Novel Recombinant Staphylokinase Derivatives and the Preparations and Applications thereof

Abstract

The present invention relates to the biotechnology field, more particularly, to novel recombinant staphylokinase (RGD/KGD-Sak) derivatives and the preparation the thereof. The derivatives, have a low polymerizing ability, low immunogenicity and a bifunctionality of thrombolytics and anticoagulant. Based on the line, structural analysis of the monomer and dimer of recombinant staphylokinases and their biochemical properties, we designed two novel bifunctional staphylokinase molecular structures. Mutant genes were constructed by PCR site-directed mutagenesis which were then recombined with a prokaryotic vector and used to transform E. coli . Engineered strains with a high expression level were selected by screening and propagated by fermentation, followed by disruption of the cells, centrifugation to collect inclusion bodies, renaturation, and purification of RGD/KGD-SAK through a two-step method. After lyophilized, the polymerizing ability and immunogenicity of the products decreased significantly. The derivatives can not only activate fibrinogen to lyse thrombus, but also significantly inhibit the platelet aggregation induced by ADP, suggesting that they have the bifunctionality of thrombolytics and anticoagulant.

1 - 15 . (canceled) 
     
     
         16 . A method for preparing a recombinant staphylokinase derivative with bifunctionality of thrombolytics and anticoagulant said method comprising the following steps: a) Preparing a DNA fragment comprising, at least the coding sequence of a bioactive staphylokinase; b) Performing in vitro site-directed mutagenesis on the DNA fragment to substitute one or more codons for in that one or more amino acid residues between amino acid residue 104 and 113 (HR2) of the wild-type staphylokinase is substituted with other amino acids, resulting in an RGD sequence or KGD sequence in said derivative, and thus its polymerizing ability and cellular and humoral immunogenicity was significantly decreased as compared with the wild-type staphylokinase; c) Cloning the mutated DNA fragment into a suitable vector; d) Transforming or transfecting a suitable host cell with the recombinant vector; and e) Culturing the host cell under conditions suitable for the expression of the DNA fragment; and f) recovering and purifying the desired staphylokinase derivative 
     
     
         17 . The method according to  claim 16 , wherein the site-directed mutagenesis is carried out by three rounds of PCR amplification with pST-Sak as template, using forward-primer, mutating-primer and backward-primer as primers. 
     
     
         18 . The method according to  claim 16 , wherein the vector used is a prokaryotic expression vector pLY-4. 
     
     
         19 . The method according to  claim 16 , wherein the host cell used is  E. coli  K802. 
     
     
         20 . The method according to  claim 16 , wherein the host Cells are cultured at 30-42° C. at a pH of about 6-8, with stirring speed decreasing as the OD increasing. 
     
     
         21 . The method according to  claim 16 , wherein the recombinant staphylokinase final product is obtained by disrupting the cell by high pressure after fermentation, collecting the inclusion bodies by centrifugation, and then isolating and purifying in a two-step way. 
     
     
         22 . A DNA construct, comprising the nucleotide sequence coding for a staphylokinase derivative in that one or more amino acid residues between amino acid residue 104 and 113 (HR2) of the wild-type staphylokinase is substituted with other amino acids, resulting in an RGD sequence or KGD sequence in said derivative, and thus its polymerizing ability and cellular and humoral immunogenicity was significantly decreased as compared with the wild-type staphylokinase. 
     
     
         23 . The DNA construct according to  claim 22  comprising the nucleotide sequence set forth in SEQ ID NO: 2. 
     
     
         24 . The DNA construct according to  claim 22  comprising the nucleotide sequence set forth in SEQ ID NO 4. 
     
     
         25 . A recombinant vector, comprising the DNA construct according to  claim 22 . 
     
     
         26 . A host cell, containing the recombinant vector according to  claim 25 . 
     
     
         27 . A pharmaceutical composition, comprising a therapeutic effective amount of the staphylokinase derivative in that one or more amino acid residues between amino acid residue 104 and 113 (HR2) of the wild-type staphylokinase is substituted with other amino acids, resulting in an RGD sequence or KGD sequence in said derivative, and thus its polymerizing ability and cellular and humoral immunogenicity was significantly decreased as compared with the wild-type staphylokinase and a pharmacological acceptable vehicle. 
     
     
         28 . A method for the treatment of arterial thrombosis, intraocular hematocele and oozing of blood, comprising administrating the pharmaceutical composition according to  claim 27  to a patient.