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A method for preparing a recombinant Prevotella asparaginase

A technology of asparaginase and Wordella, which is applied in the field of asparaginase preparation, can solve the problems of low ratio of target enzyme, difficult quality control, and many process steps, so as to achieve easy product quality, control product quality, The effect of fewer process steps

Inactive Publication Date: 2013-01-02
范铭琦
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The traditional asparaginase preparation method is mostly directly extracted from the bacteria, but the proportion of the target enzyme in the bacteria is not high, and this method involves the use of organic solvents, many process steps, and difficult quality control
The asparaginase of Wormella was originally extracted directly from the bacteria, but the bacteria are anaerobic bacteria, which are more difficult to cultivate than aerobic bacteria, and also have the defect of low yield

Method used

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  • A method for preparing a recombinant Prevotella asparaginase
  • A method for preparing a recombinant Prevotella asparaginase
  • A method for preparing a recombinant Prevotella asparaginase

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Embodiment 1

[0031] PCR Amplification of Asparaginase DNA from Wormella spp.

[0032] Such as figure 1 Shown, the primer nucleotide sequence that is used for PCR amplification is as follows:

[0033] 5'-GGTAC CATATG AATGCATGGAAAAAAAC-3'

[0034] The Nde I site is underlined.

[0035] 5'-GAATTC GTC GAC TTAATAGGTGGAGAAGATCT-3'

[0036] The Sal I site is underlined.

[0037] Such as figure 2 As shown, the PCR amplification reaction was set up as follows: In a 200 μl PCR test tube, add 32.5 μl of water, 10 μl of 5-fold buffer, 1 μl of dNTP (10 mM), 2.5 μl of each of the above primers (10 μM), and 250 ng of Walter’s bacterial genomic DNA, 0.5u of PCR polymerase. The amplification reaction was carried out for 30 cycles, and 25 μl of the reaction solution was taken for electrophoresis on 0.9% agarose, and a single amplified band of about 1 kb was detected under ultraviolet light. Such as figure 2 As indicated, the corresponding 1 kb DNA fragment was excised with a clean blade, and t...

Embodiment 2

[0039] Cloning of Asparaginase DNA from Wormella spp.

[0040] The asparaginase gene of Worella spp. was cloned using the pJET1.2 kit (Fermentas). Take 1 μl of the recovered DNA fragment of asparaginase from Wormella spp., add 10 μl of 2-fold reaction buffer, 50 ng of pJET1.2 blunt-end vector, 7 μl of water, and 1 μl of T4 DNA ligase. Stand at room temperature for 30 min, take 2 μl of the reaction product and transform it into E. coli.

[0041] Pick the transformed bacteria, culture them overnight at 37°C in about 5ml of LB medium (containing 100μg / ml of ampicillin), extract the plasmids, digest them with Nde I and Sal I, and carry out DNA analysis on the plasmids containing 1kb DNA inserts. sequencing.

Embodiment 3

[0043] Expression of Recombinant Wormella Asparaginase

[0044] see image 3 and Figure 4 After verification in the above example 2, it was determined that the plasmid containing the 1kb Asparaginase DNA fragment of Wordella was determined, then digested with Nde I and Sal I (37°C, 1hr), and the 1kb DNA fragment was cut out and recovered with DNA agarose The kit (Shanghai Jierui Bioengineering Co., Ltd.) was used to purify the target DNA fragment. The purified DNA fragment was inserted between the corresponding Nde I and Sal I of the expression plasmid pET21, and ligated with T4 DNA ligase. The ligation product was transformed into E. coli. Pick the transformant and culture it in the medium containing 100 μg / ml ampicillin (37°C), at OD 600 After reaching 0.4-0.8, add 1mM IPTG to induce the expression of asparaginase, and continue to culture for 2-4hr. The culture was harvested, centrifuged at 6000 g for 20 min, the supernatant was discarded, and the pellet was resuspende...

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Abstract

The invention relates to the biomedical field and the field of gene recombination, more specifically, to a preparation method of a recombinant Prevotella asparaginase, which includes the steps of amplifying full length or fragment DNA of asparaginase from the genomic DNA of Prevotella using PCR technique, performing ligation of the obtained DNA and a cloning plasmid with ligase, cutting out the full length or fragment DNA of the asparaginase from the cloning plasmid and then inserting into an eukaryotic expression vector, transforming into Escherichia coli, culturing transformed Escherichia coli in a selective medium containing antibiotic to amplify the expression vector containing the full length or fragment DNA of asparaginase, collecting transformed bacteria, and centrifugating to separate the bacteria; disrupting the bacteria, and releasing the recombinant asparaginase; and performing two-step column chromatography to purify the recombinant Prevotella asparaginase. The inventive method has the advantages of few process steps, controllable quality and large production quantity.

Description

[technical field] [0001] The invention relates to the field of gene recombination of biomedicine, in particular to a method for preparing asparaginase of recombined Wormella bacterium. [Background technique] [0002] Asparaginase is a class of enzymes that can convert L-asparagine (L-asparagine) into aspartic acid (aspartic acid); in addition, the enzyme usually has a lower glutaminase activity (glutaminase activity ). [0003] Asparaginase is mainly used in the treatment of acute lymphocytic leukemia and other blood cancer diseases, and is an important drug in the treatment of acute lymphocytic leukemia. In addition, it also has a good curative effect on malignant lymphoma. The main mechanism is: cancer cells cannot produce asparagine, but normal cells can; asparaginase can degrade asparagine into aspartic acid, so that cancer cells die due to lack of necessary asparagine. Most of the products currently used clinically are asparaginase derived from Escherichia coli (E.co...

Claims

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Application Information

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IPC IPC(8): C12N15/55C12N15/70C12N9/82
Inventor 范铭琦
Owner 范铭琦
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