【摘要】 以针对人活化血小板表面糖蛋白ＧＭＰ１４０的单克隆抗体ＳＺ５１的单链抗体作为导向分子，以单链尿激酶３２ｋｄ变体（ｓｃｕＰＡ３２ｋ）作为效应分子，构建了一种新型的导向性溶栓剂。通过聚合酶链式反应（ＰＣＲ），分别从ＳＺ５１的Ｆａｂ片段基因中扩增出ＶＫ和ＶＨ区；从尿激酶原基因中扩增出ｓｃｕＰＡ３２ｋ基因。通过合适的ｌｉｎｋｅｒ及酶切位点，将ＶＫ，ＶＨ及ｓｃｕＰＡ３２ｋ基因相连接并装入表达载体ｐＥＴ５ａ中的ＮｄｅＩ位点，在大肠杆菌中经ＩＰＴＧ诱导表达了重组蛋白。ＷｅｓｔｅｒｎＢｌｏｔｔｉｎｇ检测到在８ｍｏｌ／Ｌ尿素存在下，该重组蛋白与抗尿激酶的多克隆抗体之间仍有弱的结合反应。表达产物经变复性处理和部分分离纯化后，在纤维蛋白平板上表现有较好的纤溶活性，且这种活性是通过激活纤维蛋白溶酶原为纤溶酶而实现的。重组蛋白纤溶活性的比活达到１７５００ＩＵ／ｍｇ，较好地保留了尿激酶的纤溶活性，重组蛋白的产率约每１００ｇ湿菌为１５ｍｇ。更多还原

【Abstract】 A novel plasminogen activator was constructed using scFv of SZ51 as targeted mo lecule, and scu PA 32k as effect molecule. SZ51 was a monoclonal antibody of G MP140 on activated human platelets. Polymerase chain reaction (PCR) was used to amplify the region of V K and V H from Fab of SZ51, and scu PA 32k(leu144 l eu411) from urokinase gene, respectively. Through suitable linker and appropriat e restriction sites, these fragments were joined together and inserted into the expression vector, pET 5a, via Nde I site. After transforming in E.coli BL2 1 (DE3) plyS and inducing with IPTG, the recombinant protein was expressed in in clusion bodies. It was shown in Western Blotting that the protein could interac t weakly with the multiple clonal antibody of urokinase in 8 mol/L of Urea. Afte r renaturation and partial purification, the product had a strong fibrinoly tic activity through activating plasminogen on fibrin plate, the specific activi ty was about 17?500 IU/mg, which showed the recombinant protein restored the ac tivity of u PA quiet well. The yield was almost 1.5mg/100g wet bacteria.更多还原