PEGylated uricoxidase compound, preparing method, preparation and use thereof
A technology of uric acid oxidase and pegylation, which is applied in the direction of oxidoreductase, drug combination, and pharmaceutical formula, can solve the problems of poor uniformity, low immunogenicity, systemic allergic reactivity, and difficulty in separation and purification, and achieve uniformity Improvement of sex, low anaphylaxis, easy purification effect
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Embodiment 1
[0041] Modification reaction between polyethylene glycol and uric acid oxidase
[0042] The uric acid oxidase described in the present invention is a recombinant Aspergillus flavus uric acid oxidase obtained from genetic engineering technology, the purity is greater than 95%, and it maintains relatively high enzyme activity.
[0043] Dissolve uric acid oxidase (UOX) in pH 6.0, 100mM phosphate buffer, add NaCNBH after dissolving according to the ratio of UOX:mPEG active ester=1:5 (w / w) 3 Make the final concentration 10mM, and react at room temperature for 24 hours.
Embodiment 2
[0045] Separation and purification of PEGylated urate oxidase drug
[0046] The product obtained from the modification reaction of polyethylene glycol and uric acid oxidase was subjected to preparative cation exchange chromatography on the AKTA-Primer protein chromatography system (product of Amersham Pharmacia). The cationic chromatography gel is SP-Sepharose Fast Flow, and the column bed volume is 50ml. The chromatographic column was equilibrated with 20mM PB, pH8.0 buffer solution for at least 5 column bed volumes to make the pH of the effluent liquid the same as the pH value of the buffer solution, and after the balance, adjust the pH to 8.0. / min) sample loading. After sample loading, continue to equilibrate with equilibration buffer until the UV absorption returns to the baseline, and then use 0-0.5M NaCl gradient elution. When the NaCl concentration is 0.2-0.3mol / L, the protein peak that appears is mainly a polyethylene glycol Alcohol molecules are linked to a UOX mol...
Embodiment 3
[0048] Modification reaction of polyethylene glycol and uric acid oxidase in different ratios
[0049] Dissolve urate oxidase in pH 6.0, 100mM phosphate buffer, add UOX to mPEG active ester at the ratio of 1:4 (w / w), 1:5, 1:6.5, 1:8 After dissolving, add NaCNBH3 to make the final concentration 10mM, react at room temperature for 16-24 hours, analyze the ratio of the modified product and the specific enzyme activity by SDS-PAGE, the results are shown in the table below (the enzyme specific activity of unmodified urate oxidase is 17.6EAU / mg).
[0050]
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