Reformed lyophylization preparation of recombinant staphylokinase (r-Sak), its preparing method and application

Abstract

A freeze-dried preparation of recombinant glucokinase in the form free dried powder, injection, lipoplasm, or microcapsule for thrombolytic purpose contains the glucokinase with mutation of amino acids at positions 7, 3 and 43, and one or more of mannitol, phosphate, EDTA and sodium chloride. Its preparing process is also disclosed.

Description

technical field

[0001] The invention relates to a recombinant staphylokinase freeze-dried preparation, its preparation method and application as a thrombolytic drug, belonging to the technical field of biopharmaceuticals. Background technique

[0002] Cardiovascular and cerebrovascular diseases are currently the number one killer that endangers people's health, and thrombotic diseases such as acute myocardial infarction are the main causes of death of patients. Although there are some commercially available thrombolytic drugs in clinical practice, there are still many problems to be solved urgently in terms of their thrombolytic effect, specificity of thrombolysis and drug price. Staphylokinase (Staphylokinase, referred to as SAK) is a protein produced by Staphylococcus aureus with a molecular weight of 15.5KD. It can specifically bind to plasminogen, form a complex, and activate plasminogen to become Plasmin specifically degrades fibrin and dissolves thrombus. According t...

Examples

Embodiment Construction

[0025] The technical content of the present invention is described in detail below in conjunction with embodiment:

[0026] (1), construction of recombinant staphylokinase engineering bacteria:

[0027] The present invention chooses to use Staphylococcus aureus 1697 (taken from the strain preservation center of Institute of Microbiology, Chinese Academy of Sciences) to carry out the isolation of chromosomal DNA, and takes 0.5ml of Staphylococcus aureus 1697 to inoculate in 100ml of LB culture medium, shake at 37°C Cultivate overnight, collect the cells by centrifugation at 7000rpm at 4°C for 8 minutes, wash the cells once with 20ml TE, resuspend the cells in 20ml TE, add 120μl proteinase K and mix well, add 2.2ml 10% SDS and mix well, incubate at 37°C for 1 hour, Extract once with an equal volume of phenol (mix upside down for 1 minute), use 0.1 volume of 3M NaAc (pH5.2), and an equal volume of isopropanol, precipitate for 2 hours, centrifuge at 12000 rpm at 4 ° C for 10 minut...

technical field

[0001] The invention relates to a recombinant staphylokinase freeze-dried preparation, its preparation method and application as a thrombolytic drug, belonging to the technical field of biopharmaceuticals. Background technique

[0002] Cardiovascular and cerebrovascular diseases are currently the number one killer that endangers people's health, and thrombotic diseases such as acute myocardial infarction are the main causes of death of patients. Although there are some commercially available thrombolytic drugs in clinical practice, there are still many problems to be solved urgently in terms of their thrombolytic effect, specificity of thrombolysis and drug price. Staphylokinase (Staphylokinase, referred to as SAK) is a protein produced by Staphylococcus aureus with a molecular weight of 15.5KD. It can specifically bind to plasminogen, form a complex, and activate plasminogen to become Plasmin specifically degrades fibrin and dissolves thrombus. According t...

Examples

Embodiment Construction

[0025] The technical content of the present invention is described in detail below in conjunction with embodiment:

[0026] (1), construction of recombinant staphylokinase engineering bacteria:

[0027] The present invention chooses to use Staphylococcus aureus 1697 (taken from the strain preservation center of Institute of Microbiology, Chinese Academy of Sciences) to carry out the isolation of chromosomal DNA, and takes 0.5ml of Staphylococcus aureus 1697 to inoculate in 100ml of LB culture medium, shake at 37°C Cultivate overnight, collect the cells by centrifugation at 7000rpm at 4°C for 8 minutes, wash the cells once with 20ml TE, resuspend the cells in 20ml TE, add 120μl proteinase K and mix well, add 2.2ml 10% SDS and mix well, incubate at 37°C for 1 hour, Extract once with an equal volume of phenol (mix upside down for 1 minute), use 0.1 volume of 3M NaAc (pH5.2), and an equal volume of isopropanol, precipitate for 2 hours, centrifuge at 12000 rpm at 4 ° C for 10 minut...