一种融合抗体ScFv-Fc通用表达载体的构建

为了构建一个可供自由替换的ScFv区,表达人小分子融合抗体ScFv-Fc的通用载体,利用RT-PCR技术扩增人抗体IgG1的Fc片段克隆至毕赤酵母表达载体pPICZα,将一段人工合成的互补寡核苷酸链插入重组载体pPICZα/Fc中Fc区的上游,引入2个可供小分子抗体ScFv-Fc的ScFv区自由替换的限制性酶切位点。分别扩增人抗狂犬病毒以及抗乙型肝炎表面抗原的ScFv片段,克隆至已构建的通用载体pPICZα/Fc,在毕赤酵母中诱导表达。进一步在1L条件下对活性抗体进行发酵,并利用protein A亲和层析柱进行纯化。应用酵母基因组PCR、ELISA、Western blotting、活性检测等试验对此小分子抗体的表达进行生物学及免疫学分析。结果表明具有狂犬病毒抗原结合活性以及乙肝表面抗原结合活性的人源抗体分子均获得成功表达,1L发酵条件下表达量达到20~30mg/L, protein A亲和层析纯化后纯度＞95%。研究构建了可用于功能性抗体分子ScFv-Fc筛选和表达的通用载体并对其发酵、纯化条件进行了摸索,为重组抗体分子诊断、治疗试剂的开发以及抗体的人源化奠定了物质基础。

A Vector System for the Production of Single-chain Fv-Fc Fusions in Pichia pastoris

Recombinant antibodies, especially ScFv fragments, can be applied as detection reagents and even substitute for some reagents used in immunoassays such as antibody-enzyme conjugates. For ScFv fragments, a universal system available is necessary. A vector system was constructed based on pPICZα/Fc, in which the hinge, CH2 and CH3 domains (Fc fragment) of human IgG1 and His-tag were cloned into the Pichia expression vector pPICZα. Two fragments of ScFv were introduced into pPICZα/Fc, which can bind HBsAg and rabies virus antigen, to yield the expression cassette pPICZα/ScFv-Fc. Following fermentation in a 1-liter reactor, the fusions were expressed at high levels in the methylotrophic yeast Pichia pastoris, secreted as dimeric forms in the culture, and purified by protein A column chromatography. The expression yield can reach 20~30mg/L of culture medium. The ScFv-Fc fusion proteins retain the biological binding ability of the parent ScFv. Furthermore, the successful expression and maintenance of the binding activity verify the efficacy of the vector system for use as detection reagents in vitro, by reacting with the specific antigens and being readily detected using general anti-human IgG antibodies.