诊断用苛求芽孢杆菌尿酸酶的剂型与储存方法

Formulation and storage of bacillus fastidious uricase as diagnosis enzymes

摘要:

目的：考察重组苛求芽孢杆菌尿酸酶作为诊断试剂的剂型和储存方法。方法：将此重组尿酸酶制成干粉和液体剂型于-18℃和4℃储存，定期测定酶活性；在干粉制剂时加入保护剂于-18℃储存，在液体制剂时加入防腐剂于4℃储存，间隔适当时间监测酶活性。聚丙烯酰胺凝胶电泳分离此尿酸酶四聚体和二聚体，染色检测蛋白和酶活性。结果：此尿酸酶干粉和液体制剂在4℃储存28 d后对应的剩余酶活性分别为12%和100%，在-18℃冻存28 d后对应的剩余酶活性分别为24%和92%。在干粉制剂时加海藻糖、蔗糖或甘露醇作为保护剂于-18℃储存56 d后，剩余酶活性对应分别为100%、76%和43%。加叠氮钠或苯甲酸的液体制剂，在4℃储存56 d后对应的剩余酶活性分别为99%和82%。在-18℃储存至无活性的冻干粉制剂经非变性聚丙烯酰胺凝胶电泳分析，发现此尿酸酶主要以无活性二聚体存在而活性四聚体很少。结论：此尿酸酶作为诊断试剂，可加入叠氮钠制成液体剂型在4℃储存。

Abstract:

Objective：To explore the formulation and storage methods for bacillus fastidious uricase as a diagnosis enzyme. Methods： Lyophilized powders of this uricase containing protective additives，and solutions of this uricase containing preservatives，were stored at -18℃ or 4℃. Enzyme activity was monitored at proper intervals. Homotetramers and homodimers of this uricase were resolved by polyacrylamide gel electrophoresis（PAGE），and were stained separately for both proteins and enzyme activities. Results：The lyophilized powders and solution preparations of this uricase yielded the corresponding residual enzyme activities of 12% and 100%，respectively，after storage at 4℃ for 28 days，and the residual enzyme activities of 24% and 92%，respectively，after storage at -18℃ for 28 days.The use of trehalose，sucrose and mannitol as the protective additives in lyophilized powders gave the residual enzyme activities of 100%，76% and 43%，respectively，after storage for 56 days at -18℃. The use of NaN3 and benzoic acid as the preservative in the solutions gave the residual enzyme activities of 99% and 82%，respectively，after storage for 56 days at 4℃. By PAGE analyses of lyophilized powders of low activity，less active homotetramers but more inactive homodimers were observed. Conclusion：This uricase as a diagnosis reagent can be prepared in solutions containing NaN3 for storage at 4℃.