摘要
目的研究血型改造工具酶,重组咖啡豆α-半乳糖苷酶在不同保存条件下的稳定性。方法以酶解B型红细胞的活性和纯度作为主要检测指标,考察重组α-半乳糖苷酶的热稳定性。重组α-半乳糖苷酶在70℃条件下保存1d,37℃保存60d,室温、4、-20、-70℃分别保存6个月,定期取样观察样品的外观,检测样品的pH值、纯度、酶活性,并进行无菌试验;为确保临床使用中的安全性和有效性,我们对4℃保存的样品进行了长达3年的观察。结果重组α-半乳糖苷酶在70℃保存1d后失活,37℃保存2个月后活性降低了11.5%,室温保存6个月后活性降低了19%,-70℃保存6个月后活性降低了13%,4℃、-20℃保存的样品6个月内各项指标均正常,均未见蛋白明显降解,蛋白活性保持稳定。4℃保存3年的样品酶活性保持稳定,可按原剂量在26℃,2h内成功地将B型红细胞改造成O型红细胞。结论重组α-半乳糖苷酶对热不稳定,应保存4℃或-20℃,在此条件下,有效期至少2年,能满足常规临床要求。
Objective To investigate the stability of recombinant α-D-galactosidase which is used for converting human erythrocytes from group B to group O. Methods The enzyme activity and stability were measured by the conversion efficiency of B to O blood conversion after the α-D-galactosidase was preserved under the following conditions： 70 ℃ for 1 day, 37 ℃ for 60 days, room temperature, 4 ℃, -20 ℃, and -70 ℃ for 6 monthes each. In addition, the biochemical characters of α-D-galactosidase, including appearance, pH, sterility test and α-D-galactosidase activity were identified by periodic sampling. The 4 ℃ preserved α-D-galactosidase was followed up for 3 years for studying its clinical safety and efficacy. Results The α-D-galactosidase activity was decreased by 11.5% at 37 ℃, 19% at room temperature, 13% at -70 ℃, and little activity at 70 ℃. In contrast, the biochemical characteristics of α-D-galactosidase were normal after 6 monthes storage at 4℃ and -20 ℃. After 3 years of storage at 4 ℃, α-D-galactosidase successfully converted human erythrocytes from group B to group O in 2 hours at normal does. Conclusion The recombinant α-D-galactosidase is unstable at high temperature and the optimal storage conditions is 4 ℃ and -20 ℃. The shelf life of the recombinant α-D-galactosidase should be 2 years at least to meet general clinical requirement.
出处
《中国输血杂志》
CAS
CSCD
北大核心
2010年第4期244-246,共3页
Chinese Journal of Blood Transfusion
基金
国家973项目(2002CB713804)
关键词
重组α-半乳糖苷酶
稳定性
活性测定
血型改造
Recombinant α-D-galactosidase
Stability
Activity assay
Blood group conversion
