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Pegylation modification of staphylokinase epitope and application

A technology of PEGylation and polyethylene glycol, which is applied in the field of genetic engineering, can solve problems such as restrictions on use, and achieve the effect of reducing immunogenicity and retaining thrombolytic activity

Inactive Publication Date: 2017-03-22
CHONGQING MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] As an excellent thrombolytic agent, SAK has the disadvantage of rapidly inducing high-titer antibodies after entering the body, which greatly limits its clinical use.

Method used

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  • Pegylation modification of staphylokinase epitope and application
  • Pegylation modification of staphylokinase epitope and application
  • Pegylation modification of staphylokinase epitope and application

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preparation example Construction

[0031] Two, the preparation method of pegylated staphylokinase

[0032] The preparation method of pegylated staphylokinase of the present invention comprises:

[0033] (1) Provide mutant staphylokinase;

[0034] (2) Reacting the mutant staphylokinase with maleimide-modified polyethylene glycol to obtain pegylated staphylokinase.

[0035] In a preferred embodiment of the present invention, in step (1), the amino acid sequence of the mutant staphylokinase is shown in SEQ ID NO.6.

[0036] Step (1) includes: constructing an expression vector containing the mutant staphylokinase gene sequence, then transforming the expression vector containing the mutant staphylokinase gene sequence into a host cell to induce expression, and isolating the mutant staphylokinase from the expression product. In a preferred embodiment of the present invention, the mutant staphylokinase gene sequence is shown in SEQ ID NO.5.

[0037] In the above preparation method, when the thiol and the C═C double...

Embodiment 1

[0051] Expression and purification of embodiment 1 recombinant staphylokinase

[0052] 1. Amplification of target gene and construction of mutant plasmid

[0053] 1.1 Primer synthesis

[0054] Mutation primers for SAK were designed using the software Premier 5. The primers used the full-length SAK-PET28a as a template, and bidirectionally amplified around the 82nd amino acid site (Table 1) to obtain the mutant SAK-PET28a plasmid sequence. Primers were synthesized by Beijing Huada Gene Technology Co., Ltd.

[0055] Table 1 Mutation primer sequence

[0056]

[0057] 1.2 Amplification of target gene fragments

[0058] SAK-cys-82-F and SAK-cys-82-R are SAK forward and reverse primers, and the underlined part in SAK-cys-82-F and SAK-cys-82-R is the cysteine ​​mutation site of SAK point. The wild-type SAK template was amplified by PCR with primers, and the reaction conditions were: 94°C pre-denaturation for 5 minutes, 94°C denaturation for 30 seconds, 57°C annealing for 1 m...

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Abstract

The invention belongs to the technical field of genetic engineering and particularly relates to pegylated staphylokinase and preparation and application thereof. The pegylated staphylokinase structurally comprises polyethylene glycol and mutated staphylokinase which are coupled with each other. Compared with current similar products, the pegylated staphylokinase has the advantages that the immunogenicity of natural staphylokinase is effectively reduced, and sufficient thrombolytic activity is maintained.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to pegylated staphylokinase and its preparation and application. Background technique [0002] Natural staphylokinase (staphylokinase, SAK) is an extracellular protein synthesized by lysogenic phage of Staphylococcus aureus. It consists of 136 amino acid residues and has a molecular weight of 15.5KD. Staphylokinase itself has no protease activity and is an "indirect" plasminogen activator, which specifically binds to plasminogen (Flg) at a ratio of 1:1 to form an inactive SAK-Flg complex Under the action of trace plasmin (Flm) on the thrombus surface, it is further converted into an active SAK-Flm complex, which catalyzes another molecule of plasminogen to form plasmin, thereby activating the fibrinolytic system. AK has the advantages of certain fibrin specificity, will not deplete plasminogen, and has a significant effect on platelet-based white thrombus, m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/49A61K47/59A61P7/02C12N9/64
CPCA61K38/49C12N9/6456C12Y304/21
Inventor 周建中汪德强徐彦颖白垒师悦嫄雷寒黄爱龙何泉
Owner CHONGQING MEDICAL UNIVERSITY
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