T7启动子促发下的人尿激酶原在大肠杆菌中的高效表达

将人工全化学合成的尿激酶原（ｐｒｏ－ｕｒｏｋｉｎａｓｅ）ｃＤＮＡ克隆到表达载体ｐＥＴ３ｄ中，转化大肠杆菌ＢＬ２１（ＤＥ３）ＬｙｓＳ，经ＩＰＴＧ诱导，获得了占菌体总蛋白１８％的高表达。经ＳＤＳ－ＰＡＧＥ和Ｗｅｓｔｅｒｎ－ｂｌｏｔ鉴定，表达产物主要为单链尿激酶，并且在菌体内基本以无活性的包涵体形式存在。经体外变复性，从ＩＬ培养基中可获得３０００００单位的活性尿激酶原。

HIGH EXPRESSION OF HUMAN PRO-UROKINASE PROMOTED BY T7 PROMOTER IN EXCHERICHIA COLI

Pro-urokinase (Pro-UK),the second generation of thrombolytic agent,was superior to urokhoe by its more specific sanity to fibrin and mild enzymatic activity,whichmeant that it had less tendency to cause haemorlhage when injected with a large dale.Hut because Of its rare natural sources,Pro-UK could not be enough for cjjnic theral)y unless it wasproduce by genetic engineering methods.As reported up to now, the expression level Of Pro-UK cDNA in E.colt bed hardly been more than 2% of total bacterias proteins.In this article,a humam Pro-UK cDNA controlled by T7 promoter was reported to besuccessfully expressed in E. Colt with 18% expression level.To create pET3d/Pro-UK cDNA,fragment of Pro-UK gene daft from PUCg /Pro-UK plasmid by Hindlll (Klellow blunted)and BspHI,was adverted into pET3d expressionvector,whiCh was cut with BamHI (Klenow blunted) and Ncol.The positive recombinat.edplasmid was confirmed by cuting with several restriCtion enzymes,and was transformed intoE.coli BL21(DE3) LysS.Induced by 1 mmol/L IPTG,Pro-UK cDNA was expressed withan extra band of 43KDa recognbo .on SDS-PAGE, and the band was proved to be humanPro-UK by western blotting analysis.The densitometric scanning result.revealed that the expression level of Pro-UK was 18% of total cellular proteins.The exprssion bactetia were pollinated and the resultant lysate and sediment were collected separately. After denaturation andrenatulation in vitro,the sediment part disptayed fibrinolytic activity about 300,000 IU perLiter culture medium,while the supernatant rarely had the same activity.This Tesult.suggested that the expression product of PET 3d /Pro-UK was mainly in the form of inclusion btulies.