从人源单链抗体库中筛出具有抗原结合活性的重链可变区和更小抗体片段

胡志伟　段招军　朱建高　周国华　胡锦跃　孙去病

　　中国图书分类号　R730.3
　　摘　要　目的：用噬菌体呈现技术结合体外致敏法构建了人源单链抗体库，并用大肠癌相关抗原CA-Hb3筛选出抗大肠癌噬菌体抗体克隆EL5D、EL6D和Ca12，测定3个克隆的核苷酸序列，并研究其免疫活性。方法：采用抗体克隆扩增、测序分析、免疫组化法测定活性。结果：抗体基因插入片段分别为220、500、500 bp。测序结果表明，EL6D和Ca12属VH5-D-JH4，仅为VH结构亚单位，而EL5D属Vκ的Jκ1，仅为FR3-CDR3-FR4结构。三者均能与人结直肠癌细胞和组织反应，与其亲本鼠源单抗Hb3的结合特性一致。3个抗体克隆已列入国际基因库，序号为AF051165-AF051167。结论：短的人源抗体片段具有潜在临床应用价值。
　　关键词　人免疫球蛋白基因　单链抗体库　大肠癌靶向　重链可变区　互补决定区

VH and shorter fragments with antigen-binding activities biopanned from human repertoire single-chain Fv libraries
HU Zhi-Wei,DUAN Zhao-Jun,ZHU Jian-Gao et al. Laboratory of Tumor Immunobiology,Cancer Research Institute,Hunan Medical University,Changsha 410078

　　Abstract Objective:Phage display technology is the best method for humanizing mouse-original monoclonal antibodies.Human repertoire single-chain Fv libraries had been constructed with phage display technology in combination of immunization in vitro,and three phage antibody clones(EL5D,EL6D and Ca12)were biopanned with an colorectal-carcinoma associated antigen CA-Hb3.The object of this study is to detect the sequence of these antibody clones and study immunological activity.Methods:The antibody sequences were stadied and it s immune activities were detected by immunohistochemistry.Results:The inserts of EL5D,EL6D and Ca12 were of 220,500 and 500 bp,respectively.The sequences of EL6D and Ca12 belonged to subgroup VH5-D-JH4 and were just VH region.The sequence of EL5D,however,belonged to Vκ and just were Jκl region containing partial FR3-CDR3-FR4 of kabba light chain.They could specifically bind with colorectal carcinoma cell lines and tissues,which were identical with their parent mouse monoclonal antibody Hb3.The accession numbers of international genbank are AF051165-AF051167.Conclusion:The shorter antibody fragments would have better clinical application.
　　Key words Human immunoglobulin genes　Single-chain Fv library　Colorecta

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