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
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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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