%0 Journal Article
%T Single-chain Urokinase-type Plasminogen Activator (scu-PA) Purification by Immuno-affimity Chromatography<br>免疫亲和层析法纯化单链尿激酶型纤溶酶原激活剂
%A GAO Li-Hua
%A HU Xian-Wen WU Qing-Fa XIAO Cheng-Zu XU Zhao-Ping ZHANG Zheng-Guang
%A <br>高丽华
%A 胡显文
%A 吴清法
%A 肖成祖
%A 胥照平
%A 张正光
%J 生物工程学报
%D 2002
%I 
%X The only difference of primary structure between single-chain prourokinase (pro-UK or scu-PA) and two-chain urokinase (UK or tcu-PA) is the cleavage of a single peptide bond (Lys158-Ile159) and transform scu-PA into its active two-chain form. A 13-peptide (Thr-Leu-Arg-Pro-Arg-Phe-Lys-Ile-Ile-Gly-Gly-Glu-Cys), which spans the cleavage peptide bond, was synthesized and linked to KLH (Keyhole limpet hemocyanin). The Balb/c mice were immunized by the conjugated protein with proper adjuvant. According to the Kohler and Milstein's methods, a hybridoma cell line G7 secreting monoclonal antibody specific for scu-PA was obtained. The anti-scu-PA McAb, purified from the supernatant of porous microcarrier hybridoma cell culture, was conjugated to CNBr-activated Sepharose 4B to prepare an immuno-affinity chromatography column. The u-PA was purified only by this affinity column from the supernatant of cultivating the u-PA-producing recombinant CHO cell, the u-PA recovery ratio is 90.4%, the purification factor was about 50, with the specific activity of 1.2 x 10(5) IU/mg, the scu-PA ratio in the u-PA product was 96.3%. Compared to immuno-affinity chromatography, the 3-step process for purifying u-PA (cation-exchange column, gel filtration column and benzamidine affinity column) has a u-PA recovery ratio of about 65%, with a specific activity of 1.0 x 10(5) IU/mg, and an scu-PA ratio of about 90%. These results showed that immuno-affinity chromatography is simple to recover u-PA and effective to separate scu-PA from tcu-PA.
%K prourokinase
%K monoclonal antibody
%K immuno-affinity chromatography
%K hybridoma cell culture
%K porous microcarrier<br>尿激酶原，单克隆抗体，免疫亲和层析，杂交瘤细胞培养，多孔微载体
%U http://www.alljournals.cn/get_abstract_url.aspx?pcid=90BA3D13E7F3BC869AC96FB3DA594E3FE34FBF7B8BC0E591&jid=A66E90C274451689E69F6F0291467824&aid=B8E960BB3E6DD862&yid=C3ACC247184A22C1&vid=13553B2D12F347E8&iid=38B194292C032A66&sid=35E8A259891FB32F&eid=3356A7630A93A219&journal_id=1000-3061&journal_name=生物工程学报&referenced_num=1&reference_num=17