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Preparation method for high purity lumbrukinase and pharmaceutical preparation made therefrom

A lumbrokinase, high-purity technology, applied in the field of preparation of high-purity lumbrokinase

Inactive Publication Date: 2005-04-06
BEIJING SAISHENG PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the past 20 years, researchers in many units have been trying to push the research of lumbrokinase to practical application, and various separation and purification techniques have come out successively, but up to now, there is no mature set of methods for preparing high-purity lumbrokinase. Kinase technology truly translates to production

Method used

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  • Preparation method for high purity lumbrukinase and pharmaceutical preparation made therefrom
  • Preparation method for high purity lumbrukinase and pharmaceutical preparation made therefrom
  • Preparation method for high purity lumbrukinase and pharmaceutical preparation made therefrom

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Lumbrokinase 50 μg / ml purified by high performance liquid chromatography and mixed with an equal volume of complete adjuvant and emulsified as a stimulus, immunized 7-week-old BALB / c mice subcutaneously, 0.2ml each time, once a week , a total of 6 times of immunization. Three days before the fusion, 0.2ml stimulator mice were injected into the tail vein for booster immunization.

[0043] Take the immunized mouse splenic B lymphocyte suspension and mix it with mouse sp2 / 10 myeloma cells at a ratio of 5:1, add 50% polyethylene glycol with a molecular weight of 40000, and inoculate the cell mixture in multiple 96 wells in the wells of the cell culture plate.

[0044] Place the cell culture plate inoculated with the cell mixture in CO 2 Cultured in an incubator. The HAT medium containing 15% fetal bovine serum was used for the 1st to 4th day, the HT medium was used for the 5th to 10th day, and the RPM1-1640 medium containing 10% fetal bovine serum was used for cultivatio...

Embodiment 2

[0047] Lumbrokinase 250 μg / ml purified by high-performance liquid chromatography and emulsified with an equal volume of complete adjuvant was mixed and emulsified as a stimulus, and immunized by subcutaneous injection to 7-week-old BALB / c mice, 0.2ml each time, once a week , a total of 6 times of immunization. Three days before the fusion, 0.2ml stimulator mice were injected into the tail vein for booster immunization.

[0048] Take the immunized mouse splenic B lymphocyte suspension and mix the mouse sp2 / 10 myeloma cells at a ratio of 8:1, add 50% polyethylene glycol with a molecular weight of 50000, and inoculate the cell mixture in multiple 96 wells in the wells of the cell culture plate.

[0049] Place the cell culture plate inoculated with the cell mixture in CO 2 Cultured in an incubator. The HAT medium containing 15% fetal bovine serum was used for the 1st to 4th day, the HT medium was used for the 5th to 10th day, and the RPM1-1640 medium containing 10% fetal bovine...

Embodiment 3

[0052] Lumbrokinase 500μg / ml purified by high performance liquid chromatography and emulsified with an equal volume of complete adjuvant was emulsified as a stimulus, and immunized by subcutaneous injection of 0.2ml each time to 7-week-old BALB / c mice, once a week , a total of 6 times of immunization. Three days before the fusion, 0.2ml stimulator mice were injected into the tail vein for booster immunization.

[0053] Take the immunized mouse splenic B lymphocyte suspension and mix it with mouse sp2 / 10 myeloma cells at a ratio of 10:1, add 50% polyethylene glycol with a molecular weight of 60000, and inoculate the cell mixture in multiple 96 wells in the wells of the cell culture plate.

[0054] Place the cell culture plate inoculated with the cell mixture in CO 2 Cultured in an incubator. The HAT medium containing 15% fetal bovine serum was used for the 1st to 4th day, the HT medium was used for the 5th to 10th day, and the RPM1-1640 medium containing 10% fetal bovine ser...

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Abstract

The invention discloses a high-purity earthworm activating enzyme preparation method and take it as raw material preparation. The invention through uses the monoclonal antibody technology realization earthworm activating enzyme, this method characteristic is attains the enzyme the shift strongly, the activeness is high, achieves above each milligram protein 200,000 units; After the highly effective liquid chromatography test is the sole component; Swims in the Sds- polyacrylamide gelatin electricity presents a belt, the molecular weight is 32000+ / -2000 Dalton; The amino acid sequence analysis, the N- terminal 10 amino acids sequences are: He-Val-Gly-Gly-He-Glu-Ala-Arg-Pro-Tyr, take states the high-purity earthworm activating enzyme to be allowed to make as the raw material medicine may supply to take orally, intermuscular injection, as well as the intravenous injection and the venous transfusion many kinds of ways for the medicine preparation, uses in the thrombus disease treatment.

Description

technical field [0001] The invention relates to a preparation method of lumbrokinase, more specifically, to a preparation method of high-purity lumbrokinase and pharmaceutical preparations prepared therefrom in various dosage forms for oral administration, intramuscular injection and intravenous administration. Background technique [0002] The high morbidity, high mortality and high disability of thrombotic diseases seriously threaten the survival of human beings, and thrombolytic drugs are the first choice for the treatment of this disease. At present, there are common side effects that easily cause bleeding in the thrombolytic drugs used clinically; some are expensive and unbearable for ordinary people; some have poor stability and require high storage conditions, which brings a lot of inconvenience to drug users; The kind of drug administration mode is single, only has oral, or only has intravenous or intramuscular injection, and the scope of use medicine is very limited...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/48A61P7/02C12N9/48
Inventor 马骉魏化伟吴丹宋梦薇张颖
Owner BEIJING SAISHENG PHARMA
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