Thrombin activity based chimeric peptide with double targeting effects and application thereof

A chimeric polypeptide and dual-targeting technology is applied in the field of protein and polypeptide preparation to achieve the effects of strong pertinence, reducing the number of medications and prolonging the half-life

Inactive Publication Date: 2012-05-09
GUANGZHOU DONGLAI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the two polypeptides of A-box and Exendin-4 are connected with a sequence containing a thrombin cleavage site to form a chimeric polypeptide, so that it can be rapidly decomposed into A-box and Exendin in patients with increased thrombin activity. -4 Two kinds of polypeptides, respectively play the functions of anti-inflammation and lowering blood sugar, and achieve the effect of dual-target therapy. This kind of chimeric polypeptide has not been reported at home and abroad.

Method used

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  • Thrombin activity based chimeric peptide with double targeting effects and application thereof
  • Thrombin activity based chimeric peptide with double targeting effects and application thereof
  • Thrombin activity based chimeric peptide with double targeting effects and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] 1. Preparation of chimeric polypeptide of the present invention

[0038] according to figure 2 Arrangement design, using genetic engineering technology to generate a chimeric peptide sequence consisting of 83 amino acids at the N-terminal of human HMGB1, a connecting fragment of 5 amino acids, and Exendin-4, the sequence is as follows:

[0039] MGKGDPKKPRGKMSSYAFFVQTCREEHKKKHPDASVNFSEFSKKCSERWKTMSAKEKGKFEDMAKADKARYEREMKTYIPPKG VPRGP HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS (SEQ ID No: 3)

[0040] In the above sequence, the underlined part is the connection segment, and the P behind the G in the connection segment can also be A, S, V, L.

[0041] The specific steps for preparing the above sequence are as follows:

[0042] (1) Obtain the DNA sequence of 83 amino acids at the N-terminal of human HMGB1 and 5 amino acids containing thrombin and dipeptidase cleavage sites by PCR method or chemical synthesis method;

[0043] (2) Obtain the DNA sequence of the connecting f...

Embodiment 2

[0055] according to figure 2 According to the arrangement design of human HMGB1, a chimeric polypeptide consisting of 60 amino acids at the N-terminal of human HMGB1, a connecting fragment of 6 amino acids, and Exendin-4 was synthesized by chemical synthesis, and then purified and prepared according to conventional methods. drug ingredients.

[0056] The sequence of the chimeric polypeptide in this example is:

[0057] MGKGDPKKPRGKMSSYAFFVQTCREEHKKKHPDASVNFSEFSKKCSERWKTMSAKEKGKF LVPRGP HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS (SEQ ID No: 4)

[0058] In the above sequence, the part shown by the underline is the connection segment, L and V in the connection segment can also be A, I, M, F, P, W, etc., and the P after the G in the connection segment can also be A, S, V, L.

Embodiment 3

[0060] according to figure 2 According to the arrangement design of human HMGB1, a chimeric polypeptide composed of 40 amino acids at the N-terminal of human HMGB1, 12 amino acid connecting fragments, and Exendin-4 was synthesized by chemical synthesis, and then purified and prepared according to conventional methods. drug ingredients.

[0061] The sequence of the chimeric polypeptide in this example is:

[0062] MGKGDPKKPRGKMSSYAFFVQTCREEHKKKHPDASVNFSE LVPRGP GVPRGP HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS (SEQ ID No: 5)

[0063] In the above sequence, the part shown by the underline is the connection segment, L and V in the connection segment can also be A, I, M, F, P, W, etc., and the P after the G in the connection segment can also be A, S, V, L.

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Abstract

The invention provides a thrombin activity based chimeric peptide with double targeting effects, a preparation method and an application thereof. The chimeric peptide provided by the invention contains three segments: segment 1 has an amino acid sequence having 20th to 90th amino acid from N-terminal of HMGB1 protein; segment 2 has an amino acid sequence of Exendin-4 protein; and segment 3 which contains cleavage sites of thrombin and dipeptidyl peptidase is a junction fragment of segment 1 and segment 2. According to the preparation method provided by the invention, the thrombin activity based chimeric peptide with double targeting effects can be obtained by synthesis of a polypeptide synthesis technology or construction of an expression vector which contains nucleotide sequence encoding the chimeric peptide, expression of the protein in a gene engineering strain and purification of the protein. The chimeric peptide provided by the invention has double targeting effects of inhibiting inflammatory response and decreasing blood glucose, and can be applied in the preparation of anti-inflammatory and/or hypoglycemic drugs.

Description

technical field [0001] The present invention relates to the preparation and application of protein polypeptides, more specifically, the present invention relates to a dual-targeted thrombin activity-based antimicrobial agent comprising the N-terminal amino acid sequence of HMGB1 (high mobility group protein 1) and the amino acid sequence of Exendin-4. Functional chimeric polypeptides and their applications. Background technique [0002] High mobility group box 1 (HMGB1) is widely distributed in lymphoid tissues and nucleated cells in brain, liver, lung, heart, spleen, kidney and other tissues. In the cells of liver and brain tissue, HMGB1 mainly exists in the cytoplasm, but in most other tissue cells, it mainly exists in the nucleus, and its amino acid sequence is highly conserved during evolution. [0003] The human HMGB1 gene is located on chromosome 13q12, including 5 exons and 4 introns, encoding a 215 amino acid sequence (SEQ ID No: 1, see GenBank CAG33144.1). The HMG...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/63A61K38/17A61K38/22A61P29/00A61P3/10
Inventor 刘誉
Owner GUANGZHOU DONGLAI BIOTECH CO LTD
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