| PEG化重组尿酸氧化酶蛋白含量测定方法研究 |
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| 引用本文： | 李晶,;苑方园,;程速远,;张慧,;梁成罡.PEG化重组尿酸氧化酶蛋白含量测定方法研究[J].生物技术通讯,2014(4):542-546. |
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| 作者姓名： | 李晶 ;苑方园 ;程速远 ;张慧 ;梁成罡 |
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| 作者单位： | [1]中国食品药品检定研究院,北京100050; [2]昆明理工大学,云南昆明650504 |
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| 基金项目： | “重大新药创制”科技部重大专项（2012ZX09304010）;中检院学科带头人项目（2011X1） |
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| 摘 要： | 目的:以未修饰的原型蛋白为对照品,建立准确测定2种聚乙二醇化重组尿酸氧化酶(PEG-rUOX)含量的方法。方法:以原型蛋白rUOX为对照品,利用凝胶色谱法测定PEG-rUOX中的蛋白含量,通过详细的专属性、精密度、准确性等方法学考察,建立该方法。色谱条件:采用TSK Gel 5000PWxl(300 mm×7.8 mm)凝胶色谱柱,以含0.15mol/L氯化钠的0.05 mol/L的Tris-HCl溶液(pH9.0)为流动相,流速0.5 mL/min,柱温为室温,检测波长280 nm。结果:在280 nm下,2种rUOX在200~1000μg/mL浓度范围内线性关系良好(r2分别为1和0.9999),其高、中、低平均回收率均较高,为98.33%~99.96%。结论:本方法专属性、精密度和准确性良好,可作为PEG-rUOX的含量测定方法。
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| 关 键 词： | 聚乙二醇化重组尿酸氧化酶 凝胶色谱 蛋白含量 |
Studies on Determination of Protein Content in PEG-rUOX |
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| Institution: | LI Jing,YUAN Fang-Yuan,CHENG Su-Yuan,ZHANG Hui,LIANG Cheng-Gang(1. National Institutes for Food and Drug Control, Beijing 100050; 2. Kunming University of Science and Technology, Kunming 650504; China) |
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| Abstract: | Objective： To develop an assay method for 2 kinds of pegylated recombinant urate oxidase（PEG-rUOX） by using unmodified protein as a reference substance. Methods： rUOX and a SEC-HPLC method were used to determine the protein content of PEG-rUOX. This method was well established by validation on the specif-ity, precision and accuracy. The assay was performed on a TSK Gel 5000PWxl Column（300 mm × 7.8 mm） at 25℃ with the mobile phase of 0.15 mol/L NaCl-0.05 mol/L Tris-HCl solution（pH9.0）. The flow rate was 0.5 mL/min and the detection wavelength was set at 280 nm. Results： Two kinds of rUOX showed a good linear relation-ship in the range of 200~1000 μg/mL（r2 were 1 and 0.9999, respectively） and their average recoveries were good （98.33%~99.96%）. Conclusion： The method is specific, precise and accurate, and can be used as a protein con-tent assay for PEG-rUOX. |
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| Keywords: | pegylated recombinant urate oxidase SEC-HPLC protein content |
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