抗氨基末端脂多糖结合蛋白基因工程抗体库的构建及体外表达筛选  

Construction of Single Chain Antibody Library Against Human NH-lipopolysaccharide Binding Protein(NH-LBP) and In Vitro Expression of NH-LBP Antibody

作　　者：王长松[1] 刘友生[1] 李红[1] 葛晓冬[1] 邓军[1] 陈燕平[2] 

机构地区：[1]第三军医大学西南医院病理学研究所,重庆400038 [2]第三军医大学西南医院国际合作实验室,重庆400038

出　　处：《中国生物制品学杂志》2005年第3期177-181,共5页Chinese Journal of Biologicals

基　　金：国家自然科学基金资助项目(30271342).

摘　　要：目的构建人源化的单链可变区抗体库,运用体外表达技术翻译并筛选特异性的抗NHLBP抗体。方法分离健康人外周血单核细胞,抽提总RNA设计引物,扩增抗体轻重链的可变区,通过一段柔性片段将其连接成单链抗体库;应用体外表达技术翻译抗体库,并筛选出与NHLBP高度结合的单链抗体片段。结果扩增的单链抗体可变区片段分别为340bp和325bp,经连接后约为750bp。经过体外表达得到了约27000的抗体片段,经过5轮筛选进化,得到了可与NHLBP高度结合的抗体。结论已成功构建了人源化的单链抗体库;通过体外表达技术筛选到可与NHLBP高度结合的抗体。Objective To construct a single chain Fv library and screen specific ScFv against humanized NH-lipopolysaccharide binding protein(NH-LBP) by coupled transcription/translation system in vitro.Methods Isolate peripheral blood mononuclear cells(PBMC) from healthy donors,extract total RNA,design primers and amplify the variable regions of light and heavy chain of antibody library by RT-PCR.Link heavy chain and light chain by a flexible fragment(Gly_4Ser_1)_3.The single chain antibody fragments were linked with T7 promoter and AUG by PCR,and expressed by coupled transcription/translation system in vitro,then specific single chain antibody against NH-LBP was screened.~Results The lengths of V_H and V_L of antibody were 340 bp and 325 bp respectively,and that of ScFv was 750 bp.A antibody fragment binding to NH-LBP,with a relative molecular weight of 27 000,was obtained by coupled transcription/translation system in vitro.Conclusion A single chain Fv library was successfully constructed,and a antibody fragment binding to NH-LBP was screened by coupled transcription/translation.

关 键 词：体外表达 脂多糖结合蛋白 抗体库 筛选 构建 基因工程 外周血单核细胞 氨基 单链抗体 可变区 总RNA 人源化 片段 特异性 技术 健康人 高度 翻译 连接 引物 进化 

分 类 号：Q75[生物学—分子生物学] Q513