噬菌体展示EpCAM单链抗体的免疫原性初步研究 - 第三军医大学学报.DOCVIP

噬菌体展示EpCAM单链抗体及免疫原性初步研究 刘晓丽1，张岸梅1，林 盛1，王欣欣1，廖荣霞2，孙建国1* 陈正堂1*（400037重庆，第三军医大学全军肿瘤TG1,随机挑取5个克隆，经辅助噬菌体超感染，形成鼠抗人EpCAM噬菌体单链抗体，用ELISA(以P／N2判定为阳性)和免疫细胞化学法测定其抗原结合活性。结果 成功获得pCANTAB-5E/EpCAMScfv重组克隆，经ELISA和免疫细胞化学测定，噬菌体展示阳性率为20%，展示的单链抗体能与EpCAM抗原和A549细胞特异性结合。结论 噬菌体展示技术制备EpCAM单链抗体具有方法简便、省时、高效等特点，有望成为鉴定上皮来源肿瘤细胞及癌干细胞的重要方法。 [关键词] 单链抗体；噬菌体展示；EpCAM；免疫；肿瘤[中图分类号] 　 　　　[文献标码] Preliminary study on immunogenicity of phage-displayed mouse-anti-human EpCAM single-chain antibody Liu Xiaoli1，Zhang Anmei1, Lin Sheng1, Wang Xinxin1，Liao Rongxia2，Sun Jianguo1，Chen Zhengtang1 (Cancer Institute of PLA, Xinqiao Hospital, Third Military Medical University, Chongqing, 400037) [Abstract] Objective To express mouse-anti-human EpCAM single-chain antibody (ScFv) specifically targeting tumor cells or cancer stem cells of epithelial origin via phage display technology, and to identify its immunological activity, Methods Total gene synthesis method was used to synthesize mouse-anti-human EpCAM ScFv gene with a structure of VL + Linker + VH. The gene was cloned to pMD19-T Simple Vector and then was digested, after being purified, was cloned to phage vector pCANTAB 5E. Competent E. coli TG1 cells were transformed with pCANTAB 5E containing ScFv gene. Mouse-anti-human EpCAM ScFv was produced by helper phage M13K07 superinfection with 5 randomly selected clones, and the antigen binding activity was identified by ELISA and immunocytochemistry. Results Recombinant pCANTAB5E/EpCAM ScFv was constructed successfully, with a positive antigen-binding rate of 20% proven by ELISA and immunocytochemistry. The selected ScFv could specifically bind to EpCAM antigen and human lung adenocarcinoma cell line A549. Conclusion Mouse-anti-human EpCAM ScFv produced via phage display technology has advantages such as simple, highly efficient, and time-saving ,which is expected to be helpful in diagnosing tumor cells and cancer stem cells of epithelial origin. [Key words] single-chain antibody；phage display；EpCAM；immunity；cancer