大容量人源Fab噬菌体展示抗体库的构建及抗CD276抗体筛选

Construction of a large human Fab phage display antibodies library and screening of phage antibodies against CD276

目的构建大容量人源Fab噬菌体库,筛选并鉴定抗CD276的特异性抗体。方法通过采集10位健康成人的外周血淋巴细胞,提取总RNA,利用RT-PCR扩增人Fab抗体基因片段,插入噬菌体载体pCANTAB5H中,构建人源Fab噬菌体抗体库。通过固相化的抗原对抗体库进行3轮筛选后,随机挑取96个单克隆进行phage-ELISA鉴定,筛选出与抗原具有较强结合性的噬菌体克隆。结果成功构建库容为5×1010的噬菌体抗体库,从中筛选到11株阳性克隆,对其进行测序鉴定,表明该11株克隆的序列均一致,ELISA证实其对抗原CD276具有较强的结合性。结论构建了大容量人源Fab抗体库,从中获得具有抗CD276的人源Fab抗体片段,为进一步的研究和应用提供实验基础。

Objective To construct a large human Fab antibodies library and to screen and identify the anti-CD276 phage antibodies from the library. Methods Total RNA was extracted from peripheral blood lymphocytes of 10 healthy donors, and the Fab antibody genes were amplified by RT-PCR. The amplification products were sequentially cloned into phage vector pCANTAB5E to construct a human Fab phage antibodies library. Antibodies against CD276 were screened using immobilized antigen. After three rounds of screening, 96 randomly selected clones were identified by phage-ELISA to select specific ones with high affinity for CD276. Results A large human Fab phage-display library consisting of 5 × 10^10 members was successfully constructed. From the phage library, we obtained eleven positive clones which had specificity and binding reactivity towards CD276. By sequencing, all of the 11 clones had the same sequences, and their specificity was confirmed by ELISA. Conclusion We successfully constructed a large human Fab phage antibodies library and isolated the specific human anti-CD276 Fab antibodies, which provided an experimental foundation in future study and application.