Medical composition of recombinant Wautersiella asparaginase and preparation method and application thereof
A technology of asparaginase and Worella, applied in the field of biomedicine, can solve the problem of not directly elucidating the anti-cancer activity, etc., and achieve the effect of high-level expression
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Embodiment 1
[0025] Expression of Recombinant Wormella Asparaginase
[0026] Plasmid (pJET / WAP) containing a 1kb DNA fragment of Asparaginase from Wormella spp., digested with Nde I and Sal I (37°C, 1hr), cut out a 1kb DNA fragment, and purified the target DNA with a DNA agarose recovery kit fragment. The purified DNA fragment was inserted between the corresponding Nde I and Sal I of the expression plasmid pET30a, and ligated with T4 DNA ligase. The ligation product was transformed into Escherichia coli BL21 (DE3). Spread on LB+Agar medium containing 30 μg / ml kanamycin; culture at 37°C for 12 hours until a single colony appears, pick the transformant, and place in LB medium containing 30 μg / ml kanamycin Cultivate at 37°C for 4 hours; then in 3L expression medium (peptone 12g / L; yeast extract 24g / L; KH 2 PO 4 0.17mol / L; K 2 HPO 4 0.72mol / L; glycerol 4ml / L; glucose 0.05% (w / v); lactose 0.2% (w / v); magnesium sulfate 2mmol / L; 30μg / ml kanamycin) for 16-18hr. Centrifuge at 6,000g for 20m...
Embodiment 2
[0028] Purification of Recombinant Asparaginase from Wormella
[0029] Take the bacterium suspension described in the above example 3, break it with an ultrasonic cell disruptor, centrifuge again, take the supernatant and put it on a chromatographic column filled with 1.2L of DEAE agarose resin, and use 50mM Tris , pH 7.5, 50 mM NaCl buffer to elute asparaginase. The eluate containing asparaginase was collected and concentrated by ultrafiltration. The concentrate was further purified by 125ml Phenyl Sepharose hydrophobic column chromatography, gradient elution was carried out with phosphate buffer containing 1M ammonium sulfate to 0M ammonium sulfate, and the components containing asparaginase were collected, concentrated by ultrafiltration, The concentrate was dialyzed and the final recombinant asparaginase was stored in 0.1M phosphate buffer (pH 8.0). Use the Bradford method to measure the protein concentration, use the Nestle method to measure the enzyme activity, and use...
Embodiment 3
[0031] Preparation of Recombinant Wormella Asparaginase for Injection
[0032] Take 10,000 units per bottle of the recombinant Wormella asparaginase prepared in Example 2, add 80 mg of mannitol, filter aseptically with a 0.22 μm filter membrane, add to a sterile vial, freeze-dry, and obtain injection Recombinant Asparaginase was used.
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