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Low antigen recombination glucokinase mutant and its preparation method

A technology with low antigenicity and mutants, applied in the field of mutation or genetic engineering, can solve the problems of curative effect impact, limit the application of prevention or recovery treatment, etc., and achieve the effect of widening application range, good thrombolytic activity and huge economic benefits

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

AI Technical Summary

Problems solved by technology

However, like many foreign proteins with special functions, SAK also causes some immune reactions in clinical applications, especially when it is used repeatedly, the neutralizing antibodies it induces have a great impact on the curative effect
At the same time, the immunogenicity of SAK limits the application of daily prophylaxis or recovery therapy

Method used

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  • Low antigen recombination glucokinase mutant and its preparation method
  • Low antigen recombination glucokinase mutant and its preparation method
  • Low antigen recombination glucokinase mutant and its preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0050] Activity assay of recombinant SAK mutants

[0051] 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.

[0052] Dilution of standards and samples to be tested: standard dilutions are performed as follows:

[0053] Tube No. 1 2 3 4 5

[0054] Normal saline (μl) 500 500 500 500

[0055] Reference solution (μl) 1000 500 500 500 500

[0056] Reference product activity (AU / ml) 250 125 62.5 31.25 15.625

[0057] The sample to be tested was diluted to a concentration of 5 μg / ml according to the labeled amount, ready for use.

[0058] Punching, spotti...

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Abstract

This invention belongs to mutation or genetic engineering field, which discloses a preparation of low antigen mutation of staphylokinase and its usage. The front six amino acids of SAK have nothing with thrombolytic activity, but it can influence their stability in some prokaryotic cells. Meanwhile, the segment where 35,74,80,82,130,135 and 136 amino acid exist has lots of B-lymphocyte epi-position, therefore, removing the front six amino acids of SAK, constructing N-terminal deletion, SAK mutant of the intermediate and C-terminal segments multi-point modified by the methods of fragments missing, point mutation and so on. Although there is no significant change with thrombolytic activity of the mutant, antigenicity of the mutant decreases significantly. The mutant of this invention fits for expression and the preparation in the prokaryotic cell, bringing the convenience for industry. Its biological activity has no significant difference compared with wild type, so the mutant can apply for dissolving internal clot, and its characteristic of decreasing antigenicity degradation may make it possible for application many times.

Description

technical field [0001] The invention belongs to the field of mutation or genetic engineering. Background technique [0002] Myocardial infarction is a great hazard to human health, and the mortality rate is very high. Thrombolytic therapy is one of the main means of clinical emergency and routine treatment. Studies have shown that among numerous thrombolytic drugs, staphylokinase (SAK) is currently the only new thrombolytic drug comparable to tPA in terms of thrombolytic effect and specificity. Natural SAK is a single-chain protein synthesized by lysogenic phage of Staphylococcus aureus, consisting of 136 amino acids with a molecular weight of 15.5kDa. In the 1990s, the fibrin selectivity of SAK was discovered, which attracted widespread attention. Domestic units have obtained new drug certificates, and due to the use of prokaryotic cell fermentation and purification production, its market price will be relatively low, so it has a certain competitive advantage in clinical ...

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王园园王嘉玺
Owner INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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