PEG化重组尿酸氧化酶蛋白含量测定方法研究  

Studies on Determination of Protein Content in PEG-rUOX

作　　者：李晶[1] 苑方园 程速远[1] 张慧[1] 梁成罡[1] 

机构地区：[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

出　　处：《生物技术通讯》2014年第4期542-546,共5页Letters in Biotechnology

基　　金："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)

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0.15 mol/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。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.

关 键 词：聚乙二醇化重组尿酸氧化酶 凝胶色谱 蛋白含量 

分 类 号：Q503[生物学—生物化学]