中国输血杂志2012，Vol.25Issue(6)：560-564,5.
亲和凝胶分离纯化人α1-抗胰蛋白酶的研究
Isolation and purification of human α1-antitrypsin by a new immunoaffinity chromatography
摘要
Abstract
Objective To develop an effective process for isolating and purifying α1 -antitrypsin from Cohn Fraction IV by a new immunoaffinity chromatography and to achieve an improved utilization of the valuable source material fresh frozen plasma. Methods Cohn Fraction IV was dissolved with Tris solution in a ratio of 1:12 for 0. 5 hour at 241, and the suspension was centrifuged for 15min (24℃ ,4 750 x g). After treated with HL-380 (a fumed silica) and centrifugation,the supernatant was loaded through 1.2μm filters sequentially. With a new immunoaffinity chromatography medium-" Alpha-1 Antitrypsin Select" ,al -antitrypsin was purified further. Characterization of al-antitrypsin was performed by SDS-PAGE、 Western Blot and tandem mass spectrum. Total of protein content was determined by the method of Bradford under Visible light absorption at 595nm. The activity of α1-antitrypsin was determined using chromogenic substrate assay. Results The purified αl-antitrypsin concentrate showed two protein bands on gels by SDS-PAGE, One of the molecular weight was calculated 54kD;the others was calculated 140 kD approximately,After reducing SDS-PAGE,large molecular weight polymer protein band of α1-antitrypsin was depolymerized, the result of Western Blot analysis could be related to changes in reaction. Pretreatment process and immunoaffinity chromatography step achieved a (60. 35 ± 1. 39)% yield. Thus,an overall (71. 68 ± 1. 32) fold increase in purity and a (41. 88 ±6. 98)% yield was obtained approximately from plasma, α1-antitrypsin had a specific activity of about (1. 00 ~ 1. 05)pU/mg protein. Conclusion High purity and specific activity of α1-antitrypsin achieved with this process which should be an efficient and scale-up method for α1-antitrypsin purification from ;, Cohn Fraction IV.关键词
Cohn Ⅳ组份/α1-抗胰蛋白酶/分离/纯化/免疫亲和凝胶Key words
Cohn Fraction IV/α1-antitrypsin/Immunoaffinity chromatography/Specific activity分类
医药卫生引用本文复制引用
张学俊,叶生亮,曹海军,王宗奎,杜晞,谢亦武,李长清..亲和凝胶分离纯化人α1-抗胰蛋白酶的研究[J].中国输血杂志,2012,25(6):560-564,5.基金项目
四川省科技支撑计划项目(2009SZ0217) （2009SZ0217）
