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Mutant of recombined glucokinase for anti blood platelet collecting and low immunogencity

An anti-platelet aggregation and immunogenicity technology, applied in the biological field, can solve the problems of decreased immunogenicity, fibrinolytic activity and specificity, and achieve low immunogenicity, anti-platelet aggregation immunogenicity, and simple preparation process Effect

Inactive Publication Date: 2006-11-29
FUDAN UNIV
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Problems solved by technology

After mutating the key amino acids of these two antigenic determinants, the immunogenicity of the mutant was significantly reduced, but its fibrinolytic activity and specificity also decreased (Collen D et al. Circulation.94.197-206. (1996); Collen D et al. Circulation. 94.207-216. (1996))

Method used

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Embodiment 1

[0015] 1. Rationale Design of Mutants

[0016] The loop region of Sak 1(D 33 G 34 K 35 G 36 ) link β sheet 1 (G 21 -D 32 ) and β sheet 2 (N 37 -I 49 ), exposed to the molecular surface of Sak. The RGD sequence is constructed in this loop region, and it is expected to combine with platelet surface membrane receptor GPIIb / IIIa to prevent platelet aggregation. At the same time, this region is far away from the active center of Sak, and the change of its conformation has little effect on the fibrinolytic activity. Furthermore, this region is the region where Sak antigen epitope 3 is located, and mutations may lead to a conformational change in this region, thereby reducing its immunogenicity. Therefore, the present invention designs a mutant molecule RL1, which mutates loop region 1 into D 33 G 34 R 35 G 36 d. Based on the hypothesis that the recognition of RGD by the platelet surface membrane receptor GPIIb / IIIa depends on the spatial conformation rather than the pr...

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Abstract

The invention relates to an anti-bloodpatelet aggregation, low-immunogenicity recombinant staphylokinase mutant and its preparing method. The invention analyses the structures of recombinant staphylokinase monomer and micro-lumbrokinase-staphylokinase-micro- lumbrokinase compound crystal, and designs a novel molecular structure of staphylokinase mutant, which is recombined with a prokaryotic expression carrier pLY-4 after constructing mutant gene by using PCR fixed-point mutation, converting Escherichia coli, and screening high-expression engineering bacteria; by fermenting and amplifying, crushing the bacteria, centrifuging and collecting, then making two-step purification, and freeze-drying and obtaining the product, which basically maintains fibrinolytic activity of wild staphylokinase, can inhibit the bloodpatelet aggregation and has remarkable function of preventing and curing blood acid and its immunogenicity is remarkably reduced.

Description

technical field [0001] The invention belongs to the field of biological technology, and in particular relates to a recombinant staphylokinase mutant with anti-platelet aggregation and low immunogenicity and its preparation method and application. Background technique [0002] Natural staphylokinase (Staphylokinase, Sak) is a proteolytic enzyme synthesized by Staphylococcus aureus lysogenic phage, consisting of 136 amino acids. Sak itself is not an enzyme, it forms a 1:1 complex with plasminogen (plg) in human plasma, and this complex is activated to Sak plm by trace amounts of plasmin (plm) on the surface of blood clots, Sak·plm can efficiently activate plg in plasma to form plm, and plm catalyzes the degradation of fibrin, the main matrix of thrombus, thereby dissolving thrombus. In plasma, Sak·plm is quickly inhibited by α2-antiplasmin, and when plg binds to fibrin through the lysine binding site, the inhibition speed of α2-antiplasmin on Sak·plm will decrease by 100 tim...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/70C12N15/58A61K38/43A61P7/02C12N9/00C12P21/00
Inventor 宋后燕苏华波
Owner FUDAN UNIV
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