应用一步亲和层析法纯化转基因山羊乳腺定位表达的组织型纤维蛋白溶酶原激活剂

对转基因山羊乳腺定位表达的羊奶中组织型纤维蛋白溶酶原激活剂 ( t PA)的分离纯化方法作了研究 ,摸索出一种简单可行的纯化方法。羊奶中 t PA先经冰乙酸和丙酮沉淀后 ,再通过 Benzamidin Sepharose 6B柱一步亲和层析 ,纯化倍数达 671 4.3倍 ,比活性 1 880 0 0 U/mg,活性回收率 2 6%。经 SDS-PAGE还原电泳分析 ,t PA纯度大于 95%,其中低分子双链蛋白占 90 %以上 ;Western-blotting检测证实 ,它和天然 t PA具有相同的抗原性 ;用纤维蛋白平板法检测 ,其比活性与天然 t PA相近 ,并且都可被 t PA单克隆抗体所抑制

One-step Purification of Tissue-type Plasminogen Activator Derived from Expression by Milk Gland of Transgenic Goat Using Affinity Chromatography

A tissue type plasminogen activator derived from expression by milk gland of transgenic goat was isolated and purificated. Precipitated with acetate acid and acetone in advance, the t PA in the milk was purificated using one step affinity chromatography with benzamidin sepharose 6B. The final product had a specific activity of 188 000 IU/mg protein. The overall activity recovery was about 26% with 6 714.3 fold increase in specific activity. Analysis by SDS PAGE electrophoresis in the presence of a reducing agent, the purity of t PA was over 95% with the two chain t PA to be occupied over 90% of it. Western blotting analysis showed that the product was possessed of the natural t PA antigen characteristics. Analysed with fibrin plate, the product had the same specific activity and molecular weight as natural t PA and could be inhibited by the t PA monoclonal antibody.