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Hypoglycemic polypeptide fused protein, structure and use of derivate thereof

A fusion protein and hypoglycemic polypeptide technology, applied in the field of bioengineering, can solve the problems of short half-life of the original polypeptide, poor patient compliance, and short action time, and achieve the effects of high bioavailability, low immunogenicity, and improved stability.

Inactive Publication Date: 2008-12-24
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above characteristics make the GLP-1 molecule an ideal drug candidate for the treatment of diabetes, but the natural GLP-1 has not been used clinically for many years, mainly because the GLP-1 has a very short action time in the body and its plasma half-life is less than 2 minutes. After intravenous administration, the endogenous dipeptidyl peptidase (DPPIV) cuts off 2 amino acids from the N-terminus to inactivate, and the fragment GLP-1 produced after enzymatic hydrolysis 3-30 Is an antagonist of natural GLP-1, further inhibits the effect of GLP-1
[0007] At present, although great progress has been made in the research of these two polypeptides and their analogs, there are still problems in the following aspects: (1) The preparation of such polypeptides exists whether they are prepared by ordinary solid-phase synthesis or recombinant expression. Higher cost and greater difficulty; (2) Such polypeptides themselves are not stable, but they are easily inactivated by forming aggregates in the solution state, especially in acidic solutions; (3) The half-life of the original form of such polypeptides It is relatively short, but injections are often used in clinical administration, and patient compliance is extremely poor

Method used

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  • Hypoglycemic polypeptide fused protein, structure and use of derivate thereof
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  • Hypoglycemic polypeptide fused protein, structure and use of derivate thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Preparation of hypoglycemic polypeptide 1 fusion protein

[0033] Step 1: According to the amino acid sequence of hypoglycemic polypeptide 1, its gene sequence was synthesized with reference to the codon preference of Escherichia coli, and the DDDDK sequence was designed upstream of the gene, and the recognition sequences of Kpn1 and Hind3 were added at both ends. The amino acid sequence of the gene fused with TrxA is as follows: TrxA-HisTag-DDDDK-HGEGT FTSDV SSYLE GQAAK EFIAW LVKGR PSSGA PPPS Step 2: Digest the synthesized gene and pET32a vector with Kpn1 and Hind3 respectively. The products were ligated at 16°C for 8 hours, and the ligated products were transformed into E. coli competent cells according to the standard method given in "Molecular Cloning" and positive clones were selected. Sequencing verifies the accuracy of the inserted gene.

[0034] Step 3: Cultivate positive clones at 37 degrees overnight in LB medium to make seed solution, and add 50 μg / ml ampici...

Embodiment 2

[0038] Preparation of hypoglycemic polypeptide 2 fusion protein, single-chain PEGylated fusion protein derivatives, and enzymatic digestion products of derivatives and determination of hypoglycemic activity.

[0039] According to the amino acid sequence of hypoglycemic polypeptide 2, its gene sequence was synthesized with reference to the codon preference of Escherichia coli, the DDDDK sequence was designed upstream of the gene, and the recognition sequences of Kpn1 and Hind3 were added at both ends. The amino acid sequence of the gene fused with TrxA is as follows: TrxA-HisTag-DDDDK HGEGT FTSDV SSYLE GQAAK EFIAW LVKGRC

[0040] The recombinant construction, transformation, expression and purification of the gene of the TrxA-GLP-1 (Gly8, Cys37) fusion protein are the same as in Example 1. SDS-PAGE detection shows that the purity of the purified fusion protein is higher than 95%. Dilute the purified protein to 1 mg / ml, add maleimidated single-chain polyethylene glycol 5000 at a...

Embodiment 3

[0044] Preparation of hypoglycemic polypeptide 3 fusion protein and biotinylated fusion protein derivatives and determination of hypoglycemic activity.

[0045] Step 1: According to the amino acid sequence of hypoglycemic polypeptide 3, its gene sequence was synthesized with reference to the codon preference of Escherichia coli, an Arg codon was designed upstream of the gene, and recognition sequences of BamH1 and Hind3 were added at both ends. The amino acid sequence of the fusion protein composed of the gene and HisTag is as follows:

[0046] GSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSRHGEGTFTSDVSSYLEGQAAQEFIAWLVKGR

[0047] Step 2: Double digest the synthesized gene and the pET28a vector with BamH1 and Hind3 respectively, and connect the products after digestion at 16°C for 8 hours, and transform the ligated products into Escherichia coli to sense cells and select positive clones. Sequencing verifies the accuracy of the inserted gene.

[0048] Step 3: Cultivate positive clones at ...

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Abstract

The invention relates to a structure and application of a hypoglycemic polypeptide fused protein and derivates thereof, belonging to the bioengineering technical field. The invention provides the technical proposal that: a fused polypeptide obtained obtains high solubility expression in a bacillus coli system through addition of special polypeptide sequences. The fused polypeptide and the derivatives thereof have high stability and activity compared with the natural hypoglycemic polypeptide, and can generate obvious hypoglycemic activity when people take or are injected a certain dosage.

Description

technical field [0001] The invention relates to the structure and application of a hypoglycemic polypeptide fusion protein and its derivatives, belonging to the technical field of bioengineering. Background technique [0002] Diabetes Mellitus (DM) is a term that describes a metabolic disease with multiple etiologies, characterized by chronic hyperglycemia, accompanied by disorders of sugar, fat and protein metabolism caused by defects in insulin secretion and / or action. The disease can be divided into the following two types: type 1 diabetes, also known as insulin-dependent diabetes; type 2 diabetes, also known as non-insulin-dependent diabetes. According to WHO statistics, the current incidence of diabetes is 2.8%, and it is expected that this value will increase to 4.8% by 2030. According to the CDCP report of the United States, diabetic patients account for 6.8% of the entire American population, and 90-95% of them are type 2 diabetic patients. With the improvement of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C07K14/605A61K38/26A61P3/10A61P3/04A61P9/10A61P25/28
Inventor 姚文兵高明明田浤高向东
Owner CHINA PHARM UNIV
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