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

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37oC PBS(0.2% HAS)中孵育1 h后活性即开始下降, 24 h后活性完全丧失; 而dsCD3-diabody孵育72 h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。

Abstract:

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°C 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.