嵌合分子ut-PA(1)sf-9表达产物的纯化及其性质的初步鉴定

嵌合蛋白 ut- PA( u- PA与 t- PA的嵌合型纤溶酶原激活剂 )基因删除编码 u- PA中 R1 78—R1 81的 1 2个核苷酸后 ,得到的嵌合分子 ut- PA( 1 )在昆虫细胞 sf- 9中表达 ,通过苯甲脒 -Sepharose 6B亲和柱层析对表达产物进行纯化 .得到的纯品走 SDS- PAGE,结果显示 ,其分子量为60 k D.血凝块溶解实验结果表明 ,嵌合分子 ut- PA( 1 )有良好的体外溶栓能力 .在不同剂量 PAI- 1的抑制条件下测定 ,亲本嵌合蛋白 ut- PA的活力分别下降 2 2 .7%和 1 3.8%时 ,嵌合分子 ut- PA( 1 )只分别下降了 1 2 .9%和 9.1 % .说明突变体 ut- PA( 1 )具有一定的对 PAI- 1抑制作用的抗性

Purification and Characterization of ut-PA(1) Expression Product in sf-9 Cells

A chimeric recombinant ut PA(1) in which the 12 necleotide acids encoding R178—R181 of chimeric protein ut PA had been constructed previously. The deleted amino acids were said to be related to the binding of u PA to PAI 1 (plasminogen activator inhibitor 1). The expression products of the chimeric gene ut PA(1) in sf 9 cells were isolated and purified by benzamidine Sepharose 6B. The molecular weight of ut PA(1) determined by non reduced SDS PAGE was about 60 kD. Clot lysis analysis showed the mutant ut PA(1) had the ability of lysis of clot in vitro. When inhibited by PAI 1, while the activity of the parent chimeric protein ut PA decreased by 22 7% and 13 8%, that of the mutant ut PA(1) decreased by 12 9% and 9 1%, respectively. The result showed that the mutant chimeric protein obtained the resistance to the inhibition of plasminogen activator inhibitor 1 (PAI 1).