摘要
目的采用低温乙醇法分离正常人血浆组分Ⅳ沉淀(FractionⅣ,FⅣ)中提取α1-抗胰蛋白酶(α1-Antitrypsin,α1-AT),制备较高纯度的α1-AT制剂。方法 FⅣ沉淀经过滤、ConA Sepharose 4B亲和层析、DEAE Sepharose Fast Flow一步离子交换层析提纯α1-AT,采用SDS-PAGE及区带扫描法分析纯化α1-AT的纯度;紫外分光光度计测定纯化α1-AT蛋白的浓度及回收率;Western blot检测其反应原性;胰蛋白酶抑制实验(TICT)检测纯化α1-AT的活性。结果纯化α1-AT的纯度为97.3%;纯化过程中α1-AT蛋白的平均回收率为20.4%;纯化的α1-AT能与抗人α1-AT抗体特异性结合;平均比活为0.833 PU/mg。结论 从FⅣ中成功纯化获得了纯度较高、具有生物学活性的α1-AT制剂。
Objective To extract α1-antitrypsin（α1-AT） from the Cohn fraction Ⅳ（FⅣ） of healthy human plasma and prepare into a highly purified product.Methods α1-AT was purified from the precipitate of FⅣ by filtration,ConA Sepharose 4B affinity chromatography and DEAE Sepharose Fast Flow one-step ion exchange chromatography,and analyzed for purity by SDS-PAGE and zone scanning,for concentration and recovery rate by UV spectrophotometry,for reactogenicity by Western blot,and for activity by trypsin inhibition test（TICT）.Results The purity of purified α1-AT was 97.3%.The mean recovery rate of α1-AT during purification was 20.4%.The purified α1-AT showed specific binding to anti-human α1-AT antibody,of which the mean specific activity was 0.833 PU / mg.Conclusion The α1-AT with high purity and biological activity was successfully purified from FⅣ.
出处
《中国生物制品学杂志》
CAS
CSCD
2011年第9期1090-1093,共4页
Chinese Journal of Biologicals
关键词
Α1抗胰蛋白酶
层析
活性
α1-Antitrypsin（α1-AT）
Chromatography
Activity
