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

目的探索通过免疫亲和层析从Cohn法组份Ⅳ分离纯化人α1-抗胰蛋白酶浓缩物的方法。方法 24℃下,将Cohn氏法组份Ⅳ用Tris缓冲液抽提0.5 h后,离心4 750 g×15 min,上清液加入亲水型气相二氧化硅脱脂后,离心6 000 g×15 min,再用1.2μm的滤膜过滤;通过GE公司新型的Alpha-1 Antitrypsin Select免疫亲和凝胶层析柱,对α1-抗胰蛋白酶进行纯化。用考马斯亮蓝法通过紫外分光光度计测定总蛋白含量;用SDS-PAGE鉴定α1-抗胰蛋白酶的纯度,并用Western Blot和串联质谱分析鉴定纯化的蛋白;通过发色底物法测定α1-抗胰蛋白酶活性。结果纯化的α1-抗胰蛋白酶在非还原性SDS-PAGE图谱上呈现1条54 kD左右的蛋白带和1条约140 kD的多聚体蛋白带,经过还原性SDS-PAGE多聚体条带解聚,Western Blot也可以反应相关变化;在经过前处理和亲和层析后,α1-抗胰蛋白酶活性回收率为(60.35±1.39)%,α1-抗胰蛋白酶总活性回收率为(41.88±6.98)%,纯化倍数为(71.68±1.32)倍,比活性大约(1.00～1.05)PU/mg。结论 GE公司Alpha-1 Antitrypsin Select免疫亲和层析亲和层析可以从Cohn氏法组份Ⅳ中特异性捕获α1-抗胰蛋白酶浓缩物。

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 24℃, and the sus- pension was centrifuged for 15min （24℃ ,4 750 × g）. After treated with HL-380 （a fumed silica） and eentrifugation,the supernatant was loaded through 1.2μm filters sequentially. With a new immunoaffinity chromatography medium-＂ Alpha-1 Antitrypsin Select＂, α1-antitrypsin was purified further. Characterization of α1-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 ehromogenie substrate assay. Resulls The purified α1-antitrypsin concentrate showed two protein bands on gels by SDS-PAGE, One of the molecular weight was calcu- lated 54kD; the others was calculated 140 kD approximately, After reducing SDS-PAGE, large molecular weight polymer pro- tein band of α1-antitrypsin was depolymerized, the result of Western Blot analysis could be related to changes in reac- tion. 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-anti- trypsin 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 efiqeient and scale-up method for etl-antitrypsin purification from Cohn Fraction Ⅳ.