中国免疫学杂志2012，Vol.28Issue(2)：146-149,4.DOI:10.3969/j.issn.1000-484X.2012.02.011
人源Fab噬菌体抗体库的构建与抗精氨酸加压素抗体的筛选
Construction of a human na(i)ve Fab library and screening of phage antibody against arginine vasopressin
摘要
Abstract
Objective: To construct a naive human Fab phage display library, screen and identify arginine vasopressin Fab antibody from the library. Methods: Total RNA was extracted from peripheral blood lymphocytes of 18 healthy donors, and the light chain and heavy chain Fd genes were amplified by RT-PCR. Then the amplification products were sequentially cloned into phagemid vector pComb3XSS to construct a human Fab phage antibody library. The insertion of the light chain or heavy chain Fd genes were identified by cutting with endonucleases and PCR amplification. Arginine vasopressin was used as target antigen to pan the original Fab antibody library. After five rounds of panning were carried out,fifty randomly selected clones were assayed by phage-ELISA analysis. The positive clones were analyzed by DNA sequencing. Results: A large human Fab phage antibody library consisting of 2.4 × 108 members was successfully constructed. After having been panned by AVP,we obtained six positive clones which had specificity and binding reactivity towards AVP. The C4 clone was analyzed and showed that its heavy chain belonged to IgG subvariety and its light chain to X family. Conclusion : We successfully constructed a large human Fab phage antibody library and isolated the specific human anti-AVP Fab antibodies,which provided a solid foundation for the establishment of rapid detection method of arginine vasopressin in future research.关键词
精氨酸加压素/噬菌体抗体库/Fab段/筛选Key words
Arginine vasopressin/Phage antibody library/ Fab fragment/Screen分类
医药卫生引用本文复制引用
董越华,胡占东,公倩,李常颖,畅继武,朱铁虹..人源Fab噬菌体抗体库的构建与抗精氨酸加压素抗体的筛选[J].中国免疫学杂志,2012,28(2):146-149,4.基金项目
本文为天津市自然科学基金资助项目(10JCYBJC11900) （10JCYBJC11900）
