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High activity, low antigen glucokinase derivant and preparation method

A technology of low antigenicity and derivatives, applied in the field of mutation or genetic engineering, can solve the problems of curative effect impact, allergic reaction, etc., and achieve the effect of broadening the application range and ensuring safety

Active Publication Date: 2008-12-24
INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because it is a foreign protein, its antigenicity will cause some allergic reactions in clinical use, especially when it is used repeatedly, its antigenicity has a great impact on the curative effect

Method used

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  • High activity, low antigen glucokinase derivant and preparation method
  • High activity, low antigen glucokinase derivant and preparation method
  • High activity, low antigen glucokinase derivant and preparation method

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Experimental program
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Effect test

Embodiment Construction

[0056] Activity assay of recombinant SAK mutants

[0057] Weigh 125mg of agarose (Biorad electrophoresis grade), add 23ml of normal saline, boil to dissolve, equilibrate in a water bath at 60°C, add 14μl of thrombin (100IU / ml), 280μl of plasminogen (0.5mg / ml), add as you go Shake well, add 2.2ml of human fibrinogen (6mg / ml), shake constantly, pour it into a flat plate (diameter 8cm) immediately after cloudiness, place it horizontally in a refrigerator at 4°C for at least half an hour, and wait until it is fully solidified before use.

[0058] Dilution of standards and samples to be tested: standard dilutions are performed as follows:

[0059] Tube No. 1 2 3 4 5

[0060] Normal saline (μl) 500 500 500 500

[0061] Reference solution (μl) 1000 500 500 500 500

[0062] Reference product activity (AU / ml) 250 125 62.5 31.25 15.625

[0063] The sample to be tested was diluted to a concentration of 2 μg / ml according to the labeled amount, ready for use.

[0064] Punching, spotti...

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Abstract

The invention belongs to the field of mutation or genetic engineering, and discloses a preparation method and application of SAK derivatives with enhanced thrombolytic activity and reduced immunogenicity. Studies have found that after the deletion of the first 10 amino acids of SAK, its thrombolytic activity can basically remain unchanged, and the segments where amino acids 35, 74, 82, 130, 135 and 136 are located have multiple B lymphocyte epitopes, so The first 10 amino acids of SAK were deleted by fragment deletion and point mutation methods to construct SAK mutants with N-terminal deletion and modification of the above sites, and a lysine was added to the C-terminus of SAK molecule to construct SAK derivatives. The thrombolytic activity of the mutant was significantly improved. Due to the shielding of some antigenic epitopes in the SAK molecule, the antigenicity of the mutants was significantly reduced. The mutant of the present invention is suitable for expression and preparation in prokaryotic cells. Because of its high thrombolytic activity and low antigenicity, it is expected to be developed into a new drug for treating cardiovascular and cerebrovascular diseases.

Description

technical field [0001] The invention belongs to the field of mutation or genetic engineering. Background technique [0002] Cardiovascular and cerebrovascular diseases are currently the number one killer that endangers people's health, and acute myocardial infarction is the main cause of death of patients. Although there are some clinically available thrombolytic drugs, there are still many problems to be solved in terms of thrombolytic effect, specificity, side effects and drug price. Staphylokinase (Staphylokinase, SAK) is a protein produced by lysogenic Staphylococcus aureus. Its gene encodes a protein of 163 amino acids, which is processed to form a mature protein consisting of 136 amino acids with a molecular weight of about 15kDa , no disulfide bridges. SAK is an elliptical molecule whose crystal structure has been resolved. It can specifically combine with plasminogen to form a complex to activate plasminogen into plasmin, thereby specifically degrading fibrin and ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/57C12N15/58C12N9/50C12N9/68C12N15/09C07K1/14A61K38/48A61P9/00A61P7/00
Inventor 徐东刚王旻邹民吉蔡欣徐涛刘深
Owner INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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