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Recombinant L-asparaginase and preparation method and use thereof

A technology of asparaginase and DNA sequence, applied in biochemical equipment and methods, recombinant DNA technology, enzymes, etc., can solve the problem of side effects and short half-life of drugs

Pending Publication Date: 2020-01-14
南京吉瑞康生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

L-asp has been on the market for more than 50 years, but its side effects and short drug half-life are still important factors that limit its clinical use

Method used

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  • Recombinant L-asparaginase and preparation method and use thereof
  • Recombinant L-asparaginase and preparation method and use thereof
  • Recombinant L-asparaginase and preparation method and use thereof

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Experimental program
Comparison scheme
Effect test

Embodiment

[0014] Example Expression and purification, property determination, activity research and in vitro safety research of recombinant L-asp

[0015] 1 Materials and methods

[0016] 1.1.1 Cells, plasmids and strains

[0017] The prokaryotic expression vector pET-22b, Escherichia coli clone strain Top10, and Escherichia coli expression strain BL21(DE3) are all preserved by our laboratory. Jurkat and HBE cells were purchased from the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, and Raji cells were provided by the Blood Center of Soochow University.

[0018] 1.1.2 Reagents

[0019] Tsi High-Fidelity DNA polymerase was purchased from Tianjin Jinbeisi Biotechnology Co., Ltd.; Tsi 4 Ligase was purchased from American Thermo Company; restriction endonucleases NdeI and XhoI were purchased from American Thermo Company; plasmid mini-extraction kit was purchased from Shanghai Jierui Bioengineering Co., Ltd.; DNA gel ...

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Abstract

The present invention relates to the fields of indications for biopharmaceuticals, biochemistry and drugs, and particularly relates to a preparation method of recombinant L-asparaginase (recombinant L-asp) and an application of the recombinant L-asp in preparing anti-leukemia drugs. The L-asparaginase purified by an improved pulse dilution method has a purity of 90.14%, a yield of 5.23 mg / L, and an enzyme specific activity of 201.21 IU / mg. Cell-level activity shows that IC50 of the L-asparaginase on Jurkat and Raji cells is 4.15 IU / mL and 4.65 IU / mL respectively after 48 h; and the IC50 of theL-asp on the Jurkat and Raji cells is 2.40 IU / mL and 2.01 IU / mL respectively after 72 h. Based on the above results, the efficient preparation method for the recombinant L-asp and an application of the recombinant L-asp in preparation of anti-acute leukemia drugs are provided.

Description

technical field [0001] The invention relates to the fields of biopharmaceuticals, biochemistry and pharmacology, in particular to recombinant L-asparaginase and its preparation method and application. Background technique [0002] Recombinant L-asparaginase (L-asp) is the most effective drug for the treatment of acute leukemia (ALL) at home and abroad. The drug treatment remission rate is above 50%, and the drug remission period is 1 to 9 months. Cell leukemia and acute monocytic leukemia also have a certain effect. At the same time, L-asp has the characteristics of short remission period and easy tolerance, so most of it will be used in combination with other anticancer drugs in the course of treatment. L-asp has been on the market for more than 50 years, but its side effects and short drug half-life are still important factors that limit its clinical use. Human Serum Albumin (HSA) is the most abundant protein in the human body, and it is responsible for the storage and t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/82C12N15/70A61K38/50A61P35/02
CPCC12N9/82C12N15/70C12Y305/01001A61P35/02A61K38/00Y02A50/30
Inventor 华子春徐珊珊
Owner 南京吉瑞康生物科技有限公司
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