人源核糖体展示单链抗体库的构建

目的构建人源核糖体展示单链抗体(scFV)库。方法从人外周血单个核细胞中提取总RNA,反转录为cDNA,以此为模板,设计多对具有简并性特点的引物,PCR扩增人免疫球蛋白重链可变区(VH)和轻链可变区(VL)基因,在其两端加上核糖体展示所需元件,并通过重叠PCR法将VH和VL经(Gly4Ser)3短肽连接成单链抗体,构建核糖体展示库。结果利用不同引物进行PCR时,绝大多数引物能扩增出300～400 bp的VH和VL片段;通过大引物扩增成功加上了核糖体展示所需元件,重叠PCR体外连接成约900 bp大小的scFv基因,大量扩增得到核糖体展示库。结论已成功构建了人源核糖体展示scFv库,为人源抗体药物的开发奠定了基础。

Construction of Human Ribosome Display scFv Library

Objective To construct human ribosome display scFv library.Methods Total RNA was extracted from human peripheral blood mononuclear cells(PBMCs)and reversely transcribed to cDNA,then used as a template for amplification of VH and VL genes of human IgG by PCR with several pairs of designed degenerate primers.The necessary elements to ribosome display were introduced at two terminuses of the amplified VH and VL genes which were linked through(Gly4Ser)3 short peptide by overlapping PCR to construct a ribosome display scFv library.Results The VH and VL gene fragments at lengths of 300 ~ 400 bp were ampli-fied by PCR using various primers.The scFv gene at a length of about 900 bp,with necessary elements to ribosome display intro-duced,was obtained by overlapping PCR using long primers,based on which a ribosome display scFv library was constructed by am-plification in a large quantity.Conclusion A human ribosome display scFv library was successfully constructed,which laid a foun-dation of developing human antibodies as drugs.