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目的:构建人源天然库容量大、多样性好的核糖体展示单链抗体(scFv)库。方法:采集人新鲜外周血分离淋巴细胞,提取总RNA,用RT-PCR扩增人抗体VH和VL基因,同时扩增作为间隔区的人抗体CK基因;采用重叠延伸PCR(简称SOEPCR)技术连接VH-VL-CK基因,同时引入T7启动子和核糖体结合位点序列,体外构建核糖体展示scFv库模板,连接T-Vector转化E.coliDH5α大肠杆菌,蓝白筛选,挑取阳性克隆测序以鉴定scFv组装。结果:成功构建了库容量达3.1×1013的人源性胃癌核糖体展示scFv库。结论:构建的大容量人源性胃癌核糖体展示抗体库可以成为进一步筛选多种特异性人源抗体的实验平台,为开发治疗性人源抗体奠定了很好的实验基础。
OBJECTIVE: To construct a ribosomal display single chain antibody (scFv) library with large capacity and diversity of human natural pools. Methods: Lymphocytes were harvested from fresh peripheral blood of patients and the total RNA was extracted. The VH and VL genes of human antibodies were amplified by RT-PCR. At the same time, the CK gene of human antibody was amplified. The overlap extension PCR (SOEPCR) VH-VL-CK gene, and introduced the sequence of T7 promoter and ribosome binding site. The ribosome display scFv library template was constructed in vitro. T-Vector was transformed into E.coli DH5α E.coli and blue-white screening. The positive clones were sequenced with Identify scFv assembly. Results: The human gastric cancer ribosome display scFv library with a capacity of 3.1 × 1013 was successfully constructed. CONCLUSION: The constructed large-capacity human gastric cancer ribosome display antibody library can be used as an experimental platform for further screening a variety of specific human antibodies, and has laid a good experimental foundation for the development of therapeutic human antibodies.