抗红细胞双价小抗体(Diabody)的构建及表达

Construction and Expression or Anti-RBC Diabody

单链抗体(SeFv)是一个重链可变区(V_H),一个轻链可变区V_L由连接肽(Linker)连在一起构成的单价小分子抗体(只有一个抗原结合位点),如将其改为双价,则可使抗体多位点结合抗原,提高抗体的效价,并可进一步构建双特异性抗体,在临床上有广泛的应用前景.单链抗体中V_H和V_L为顺利组合形成F_V结构,连接肽的长度一般不得少于14个氨基酸,连接肽缩短不利于同一肽链内V_H和V_L形成立体结构,则强迫不同分子间V_H和V_L结合,而形成双价F_v分子.本文对一个抗人红细咆的单链抗体进行了改造,经PCR等方法,将其连接肽由17个氨基酸[SR(GGGGS)_3]缩短为6个氨基酸(SRGGGS),强迫不同分子间的V_H和V_L组合成F_v,从而形成双价小分子抗体(Diabody).DNA序列测定证实连接肽序列编码六个氨基酸;抗体在大肠杆菌中分泌型表达后,具有血球凝集活性,证明其为双价;进一步用凝胶过滤分析,显示改造后抗体分子的分子量约为原单链抗体的两倍.因抗RBC双价Diabody可产生红细胞凝集反应,将抗红细胞与抗血清中异常成分(如抗HB-sAg等)的抗体构建成双特异性Diabody,有可能利用简单的血凝实验在临床上进行快速检测和诊断.

ScFv is univalent small Ab molecule comprising a VH domain and a VL domain con-nected by a polypeptide linker. In order to construct bivalent molecules, we modified the anti-hu-man RBC ScFv expressing vector by shortening the linker from 17 aminoacid residues 〔SR(GGGGS)3〕 to 6 aminoacid residues (SRGGGS) to force the pairing of VH and VL between two dif-ferent molecules to form bivalent antibody fragment (diabody). The bivalency of E coli expresseddiabody was proved by its ability to agglutinate human RBC. The dimerization of diabody was alsodemonstrated by gel filtration (size-exclution) chromatography.