Low-immunogenicity staphylokinase mutant and preparation method and use thereof

An immunogenicity and mutant technology, applied in the biological field, can solve the problems of energy reduction, binding ability reduction, staphylokinase activity loss, etc., to achieve the effect of reducing immunogenicity, reducing the level of antibodies, and reducing immunogenicity

An immunogenicity and mutant technology, applied in the biological field, can solve the problems of energy reduction, binding ability reduction, staphylokinase activity loss, etc., to achieve the effect of reducing immunogenicity, reducing the level of antibodies, and reducing immunogenicity

CN102108351BInactive Publication Date: 2013-04-03HEBEI NORMAL UNIV
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  • Low-immunogenicity staphylokinase mutant and preparation method and use thereof
  • Low-immunogenicity staphylokinase mutant and preparation method and use thereof
  • Low-immunogenicity staphylokinase mutant and preparation method and use thereof

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Embodiment

[0020] 1. Mutant design

[0021] The present invention selects R77 and E80 as mutation sites, and replaces them with other suitable amino acids, hoping to greatly reduce the immunogenicity of the constructed mutants while retaining the fibrinolytic activity of staphylokinase. The mutant Sak (R77N) designed by the present invention replaces R77 of wild-type Sak with N, or Sak (R77K) replaces R77 of wild-type Sak with K, and Sak (R77Q) replaces R77 of wild-type Sak with Q; Sak(E80A) replaces E80 of wild-type Sak with A, and Sak(E80S) replaces E80 of wild-type Sak with S; Sak(R77K / E80A) replaces R77 and E80 of wild-type Sak with K and A, Sak(R77K / E80S) is to replace R77 and E80 of wild-type Sak with K and S respectively, and Sak(R77N / E80A) is to replace R77 and E80 of wild-type Sak with N and A respectively, Sak( R77N / E80S) is to replace R77 and E80 of wild-type Sak with N and S respectively, Sak (R77Q / E80A) is to replace R77 and E80 of wild-type Sak with Q and A respectively, a...

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Abstract

The invention discloses a protein structure of a low-immunogenicity recombinant staphylokinase mutant and a preparation method thereof. The invention relates to the molecular structure of the low-immunogenicity recombinant staphylokinase mutant. The preparation method comprises: directly obtaining the expression plasmid of the recombinant staphylokinase mutant by a polymerase chain reaction (PCR)site-directed mutagenesis technique; transforming Escherichia coli; performing fermentation culture; inducing expression; breaking thalli; centrifuging; collecting supernate; obtaining pure protein by continuous three-step chromatographic purification; and subjecting the pure protein to freeze drying to obtain the product. The obtained staphylokinase mutant has much lower immunogenicity, retains fibrinolysis activity for dissolving thrombi and can be used in preparation of medicaments for treating thromboembolic diseases.

Description

technical field [0001] The invention relates to a staphylokinase mutant with significantly reduced immunogenicity, a preparation method and application thereof, and belongs to the field of biotechnology. Background technique [0002] Natural staphylokinase (staphylokinase, Sak) is a protein derived from Staphylococcus aureus, consisting of 136 amino acids. Sak is an "indirect" plasminogen activator, which cannot directly convert plasminogen (Plg) into plasmin (plasmin, Plm), but first binds to plasminogen in a 1:1 ratio. The inactive complex Sak·plg is formed, followed by the activation of a small amount of plasminogen, the active site of plasminogen is exposed, and the single-chain becomes double-chained plasmin, forming an active Sak·plm complex, the latter The plasminogen molecule is further activated to convert it into plasmin, which dissolves the thrombus matrix fibrin to dissolve the thrombus. In plasma, Sak·plm is quickly inhibited by α2-antiplasmin and does not act...

Claims

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Application Information

Patent Timeline
03 Apr 2013
Publication
CN102108351B
IPC
C12N9/68; C12N15/70; A61K38/49; A61P7/02; C12R1/19; C12R1/445
Inventors
贺进田; 徐瑞光