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Staphylokinase and expression vector thereof

A technology for expressing vectors and staphylokinase, which is applied in the field of biomedicine to achieve the effects of high expression, strong thrombolytic effect, and high activity

Active Publication Date: 2011-04-20
INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently commonly used clinical thrombolytic drugs include urokinase (UK), streptokinase (SK), recombinant tissue plasminogen activator (r-tPA), but all have their limitations in clinical application

Method used

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  • Staphylokinase and expression vector thereof
  • Staphylokinase and expression vector thereof
  • Staphylokinase and expression vector thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] The cloning of staphylokinase gene of the present invention and the construction of expression vector

[0040] (1), extraction of Staphylococcus aureus total DNA

[0041] Take 0.5ml of Staphylococcus aureus 1.1476 (purchased from the General Microbiology Center of China Microbiological Culture Collection Management Committee) to inoculate in 100ml LB medium, culture with shaking at 37°C overnight, then centrifuge at 4°C and 7000rpm for 8 minutes, Collect the bacteria, wash the bacteria once with 20ml TE, resuspend the bacteria with 20ml TE, add 120ul proteinase K to it, mix well, add 2.2ml 10% SDS, mix well, and incubate at 37°C for 1 hour, then Extract with an equal volume of phenol once (mix upside down for 1 minute); 0.1 volume of 3M NaAc (pH5.2), an equal volume of isopropanol, precipitate for 2 hours; centrifuge at 4°C, 12000rpm for 10 minutes, remove the supernatant , Wash the precipitate with 75% ethanol, then dissolve it in 200ul ddH2O, take 2ul sample for 1% a...

Embodiment 2

[0057] Staphylokinase thrombolytic activity test of the present invention

[0058] (1), preparation of fibrin plate Weigh 125mg of agarose (Biorad electrophoresis grade), add 23ml of normal saline, boil and dissolve, equilibrate in a 60°C water bath, add 14 μl of thrombin (100IU / ml) (purchased from the Institute for the Control of Biological Drugs), Plasminogen (purchased from the Institute for the Control of Biological Drugs) 280μl (0.5mg / ml), shake well while adding, add 2.2ml human fibrinogen (purchased from the Institute for the Control of Biological Drugs) (6mg / ml), shake well , Pour it into a flat plate (diameter 8cm) immediately after becoming turbid, place it horizontally in a refrigerator at 4°C for at least half an hour (should be used within two days), and set it aside after being fully solidified.

[0059] (2), standard substance and the dilution standard substance of sample to be tested are staphylokinase standard substance (purchased from Institute for the Contro...

Embodiment 3

[0066] Thrombolytic Effect Test of Staphylokinase of the Present Invention on Miniature Pig Coronary Thrombosis

[0067] (1) Surgical procedure and model preparation for coronary thrombosis in minipigs

[0068] Before the experiment, miniature pigs (Chinese experimental miniature pigs, provided by the China Experimental Miniature Pig Breeding Farm of China Agricultural University, male, weighing 20-30 kg) were fasted for 12 hours and had free access to water. On the day of the experiment, 3% sodium pentobarbital (20 mg kg -1) Mixed anesthesia with intramuscular injection of ketamine (10mg·kg-1), and intravenous injection of sodium pentobarbital to maintain anesthesia. Minipigs were fixed on the operating table in supine position, tracheal intubation, connected to SC-3 electric ventilator, and performed artificial positive pressure breathing; separated right common carotid artery, inserted a plastic tube filled with 0.5% heparin, and connected to TP-400T pressure On the transd...

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Abstract

The invention discloses a staphylokinase and an expression vector thereof. The staphylokinase is polypeptide with amino acid sequence shown in SEQ ID No: 1, and the gene coding sequence thereof is shown in SEQ ID No: 2. The invention also discloses an expression vector containing a staphylokinase gene. The thrombolysis of the staphylokinase has strong effect, so that the staphylokinase can be used for preparing medicine for remedying cardio-cerebrovascular diseases, in particular to medicine for remedying myocardial infarction and other thrombotic diseases; the thrombolysis activity of the staphylokinase is high, and the activity rate thereof is 4.0*10<4>AU / mg; in addition, the expression amount of the expression vector of the aphylokinase is high, and the expression amount of the aphylokinase accounts for more than 45 percent of total protein of thallus.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a staphylokinase with thrombolytic effect and an expression carrier thereof. Background technique [0002] Cardiovascular and cerebrovascular diseases seriously threaten human health and are the main fatal diseases. The formation of thrombus is the inducement of many cardiovascular and cerebrovascular diseases, and it is also the main cause of death and disability. Thrombolytic therapy is one of the main treatments for these diseases. Thrombolytic therapy has been a routine therapy for acute myocardial infarction since the 1980s. Currently, commonly used clinical thrombolytic drugs include urokinase (UK), streptokinase (SK), and recombinant tissue plasminogen activator (r-tPA), but all of them have limitations in clinical application. [0003] Natural staphylokinase (staphylokinase, SAK) is a single-chain protein synthesized by lysogenic Staphylococcus aureus (Staphylocc...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61P9/00A61P7/02C12N9/48C12N15/70A61P9/10A61K38/49C12N15/63C12N15/58C12R1/19C12N1/21
Inventor 邹民吉徐东刚王旻蔡欣
Owner INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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