at 60°C for 1 minute, extension at 72°C for 90 seconds, cycle 30 times;

[0037] The reaction product was analyzed by agarose gel electrophoresis, and the target band appeared in the loading lane, and the 1.8kb target fragment was purified with a PCR fragment gel recovery kit, and the purified target fragment and the carrier pBlue2KSP were doubl...

Embodiment 3

[0038] Embodiment 3: (GLP-1) 2 Cloning of cDNA and HSA cDNA fusion gene

[0039] (GLP-1) 2 For PCR amplification of cDNA, the primers used are as follows:

[0040] PG3: 5’-GAGA GGTACC TAAAAAGACACGGTGAAGGTACTTTCACT-3'

[0041] PG4: 5'-ACCTCACTCTTGTGTGCATCCCTTCCTTTACTAACCATG-3'

[0042] The PCR method is as follows: 50 μl reaction system was added: 1.5 μl of 10 μmol / L Pf1 and Pf2 primers, 5 μl of 2 mmol / L dNTP, 1 μl of 10×pfu Buffer, 0.5 μl of 5 U / μl pfu DNA polymerase, pBlue2KSP- (GLP-1) 2 1ng, add double distilled water to make up 50μl; PCR conditions are: denaturation at 95°C for 5 minutes, annealing at 65°C for 1 minute, extension at 72°C for 90 seconds, denaturation at 94°C for 1 minute, cycle 30 times;

[0043] PCR amplification of HSA cDNA, the primers used are as follows:

[0044] PH3: 5'-CATGGTTAGTAAAAGGAAGGGATGCACACAAGAGTGAGGT-3'

[0045] PH4: 5'-TGAA CCGCGGCCGC TTATAAGCCTAAGGCAGCTTG-3'

[0046] The PCR method is as follows: Add to the 50 μl reaction system...

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Abstract

This invention discloses a method for preparing fusion protein and its related products containing human glucagon-like peptide 1 (GLP-1) and human serum albumin (HSA). The fusion protein contains a first peptide having at least 85% homology to human GLP-1 and a second peptide having at least 85% homology to HSA, wherein the two peptides are directly linked without any linking peptide, and several amino acid residues can be replaced, deleted or inserted if not changing the property of the fusion protein. The fusion protein is obtained by linking two tandem GLP-1 cDNA ((GLP-1)2) and HAS cDNA through overlapping PCR and expressing the fusion gene ((GLP-1)2-HAS) in a host cell. The fusion protein retains the physicological property of human glucagon-like peptide 1, and has improved half-life in vivo and potential application in pharmaceutical field.

Description

technical field [0001] The invention relates to a preparation method and product of a fusion protein of human glucagon-like polypeptide-1 and human serum albumin, belonging to the technical field of long-acting fusion protein drugs. Background technique [0002] Human glucagon-like peptide-1 (GLP-1) is a polypeptide composed of 30 amino acids synthesized and secreted by intestinal endocrine L cells. In vivo and in vitro studies have shown that GLP-1 can enhance glucose-dependent insulin secretion, reduce food intake, slow down gastric emptying, and inhibit glucagon secretion, resulting in lower blood sugar. In addition, recent studies have revealed that GLP-1 can stimulate the proliferation of pancreatic β cells, promote the regeneration of islets, inhibit the apoptosis of pancreatic β cells, and promote the synthesis of insulin by pancreatic β cells. Animal experiments and clinical trials have shown that the blood sugar level of patients with type II diabetes is significan...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12N15/62C12N15/14C12N1/19C07K19/00
Inventor 金坚窦文芳张莲芬雷楗勇卢大儒
Owner JIANGNAN UNIV
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