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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。
Objective To construct a human ribosome display single chain antibody (scFV) library. Methods Total RNA was extracted from human peripheral blood mononuclear cells and reverse transcribed into cDNA. As a template, a number of pairs of primers with degenerate characteristics were designed and PCR amplified human immunoglobulin heavy chain variable region (VH) And light chain variable region (VL) genes were added to both ends with the necessary elements for ribosome display and ribosomal display was constructed by linking VH and VL to the single chain antibody via (Gly4Ser) 3 short peptides by overlapping PCR Library. Results The majority of primers could amplify VH and VL fragments of 300-400 bp by using different primers. The components required for ribosome display were successfully amplified by large primers, and the overlap PCR was performed in vitro for about 900 bp Size scFv gene, a large number of amplified ribosome display library. Conclusion The human ribosomal scFv library has been successfully constructed and laid the foundation for the development of human antibody drugs.