Low-immunogenicity staphylokinase mutant and preparation method and use thereof

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...

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Problems solved by technology

Further research found that the C3 region peptide contains two overlapping T cell epitope sequences, S1 (72-82) and S2 (75-85), among which the amino acids at positions Y73, F76, and L81 are the key amino acids of the epitope, and they are replaced by A , the binding ability of peptides S1 and S2 to HLA-DR decreases, but the activity of staphylokinase is lost; and R77 and E80 in the peptide are replaced by A, and the computer fitting results show that S1 and S2 interact with HLA-DR The energy is greatly reduced, but the activity is only maintained at about 50% of wild-type staphylokinase (Petra A M et al.Thromo Heamonst, 2002; 87(4):666-673)

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