人血浆α1-抗胰蛋白酶的纯化与鉴定

发布时间：2018-07-25 06:04

【摘要】：α1-抗胰蛋白酶(α1-antitrypsin,α1-AT)是人类血浆中最丰富的蛋白酶抑制物,主要是由肝细胞合成的一种糖蛋白,在单核巨噬细胞、肠上皮细胞、肾实质等肝外组织及某些肿瘤细胞中也能产生,它能抑制多种蛋白酶及多种丝氨酸内切肽酶。1989年,人血浆α1-AT制剂获得美国FDA批准用于严重α1-AT缺乏的患者的替代治疗。随着基因工程技术的发展,重组人血浆α1-AT获得成功表达,但目前尚无重组表达的产品被批准上市。目前经FDA批准应用于α1-AT缺乏疾病替代治疗的α1-AT制剂仍供不应求,国内尚无同类产品问世,且生产血液制剂要产生大量的Cohn FIV-1废弃组分,因此开发新血液制剂α1-AT无论在血浆综合利用理论研究及实际应用方面均具有重要意义。以Cohn FIV-1组分为原料,加适量脂蛋白去除剂(LRA)室温搅拌1小时处理后,经四种不同的层析纯化工艺路线分离纯化α1-抗胰蛋白酶,其中DEAE-Sepharose Fast Flow阴离子交换层析结合Sephacryl S-200凝胶过滤层析和Q-Sepharose Fast Flow阴离子交换层析结合Sephacryl S-200凝胶过滤层析两种纯化工艺纯化的α1-AT分别达到100%和95%。对DEAE-Sepharose Fast Flow阴离子交换层析结合Sephacryl S-200凝胶过滤层析纯化α1-AT的工艺进行初步放大,纯化的α1-AT纯度仍可达95.5%。纯化的α1-AT表观分子量为55.9kD,p14.55,为酸性蛋白质。用免疫双扩散试验、微量免疫电泳、免疫印迹分析对其进行鉴定,证明其具有良好的免疫反应性及较高的纯度。以胰蛋白酶抑制能力试验(微量法)测其生物活性,DEAE-Sepharose Fast Flow阴离子交换层析结合Sephacryl S-200凝胶过滤层析纯化的α1-AT比活性为680.67±34.58IU/mg(n=3),活性回收率为48.35±5.15%(n=3);Q-Sepharose Fast Flow阴离子交换层析结合Sephacryl S-200凝胶过滤层析纯化工艺纯化的α1-AT比活性为328.00±44.42IU/mg(n=3),活性回收率为30.40±5.23%(n=3);前一种层析纯化工艺路线初步放大试验所获α1-AT比活性为645.43±
[Abstract]:伪 1-antitrypsin (伪 1-AT) is the most abundant protease inhibitor in human plasma. It is mainly a glycoprotein synthesized by hepatocytes. It is also produced in mononuclear macrophages, intestinal epithelial cells, renal parenchyma and some tumor cells. In 1989, human plasma 伪 1-AT preparation was approved by FDA for replacement therapy in patients with severe 伪 1-AT deficiency. With the development of genetic engineering technology, recombinant human plasma 伪 1-AT has been successfully expressed, but no recombinant expression product has been approved. At present, 伪 1-AT preparations approved by FDA for 伪 1-AT lack of disease replacement therapy are still in short supply, no similar products have been produced in China, and the production of blood preparations should produce a large number of waste components of Cohn FIV-1. Therefore, the development of new blood preparation 伪-1-AT is of great significance in the theoretical study and practical application of plasma utilization. 伪 1-antitrypsin was separated and purified by four different chromatographic purification processes using Cohn FIV-1 component as raw material and lipoprotein remover (LRA) stirred for 1 hour at room temperature. The 伪 1-AT purified by DEAE-Sepharose Fast Flow anion exchange chromatography combined with Sephacryl S-200 gel filtration chromatography and Q-Sepharose Fast Flow anion exchange chromatography combined with Sephacryl S-200 gel filtration chromatography was 100% and 95% respectively. The purification process of 伪 1-AT by DEAE-Sepharose Fast Flow anion exchange chromatography and Sephacryl S-200 gel filtration chromatography was amplified, and the purity of 伪 1-AT was still up to 95. 5%. The apparent molecular weight of the purified 伪 1-AT is 55.9 kDX p14.55, which is an acidic protein. Double immunodiffusion assay, microimmunoelectrophoresis and immunoblotting analysis were used to identify it. It was proved that it had good immunoreactivity and high purity. The specific activity of 伪 1-AT purified by DEAE-Sepharose Fast Flow anion exchange chromatography combined with Sephacryl S-200 gel filtration chromatography was 680.67 卤34.58IU/mg (Nm3) by trypsin inhibition test (trace method). The recovery rate of 伪 1-AT was 48.35 卤5.15% (nun3) Q-Sepharose Fast Flow anion exchange chromatography combined with Sephacryl. The specific activity of 伪 1-AT purified by S-200 gel filtration chromatography process was 328.00 卤44.42IU/mg (Nm3), and the recovery rate was 30.40 卤5.23% (Nm3), and the specific activity of 伪 1-AT obtained from the preliminary amplification test of the previous purification process was 645.43 卤5.23%.
【学位授予单位】：北京生物制品研究所
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R341

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