阅读说明：本技术 重组l-门冬酰胺酶及其制备方法和用途 (Recombinant L-asparaginase and preparation method and application thereof ) 是由 华子春 徐珊珊 于 2018-07-06 设计创作，主要内容包括：本发明涉及生物制药、生物化学和药物的适应症领域,具体涉及重组L-门冬酰胺酶(重组L-asp)的制备方法和在制备抗白血病药物中的应用。本发明利用改良的脉冲稀释法纯化的L-门冬酰胺酶纯度为90.14％,产率为5.23mg/L,酶的比活性为201.21IU/mg。细胞水平的活性显示：作用48h后,L-门冬酰胺酶对Jurkat和Raji细胞的IC50分别为4.15IU/mL和4.65IU/mL；作用72h后,L-asp对Jurkat和Raji细胞的IC50分别为2.40IU/mL和2.01IU/mL。基于以上结果,本发明提供了一种高效的重组L-asp的制备方法并且也提供了重组L-asp在制备抗急性白血病药物中的应用。(The invention relates to the fields of biopharmaceutical, biochemical and pharmaceutical indications, in particular to a preparation method of recombinant L-asparaginase (recombinant L-asp) and application in preparing anti-leukemia drugs. The purity of the L-asparaginase purified by the improved pulse dilution method is 90.14 percent, the yield is 5.23mg/L, and the specific activity of the enzyme is 201.21 IU/mg. The activity at cellular level showed: after 48 hours of action, the IC50 of the L-asparaginase on Jurkat cells and Raji cells is 4.15IU/mL and 4.65IU/mL respectively; after 72h of action, the IC50 of L-asp on Jurkat and Raji cells was 2.40IU/mL and 2.01IU/mL, respectively. Based on the above results, the present invention provides a highly efficient method for preparing recombinant L-asp and also provides the use of recombinant L-asp in the preparation of anti-acute leukemia drugs.)

1. A recombinant L-asparaginase has a DNA sequence shown as SEQ ID N0:1 and a protein sequence shown as SEQ ID N0: 2.

2. A recombinant L-asparaginase according to claim 1, wherein said recombinant L-asparaginase has a long half-life.

3. The recombinant L-asparaginase according to claim 1, wherein said recombinant L-asparaginase is highly toxic to leukemia cells and not toxic to normal cells.

4. A preparation method of recombinant L-asparaginase is characterized by comprising the following steps:

(1) PCR cloning to obtain the DNA sequence of the recombinant L-asparaginase;

(2) inserting the DNA sequence of the recombinant L-asparaginase into a vector pET-22b and transforming Top10 competent cells to obtain recombinant pET-22b-L-asp plasmids and transforming the recombinant pET-22b-L-asp plasmids into expression strain BL21 competent cells to obtain plasmids and strains for expressing the recombinant L-asparaginase;

(3) culturing a strain expressing the recombinant L-asparaginase;

(4) breaking the bacteria, extracting protein, and purifying by adopting an improved pulse dilution method to obtain the recombinant L-asparaginase.

5. An application of recombinant L-asparaginase in preparing the medicine for treating acute leukemia.

Technical Field

The invention relates to the fields of biological pharmacy, biochemistry and pharmacology, in particular to recombinant L-asparaginase and a preparation method and application thereof.

Background

The recombinant L-asparaginase (L-asp) is used as the most effective medicament for clinically treating acute leukemia (ALL) at home and abroad, the remission rate of medicament treatment is more than 50 percent, the remission period of the medicament is 1-9 months, and the recombinant L-asparaginase also has certain curative effect on acute myelocytic leukemia and acute monocytic leukemia. Meanwhile, L-asp has the characteristics of short remission period and easy tolerance generation, so that most of L-asp can be combined with other anti-cancer drugs in the treatment process at present. L-asp has been marketed for over 50 years, but its side effects and short drug half-life remain important factors limiting its clinical use. Human Albumin (HSA) is the most abundant protein in Human body and is responsible for storage and transportation of many substances in vivo. Researches show that albumin can be used as a drug carrier, so that the drug is slowly released, and the half-life of the drug is remarkably improved. In addition, most drugs have strong affinity with HSA itself, and the HSA has the characteristic of high content of human HSA, so that the half-life of the drugs can be improved by using HSA as a drug carrier for the drugs with weak binding capacity with HSA.

The invention constructs a new recombinant L-asp and a preparation method of the recombinant L-asp, characterizes the properties of the recombinant L-asp, and detects the affinity of the recombinant L-asp and HSA by the focus of protein engineering modification, thereby obviously improving the half-life period of the L-asp.

Disclosure of Invention

The invention adopts an improved pulse dilution method to purify the recombinant L-asparaginase, adopts a Neesler reagent method to detect the enzyme activity of the purified protein, adopts dynamic light scattering to detect the particle size distribution of the recombinant L-asparaginase, adopts micro-calorimetric electrophoresis to detect the binding capacity of the protein and the human albumin, adopts CCK-8 to detect the killing effect of the recombinant protein on lymphoma cells and normal cells at the cell level, and finally detects the in-vitro plasma half-life period of the recombinant L-asparaginase.

The purity of the recombinant L-asparaginase protein purified by the improved pulse dilution method is 90.14 percent, the yield is 5.23mg/L, and the specific activity of the enzyme is 201.21 IU/mg. The average particle size of the recombinant protein is 6.25 +/-0.16 nm, and the recombinant protein is uniformly distributed. The activity at cellular level showed: after 48 hours of action, the IC50 of the L-asp on Jurkat and Raji cells is 4.15IU/mL and 4.65IU/mL respectively; after 72h of action, the IC50 of L-asp on Jurkat and Raji cells was 2.40IU/mL and 2.01IU/mL, respectively. In the concentration range of the L-asp experiment, after 24h, 48h and 72h of action, the L-asp has weak action on HBE cells, and IC50 cannot be calculated. The binding kd value of the recombinant L-asp protein to HSA was 14.15. mu.M, and the in vitro plasma half-life was 25 h.

The invention provides an expression and purification method of recombinant L-asparaginase, wherein the protein obtained by pulse dilution purification has high purity and enzyme activity, the particle size of the recombinant protein is uniform, no conglomerate is formed in buffer solution, the recombinant L-asp protein has strong killing effect on lymphoma cells, weak cytotoxicity to normal cells, certain binding capacity with HSA and in-vitro plasma half-life of 25 h.

The invention provides a high-efficiency expression and purification method of recombinant L-asp, and the obtained recombinant L-asp can be used for preparing a medicine for treating acute leukemia.

Drawings

FIG. 1 results of purification of recombinant L-asp.

FIG. 2 particle size distribution of recombinant L-asp.

FIG. 3 killing of Jurkat and Raji cells by recombinant L-asp A: killing effect of the recombinant L-asp on Jurkat cells under different concentrations; b: killing effect of the recombinant L-asp on Raji cells under different concentrations; c: killing of Jurkat and Raji cells by recombinant L-asp at various times.

FIG. 4 cytotoxic Effect of recombinant L-asp on HBE cells A: the cytotoxic effect of the recombinant L-asp on HBE cells under different concentrations; b: cytotoxic effects of recombinant L-asp (20IU/ml) on HBE cells at various times.

FIG. 5 results of analysis of binding of recombinant L-asp to HSA

FIG. 6 results of plasma metabolism of recombinant L-asp

Detailed Description