抗GPC3单链抗体的制备及其在肝细胞癌靶向检测中的应用

Preparation of anti-GPC3 single chain antibody for targeted detection of hepatocellular carcinoma

摘要:

磷脂酰肌醇蛋白聚糖3 (Glypican-3，GPC3) 是Glypican家族的关键成员，它在肝细胞癌 (HCC) 的发展、血管生成和转移中起着重要作用。大多数HCC过表达GPC3，而在正常的成人肝脏和良性肝脏病变中几乎检测不到GPC3，因此其可以作为HCC高度特异性的诊断标志和理想的治疗靶标。文中将前期筛选得到的靶向GPC3的单克隆抗体1B11轻重链基因通过柔性肽 (Linker) 连接，经原核表达纯化，成功获得抗GPC3单链抗体1B11-scFv。通过蛋白印迹法鉴定验证了单链抗体的表达和装配正确；流式细胞术检测其与GPC3阳性细胞株具有与全长抗体相当的结合活性；将单链抗体与近红外荧光探针Cy5.5-NHS偶联，发现其在荷瘤裸鼠体内可精准靶向肿瘤部位，提示其具有实现探针引导下的多方位肝癌精准手术导航的潜力。

Abstract:

Glypican-3 (GPC3) is a key member of Glypican family and plays an important role in the development, angiogenesis and metastasis of hepatocellular carcinoma (HCC). Most HCC overexpresses GPC3, but GPC3 is hardly detected in normal adult liver and benign liver lesions, so it is regarded as a highly specific diagnostic marker and an ideal therapeutic target for HCC. In this study, we cloned the heavy and light chain variable region gene from the monoclonal antibody targeted to GPC3 screened in the previous stage, and linked it with a segment of flexible peptide (Linker) to obtain the single chain antibody against GPC3. The single chain antibody gene was cloned into vector for prokaryotic expression and purified to obtain high purity protein. Detection shows that the single-chain antibody produced by us has the same binding activity with the full-length antibody, and can accurately target the tumor site of Huh7 tumor-bearing model mice after coupling Cy5.5 fluorescence, suggesting that the single-chain antibody has the potential to realize multi-directional liver cancer precise surgical navigation under the guidance of a probe.