人尿激酶原cDNA在昆虫杆状病毒真核表达系统中的高效表达

Expression of The Human Pro-Urokinase cDNA Product in Insect Cells Using Baculovirus System

人尿激酶原(pro-urokinase,pro-UK)是一种新型溶栓剂,优于尿激酶,具有血纤维蛋白特异性。为了在昆虫杆状病毒表达系统中高效表达pro-UK,我们在pAc373基础上,插入野生型AcMNPV polyhedrin启动子区-7～1碱基序列,构建了一个高表达转移载体pAcYT。分别经三次克隆将pro-UK cDNA正向插入到转移载体pAc373或PAcYT的BarnHI-KpnI位点上。用LiPofectin将pAcyT-UKDNA或pAc373-UK DNA与AcMNPV DNA共转染到昆虫Sf9细胞中,空斑法筛出重组病毒阳性克隆株。高效表达结果是:1.ELISA法确定重组病毒Sf9细胞分泌表达产物pro-UK为96mg/L培养基上清,平板法测定溶圈活性为1600IU/mL培养基上清;2.亲和层析一步法纯化表达产物,回收率选70％,以上,比活约为60000IU/mg;3.纯化的pro-UK,无论是否经还原处理,其SDS-PAGE图谱均为相同的单一条带,MR 50000;4.Western blot与SDS-PAGE图谱吻合。

A cDNA encoding human pro-urokinase (pro-UK) was inserted downstream from the polyhedrin promoter of a baculovirus Antographa californica nuclear polyhedrosis virus by recombination in rivo using a new transfer vector, pAcYT DNA. The pro-UK product was secreted from five strains of recombinant virus-infected Spodoptera frugiperda(Sf9) cells by visual screening. The yield of pro-UK was 96 mg/L medium determined by ELISA and its fibrinolytic activity was 1600 IU/mL medium. Final yield of 67mg/L was obtained from immunoaffinity chromatography with a 1000 fold purification factor and a recovery of more than 70%. The purified pro-UK product with a specific activity of 60 000 IU/mg consists of a single chain with Mr 50 000 as determined by SDS-PAGE and Western blotting.