南方医科大学学报2012，Vol.32Issue(8)：1082-1087,6.DOI:10.3969/j.issn.1673-4254.2012.08.03
系统性红斑狼疮儿童特异性全长抗体基因库的构建与分析
Construction of personalized full-length fully human mammalian display antibody library for children with systemic lupus erythematosus
摘要
Abstract
Objective To construct a personalized full-length fully human antibody mammalian display library for children with systemic lupus erythematosus (SLE). Methods The total RNA was isolated from the PBMCs of SLE children. The heavy chain variable region and kappa light chain (VH and LCk) of the antibody genes were amplified by RT-PCR and inserted into the pDGB-HC-TM vector separately to construct the heavy chain and light chain libraries. The library DNAs were transfected into 293T cells and the expression of full-length fully human antibody on the surface of 293T cells was analyzed by flow cytometry. Results Using 0.8 ug total KNA as the template, the VH and LCk were amplified and the full-length fully human antibody mammalian display library was constructed. The VH and LCk gene libraries had a size of 9.4×104 and 8.4×104, respectively. Sequence analysis of 10 clones randomly selected from the VH and LCk gene libraries each showed that 8 heavy chain clones and 7 light chain clones contained correct open reading frames, and flow cytometry demonstrated that all the 15 clones express full-length antibodies on 293T cell surfaces. 293T cells co-transfected with the VH and LCk gene libraries expressed the full-length antibodies on the cell surface. Conclusion The personalized full-length fully human antibody library for SLE children constructed allows display of the full-length antibodies on mammalian cell surfaces, thus providing a valuable platform for analyzing the autoantibodies, their etiological role, and their clinical implications in SLE.关键词
系统性红斑狼疮/儿童/哺乳动物细胞表面展示/全长人源抗体库Key words
systemic lupus erythematosus/ children/ mammalian display/ full-length fully human antibody library分类
医药卫生引用本文复制引用
周志刚,周烨,周辰,朱美华,梁中锟,陈振瑞,贺微,李长征,谭万龙,姜世勃,刘叔文..系统性红斑狼疮儿童特异性全长抗体基因库的构建与分析[J].南方医科大学学报,2012,32(8):1082-1087,6.基金项目
国家自然科学基金(31100661) （31100661）
香港Dgen Biotech Ltd科研基金(DGB12012008) （DGB12012008）
十二五科技重大专项(2011ZX09506-004) （2011ZX09506-004）
高等学校博士学科点专项科研基金(20114433120019) （20114433120019）
广东省自然科学基金(S2011040002748) （S2011040002748）
