Application of tachyplesin in preparing antihrombotic medicines and health-care products and preparation method thereof
The technology of an antithrombotic drug and a limulus peptide is applied to the application of the limulus peptide in the preparation of antithrombotic drugs and health care products and the field of preparation thereof, and can solve the problem of not finding the limulus peptide and the like.
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Embodiment 1
[0052] The lysate of Limulus hemocytes prepares the process steps of Limulus peptide as follows:
[0053] (1) Extract fresh Limulus blood with sterile non-pyrogenic equipment, centrifuge at 1000 rpm for 5 minutes, lyse the cells with 20 mmol / L HCl, break up the sticky cell clumps with a homogenizer, and obtain the crude extract.
[0054] (2) The crude liquid was centrifuged at 8000 rpm for 15 minutes, and the supernatant was collected.
[0055](3) Filter the supernatant with three layers of gauze, separate it with dextran G-50 (Sephadex G-50) (1.5cm×90cm), and elute with 20mmol / L HCl solution at a flow rate of 1.0mL / min. There are three groups of peaks, and the eluted product of the third group is collected, the solution is adjusted to pH 6.0-7.0, centrifuged at 10,000 rpm for 15 minutes, and the supernatant is freeze-dried and stored. It was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) that it was a single electrophoresis band with a mole...
Embodiment 2
[0057] The process steps of preparing limulus peptide from the remaining waste in the production of Limulus reagent are as follows:
[0058] (1) Collect the cell lysate aggregates produced during the production of LAL, add 20mmol / L HCl solution, and use a homogenizer to break up the sticky cell aggregates to obtain the crude extract.
[0059] (2) The crude liquid was centrifuged at 8000 rpm for 15 minutes, and the supernatant was collected.
[0060] (3) Filter the supernatant with three layers of gauze, separate it with dextran G-50 (Sephadex G-50) (1.5cm×90cm), and elute with 20mmol / L HCl solution at a flow rate of 1.0mL / min. There are three groups of peaks, and the eluted product of the third group is collected, the solution is adjusted to pH 6.0-7.0, centrifuged at 10,000 rpm for 15 minutes, and the supernatant is freeze-dried and stored. It was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) that it was a single electrophoresis band with ...
Embodiment 3
[0062] The process steps for preparing limulus peptide from unqualified limulus reagent raw materials are as follows:
[0063] (1) Add 20mmol / L HCl solution to the unqualified Limulus reagent raw material, and adjust the pH value of the solution to 3.
[0064] (2) The crude liquid was centrifuged at 8000 rpm for 15 minutes, and the supernatant was collected.
[0065] (3) Filter the supernatant with three layers of gauze, separate it with dextran G-50 (Sephadex G-50) (1.5cm×90cm), and elute with 20mmol / L HCl solution at a flow rate of 1.0mL / min. There are three groups of peaks, and the eluted product of the third group is collected, the solution is adjusted to pH 6.0-7.0, centrifuged at 10,000 rpm for 15 minutes, and the supernatant is freeze-dried and stored. It was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) that it was a single electrophoresis band with a molecular weight of about 2KDa.
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