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噬菌体抗体库技术是获得治疗性抗体的一条重要途径。以20份健康人外周血为样本,通过提取淋巴细胞、逆转录-PCR(RT-PCR)、抗体可变区基因的扩增、重叠PCR获得单链抗体(ScFv)基因,将ScFv克隆入噬粒载体,通过近300次的电转化获得了库容量为1.3×109的全人源天然ScFv噬菌体抗体库。通过随机挑克隆测序和用5种不同抗原筛选对抗体库进行了初步验证。随机测序表明抗体库具有较好的多样性,用5种不同抗原对其进行筛选,均获得了特异性噬菌体抗体的不同富集,表明成功构建了一个多样性良好的人源天然ScFv噬菌体抗体库。
Phage antibody library technology is an important way to obtain therapeutic antibodies. A total of 20 healthy volunteers were enrolled in this study. Single-chain antibody (ScFv) was obtained by overlapping PCR by extracting lymphocytes, RT-PCR and variable region antibody. The ScFv gene was cloned into the phagemid The granule vector, a fully human natural ScFv phage antibody library with a library size of 1.3 × 109, was obtained by nearly 300 electroporation. The antibody library was preliminarily verified by random cloning sequencing and screening with five different antigens. Random sequencing showed that the antibody library has a good diversity, with five different antigens were screened, were obtained different enrichment of specific phage antibodies, indicating the successful construction of a good diversity of human natural ScFv phage antibody library .