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

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.