二硫键稳定的抗CD3/抗Pgp微型双功能抗体的构建、表达及活性测定  

Construction and expression of disulphide stabilized anti-CD3/anti-Pgp diabody

作　　者：苏晔[1] 刘娟妮[1] 高瀛岱[1] 秦立[1] 杨铭[1] 王金宏[1] 许元富[1] 邵晓枫[1] 纪庆[1] 熊冬生[1] 杨纯正[1] 

机构地区：[1]中国医学科学院北京协和医学院血液学研究所血液病医院实验血液学国家重点实验室,天津300020

出　　处：《生物工程学报》2009年第7期1042-1048,共7页Chinese Journal of Biotechnology

基　　金：国家自然科学基金(No.30400558);津市科技发展计划项目(No.08ZCKFSH04100)资助~~

摘　　要：向抗CD3/抗Pgp(P-glycoprotein)微型双功能抗体(Diabody)中引入二硫键,使diabody两条链以共价键交联,增强其稳定性。用重叠PCR(Overlap PCR)和PCR方法,将抗CD3/抗Pgp diabody中抗CD3或抗Pgp轻重链可变区的适当部位定点突变成半胱氨酸,进行原核表达。表达产物经阳离子层析柱和抗Etag亲和层析柱分离纯化,还原性和非还原性SDS-PAGE电泳鉴定。应用间接免疫荧光和竞争性免疫荧光结合流式细胞分析技术(FCM)检测生物学活性。将改造前后的两种抗体分别置37oC含0.2%人血清白蛋白(Human serum albumin,HAS)的PBS中孵育,取多个时间点比较两者与两种把抗原的结合能力。基因重组质粒经测序证实序列正确。向抗Pgp轻重链引入突变的diabody(dsPgp-diabody)在原核表达系统中,SDS-PAGE显示纯化后的蛋白二硫键不能配对。而向抗CD3轻重链引入突变的diabody(dsCD3-diabody)能够在原核表达系统中进行可溶性表达,二硫键能够正确配对。并且与改造前的diabody相比,dsCD3-diabody的抗原结合特异性没有改变,抗原结合和竞争活性均无下降。改造前的diabody在37oCPBS(0.2%HAS)中孵育1h后活性即开始下降,24h后活性完全丧失;而dsCD3-diabody孵育72h后活性并无明显下降。本实验成功构建了dsCD3-diabody,并且能够进行可溶性表达。向diabody抗CD3轻重链区引入的二硫键在宿主菌中可以正确配对,抗原结合活性没有明显下降,而稳定性大大增强。向diabody轻重链区引入二硫键,以增强其稳定性的方法是可行的。We constructed and expressed an anti-CD3/anti-Pgp （P-glycoprotein） diabody previously. However, the two chains of diabody are associated non-covalently, resulting in being capable of dissociating. The aim of this study is to enhance the stability of the diabody. We introduced cysteine residues into the CD3 or Pgp V-domain to covalently lock the two chains together. The disulphide crosslinked diabody were expressed by Escherichia coli （E. coli） 16C9 and purified by a cation exchange column and an anti-Etag affinity chromatography. The purified proteins were verified through SDS-PAGE. Flow cytometry （FCM） was used to analyse the binding properties, competitive binding capacity and stability in vitro. The dsPpg-diabody failed to form disulphide bond properly. The designed disulphide bridge between the different chains of dsCD3-diabody was formed correctly. FCM demonstrated the dsCD3-diabody has specific antigen binding activity, the same binding activity and competitive binding activity as its parent diabody. The dsCD3-diabody retained the full activity even after 72 h incubation at 37℃ in human serum, in contrast, the parent diabody began to lose activity after only 1 h and lose all its activity 24 hours later. The induced disulphide bond in the CD3 V-domain effectively enhanced the stability of anti-CD3/anti-Pgp diabody. The method of stabilizing a diabody by introducing a disulphide bond into is practical.

关 键 词：基因工程抗体 DIABODY 二硫键 药物稳定性 

分 类 号：R392[医药卫生—免疫学]