一种具有人VEGF结合活性的人源单一重链可变区的克隆与表达

Cloning and expression of a single human immunoglobulin heavy-chain variable domain with vascular endothelial growth factor binding activity

摘要:

为避免一种来自五特征转基因小鼠的全人VEGF单克隆IgM抗体分子量大的不足，本研究探讨了该抗体单一重链可变区的功能特性。首先，PCR获得该抗体的重链可变区，将该序列克隆至pET28a表达载体内，在大肠杆菌中进行了诱导表达。通过变性纯化和复性等方法获得了具有生物学活性的16 kDa重组抗体片段——rhVVH。体外结合实验表明，rhVVH保留有完整免疫球蛋白的人VEGF结合活性。人脐静脉内皮细胞 (HUVEC) 增殖抑制实验表明：rhVVH可以剂量依赖性的抑制HUVEC的增殖。上述结果揭示了该抗体单一重链可变区保留有完整抗体的部分功能，为进一步开展全人源VEGF单克隆IgM抗体小型化研究奠定了基础。

Abstract:

In the application of therapeutic antibodies, large molecular weight of antibodies is always a problem that prevents them from penetrating into tissues or binding to antigenic determinants. To overcome this problem, we investigated the function of the heavy chain variable domain of a monoclonal anti-VEGF human IgM antibody derived from the Five-Feature Translocus Mice. We cloned the cDNA of the heavy chain variable domain, which was then inserted into pET28a vector and expressed in Escherichia coli. After purification and renaturation of the denatured recombinant protein, we obtained a 16 kDa antibody fragment, which is named as rhVVH. By immunoassaying its VEGF-binding capability in vitro, we proved that rhVVH retains this activity as the complete IgM. Importantly, rhVVH is shown to inhibit the HUVEC cell proliferation in a concentration-dependent manner. Our results indicate that the single heavy chain variable domain might inherit part of the biological function of the complete IgM antibody, which provided a valuable potential in further research on antibody miniaturisation.