重组抗体-尿激酶导向溶栓剂的基因构建及表达  

Construction and Expression of a Recombinant Antibody targeted Plasminogen Activator Composed of a Humanized Monoclonal Antibody Against Activated Human Platelets and a Single chain Urokinase

作　　者：刘征辉[1,2] 万海英[1,2] 王斌 李佩霞 茹炳根[1,2] 胡美浩 阮长耿[1,2] 

机构地区：[1]苏州医学院附属第一医院江苏省血液研究所 [2]北京大学生命科学学院

出　　处：《生物化学与生物物理学报》1998年第6期601-606,共6页

基　　金：国家"863"高科技计划

摘　　要：为了获得高效、高特异性溶栓药物，应用基因工程技术，成功的表达了由人源化抗人活化血小板单抗和单链尿激酶组成的抗体导向溶栓剂（ＳＺ５１Ｈｕ－ｓｃｕＰＡ）。通过基因重组ＰＣＲ方法将ｓｃｕＰＡ全长ｃＤＮＡ的Ｎ末端连接在ＳＺ５１重链恒区ＣＨ３末端，构建了含有目的蛋白融合基因的真核表达载体αｌｙｓ３０－ＳＺ５１ＶＨ／Ｈｕ－ｓｃｕＰＡ。采用脂转染法将表达载体导入分泌ＳＺ５１ＶＫ／Ｈｕ轻链的小鼠骨髓瘤细胞中，筛选出三株能持续分泌ＳＺ５１Ｈｕ－ｓｃｕＰＡ的高表达细胞株。培养上清中ＳＺ５１Ｈｕ－ｓｃｕＰＡ表达量约达５ｍｇ／Ｌ。纯化的表达产物在非还原条件下分子量为１６０ｋＤ。Ｗｅｓｔｅｒｎ印迹证实ＳＺ５１Ｈｕ－ｓｃｕＰＡ与亲本鼠源单抗ＳＺ５１具有相同的抗原结合特异性，酶活力为３９０００ＩＵ／ｍｇ总蛋白。上述结果表明，我们已成功地获得一个兼具抗体特异性和纤溶酶原激活特性的新型抗体导向溶栓剂，为进一步进行体内外导向溶栓研究奠定了基础。To obtain a thromblytic agent with high efficacy and specifity, we have engineered a recombinant chimeric plasminogen activator SZ51Hu scuPA, which was composed of a humanized monoclo nal antibody(SZ51Hu) directed against activated human platelets and a single chain urokinase(scuPA). The cDNA sequence encoding scuPA was fused through 5′end to 3′end of the CH3 of SZ51Hu heavy chain sequence in the expression vector αlys30(αlys30 SZ51VH /Hu scuPA ) by PCR. This construct was transfected into a murine myeloma line secreting SZ51Hu light chain with Lipofectin reagent and three lines which showed stable integration and high level expression with concentration of 5 mg/L in supernatant, were selected in the end. Purified SZ51Hu scuPA migrated as a 160 kD band on non reduced SDS/PAGE and had the same characteristic of binding to the activated platelets compared with its parent murine antibody molecule SZ51 by Western blot analysis. The fibrinolytic activity of purified products was 39 000 IU/mg. Thus, by recombinant techniques, an expressed fusion protein was capable of specific binding to activated platelets and plasminogen activation. These observations laid a solid foundation for further study of targeting of thrombolysis in vitro and in vivo.

关 键 词：活化血小板 单抗 单链尿激酶 基因表达 导向溶栓 

分 类 号：R394[医药卫生—医学遗传学]